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NORTHEASTERN UNIVERSITY

Graduate School of Arts and Sciences

Dissertation Title: Identification of a Novel 1,3-N-Acetylgalactosaminyltransferase Activity and its Unique 1,3-Homopolymer that forms the Giardia Cyst Wall

Author: Craig D. Karr Department: Biology

Approved for Dissertation Requirements of the Doctor of Philosophy Degree Dissertation Committee

______Edward Jarroll, Advisor Date

______David Newburg Date

______James Manning Date

______Wendy Smith Date

______Donald O’Malley Date

Head of Department

______Fred Davis Date

Graduate School Notified of Acceptance

______Director of Graduate Student Services Date

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Identification of a Novel 1,3-N-Acetylgalactosaminyltransferase Activity and its Unique 1,3-GalNAc Homopolymer that forms the Giardia Cyst Wall

A dissertation presented

by

Craig D. Karr

to

The Department of Biology

In partial fulfillment of the requirements for the degree of

Doctor of Philosophy

in the field of

Biology

Northeastern University Boston, Massachusetts

June 2009

2

3

 2009 Craig D. Karr ALL RIGHTS RESERVED

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Identification of a Novel 1,3-N-Acetylgalactosaminyltransferase Activity and its Unique 1,3-Homopolymer that forms the Giardia Cyst Wall

by

Craig D. Karr

ABSTRACT OF DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology in the Graduate School of Arts and Sciences of Northeastern University, June 2009

5 ABSTRACT

During the life cycle of the pathogenic protistan parasite, Giardia must differentiate into a metabolically-reduced infective form known as the cyst prior to exiting the host continuing the fecal-oral route of transmission. The mature cyst is surrounded by a thick filamentous outer cyst wall, once suggested to be composed of chitin (a 1-4-linked N-acetylglucosamine homopolymer) as in some fungal and other more closely related protozoan wall filaments. Because of the near cosmopolitan nature of chitin among eukaryotic microbes, crustaceans, insects, and some algae, and because of early lectin-binding data, this view was casually accepted for Giardia wall filaments.

However, no direct biochemical evidence was provided in support of the claim.

To address the nature of the cyst wall filaments, our direct biochemical data demonstrated the presence of a large amount of the unique cyst-specific sugar N- acetylgalactosamine (GalNAc) in isolated cyst walls with only trace amounts of the

GalNAc precursor N-acetylglucosamine (GlcNAc) found in mature cysts, and the presence of an inducible UDP-GalNAc synthesizing pathway including an inducible

UDP-N-acetylgalactosamine 4'-epimerase responsible for the synthesis of the UDP-

GalNAc precursor to the Giardia cyst wall filaments.

Now we demonstrate the presence of a novel 1,3-N-acetylgalactosaminyl transferase activity (3GaN-T) responsible for the synthesis of the unique cyst wall homopolymer of (1,3)--D-N-acetylgalactopyranosamine that comprises ca. 63% of the dry weight and essentially 100% of the carbohydrate of isolated filamentous cyst walls.

6 This newly described transferase activity catalyzes the following reaction:

UDP-GalNAc + (GalNAc-1,3-GalNAc)n (GalNAc-1,3-GalNAc)n+1 + UDP and has been given the common name cyst wall synthase (CWS) for its unique action in synthesizing the cyst wall carbohydrate homopolymer. The IUPAC-UMB systematic nomenclature proposed for CWS is UDP-N-acetylgalactosamine : (1→3)--D-N- acetylgalactopyranosamine (1→3)-N-acetylgalactosaminyltransferase (UDP-GalNAc :

(GalNAc-1-3-GalNAc)n (1→3)-N-acetylgalactosaminyltransferase). This enzyme activity may be abbreviated as 3GaN-T following current literature convention. The

3GaN-T activity was induced during encystment more than 600-fold from less than

0.001 nmol min-1 mg-1 protein (limit of detection) with peak activity at 24-36 hours post- induction and was purified 155-fold from microsomal encystment specific vesicles (ESV) by differential and isopycnic centrifugation, followed by detergent extraction. 3GaN-T co-localized with cyst wall specific proteins CWP1 and CWP2 to the microsomal ESV sub-population distinct from the lysosome-like peripheral vacuoles. The vesicle- associated Ca2+ / Mg2+-dependent activity was specific for the incorporation of [14C]-

GalNAc from UDP-[1-14C]GalNAc into an ethanol- or TCA-precipitate or filtrate with a

app app -1 -1 Km and a Vmax for UDP-GalNAc of 49.3 M and 0.701 nmol min mg protein, respectively. An endogenous or exogenous acceptor was not identified, which is consistent with other processive glycan synthases. 3GaN-T did not incorporate radiolabel from UDP-[1-14C]GlcNAc or other typical UDP-sugar substrates regardless of cofactor tested. Substrate analogs such as UDP-GlcNAc, Nikkomycin Z, Polyoxin D, and uridine 5'- tri, di, and monophosphate inhibited enzyme activity in a concentration- dependent manner.

7 The 3GaN-T reaction product was chemically-resistant in a manner similar to that of isolated cyst wall filaments, which were characterized with respect to composition, linkage, conformation, branching, substitution/modification, and chain length. Analysis revealed that the isolated filamentous cyst walls are composed of 63% dry weight N-acetylgalactopyranosamine in a 1→3 unbranched, unsubstituted, and nearly completely N-acetylated homopolymer of at least 23 residues in length, after partial acid hydrolysis.

8 ACKNOWLEDGEMENTS

To those who have helped me achieve my degree and to grow as a person and as a scientist ... I can't thank you enough.

Especially to my advisor, Ed Jarroll, I would like to express my sincere and deep appreciation for all that he has done for me. I will remember always his kindness as much as what he has taught me about research. Because of the opportunities he has provided me, I will never be the same person I was before.

I would also like to thank my committee members - Drs. David Newburg, Jim

Manning, Wendy Smith, and Don O'Malley - for their help.

No research is completed in a vacuum, and as such I would acknowledge the contributions of Drs. Stan Erlandsen and Tim Macechko (University of Minnesota

Medical School), and Bill Fowle and Rita Droste (Northeastern University) for providing expertise with electron microscopy.

For collaboration on glycan chemical analysis and structural studies, I would like to thank Drs. Hans Vliegenthart, Gerrit Gerwig, Albert van Kuik, Johannis Kamerling, and Bas Leeflang of the University of Utrecht, Netherlands.

I would also like to thank the Giardia researchers who have helped me along the way including Drs. Harry van Keulen, Don Lindmark, Paul Steimle, Tim Macechko, Paul

Manning, and Dorota Bulik.

Last, I would thank the undergraduates who worked in the laboratory on various projects for making my time their more enjoyable.

9 DEDICATION

I would like to dedicate this work to all those who helped me grow as a person and a scientist, especially my wife - Praveen, my children - Deven and Amalia, my parents - Ruth and Dale Karr, and to Ed Jarroll - my friend and advisor.

10 TABLE OF CONTENTS

ABSTRACT ...... 5

ACKNOWLEDGEMENTS ...... 8

DEDICATION ...... 9

TABLE OF CONTENTS ...... 10

LIST OF FIGURES ...... 13

LIST OF TABLES ...... 15

LIST OF ABBREVIATIONS ...... 16

CHAPTER I ...... 18

INTRODUCTION ...... 18

Giardia and Giardiasis ...... 18

Chemotherapy ...... 20

Giardia Life-cycle: Excystation and Encystment ...... 21

Cyst Wall Morphology ...... 28

Cyst Wall Biochemistry ...... 30

Biochemistry of Related Carbohydrate Polymers...... 36

Giardia Cyst Wall Synthesis: Scope of Research...... 63

CHAPTER II ...... 66

METHODS ...... 66

Reagents ...... 66

Maintenance of Parasites in Culture and Induction of Encystment ...... 66

CWS / 3GaN-T Purification Procedure ...... 67

11 Glycosyltransferase Assays ...... 69

Characterization of CWS / 3GaN-T Activity ...... 76

Sub-cellular Organelle Marker Enzyme Assays ...... 82

One Dimensional SDS-PAGE ...... 83

Preparative One Dimensional SDS-PAGE and Electroelution ...... 84

Two Dimensional SDS-PAGE ...... 85

Internal Polypeptide Digestion and Protein Microsequencing ...... 86

Autoradiography ...... 86

Western Blot ...... 87

Lectin Blot ...... 87

Production and Purification of Giardia Cyst Wall Material ...... 88

Complete Acid Hydrolysis ...... 90

Controlled Partial Acid Hydrolysis ...... 90

Monosaccharide and Amino Acid Compositional Analysis ...... 91

Polysaccharide Linkage Analysis (Methylation Analysis) ...... 92

Proteinase Digestion of CWM (Pronase) ...... 92

Deglycosylation of CWM ...... 93

Matrix Assisted Laser Desorption-Time of Flight Mass Spectrometry ...... 95

1H NMR Spectroscopy ...... 96

Antibody Production ...... 96

Electron Microscopy of CWM and CWS-containing Vesicles ...... 96

CHAPTER III ...... 99

RESULTS ...... 99

12 Identification of Induced CWS / 3GaN-T Activity ...... 99

CWS Sub-Cellular Localization and Purification ...... 99

CWS Localization ...... 103

Purification of Cyst Wall Synthase (CWS) ...... 108

Identification of cyst wall synthase (CWS) subunit(s) ...... 109

Characterization of cyst wall synthase (CWS) activity ...... 113

Acceptor Profiling of CWS Activity...... 118

Characterization of the CWS Product ...... 133

CWM Compositional Analysis ...... 133

Identification of the Protein Component of CWM ...... 136

Characterization of the Cyst Wall Material ...... 142

Structural Characterization of the CWM Filaments ...... 143

CHAPTER IV ...... 146

DISCUSSION ...... 146

REFERENCES ...... 167

13 LIST OF FIGURES

Figure 1. Low voltage scanning electron micrographs of Giardia in vivo...... 23

Figure 2. Immunofluorescence micrographs of Giardia encystment...... 25

Figure 3. Giardia cyst in the process of excystation...... 29

Figure 4. Transmission electron micrographs of the Giardia inner and outer cyst wall. 31

Figure 5. Inducible pathway of Giardia cyst wall 1,3-giardan synthesis...... 35

Figure 6. Oligosaccharides in nature based on the three domain phylogenetic tree...... 42

Figure 7. Correlation of Time Course of Encystment and Induction of CWS Activity. 100

Figure 8. Diagram of CWS purification scheme from encysting Giardia trophozoites. 101

Figure 9. Sub-cellular localization of CWS activity within encysting Giardia...... 102

Figure 10. LVSEM photomicrographs: peak CWS-containing microsomal population 104

Figure 11. LVSEM of Giardia cyst wall filaments in vivo and CWS product in vitro. . 105

Figure 12. Identification of protein bands enriched during CWS purification–I ...... 110

Figure 13. Identification of protein bands enriched during CWS purification–II ...... 111

Figure 14. Effect of pH, temperature, and divalent cations on Cyst Wall Synthase. .... 114

Figure 15. Determination of enzyme kinetics by non-linear regression analysis...... 116

Figure 16. Analysis of CWS Reaction Product by 1D-PAGE and Autoradiography-I. 119

Figure 18. (Glyco)peptide acceptor profiling...... 127

Figure 19. Solid-phase acceptor profiling of CWS and control enzymes...... 130

Figure 20. Electron micrographs of Giardia cyst walls before and after purification. .. 135

Figure 21. Isolated CWM as seen by LVSEM and indirect immuno-gold staining...... 138

Figure 22. Protein epitopes recognized by polyclonal antibodies to isolated CWM. .... 139

14 Figure 23. Elution profile of solubilized cyst wall material from partial hydrolysis. .... 144

Figure 24. Structural analysis of the Giardia cyst wall filaments...... 145

Figure 25. Model of the Giardia cyst wall polymer Giardan...... 166

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LIST OF TABLES

Table I. Current defined oligosaccharide polymers found in nature...... 37

Table II. Solution-phase biotinylated (glyco)peptide acceptors assayed...... 77

Table III. Solution-phase biotinylated neoglycoconjugate acceptors assayed...... 78

Table IV. Solid-phase biotinylated neoglycoconjugate acceptors assayed...... 81

Table V. Co-localization of ESVs and CWS activity from the detergent insoluble fraction of Peak CWS-containing microsomes...... 106

Table VI. Purification of cyst wall synthase from encysting Giardia...... 108

Table VII. Substrate specificity of Giardia cyst wall synthase (CWS) activity...... 115

Table VIII. Identification of inhibitors of Giardia cyst wall synthase (CWS) activity. ..117

Table IX. Effects of various treatments on the cyst wall synthase reaction product...... 121

Table X. Solid-phase acceptor profiling of CWS and control enzymes...... 131

Table XI. Solution-phase acceptor profiling of CWS and human β3Gn-T2...... 132

Table XII. Amino acid composition of the cyst wall protein component...... 137

Table XIII. Resistance of CWM to known glycosidases...... 141

Table XIIII. Characteristics of partially purified CWS from encysting Giardia...... 160

16 LIST OF ABBREVIATIONS

1D-PAGE - one dimensional polyacrylamide gel electrophoresis

2D-PAGE - two dimensional polyacrylamide gel electrophoresis

3GaN-T - (1-3)-N-acetylgalactosaminyltransferase

BME - -mercaptoethanol

BSA - bovine serum albumin

CWP1 - cyst wall protein 1

CWP2 - cyst wall protein 2

CWP3 - cyst wall protein 3

CWM - cyst wall material

CWS - cyst wall synthase (syn. 3GaN-T)

EC - enzyme commission

EDTA - ethylenediaminetetraacetic acid

ESV - encystment specific vesicles (syn. encystation secretory vesicles)

Fuc - L-fucose

GalNH2 - D-galactosamine

GC / MS - gas chromatography / mass spectrometry

Glc - D-glucose

GlcNH2 - D-glucosamine gpGaN-T - glycopeptide α(1-O)-N-acetylgalactosaminyltransferase

GPI - N-acetylglucosamine-6-phosphate isomerase

HPLC - high pressure liquid chromatography

17 IFM - immunofluorescence micrograph

LVSEM - low voltage scanning electron micrograph

Man - mannose

MurNAc - N-acetylmuramic acid

Neu5Ac - N-acetylneuraminic acid

1H-NMR - proton nuclear magnetic resonance spectroscopy

P-fraction - particle fraction

PFOR - pyruvate:ferredoxin oxidoreductase ppGaN-T - polypeptide-1,O-N-acetylgalactosaminyltransferase

L-Rha - L-Rhamnose

S-fraction - non-sedimentable fraction

SEM - scanning electron micrograph

SREBP - sterol regulatory element binding protein

TCA - trichloroacetic acid

TLC - thin layer chromatography

TEM - transmission electron micrograph

UDP - uridine 5'-diphosphate

UMP - uridine 5'-monophosphate

UTP - uridine 5'-triphosphate

UDP-Gal - UDP-D-galactose

UDP-GalNAc - UDP-N-acetyl-D-galactosamine

UDP-GlcNAc - UDP-N-acetyl-D-glucosamine

VSP - variant specific surface proteins

WGA - wheat germ agglutinin 18 CHAPTER I

INTRODUCTION

Giardia and Giardiasis

Giardia is a genus of exclusively pathogenic protozoa that parasitize primarily the middle small intestine - the jejunum. The organism causes infection in a wide range of hosts from fish to mammals, including man, where it is a significant burden associated with acute epidemics and chronic illness.

Giardia is spread by a simple and direct fecal-oral route of transmission through consumption of contaminated water, food or fomites, by oral/anal sexual contact, and from person-to-person. This latter mode of transmission and fomites are relevant to the day-care setting where epidemics are common and further infection of family members is possible (Kulda and Nohýnková, 1995). Unlike many bacterial or viral diarrheas that require a large inoculum, as few as 10 Giardia cysts have caused infection in clinical trials with human volunteers, and the severity of the symptoms was not correlated in a dose-dependent manner to the inoculum size (Nash et al., 1987).

A potential zoonosis, established animal reservoirs such as the beaver and muskrat (Erlandsen et al., 1988) are likely to present an obstacle to the eradication of the disease from the public water supply. The relationship between other potential animal reservoirs such as dairy cows and human epidemics is also likely (Coklin et al., 2007), but confirmation of a zoonotic link awaits development of accepted species-specific probes, such as those recently developed by Erlandsen et al. (2005), and their application in thorough epidemiological studies.

19 In the United States, infections caused by Giardia, including asymptomatic cases, is estimated at 2 million annually (Mead, et al. 1999). Estimates within the United States conducted prior to 1990 indicate that up to 20% of the population has contracted giardiasis at one point during their lifetime (Craun, 1990).

While the rate of infection within the United States is significant, the rates of infection in developing nations are far greater, approaching 20-40% annually (Farthing,

1994). Giardia is considered the most commonly isolated intestinal parasite throughout the world (Marshall et al., 1997; Craun, 1990) and infectious diarrhea accounts for 17%

(1.87 million) of global annual mortality in children under 5 years (Boschi-Pinto, 2008;

Lawn et al., 2006; Bryce et al., 2005).

Giardiasis is characterized by clinical symptoms distinct from viral or bacterial infections, intoxications, or from protozoan parasites that are invasive during the course of infection. The acute stage typically begins approximately 7-19 days post-infection and commonly reported symptoms include: 1) diarrhea (96%), 2) fatigue (72%), 3) anorexia

(62%), 4) abdominal pain (61%), 5) nausea (60%), 6) steatorrhea, a mucus filled stool

(57%), 7) flatulence (35%), 8) vomiting (29%), and 9) fever (17%) (Kulda and

Nohýnková, 1995; Farthing, 1994). The duration of the acute stage in untreated patients is variable, lasting from 3 days to months with an average of 2-4 weeks. The persistence of acute symptoms may be continuous or sporadic.

For 30-50% of patients the acute stage is commonly followed by a chronic or subacute stage (Farthing, 1994) that presents with an altered pattern of clinical symptoms.

Patients often have persisting or recurring: 1) flatulence, 2) loose, but typically not watery stool, 3) abdominal distention with mid-epigastric cramps, and 4) general malaise.

Blood or pus in diarrheic stool is rarely present in either the acute or the chronic stage

20 and a period of asymptomatic cyst-passing can occur for an indeterminate time (Farthing,

1994). At least 50% of symptomatic patients, especially children, have biochemical evidence of carbohydrate, fat, and micronutrient malabsorption, and with chronic illness, malabsorption of fat, lactose, vitamin A, and vitamin B12 have been reported, and failure of children to thrive has been noted (Gardner and Hill, 2001; Farthing, 1994).

Chemotherapy

Current prescription treatments for giardiasis include: 1) the antiprotozoal, anaerobic antibacterial 5-nitroimidazoles such as metronidazole (Flagyl®) and tinidazole

(Tindamax®, Fasigyn®), 2) the free-radical generating nitrofuran - furazolidone

(Furoxone®), 3) the poorly absorbed aminoglycoside translation inhibitor also efficacious against Entamoeba and Trichomonas - paramomycin, 4) the broad-spectrum antihelminthic cytoskeleton-disrupting benzimidazoles - mebendazole (Vermox®) and albendazole (Albenza®), 5) quinacrine (Mepacrine®, Atebrine®), whose mode of action is apparently not against nucleic acid synthesis in Giardia (Upcroft et al. 1996), and 6) nitazoxanide (Alinia®), a nitrothiazoyl-salicylamide derivative also effective against

Cryptosporidium infection (Ali and Nozaki, 2007; Gardner and Hill, 2001). In refractory cases multi-drug cocktails have proven effective in resolving infection.

Metronidazole has remained the most frequently prescribed drug for giardiasis, interestingly however, metronidazole has never been approved for the treatment of giardiasis by the United States Food and Drug Administration (Ali and Nozaki, 2007;

Gardner and Hill, 2001). This compound class affects Giardia trophozoites and anaerobic microorganisms that can generate large redox potentials, in this case from the use of an active prokaryotic-like pyruvate:ferredoxin oxidoreductase (PFOR).

21 Metabolism of the 5-nitroimidazoles by PFOR leads to selective reduction to free radical metabolites that are believed responsible for the subsequent cellular damage and ultimately to clearance of infection. However, developing resistance in vivo to metronidazole has been reported (Upcroft and Upcroft, 1993; Johnson, 1993) and has been developed with in vitro models (Pal et al., 2009; Müller et al., 2007; Upcroft et al.,

1996; Townson et al., 1994). These models suggest that resistance occurs by down- regulation of PFOR and that cross-resistance to other members of the same class of antibiotics is conferred by this same mechanism (Townson et al., 1994).

Furthermore, drug failures have been reported with all common antigiardial agents, and drug resistance to all of these drugs has been demonstrated in the laboratory

(Pal et al., 2009; Müller et al., 2008; Müller et al., 2007; Wright et al., 2003; Upcroft et al., 1996).

Giardia Life-cycle: Excystation and Encystment

The ability of Giardia to form and exit a protective cyst is crucial to survival of the parasite and is considered a virulence factor. Giardia's infective cyst stage is acquired most commonly through contaminated water supplies (Meyer, 1994). The cyst stage is relatively resistant to environmental conditions and routinely can tolerate levels of chlorine recommended for potable drinking water (Craun, 1986). Within the host, ingested cysts are stimulated to excyst in the presence of hydrogen ions of gastric secretions (Bingham and Meyer, 1979; Bingham et al., 1979) or bicarbonate of pancreatic secretions (Feely et al., 1991). Completion of excystation occurs within the upper small intestine where the emergent amoeboid excyzoite rapidly divides into two flagellated trophozoites, the vegetative pathogenic form, to begin the process of colonization.

22 The parasites occupy primarily in the distal duodenum and the jejunum by attaching themselves to the surface of the intestinal villi (Figure 1) via a unique organelle

- the ventral adhesive disk. Attachment must occur to prevent the trophozoite from being cleared from the lumen by peristalsis, and thus attachment has also been considered a virulence factor. Erlandsen et al. (2004) demonstrated that attachment can occur by means of the ventrolateral flange alone, though the percentage of adherent trophozoites was low. The mechanism of attachment in vivo is hypothesized to be an osmotic force independent of the beating of caudal flagella (Hansen and Fletcher, 2008), but may in fact be also related to flagellar action (Erlandsen and Feely, 1984).

In the upper small intestine trophozoites typically withstand exposure to fluctuations in oxygen tension, pH, ionic strength, bile, and digestive enzymes where they have relatively little competition from other microbes (Kulda and Nohýnková, 1995).

Here, the active feeding form acquires all of its preformed lipids, nucleic acids, and amino acids except alanine and valine by salvage rather than de novo synthesis (Jarroll et al., 1995).

During binary fission in vivo, trophozoites likely detach from the surface of the villi, as is the case in vitro; hypothetically as a result they are swept by peristalsis distally through the vertebrate intestinal tract. Eventually, individual trophozoites arrive in the region of the ileum - which has reduced levels of available cholesterol, lipids, fatty acids, and other nutrients - where cysts are first demonstrated (Luján et al., 1997). Cyst maturation apparently occurs during passage through the large intestines and prior to exiting the host.

23 Figure 1. Low voltage scanning electron micrographs of Giardia in vivo.

A. An intestinal villus covered by Giardia trophozoites

(courtesy of Dr. Stanley Erlandsen. bar = 10 µm).

B. Giardia trophozoite attached to intestinal microvilli.

From Hansen and Fletcher (2008) (bar = 2 µm)

24 In vitro, Giardia trophozoites are cultured in the presence of bile (1.0 mg ml-1), which enhances growth presumably by solubilizing lipids from serum supplementation.

Giardia encysts in response to an increased concentration of bovine bile (10 mg ml-1)

(Schupp et al., 1988; Gillin et al., 1987), to cholesterol deprivation (Luján et al., 1996), and responds more quickly and with a higher percentage of cyst-like forms with previously used (presumably nutrient reduced) or serum-reduced encystment medium

(unpublished data) by undergoing major morphologic and metabolic changes. While the precise mechanism remains elusive, these extracellular signals have produced in vitro encystment models which apparently mimic the cholesterol-deficient environment near the ileum and result in infectious cysts that when re-isolated from animals can excyst to complete the life cycle (Schupp et al., 1988).

The increased and excess concentration of bovine bile added to in vitro cultures was hypothesized to stimulate encystment indirectly by inhibiting trophozoite cholesterol uptake. This hypothesis was supported by Luján et al. (1997) who cultured cells in a cholesterol-deficient medium and described the production of cyst-like forms, the induction of encystment-specific vesicles (ESV) (Figure 2), and ESV-transported cyst wall proteins CWP1, CWP2, and CWP3. Further, samples of cholesterol-deprived trophozoites provided by the laboratory of Dr. Ted Nash exhibited the encystment- regulated enzymes glucosamine-6-phosphate isomerase, UDP-GlcNAc pyrophosphorylase, and weakly positive for cyst wall synthase activity (unpublished results from out laboratory) consistent with the induction of encystment.

25 Figure 2. Immunofluorescence micrographs of Giardia encystment.

A. 24 h encysting trophozoites probed with anti-cyst wall antiserum stain ESVs.

(Courtesy of Drs. Janiel Shields and Harry van Keulen)

B. Encysting trophozoites (72 h) probed with anti-cyst wall antiserum stain cyst walls.

(Courtesy of Drs. Janiel Shields and Harry van Keulen)

26 Luján et al. (1997) suggested that transcriptional regulation by the sterol regulatory element binding proteins (SREBP) to SRE promoter regions of DNA encoding both the

LDL receptor and cholesterol synthesizing enzymes in mammalian cells could be functionally analogous to Giardia encystment noting that 14 nucleotides of a 120- nucleotide 5’-untranslated region of two cyst wall proteins, CWP1 and CWP2, induced during encystment are similar between the two genes, though they bear no sequence similarity to the reported human SRE promoter.

Since cholesterol starvation induced the transcription of SREBP-dependent genes important in lipid and membrane homeostasis in mammalian cells, Worgall et al. (2004) tested if a similar mechanism existed in Giardia. These authors' transfected Chinese hamster ovary cells with the upstream regulatory region of the CWP2 gene as a fusion with luciferase (luc). The cwp2::luc reporter system was transcriptionally active (induced

3-fold) under conditions of cholesterol deprivation (vs. incubation in 10% fetal bovine serum), in the presence of cholesterol synthesis inhibitors, and in response to SREBPs overexpression; the transcriptional activity was reduced by addition of cholesterol (34%) or oleic acid (46%) in serum-free medium (Worgall et al., 2004). In addition, the cwp2::luc system was not regulated in a cholesterol auxotroph deficient in SREBP processing, indicating that cholesterol suppression of CWP2 mediated gene transcription is dependent on physiological processing of SREBPs. To directly assess the interaction of the Giardia promoter region with SREBPs, promoters as minimal as 64 base pair were functionally capable of CWP2 transcription, but promoters of at least 72 base pairs were required for regulation by sterols and contain 2 of 3 unique sterol regulatory element binding sites to which SREBPs bind (as identified in DNA footprinting assays) (Worgal et al., 2004).

27 In addition, cholesterol sequestration in the in vitro encystment medium by an increase in bile may result in reduced cholesterol-receptor signaling. This hypothesis is supported by similar findings in cultured intestinal epithelial tissue where extracellular cholesterol-receptor-Ck-dependent binding with intrinsic receptor tyrosine protein kinase activity has been demonstrated (Goel et al., 1997). This model was confirmed in Giardia when Kaul et al. (2001) demonstrated that trophozoites have the ability to express genes coding for receptor-Ck and SREBP, and that inhibition of cholesterol-dependent activation of receptor-Ck results in the upregulation of CWP1 gene expression leading to encystment.

An additional hypothesis that could link the reduction of available cholesterol to signaling events involves changes in membrane fluidity. A reduction in cholesterol disrupts cholycystokinin receptor activity (Gimpl et al., 1997) and GPI-anchored protein- dependent calcium signaling in response to decreasing cholesterol beyond the stabilization of membrane microdomains (Stulnig et al., 1997). While it is possible that cholesterol starvation has the potential to cause increased membrane fluidity, Ellis et al.

(1996) quantified the changes in lipids between vegetative trophozoites and encysting

Giardia cells. They found fatty acid desaturase activity and a significant change in the level of desaturated fatty acids which could increase membrane fluidity, but failed to show a reduction in total cholesterol in encysting cells (Ellis et al., 1996).

Thus, an initial extracellular signal for encystment of Giardia appears to be the reduction of available extracellular cholesterol. In an organism that does not synthesize cholesterol de novo this stimulus seems to serve as a plausible hypothesis for the induction of encystment and the timing of cyst formation prior to exiting the host's intestinal tract. The ultimate result of encystment is the formation of a metabolically

28 reduced protective cyst stage with an extremely resistant cyst wall. Infective cysts are then passed from the host in the feces ready to begin the cycle of transmission again.

Cyst Wall Morphology

Giardia cysts are 7-10 m long, oval in shape (Kulda and Nohýnková, 1995)

(Figure 3), and lack a noticeable suture line or an operculum that demarcates the site of excystation in other intestinal parasite ova stages. The Giardia cyst contains four nuclei of two undivided trophozoites halted apparently after karyokinesis and prior to cytokinesis. The trophozoites within are separated from the external environment by an inner and an outer cyst wall plus the associated periplasmic regions.

The inner cyst wall consists of two double lipid bilayers, one of which is closely opposed to the outer filamentous layer (Chávez-Munguía et al., 2007). In total, three lipid bilayers are visible between the trophozoite cytosol and the filamentous layer of the cyst wall and one of these is the original plasma membrane. Chávez-Munguía et al.

(2007), who used the combination of osmium tetroxide and ruthenium red for greater contrast of the filamentous cyst wall, clearly demonstrated by TEM that these three membranes surround the trophozoites of maturing cysts (Figure 4).

The biogenesis of two of these double lipid bilayers remain unknown, but may be the result of: 1) the fusion of peripheral vesicles (PV), which would suggest that the original plasma membrane becomes the outermost cyst membrane (Chávez-Munguía et al., 2007), 2) the fusion of PV and the large encystment specific vesicles (ESV), which are induced during encystment, or 3) the fusion of empty ESVs, after they release their

29 Figure 3. Giardia cyst in the process of excystation.

A. Excysting trophozoite emerging from one pole of cyst.

(from Erlandsen et al., 1989. b = bacterium, t = emerging trophozoite)

B. Empty cyst wall remnant remaining after excystation.

(courtesy of Dr. Stanley Erlandsen. bar = 5 µm.)

30 cargo onto the pre-cyst wall during encystment. In support of the PV or PV/ESV fusion hypothesis for forming the internal most two membranes and pushing the PM towards the exterior, Chávez-Munguía et al. (2004) showed that monoclonal antibodies against plasma membrane-associated variant surface proteins localized only to the external membrane by indirect immunogold TEM. They further showed in co-localization studies that monoclonal antibodies to cyst wall protein recognized specifically the inner most two membranes (Chávez-Munguía et al., 2007). Complicating these models is the finding by

Erlandsen et al. (1996) demonstrated the release of the ESV contents onto the surface of encysting trophozoites using indirect immunogold labeling in high resolution field emission SEM and antibodies to cyst wall protein. However, it is unclear how release and degranulation of ESV contents onto the external surface is accomplished if the ESVs form the inner most two membranes, and the complete understanding of the biogenesis of the inner cyst wall and the function of the multi-membrane system will require additional studies.

The outermost layer of the cyst wall is the filamentous layer which, typical of high polysaccharide content, contrasts poorly in TEM images using osmium tetroxide alone. The outer filamentous cyst wall is composed of a tightly packed meshwork of 7-

20 nm wide microfibrils (Erlandsen et al., 1989) (Figure 3), is approximately 0.3-0.5 m in thickness, and contrast is enhanced with the addition of ruthenium red (Figure 4).

Cyst Wall Biochemistry

As early as the 1930's researchers inferred the presence of a protein component of the Giardia cyst wall from cytochemical studies following proteinase treatment (Kofoid

31 Figure 4. Transmission electron micrographs of the Giardia inner and outer cyst wall.

A. TEM of three double lipid bilayers (inner wall) and filamentous outer cyst wall.

(from Chávez-Munguía et al., 2007. bar = 0.5 µm; cyst membranes = CM1-3).

B. TEM of cyst wall architecture after acid-induced excystation.

(from Chávez-Munguía et al., 2007. bar = 0.5 µm; cyst membranes = CM1-3).

32 et al., 1932). In 1952 and 1965 Filice and Kofoid, respectively, expanded on this work and suggested the wall was composed of a carbohydrate-protein complex, but could not reproduce Kofoid's results with proteinase treatment.

Later however, cyst specific proteins localized to the outer cyst wall by immunogold electron microscopy (Erlandsen et al., 1990; Reiner et al., 1990), and the presence of amino acids was confirmed by total amino acid analysis from hydrolysates of cyst wall preparations even after proteinase treatment (Manning et al., 1992; Gerwig et al., 2002). Three of these encystment specific proteins, CWP1-3, were later identified

(Luján et al., 1995; Mowatt et al., 1995; Sun et al., 2003), and the proteinase-resistant nature of CWP1 (recombinantly expressed) was subsequently confirmed (Boone et al.,

1999). However, if other proteins are associated with the wall and/or the nature of the protein association(s) remains unclear.

Though Giardia cysts were histochemically shown to be Feulgen stain negative

(Filice, 1952; Dutta, 1966; Erlandsen and Rasch, 1994) and did not appear to contain chitin, cellulose, or lipids (Filice, 1952), it was proposed that a major portion of the cyst wall was composed of chitin based upon lectin binding studies (Ward et al., 1985; 1988).

In these works the lectin wheat germ agglutinin (WGA) was used which has an affinity for terminal N-acetylglucosamine (GlcNAc), the sole carbohydrate substituent of the chitin homopolymer, and parasite binding by WGA was abolished by pretreatment with chitinase. Later, studies by Ortega-Barria et al. (1990) demonstrated the presence of

GlcNAc-containing components of encysting homogenates isolated by WGA-affinity chromatography and provided this as evidence of the presence of chitin. However, the column purification procedure used would have excluded the insoluble filamentous cyst walls from the analysis and most likely isolated the variant specific surface proteins

33 (VSPs) which have been characterized as containing a novel type of short O-linked sugar chain composed of GlcNAc and glucose residues (Hilpold, et al, 2000; Papanastasiou et al., 1997; Banerjee et al., 2009), and/or the truncated N-linked di-GlcNAc modifications common in Giardia (Ratner et al., 2008; Samuelson et al., 2005) that were isolated and characterized using the same WGA-affinity chromatography approach. Thus, until the publication of the first cyst wall compositional analysis in 1989 by Jarroll et al. and later by Manning et al. in 1992 no direct biochemical data were available to begin to elucidate the carbohydrate nature of the Giardia cyst wall.

To address the biochemical nature of the Giardia filamentous cyst wall, studies conducted by gas chromatography and mass spectrometry (GC/MS) on intact cysts after complete acid hydrolysis revealed that the major cyst wall carbohydrate present was galactosamine, and that this sugar was a cyst-specific marker for both encystment and the biochemical presence of cyst wall material (CWM) derived from G .intestinalis (MR4) in vitro and in vivo, and from G. muris in vivo (Jarroll et al., 1989; Manning et al., 1992). In addition, after treatment of intact cysts sequentially with chloroform / methanol extractions, sodium dodecyl sulfate (SDS) extractions, and amyloglucosidase to enrich for the outer filamentous cyst walls, reacetylated hydrolysates revealed that N- acetylgalactosamine (GalNAc) was present as 86% of the total carbohydrate, while N- acetylglucosamine (GlcNAc), as would be expected in hydrolysates of chitin, was below the levels of detection by GC/MS from 11 mg of cyst walls (Jarroll et al., 1989; Manning et al., 1992). Untreated intact cysts analyzed similarly for total carbohydrate present contained less than three percent GlcNAc compared to 71% for GalNAc (Manning et al.,

1992). To address the state of N-acetylation of the galactosamine found in both untreated intact and treated cyst wall material (CWM), binding of Phaseolus limensis lectin

34 (affinity for terminal GalNAc) to intact cysts and treated cyst walls was tested. This study suggested that the galactosamine (from cyst wall hydrolysates) re-N-acetylated prior to GC/MS compositional analysis is actually present as GalNAc in intact filamentous walls (Jarroll et al., 1989).

As a result of these unique findings, radiolabeling studies by Macechko et al.

(1992) using precursors of UDP-hexosamine synthesizing pathways known in other systems revealed that the GalNAc detected in the cyst wall is incorporated predominately from endogenous glucose reserves, probably from the Giardia glycogen-like cytoplasmic granules (Ladeira et al. 2005; Machechko et al., 1992), and not from other endogenous or exogenous precursors. Additionally, the enzymes responsible for the production of uridine diphospho-N-acetylgalactosamine (UDP-GalNAc) from glucose have been identified, purified, and characterized and constitute a pathway (Figure 5) whose enzyme activities are induced during the encystment process (Macechko et al., 1992; Jarroll et al.,

2001; Karr and Jarroll, 2004; Bulik et al., 2000; Bulik et al., 1998; van Keulen et al.,

1998; Steimle et al., 1997). The detection of the enzyme activities in this pathway, their regulation, and the detection of GalNAc were suitable encystment-specific markers to use to follow the encystment process. Perhaps the most important finding of these studies is that GalNAc is undetectable by GC/MS in nonencysting cells and consequently that the demonstrated enzyme activities increase up to 4000-fold during cytodifferentiation

(Macechko et al., 1992). Further studies by Steimle et al. (1998) and van Keulen et al.,

(1998) have shown that the first enzyme unique to this pathway, N-acetylglucosamine-6- phosphate isomerase (GPI), is undetectable in non-encysting cells and all five pathway- specific enzymes responsible for the synthesis of UDP-GalNAc are induced at the transcriptional level during Giardia encystment (Lopez et al. 2003).

35 Figure 5. Inducible pathway of Giardia cyst wall 1,3-giardan synthesis.

Glucose

Glucose-6-PO4

Fructose-6-PO4

NH3 Glucosamine-6PO4 -isomerase

Glucosamine-6-PO4

Glucosamine-6PO4-N-acetylase

N-acetylglucosamine-6-PO4

+ Phosphoacetylglucosamine mutase N-acetylglucosamine-1-PO4

UDP-N-acetylglucosamine Pyrophosphorylase O-GlcNAc O-glycosylation UDP-N-acetylglucosamine transferase N-glycosylation UDP-N-acetylglucosamine- chitin 4'-epimerase UDP-N-acetylgalactosamine

Polyoxin D, Nikkomycin Z Cyst Wall Synthase UDP-GlcNAc, EDTA, UDP

(1→3)-2-acetamido-2-deoxy-β-D-galactan (giardan) [-3)-D-GalpNAc--(1-3)-D-GalpNAc--(1-]n

36 In contrast, the enzyme UDP-N-acetylglucosamine pyrophosphorylase activity, also of this encystment-specific pathway, is up-regulated and activated by the biochemical product of the GlcNAc Isomerase reaction - N-acetylglucosamine-6- phosphate - by feed-forward regulation (Bulik et al., 2000; Jarroll et al., 2001).

Biochemistry of Related Carbohydrate Polymers

Given that the majority of the carbohydrate detected in hydrolysates of in vivo- derived G. muris cyst walls is GalNAc (16% of intact cysts and 66% of cyst wall dry weight), at least one glycosyltransferase activity should be present that produces a cyst wall structural polymer composed of GalNAc, likely from UDP-GalNAc. Based upon the resistant nature of the cyst wall to chemical or enzymatic (chitinase, N- acetylhexosaminidase) degradation (Manning et al., 1992; Jarroll et al., 1989), lack of cyst wall staining with Calcofluor White (Jarroll et al., 1989), and lack of periodic acid-

Schiffs staining (Manning et al., 1992) which requires adjacent hydroxyls only possible with 1,6-linked N-acetylhexosamines or 1,6- or 1,4-linked hexosamine (Krishnamurthy,

1999), then a polymer with 1,3-linkages is likely present.

However, prior to these studies there has been no publication of: 1) a 1,3-linked

GalNAc homopolymer, 2) a glycosyltransferase that forms GalNAc-β1,3-GalNAc bonds, nor 3) a glycosidase identified that acts on GalNAc-β1,3-GalNAc bonds. Thus, there exists no direct model for the present work. Therefore, to gain insights into the biochemistry of cyst wall synthesis it was necessary to examine research conducted in other systems involving the synthesis of structural polymers (Table I, Figure 6), especially those of microbial origin related to formation of a cyst or cell wall.

37 Table I. Current defined oligosaccharide polymers found in nature. Primary Oligosaccharide Oligosaccharide found in: Common Name (core / backbone) Archaea & Bacteria a Protista Fungi b Algae a Plantae Animalia c Amylose -[α1,4-Glc- α1,4-Glc-α1,4-Glc]n- (2:1) E green ‡ ― c Amylopectin -[α1,4-Glc-(α1,6-Glc)α1,4-Glc]n- (22:1) N ‡ ‡ ‡ green, red ‡ ― E Glycogen -[α1,4-Glc-(α1,6-Glc)α1,4-Glc]n- (12:1) R ― ― ‡ c Laminaran -[β1,3-Glc-(β1,6-Glc)β1,3-Glc]n- (3:1) G Phaeophyta Y Chrysolaminaran -[β1,3-Glc-(β1,6-Glc)β1,3-Glc]n- (11:1) phytoplankton d d Gluconacetobacter Oomycetes Acytostelium d d d Cellulose -[β1,4-D-Glc-β1,4-D-Glc] - ‡ ‡ Urochordata n & other genera d Acanthamoeba d Dictyostelium d Cellulose- -[β1,4-D-Glc-β1,4-D-Glc*] - n Gluconacetobacter e copolymer *(NAc/NH2), (~5:1) f f β1,6Glucan -[β1,6-D-Glc-β1,6-D-Glc]n- (3-branch) Actinobacillus ‡ g Pustulan -[β1,6-D-Glc-β1,6-D-Glc]n- (2,3,4-) ― Lichens Aureobasidium h Pullulan -[α1,4-D-Glc-α1,6-D-Glc] - (2:1) n Lichens g -[α1,6-D-Glc-α1,6-D-Glc] - Leuconostoc, Lactobacillus, Dextran n Rhizopus i (linear or var. 2,3,4 branched) Streptococcus i β1,3Glucan j Saprolegnia, j j -[β1,3-D-Glc-β1,3-D-Glc] - multiple genera ‡ (laminaran) ‡ "Callose" n Achlya j k -[β1,2-D-Glc-β1,2-D-Glc]n- (cyclic) multiple genera Crown Gall k -[β1,2-D-Glc-β1,2-D-Glc]n- (linear) multiple genera l m Nigeran -[α1,3-D-Glc-α1,4-D-Glc]n- (1:1) ― Aspergillus Lichens l n o Pseudonigeran -[α1,3-D-Glc-α1,3-D-Glc]n- Streptococcus sp. ‡ Lichens p Lichenan -[β1,3-D-Glc-β1,4-D-Glc]n- (1:1)xxx. Aspergillus p Isolichenan -[α1,3-D-Glc-α1,4-D-Glc]n- (1:3-3:7) Lichens c c β1,4Mannan -[β1,4-D-Man-β1,4-D-Man]n- ― ‡ ‡ c β1,4GlucoMannan -[β1,4-D-Glc - β1,4-D-Man]n- (1:1 - 3:1) ― ― ‡ c α1,6Mannan -[α1,6-D-Man-α1,6-D-Man]n- (α1,2-D-Man branching) Candida ― ― c β1,4Xylan -[β1,4-D-Xyl-β1,4-D-Xyl]n- (1,3&1,4)Rhodophyta ‡ c β1,3Xylan -[β1,3-D-Xyl-β1,3-D-Xyl]n- (1,3)Chlorophyta ― q Pectin -[α1,4-D-GalA-α1,4-D-GalA]n- (homopolymer) or Diatoms flowering q q "block copolymer" -[α1,4-D-GalA*]n-[α1,2-D-Rha]n - (*-OCH3 / -OAc; 80%) Chlorophyta plants

38 a b a Common Name Primary Oligosaccharide Archaea & Bacteria Protista Fungi Algae Plantae Animalia c c β1,4Galactan -[β1,4-D-Gal-β1,4-D-Gal]n- Chlorophyta Lupinus marine c c β1-3,4Galactan -[β1,3-D-Gal(2/4SO3)-β1,4-D-Gal]n- (copolymer) Rhodophyta Ruppia inverts

r Murein -[β1,4-D-GlcNAc-β1,4-D-MurNAc ] - Eubacteria n

s Pseudomurein -[β1,3-D-GlcNAc-β1,3-L-TalANAc]n- Gram-+ Archaea

t u t t Streptomyces t t Viral , diatoms Arthropoda Chitin -[β1,4-D-GlcAc-β1,4-D-GlcNAc] - t Entamoeba ‡ n Rhizobium Oomycetes t Mollusca t t t Mucor rouxii, Arthropoda Chitosan -[β1,4-D-GlcNH -β1,4-D-GlcNAc] - Entamoeba t t 2 n & other genera Mollusca

v Giardan -[β1,3-D-GalNAc-β1,3-D-GalNAc]n- Giardia w w Poly(GalNAc) -[ (15%) GalNH2 - (85%) GalNAc]n- Myxococcus Physarum ― x x x α1,4Poly(GalNAc) -[α1,4-D-GalNH2-α1,4-D-GalNAc]n- Neisseria ― Aspergillus Penicillium y -[?x,x-D-Gal- ?x,x-D-Gal(NAc/NH2)]n- ?(1:3) Amoebidium ― z Gal/GalNAc-rich -[α1,4-D-Gal-α1,4-D-Gal(NAc/NH2)]n- (2:1) ― Aspergillus aa -[β1,3-D-Gal-α1,3-D-*GalNAc]n- (*β1,6-D-Gal-branches) Dictyostelium ab Trebouxia photobiont Galactofuranan -[β1,5-D-Gal -β1,6-D-Gal ] - Mycobacterium f f n of Ramalina gracilis ac

ad ad Carrageenan -[β1,3-Gal(SO )-α1,4-D-3,6-anhydro-Gal(SO )] - Rhodophyta 3 3 n ae ae Agar -[β1,3-Gal-α1,4-L-3,6-anhydro-Gal]n- Rhodophyta ae ae Agaropectin -[β1,3-Gal-α1,4-L-3,6-anhydro-Gal]n- Rhodophyta af af h Azotobacter Rhodophyta Alginate -[β1,4-ManA-α1,4-L-GulA] - n Pseudomonas af Phaeophyta af ag Streptococcus u ai Hyaluronan -[β1,4-L-GlcA-β1,3-GlcNAc] - (~80%) ag Chlorella-virus ‡ n Pasturella Pasteurella ag Heparosan -[α1,4-L-GlcA-β1,4-GlcNAc] - ag n E. coli ag -[β1,4-L-GlcA-β1,3-GalNAc]n- E. coli Chondroitin ah -[β1,4-L-GlcA-β1,3-GalNAc]n- Gram-+ Archaea Methanochondroit. -[β1,4-L-GalA-β1,3-GalNAc] - Gram-+ Archaea ah n 39 a b a Common Name Primary Oligosaccharide Archaea & Bacteria Protista Fungi Algae Plantae Animalia Chondroitin SO A -[β1,4-L-GlcA-β1,3-GalNAc(6SO )] - 3 3 n ‡ ai Chondroitin SO3 C -[β1,4-L-GlcA-β1,3-GalNAc(4SO3)]n-

Dermatan SO or -[β1,4- L- IdoA(2SO )-α1,3-GalNAc(4SO )] - 3 3 3 n ‡ ai Chondroitin SO3 B -[β1,4-D-GlcA- -β1,3-GalNAc(4SO3)]n-

ai Keratan SO3 -[β1,3-Gal(6SO3)-β1,4-GlcNAc(6SO3)]n- ‡

ah -[α1,4 -L- IdoA(2SO3)-α1,4-GlcN(Ac-or-SO3)-(6SO3)]n- (~30%) aj Heparan SO3 ah ‡ -[α1,4-D-GlcA(2SO3)-β1,4-GlcN(Ac-or-SO3)-(6SO3)]n- (~70%) ai -[α1,4-L- IdoA(2SO3)-α1,4-GlcN(Ac-or-SO3)-(6SO3)]n- (~80%) aj Heparin SO3 ai ‡ -[α1,4-D-GlcA(2SO3)-β1,4-GlcN(Ac-or-SO3)-(6SO3)]n- (~20%)

‡ Indicates that the glycan has been widely detected. a For this Table the eukaryotic algae are treated separately. The Prokaryotic algal-like cyanobacteria are included under "Archaea & Bacteria". b Lichens, fusions of a fungus and algal cell, are considered in this Table as Fungi, unless a polymer was isolated from the cultured photobiont. c For algae and plants (see Davis et al., 2003; Estevez et al., 2009; Aquino et al., 2005); for lichenized-fungus Dictyonema (see Carbonero et al., 2002); for Fungi; Candida with α1,2-branching (Shibata et al., 2007; 2003); GlucoMannan is synthesized by a dual synthase (Liepman et al., 2005; Suzuki et al., 2006). d Cellulose is synthesized in: 1) Bacteria; Gluconacetobacter, Aerobacter, Achromobacter, Agrobacterium, Alacaligenes, Azotobacter, Pseudomonas, Rhizobium, and Sarcina (Ross et al., 1991), 2) Oomycetes; Phytophythora, Pythium, Peronospora, and Pseudoperonospora (Ross et al., 1991), 3) Amoebazoa; Acytostelium and Dictyostelium (Eichinger et al, 2005; West 2003) and Acanthamoeba (inner cyst wall) (Tomlinson & Jones, 1962; Linder et al, 2002), 4) algae, theca of dinoflagellates, and land plants (Davis, 2003; Niklas, 2004), and 5) the Urochodata (Anamalia) (Nakashima et al., 2004). e Lee et al. (2001) fed Gluconacetobacter xylinus cultures with glucosamine (GlcNH2) or GlcNAc as primary carbon sources and analyzed cell wall glycans finding GlcNH2 and GlcNAc constituted 19% and 18% of cell wall copolymers, respectively. f β1,6Glucan in Saccharomyces, Candida (Aimanianda et al., 2009; Iorio et al., 2008; Manners et al., 1973); in Bacteria; Actinobacillus (Monteiro et al., 2000). g (Carbonero et al., 2005) h Pullulan from chlamydospores and swollen cell forms of Aureobasidium (Leathers, 2005; Campbell, et al., 2004) and Lichens (Carbonero et al., 2005). i Dextran, considered >50% 1,6-linked linear glucose, is produced by: 1) Bacteria; Leuconostoc, Streptococcus, Gluconobacter, Lactobacillus and 2) Fungi; Rhizopus, nearly linear or with branching; 0-35% (1-2), 0-50% (1-3), and/or trace amounts (1-4) (see Khalikova et al., 2005). j β1,3Glucan: 1) Bacteria; Agrobacterium spp. (7 of 17 strains), Rhyzobium, Cellulomonas, Bradyrhizobium, Azorhizobium, Sinorhizobium, and Streptococcus pneumoniae (McIntosh et al., 2005), 2) Fungi (West, 2003), and 3) Oomycetes; Saprolegnia, Achlya (Briolay et al., 2009; West, 2003) and Algae (laminaran). k Crown Gall or cyclic β1,2Glucan (degree of polymerization ca. 20, non reducing terminus) has been found in Agrobacterium, Rhizobium, Xanthomonas, Brucella, and linear β1,2Glucan has been detected in Rhizobium, E. coli, Xanthomonas, and Gluconacetobacter (Breedveld & Miller, 1994; Harada, 1983). l Streptococcus mutans produces a pseudonigeran-type (syn. mutan) polymer with 9:1 ratio of α1,3-to-α1,6 linkages (see Khalikova et al., 2005).

40 m Nigeran was first identified in Aspergillus niger and Penicillium expansum (Dox & Neidig 1914). Bobbitt et al. (1977) found that the amount of nigeran is enhanced by culture in nitrogen-limited medium (7-fold), and later characterized the structural oligosaccharide (Nordin & Bobbitt, 1982). Nigeran was also found in Lichens with a (1:1) ratio in Cladonia, Cladina, Letharia, Parmelia, and Ramalina. n Aspergillus, Histoplasma, Paracoccidioides have α1,3- instead of β1,6-glucan (Damveld, 2005; Arana et al., 2008; Latgé et al., 2005; Sorais et al., 2009). o Pseudonigeran was characterized from the lichenized-fungus Dictyonema glabratum along with β1,4-xylan and β1,6-mannan (Carbonero et al., 2005; 2003). p Lichenan from Cetraria islandica, C. richardsonii, and Usnea rubescens has a linkage ratio of 3:7, while polymers from Usnea sp., Parmelia, Parmotrema, and Rimelia have a ratio of 1:3.1 (Carbonero et al., 2005; 2003). Aspergillus fumigatus lichenan has a ratio of 1:1 (Fontaine et al., 2000). q Pectin in diatoms and plants (Wustman et al. 1998; Jarvis, 1984). r Structural data and synthesis reviewed in Vollmer et al. (2008), Price and Momany, (2005), and Heijenoort, (2007). s L-TalANac = L-N-acetyltalosaminuronic acid. See König et al. (1983), Leps et al. (1984a; 1984b), Kurr et al., (1991), and Kandler and Konig (1998). t 1) fungal chitin (and chitosan - Mucor, Rhizopus, Rhizomucor, Phycomyces, Schizosaccharomyces), (Latgé, 2007; Bowman & Free, 2006; Ruiz-Herrera & Martinez-Espinoza, 1999), 2) in molting Ecdysozoa (arthropods) (Falini & Fermani, 2004; Merzendorfer, 2006), 3) biomineralization in mollusc shells and radula (Furuhashi et al., 2009), 4) Bacteria; Rhizobium, Streptomyces spores (Gomes et al., 2008), 5) chitin and pectin in the diatom frustule (Tesson et al., 2008; Wustman et al. 1998; Hecky et al. 1973), 6) in the oomycete Saprolegnia (Briolay et al., 2009), 7) chitin (Arroyo-Begovich et al., 1978; Campos- Góngora et al., 2004) and chitosan (Das et al., 2006) in the protist Entamoeba, 8) the chitooligosaccharides in embryos of the vertebrate Xenopus (Semino et al., 1996), 9) in tubes of the annelid tubeworm Lamellibrachia (Imai et al., 2003), and 10) nematode eggshells (Veronico et al., 2001; Dubinský et al., 1986). u Chlorella-virus encoded hyaluronan synthase (HAS), 50% identity to vertebrate HAS (de Angelis et al., 1997), produces hyaluronan on infected cell surfaces (Graves et al., 1999) and in Chlorella-virus genomes analyzed, HAS was found alone, together with chitin synthase (Chs), or Chs was found alone, and chitin production on infected and transfected cell surfaces was also detected (Kawasaki et al., 2002; Yamada et al., 2005, 2006). Viral-encoded chitin synthase (Chs) has also been sequenced from Ectocarpus siliculosus-virus EsV-1(Delaroque et al., 2001). v In the Giardia cyst wall, giardan was characterized as a linear unbranched unmodified homopolymer of 1,3-linked-β-D-GalNAc (see Gerwig et al., 2002). w 2) Myxococcus xyantus, a δ-proteobacteria with a complex lifecycle similar to cellular and plasmodial slime molds such as Dictyostelium, Polysphondylium, and Physarum, can be induced to form myxospores whose walls contain a GalNAc polymer; 50% dry weight and >85% N-acetylated (Filer et al., 1977). A pathway of inducible enzyme activities was demonstrated in cultures induced to form myxospores and six of seven enzymes required to convert fructose-1,6- bisphosphate to UDP-GalNAc were identified and their activities were induced 4-8 fold (White et al., 1977). The galactosamine polymer was considered separate from α1,3glucan after treatment with Aspergillus α1,3glucanase (Sutherland et al., 1977) or peptidoglycan after treatment with lysozyme (White et al., 1968). White et al., (1977) An acid insoluble material was radiolabeled after providing cell free extracts UDP-[14C]GalNAc, which upon hydrolysis yielded only [14C]galactosamine, and unlabeled glucose and acetate. 2) In Physarum polycephalum, a galactosamine polymer(s) was identified as the major constituent of the walls of spherules (95.5%) and spores (97.5% of carbohydrate, 82% including melanin) upon partial and complete hydrolysis (McCormick et al., 1970) and UDP-GlcNAc 4'-epimerase was detected (Hiatt & Whiteley, 1974), however, determination of N-acetylation was not attempted. x In Neisseria, a branched α1,4/α1,6-linked GalNAc polymer was isolated from sonicated pronase- & SDS-treated cell walls (Adams & Chaudhari, 1972). In Aspergillus; Distler & Roseman (1960): 1) partially characterized poly(GalNAc) polymer (>60% deacetylated) from A. parasiticus QM 884 secreted into the culture medium, which produced only GalNH2 with an α-optical rotation (no other sugar or amino acid) upon hydrolysis, 2) identified an alkali-extractable GalNH2 (only) polymer from mycelia, and 3) demonstrated GalNH2 in young mycelia of Aspergillus niger (4.0%), A. oryzae (2.7%), A. niger (2.3%) and lesser amounts in Helminthosporium sativum, Neurospora spp., and Rhizopus sp.. Poly(GalNAc) was further characterized from Aspergillus parasiticus

41 AHU 7165 by Araki et al. (1979) who: 1) generated oligosaccharides of n=50 maximum chain length by partial acid hydrolysis, 2) demonstrated enzymatic de-N-acetylation of poly(GalNAc) by a purified Aspergillus deacetylase with a preference for chain lengths >14 and inactive with chitin, 3) showed that ammonium tartrate stimulated polymer synthesis and deacetylase activity up to 7-fold, and 4) identified poly(GalNAc) deacetylase activity in other species. Takada et al. (1981) characterized the linear α1,4-polyGalNH2 polymer (55-65% random N-deacetylated) with an average degree of polymerization of 200. Fontaine et al. (2000) identified a β-1,3-glucanase and chitinase-resistant insoluble polyGalNAc polymer from A. fumigatus cell walls and culture supernatants, with 1-4 linkages as determined by GLC-MS of the partially methylated methyl-glycosides. In Penicillium stoloniferum, an uncharacterized Gal(NH2/NAc) polymer was found in a virus-containing strain (18% of cell wall) (Buck et al., 1969). y In Amoebidium parasiticum, Trotter and Whisler (1965) found neither chitin nor cellulose in cell walls as acid hydrolysates of the wall material showed no evidence of glucose or glucosamine. Compositional analysis showed 30% galactosamine, 10% galactose, 3% xylose, and 30% protein. The apparent lack of chitin, glucan, and cellulose raised the possibility of a lack of relationship of A. parasiticum with other groups of fungi, and recent DNA evidence confirms that A. parasiticum is a protozoan (Benny & O'Donnell, 2000; Ustinova et al., 2000). z In Aspergillus niger, Bardalaye and Nordin (1976) found an insoluble α1,4-linked Gal (70%) / GalNH2 (20%) -rich copolymer(s) with an average degree of polymerization of 100 following alkali extraction to remove cell wall chitin and glucans from young mycelia. In Aspergillus nidulans, Gorin and Eveleigh (1970) characterized an α1,4-linked Gal (65%) / GalNAc (35%) -rich copolymer(s) as determined by GLC-MS of the partially methylated methyl-glycosides. aa Galactosaminoglycan core structure from Dictyostelium was determined in detail to be a repeating block of [β1,3Gal-α1,3GalNAc] with β1,6Gal-branching and is a component of the spore and stalk walls associated with cellulose deposition (Sakurai et al., 2002). ab β(1-5/1-6)Galactofuranan was characterized, an enzyme activity using UDP-Galf detected, and the transferase was sequenced (Brenan & Crick, 2007). ac β(1-5)Galactofuranan was extracted from the aposymbiotically cultured photobiont, Trebouxia, of the lichen Ramalina gracilis (Cordeiro et al. 2005). ad Carrageenan is a copolymer class from red algae (Rhodophyceae). The backbone is -[3)-D-Gal*β(1-4)-D-Gal**α(1-]n- where *the α(1-3)- linked galactose unit can be D-Gal, D-Gal-2-SO3, D-Gal-4-SO3, D-Gal-6-SO3, or 4,6-0-(1'-carboxyethylidene)-D-Gal-2-SO3.and where **the β(1-4)-linked galactose unit can be 3,6-anhydro-D-galactose, 3,6-anhydro-D-galactose-2-SO3, D-Gal-2-SO3, D-Gal-6-SO3, D-Gal(2-SO3)-6-SO3 (see Chaoyuan, 1990; Rao et al. 1998). ae Agar is a neutral unbranched structural component of some red algae (Rhodophyceae) cell walls; Agaropectin, from the same organisms is highly modified with sulfate, methoxy, pyruvate, and/or glucuronate. Commercial "Agar" is considered a 7:3 mix of Agar and Agaropectin (see Chaoyuan, 1990). af Alginate found in all brown algae (Phaeophyceae) is composed of β1,4-D-ManA and α1,4-L-guluronate (GulA, the C5 epimer of ManA) in copolymer and alternating blocks (see Davis et al., 2003; Chaoyuan, 1990; Rao et al. 1998), and for Bacteria see Remminghorst & Rehm, (2006). ag Hyaluronan synthase/synthesis has been found in Streptococcus pyogenes, S. equistimilis, S. uberis, S. equi subsp. zooepidemicus, Pasturella multocida; heparosan synthase/synthesis in P. multocida; and heparosan synthase/synthesis and chondroitin synthesis in E. coli (Tracy et al 2007; DeAngelis et al. 2002). ah The archaeabacteria such as Methanosarcina and Halococcus aggregate within a "methanochondroitin" cell wall composed of the alternating heteropolysaccharides GlcA-GalNAc (7-20%) and GalA-GalNAc (20-40%), respectively, by dryweight (Kreisl and Kandler, 1986). ai The sulfated polymers keratan, dermatan, chondroitin, heparan, and heparin are found in vertebrates, tunicates and mulluscs (see Volpi & Maccari, 2009). Keratan sulfate chains decorate core proteoglycans, are commonly sulfated at any sugar C6, and are generally modified by fucose or Neu5Ac capping. aj Heparan sulfate and Heparin sulfate have 1-2 and 4-6 sulfates per tetrasaccharide, respectively (Rao et al. 1998). Potential sites of sulfation are noted.

42 Figure 6. Oligosaccharides in nature based on the three domain phylogenetic tree. GALACTOFURANAN CARRAGEENAN, ALGINATE, (Trebouxia photobiont) XYLAN, GALACTAN, AGAR, GALACTAN, MANNAN, PECTIN, GLUCAN CELLULOSE CELLULOSE CHITIN CELLULOSE GLUCAN (Entamoeba) CELLULOSE, GLUCAN, CHITIN polyGalNAc PECTIN, CHITIN, GLUCAN (Physarum) CELLULOSE... (Acanthamoeba). ALGINATE, Gal/GalNAc & (Dictyostelium) (Dictyostyleum)... LAMINARIN (Amoebidium) (Polysphondylium).. . GLUCAN CELLULOSE (Urochordata) GLUCAN HYALURONAN DERMITAN GLUCANS CHONDROITIN-SO MANNAN GIARDAN 3 (Giardia) HEPARAN-SO3 CHITIN HEPARIN-SO MUREIN poly[β1,3GalNAc 3 ] KERATAN-SO3 α1,4GalNAc α1,4Gal/GalNAc (Myxococcus) ISOLICHENAN (Aspergillus) polyGalNAc PUSTULAN α1,3GLUCAN PULLULAN CELLULOSE LICHENAN GLUCAN NIGERAN α1,4GalNAc (Neisseria) ALGINATE CELLULOSE METHANO– HYALURONAN CHONDROITIN CHONDROITIN HEPAROSAN

MUREIN DEXTAN PSUEDO. HYALURONAN MUREIN CELLULOSE CHITIN (Streptomyces lunalinharesii) Tree from Baldauf et al. (2004); glycan structures added. GALACTOFURANAN (Mycobacterium) 43 Table I details much of what is currently known from the literature regarding the most common structural, energy storage, and capsular polysaccharides for which there exists sufficient biochemical data to interpret. In cases where information may be of value for clarity, those data have been included in the table's footnotes. However, for brevity the table is focused on the polymer core structures rather than on polymer branching or modifications (e.g. limiting the table to peptidoglycan and not including variants of acetylated peptidoglycan now known). Emphasis has been placed on the homopolymers, block or alternating copolymers, and polysaccharides that are abundant, widely distributed, or specifically or contextually relevant to understanding the Giardia cyst wall, its synthesis, and potentially its relationship to other systems.

Figure 6 Figure 6attempts to present these data as general categories of polysaccharides and their polysaccharide synthases in relation to a macro view of phylogeny of organisms as determined from the rapid advance of bioinformatics and the ready availability of sequenced genomes. From this figure it should be clear that the pattern of distribution of a polysaccharide class is not simply confined to broad groups

(e.g. peptidoglycan is found among some Fungi), though many generalizations can be made. Until the genes for the catalytic subunits of these polysaccharide synthases are identified fully and sequence data across a spectrum of organisms are analyzed, it will not be possible to infer relationships such as which polymer or synthase arose first, if polymer classes are the result of convergent evolution or conservation of synthase sequences, or if lateral gene transfer between diverse groups has driven the distribution of polysaccharides among organisms. However, given the presence of specific polymers on cell surfaces of virus-containing fungi not found among virus-free strains of the same

species, and given that algal virus genomes have been sequenced revealing at least two 44 specific polysaccharide synthases and their accessory enzymes required for production of polymer precursors, then it is probable that lateral gene transfer has occurred and it is interesting to speculate on these relationships. Regarding the Giardia cyst wall polymer and synthase required for its synthesis, it is of interest in understanding their uniqueness and potential origins, and this information provides a basis from which to begin considering relevant molecular probes from other systems in an attempt to determine the sequence of the Giardia "cyst wall synthase."

Cellulose - a resistant homopolymer of 1,4glucose - is considered the most abundant biopolymer on Earth with an annual production of over 1011 metric tons

(Niklas, 2004; Mutwil et al., 2008) and is widely recognized as a key structural component of higher plant cell walls. However, cellulose is also synthesized by: 1)

Bacteria; Gluconacetobacter (formerly Acetobacter), Aerobacter, Achromobacter,

Agrobacterium, Alacaligenes, Azotobacter, Pseudomonas, Rhizobium, Sarcina, E. coli, and the cyanobacterium Nostoc (Ross et al., 1991), 2) the pathogenic aquatic oomycetes;

Phytophythora, Pythium, Peronospora, and Pseudoperonospora (Ross et al., 1991), 3)

Amoebazoa; Acytostelium and Dictyostelium (Eichinger et al, 2005; West 2003)

Acanthamoeba (inner cyst wall) (Tomlinson & Jones, 1962; Linder et al, 2002) and potentially Balamuthia (cyst wall) (Siddiqui et al., 2009) 4) in algae and the theca of dinoflagellates (Davis, 2003; Niklas, 2004), and 5) in the Urochodata (Anamalia)

(Nakashima et al., 2004).

Cellulose synthases (UDP-glucose : (1,4)--D-glucopyranose 1-4-glucosyl- transferase [UDP-forming], EC 2.4.1.12) consume UDP-glucose as substrate producing a linear unbranched homopolymer with β1,4-linkages and a chain length or degree of

45 polymerization reaching 8,000-15,000 (Taylor, 2008). Synthesis apparently occurs in processive fashion (i.e. once the polymer exits the synthase complex the polymer cannot be elongated further) and synthesis occurs from the non-reducing end in the bacterial system (Koyama et al., 1997), however, this remains to be tested across a range of systems. After synthesis and concomitant extrusion across the plasma membrane up to

36 individual chains aggregate forming extremely strong insoluble and crystalline microfibrils due to inter- and intra-chain hydrogen bonding of all available hydroxyl groups among adjacently aligned β1,4-glucan chains (Rao et al., 1998; Niklas 2004).

Microfibrils further aggregate into larger strands called macrofibrils that, in land plants, are held together by structural proteins (e.g., extensin), a variety of pectins (e.g., homogalacturonan, rhamnogalacturonan, and arabinan), and hemicelluloses (e.g., xyloglucan, arabinoxylan, glucomannan, and xylan) (Niklas, 2004). Macrofibril dimensions appear to vary based on the number of cellulose synthases within and the arrangement of plasma membrane terminal complexes producing thinner sheets of cellulose (1.5 x 100 µm) extruded from the bacterium Gluconacetobacter xylinus and thicker formations (10 x 25 µm) on surfaces such as of the green algae Oocystis (Niklas,

2004). The functional role of the multi-subunit plasma membrane terminal complex or rosette was demonstrated in temperature sensitive mutants that at restrictive temperatures lost the ability to synthesize ordered cellulose and correlated with dissociation of the rosettes into monomers (Taylor, 2008).

To date, between 9-12 cellulose synthase genes (CesA) or CesA-like genes have been identified per species of plant examined revealing a protein of between 100-120 kDa (prior to proteolytic processing) with a conserved secondary structure including

eight transmembrane domains and a conserved D,D,D-QxxRW motif necessary for 46 catalytic activity located on the cytosolic face of the plasma membrane (Saxena et al.,

2001; Saxena and Brown, 2005). The requirement for other components of the functional cellulose synthase terminal complex has been suggested based on two lines of evidence.

First, Peng et al. (2002) identified radiolabeled Glc-β-sitosterol and short glucan- derivatives up to Glc4-β-sitosterol from plant cell membrane extracts when assayed in the presence of UDP-[14C]Glucose, and suggested that glucan-β-sitosterols might serve as primers for cellulose synthesis. Incubation of membrane extracts in the presence of

[14C]glucan-β-sitosterols and subsequent incorporation of [14C]glucose into cellulose would have definitively shown this hypothesis to be true. However, no additional publications have addressed this potential lipid-linked intermediate or primer. Second, a

β1,4glucanase is believed required for synthesis of plant (Taylor, 2008) and bacterial

(Molhoj et al., 2002) cellulose. Taken together, the β1,4glucanase may function in cleaving glucan residues from a sitosterol-linked glucan primer that is then incorporated into the growing cellulose chain (Peng et al., 2002), or that β1,4glucanase functions in the recycling of these sterol glucoside primers (Robert et al., 2004).

Due to the high redundancy in cellulose synthase (CesA) and CesA-like genes, the insoluble nature of the cellulose synthases, localized together with β1,3glucan synthases in detergent-resistant membrane microdomains (Bessueille et al., 2009), the potential for a glucan-β-sitosterol primer, and the requirement for other proteins such as

β1,4glucanase (cellulase) for the "synthesis" of cellulose, the precise mechanism of cellulose synthesis and the number of different proteins, CesA subunits, and activating factors has not yet been fully determined.

47 An additional cellulose synthase class (GDP-glucose : (1,4)--D-glucopyranose

1-4-glucosyltransferase [GDP-forming], EC 2.4.1.29) consuming GDP-glucose as substrate, but still producing a linear unbranched glucan homopolymer with β1,4- linkages has been reported (Flowers et al., 1969; Chambers and Elbein, 1970). However, a partially purified particulate activity incorporating both glucose and mannose from

GDP-Glc and GDP-Man into a polymer characterized as β1,4glucomannan was reported in 1969, as well (Elbein, 1969). Recently, the search for polysaccharide synthases in plant genomes produced few results other than the numerous cellulose synthase-like (Csl) genes. When these Csl proteins were expressed recombinantly in an insect cell line and microsomal preparations were assayed for synthase activity, they variably synthesized

β1,4glucan, (cellulose), β1,4mannan, or mixed β1,4glucomannan (~2:1 Glc:Man ratio) depending on the pool of radiolabeled substrate(s) provided (Leipman et al., 2005; Suzuki et al., 2006). The preferential incorporation of Glc over Man (2:1) into β1,4- glucomannan is likely the result of a lower Km for GDP-Glc, as was suggested by Elbein

(1969). In addition, the recombinant Csl synthases produced polymers with a degree of polymerization of 800-6000 and did so without the addition of an exogenous acceptor to the assay. Thus, it would appear that a single synthase can produce multiple polymers, potentially without an initial acceptor, and suggests that the previously reported GDP- forming cellulose synthases (EC 2.4.1.29) are actually a class of hemicellulose synthases producing β1,4glucomannan in plants under physiological conditions.

Chitin is the second most widespread aminosugar oligosaccharide in biomass production of any carbohydrate polymer (by an order of magnitude), in nature

(Merzendorfer, 2006). Chitin is a linear unbranched and unmodified homopolymer of N-

48 acetylglucosamine (GlcNAc) with β1,4-linkages. Individual chains of chitin participate in interchain hydrogen bonding resulting in strong, resistant and insoluble microcrystalline fibrils (Rao et al., 1998) similar to cellulose. The chain length or degree of polymerization reported from partial acid hydrolysates from Saccharomyces suggests chains up to 100 residues in length (Kang et al., 1984). In Fungi, microfibrils are estimated to be composed of 20-400 sugar chains associated through hydrogen bonding

(Ruiz-Herrera and Martinez-Espinoza, 1999) and are found in covalent association with the homopolymer 1,3glucan, having one 1,4-chitin-glucan bond per 8,000 hexose units

(Kollár et al., 1995). The chitin content of fungal cell walls is variable, composing as little as 1-2% of the dry weight in the yeast Saccharomyces (predominantly associated with bud scars), 10–20% in the walls of filamentous fungi, such as Neurospora and

Aspergillus, and as much as 40% in Aspergillus nidulans (Specht et al., 1996).

Chitin has been identified: 1) extensively in Fungi (Latgé, 2007; Bowman and

Free, 2006), 2) in molting Ecdysozoa (arthropods) (Gagou et al. 2002; Falini & Fermani,

2004; Merzendorfer, 2006), 3) to participate in biomineralization in mollusc shells and radula (Furuhashi et al., 2009), 4) in bacterial spores of Streptomyces lunalinharesii and chitooligosaccharides of Rhizobium (Gomes et al., 2008), 5) in the diatom frustule

(Tesson et al., 2008; Wustman et al. 1998; Hecky et al. 1973), 6) in the oomycete

Saprolegnia (Briolay et al., 2009), 7) in the cyst walls of the protist Entamoeba (Arroyo-

Begovich et al., 1978; Campos-Góngora et al., 2004), Opisthonecta (Calvo et al., 2003) and Hyalophysa (Landers, 1991), 8) in eggshells of nematodes (Dubinský et al., 1986;

Veronico et al., 2001), 9) from Xenopus embryos (Semino et al., 1996), 10) in the annelid tubeworm Lamellibrachia (Imai et al., 2003), and 11) on surfaces of infected and

49 transfected cells from viral-encoded synthases of Ectocarpus siliculosus-virus EsV-1

(Delaroque et al., 2001) and the Chlorella-viruses PBCV-1 and CVK2 that infect the algal Chlorella symbiont of the ciliate protist Paramecium bursaria (Kawasaki et al.,

2002; Yamada et al., 2005, 2006). The related polymer of chitosan is the partial (>50%) enzymatic N-deacetylated variant of chitin and is a large component of walls of some fungi (Mucor, Rhizopus, Rhizomucor), the cyst wall of Entamoeba (Das et al., 2006), in insect cuticles (Anax, Apis, Oecophila), in crustaceans' shells (Lepas, Sacculina,

Neptunes), and in arachnids (Ruiz-Herrera and Martinez-Espinoza, 1999).

Chitin synthase [UDP-N-acetyl-D-glucosamine: chitin 4-β-N-acetylglucosamine transferase, EC (2.4.1.16)], formerly chitin synthetase, was first identified in 1957 in

Neurospora by Glazer and Brown, was demonstrated to form microfibrils in vitro in 1969 by Bartnicki-Garcia and Lippman, and the first chitin synthase gene was described in

1986 from Saccharomyces by Bulawa et al. Since 1986, the number of genes coding for demonstrated and putative chitin synthases has grown dramatically. Among the Fungi the number of chitin synthases per species is variable, from one gene in the ancestral fungus Encephalitozoon cuniculi to more than 20 genes in Rhizopus oryzae (Latgé, 2007) and loss of two of three genes is typically not lethal (Bulawa et al., 1993).

To date, all chitin synthase complexes characterized contain multi-subunit integral membrane proteins with multiple membrane-spanning domains, the enzyme complex has a native molecular weight of 500-600 kDa, are functional when activated and localized to the plasma membrane, and possess a cytosolic active site and an extracellular product, suggesting the formation of a pore complex (Roncero, 2002; Latgé, 2007; Cabib, 1987;

Ruiz-Herrera and Martínez-Espinoza, 1999). Activation by proteolysis has been noted

from many fungal systems (Cabib, 1987), but lacking in others such as chitin synthase III 50 of Saccharomyces (Merz et al., 1999). If an inhibiting subunit is removed or if the enzyme is modified like other proenzymes remains undetermined.

Chitin synthases, like cellulose synthases, also appears to be active within membranes under specific conditions. Support for this was provided by Duran and Cabib

(1978) who showed that: 1) the synthase is activated in the presence of specific lipids; in this case phosphatidylserine or lyso-phosphatidylserine, 2) pretreatment with phospholipase A resulted in complete inhibition of activity, and 3) activity can be abolished upon addition of unsaturated, but not saturated, fatty acids (Duran and Cabib,

1978).

Up to 80% of fungal chitin synthases may be found as inactive zymogens in fungal Golgi-derived chitosome vesicles with a uniquely different lipid composition from the plasma membrane heavily incorporating sterols and glycolipids (Bartnicki-Garcia,

2006; Ruiz-Herrera and Martínez-Espinoza, 1999; Cohen, 2001; Cabib, 1987). Recently, both chitin synthase and β1,3glucan synthase from the oomycete Saprolegina (Briolay et al., 2009), as was also determined for both β1,3glucan synthase and cellulose synthase from a higher plant (Bessueille et al., 2009), reside together in detergent-resistant membrane microdomains which is consistent with the lipid profile of chitosomes.

Chitin synthases are not inhibited by tunicamycin (Cabib, 1987), an inhibitor of the lipid-linked precursor dolichol-phosphate-GlcNAc involved in eukaryotic N- glycosylation and undecaprenyl-phosphate-MurNAc-(pentapeptide) involved in bacterial peptidoglycan synthesis (Price and Momany, 2005; Heijenoort, 2007), and [14C]GlcNAc was not incorporated into extractable lipid components (Cabib, 1987) indicating that a lipid-intermediate is not involved in chitin synthesis from yeast.

51 In addition, chitin synthases from most sources do not elongate exogenous chitooligosaccharides in vitro (Duran and Cabib, 1978; Cabib, 1987), and thus are generally considered processive enzymes that do not require an acceptor. In this model the sugar residue is transferred from the nucleotide-activated sugar to the non-reducing end of the glycan resulting in the cleavage of the nucleotide diphosphate and elongation continues until the polymer is released; and cannot be elongated further. However, both a recombinantly expressed chitooligosaccharide synthase from Mesorhizobium loti (NodC) and a similar activity from Xenopus embryos whose protein sequence is similar to NodC incorporated free [14C]GlcNAc and p-nitrophenyl-β-N-acetylglucosaminide (pNP-

GlcNAc; linked to C1 of GlcNAc - the reducing terminus) into short chitooligosaccharides (n5), indicating that they do serve as a primer and that chain elongation occurs at the non-reducing terminus (Kamst et al., 1999). These results are consistent with the known synthesis of lipo-chitin (Nod factor), which is characterized primarily as a chitopentaose glycan. Since both the Mesorhizobium and Xenopus systems produce short chitooligosaccharides and not the extended polysaccharide typical of this polysaccharide synthase family, these results should be considered in context.

In the yeast, the addition of GlcNAc to in vitro assay systems was either mildly stimulatory or inhibitory when homogenates were used as enzyme source, but was not required for activity (Duran and Cabib, 1978; Cabib, 1987; Kang et al., 1984). When chitin synthase from Saccharomyces was purified 10,700-fold by product entrapment, the enzyme preparation was insensitive to added GlcNAc (Kang et al., 1984). In addition, the NodC chitooligosaccharide synthase and the Xenopus activity initiated elongation with GlcNAc, but not chitooligosaccharides larger than the monomer as a primer (Kamst

52 et al., 1999), while in the yeast even the monomer was not incorporated into chitin in vitro (Duran and Cabib, 1978; Cabib, 1987; Kang et al., 1984). Use of a novel microscopy approach has suggested that chitin from the tubeworm Lamellibrachia is synthesized from the non-reducing end in vivo (Imai et al., 2003), as well.

A second important structural glucan to be discovered in cell walls is 1,3glucan.

The 1,3glucan polymer has been extensively studied from many diverse genera of Fungi and plants and is also found as an energy reserve (known as laminaran) and a cell wall constituent in many algae. In plants the polymer is commonly called callose and from

Agrobacterium the polymer is referred to as curdlan.

The 1,3glucan synthases (UDP-glucose : (1,3)--D-glucopyranose 1-3-glucosyl transferase, EC 2.4.1.34) consume UDP-glucose in the production of a linear unbranched glucose homopolymer. Callose (syn. 1,3glucan) synthases of plants are generally involved in wound healing or in response to infection, while fungal β1,3glucan synthases produce structural wall components during growth and wall turnover. These synthases are typically dependent on the divalent cations Ca2+ and Mg2+ [nearly equally effective in

Daucus carota (Lawson et al., 1989)], the substrate UDP-Glc; where other nucleotide diphosphate-sugars (ADP-, CDP-, GDP-Glc; UDP-Man, UDP-GlcNAc, UDP-GlcA,

UDP-GalA, UDP-Gal) were ineffective (Wu et al., 1991), and on GTP (10 µM GTPγS stimulates partially purified Aspergillus β1,3glucan synthase 20-fold in vitro; Kelly et al.,

1996) through a GTP-binding regulatory subunit (Rho1p in Saccharomyces). In vitro, the

1,3glucan isolated from partially purified enzyme from Aspergillus nidulans produced linear glucan chains up to 1,500 residues in length (Kelly et al., 1996) that is similar to alkali-extracted filaments from fungal cell walls (Manners et al., 1973; Fleet and

53 Manners, 1976). This fungal cell wall glucan was determined to be present in a 10:1 ratio to chitin (Kollár et al., 1995), and in covalent association to chitin via 1,6glucan (Kollár et al., 1997).

For several decades, attempts to purify to homogeneity β1,3glucan synthase enzyme complexes failed as these activities are associated with large insoluble membrane proteins that exist in association with accessory and regulatory subunits. In addition, while the spatial separation of cellulose and β1,3glucan deposition in pollen tube growth suggested the existence of two different glucan synthase complexes (Kudlicka and

Brown, 1997), differentiating between each complex was complicated since during the purification of the structurally related synthases partially purified from membrane fractions, the cellulose synthase produced β1,3glucan instead of β1,4glucan (cellulose)

(Mio et al., 1997) and this was true for the Dictyostelium CesA (cellulose synthase) gene product as well (Blanton and Northcote, 1990; Blanton et al., 2000). However, it is now known that β1,3glucan synthase and cellulose synthase are localized together in detergent-resistant membrane microdomains (Bessueille et al., 2009), which may explain the anomalous results of apparent polymer switching during purification and the intractable nature of the associated subunits to attempted detergent extraction. Finally, the use of photoaffinity probes to attempt to determine the catalytic or substrate-binding subunit(s) produced mixed results identifying multiple polypeptides of common masses among the Fungi tested, but also labeling numerous other polypeptides even in protection assays with unlabeled substrate. Perhaps the most convincing evidence for the identification of an active substrate-binding subunit was accomplished by non-radioactive photoaffinity labeling from Neurospora crassa by protein microsequencing performed on

54 peptide fragments containing cross-linked probe, which identified FKS (Schimoler-

O'Rourke, 2003).

With the application of molecular techniques, studies with Saccharomyces revealed that the β1,3glucan enzyme complex consists of at least two components; a catalytic and a regulatory subunit. The regulatory component essential for β1,3-glucan synthase activity has been identified as a GTP-bound active geranylgeranyl-modifed

Rho1p (S. cerevisiae), which is itself activated when localized to the cytosolic face of the plasma membrane by a GDP/GTP exchange factor (Rom2p in Saccharomyces) and physically associates with putative catalytic subunits (Mio et al., 1997; Latgé, 2007).

Besides the regulatory component, gene products required for β1,3glucan synthase activity generally considered as probable catalytic subunits have been identified from Saccharomyces (FKS1-2), Candida (GSC1, GSL1-2), Aspergillus (FKSA),

Schizosaccharomyces (BGS1-4), and cotton (GSL, glucan synthase-like) by photoaffinity labeling, by co-localization of enzyme activity during immunoprecipitation and product- entrapment, by selective detergent extraction, and gene disruption (Kelly et al., 1996;

Mio et al., 1997; Doblin et al., 2001; Cortés et al., 2007). Kelly et al. (1996), using most of these techniques in Aspergillus nidulans identified specific polypeptides of 200 kDa

(similar to Saccharomyces Fks1p and molecular masses found in other systems), 57 kDa

(within the range common to other β1,3glucan synthase complexes), 31 kDa (annexin- like proteins; Bouzenzana et al., 2006; Bulone and Fevre, 1996; Andrawis et al., 1993), and 20 kDa (Rho1p orthologs), similar to polypeptide masses identified for other

β1,3glucan synthases that have been partially purified. A similar series of subunits was proposed from A. fumigatus including a 180-kDa probable active subunit, a 21 kDa

Rho1p, a 55 kDa band, and a 160 kDa polypeptide found to be similar after peptide 55 analysis to Rhizobium and Agrobacterium β1,2glucan transporters and the 160 kDa band specifically co-purified only during product entrapment (i.e. synthesis of the glucan)

(Beauvais et al., 2001), though numerous other bands were labeled with a photoaffinity probe. However, purification to homogeneity has remained elusive and the mechanism of action of the synthase complex remains an active area of research.

The most common and abundant structural hexose-containing glycans known are cellulose and β1,3glucan, composed of glucose. The widely distributed aminoglycans chitin and chitosan are also glucose derivatives composed of GlcNAc or its post-synthesis

N-deacetylated variant, respectively. Other Glc-derivative polymers include: 1) murein in the Bacteria composed of alternating β1,4-linked GlcNAc and N-acetylmuramic acid

(MurNAc, the ether of lactic acid and GlcNAc) and 2) pseudomurein in the gram-positive methanogenic Archaea, composed of alternating β1,3-D-GlcNAc and β1,3-L-TalANAc

(N-acetyltalosaminuronic acid).

Murein precursor biosynthesis starts by the generation of two UDP-GlcNAc molecules. One UDP-GlcNAc is converted to MurNAc with the subsequent addition of amino acids resulting in an activated MurNAc-pentapeptide. The N-acetylmuramic acid pentapeptide is then linked via a phosphate bond (by MraY) to the isoprenoid undecaprenyl to which the second GlcNAc is added (by MurG) (van Heijenoort 2007;

Holtje, 1999). The undecaprenyl-phosphate-GlcNAc-MurNAc-pentapeptide is then translocated to the extracellular surface of the plasma membrane and assembled into existing murein chains by the murein synthase complex (van Heijenoort 2007; Holtje,

1999). Recently, a likely candidate for a flippase (MurJ) required for translocation of the lipid-linked precursors has been identified through bioinformatics and partially

characterized in an inducible-promoter system in E. coli, and homologues have been 56 identified from the methanogenic archaebacteria Methanobacterium and Methanosarcina

(Ruiz, 2008), which produce pseudomurein and in a similar fashion, methanochondroitin, respectively.

A variation on the synthesis of murein, the biosynthesis of pseudomurein starts with the nucleotide-activated disaccharide UDP-GlcNAc-(1,3)-GalNAc, but not UDP-

GlcNAc-(1,3)-TalANAc as would be expected (Kreisl and Kandler, 1986; König et al.,

1989; Hartman and König, 1991; Kandler and König, 1998), and it has been suggested that UDP-GlcNAc-(1,3)-TalANAc is formed by the oxidation and epimerization of the terminal GalNAc of the nucleotide-disaccharide (König et al., 1989). The biosynthesis of the pseudomurein peptide moiety begins with a UDP-activated glutamic acid to which L- amino acids are connected individually (König and Kandler, 1979b; Hartmann et al.,

1990; Hartmann and König, 1994; König et al., 1994). The disaccharide and pentapeptide are finally combined in a UDP-activated precursor which becomes linked to the polyprenol undecaprenyl-phosphate as in murein synthesis (Hartmann and König,

1990).

Nucleotide-activated oligosaccharides from the archaebacteria Methanosarcina and Hallococcus have also been identified as UDP-GalNAc-(1,3)-GlcA and UDP-

GalNAc-(1,4)-GalNAc-(1,3)-GlcA, and were characterized as structural elements in the biosynthesis of methanochondroitin (Kreisl and Kandler, 1986; Kandler and König,

1998). However, the enzymes involved in these biosynthetic routes for the synthesis of pseudomurein or methanochondroitin have not yet been identified.

In addition to the synthesis of murein among the Bacteria, and pseudomurein and methanochondroitin among the Archaea, several pathogenic prokaryotes (or strains of

typically non-pathogenic species) synthesize polysaccharide capsules that mimic 57 polymers of vertebrates including: hyaluronan (Streptococcus pyogenes and Pasteurella multocida type A), heparan/heparin (as the unsulfated heparosan; Pasteurella multocida type D and E. coli K5), and chondroitin (Pasteurella multocida type F and E. coli K4)

(DeAngelis, 2002). However, with the exception of the unmodified vertebrate hyaluronan, these microbial polymers lack sulfation or other modifications typical of the host polymers.

Transposon insertional mutagenesis was used to identify hyaluronan synthases

(HAS) from Streptococcus pyogenes (spHAS) (DeAngelis et al., 1993) and Pasteurella multocida (pmHAS) (DeAngelis et al., 1998). The transposon-disrupted DNA was used to generate probes for obtaining intact synthase sequences, which were subsequently expressed in E. coli. In all HAS known from the Bacteria, viruses, and vertebrates the synthesis of hyaluronan, composed of alternating GlcA and GlcNAc, occurs from a single protein with two glycosyltransferase activities (Jing and DeAngelis, 2000; DeAngelis,

1993, 1998) and are active as monomers (Pummill et al., 2007). The reported acceptor specificity of pmHAS is highly specific for UDP-GlcA and UDP-GlcNAc, with minimal activity reported with UDP-GalA, UDP-GalNAc, or UDP-Glc (DeAngelis, 2002).

While the streptococcal and Pasteurella hyaluronan synthases produce the same polymer, their sequences and mechanism of synthesis are distinctly different. The streptococcal HAS (type Class I) is more similar to vertebrate enzymes with multiple transmembrane spanning domains and the HAS from Streptococcus equisimilis (seHAS) is activated and protected from detergent-inactivation in the presence of tetraoleoyl (18:1) cardiolipin-containing liposomes (Weigel et al., 2006), while the Class II Pasteurella

HAS (type Class II) associates with membranes through a C-terminal tail region and

when cleaved produces a soluble active enzyme monomer (Weigel et al., 1997). The 58 pmHAS has been shown to elongate exogenous acceptors specifically (hyaluronan oligosaccharides) or non-specifically (chondroitin, up to 10,000 sugars, or variation in the orientation of the terminal residue's C-4 hexosamine hydroxyl, the C-5 of the uronic acid, or presence of C-6 hexosamine sulfation) (Tracy et al., 2007). In contrast, spHAS and vertebrate enzymes are not known to elongate acceptors (DeAngelis, 1999), and are thought to directly incorporate sugars from their nucleotide donors into elongating chains.

Also different between the two classes of HAS is the directional synthesis of hyaluronan chains. The Class II pmHAS elongates short tri- or tetra-saccharides of hyaluronan from the non-reducing end (Williams et al., 2006), whereas the Class I spHAS and vertebrate

HAS elongate chains from the reducing end (Tlapak-Simmons et al., 2005; Prehm, 2006).

In hyaluronan synthesis by Class I enzymes, the growing chain is always attached to

UDP (i.e. UDP-hyaluronan), and cleavage of the UDP-hyaluronan linkage at the reducing end results in chain termination (Weigel et al., 2006). This implies that a UDP-sugar serves as both the substrate and primer for the reaction.

The bulk of the polysaccharide synthases discussed thus far have been processive glycosyltransferases or utilize UDP-glucose or its derivatives (i.e. UDP-GlcNAc, UDP-

GlcA, UDP-MurNAc) in the formation of β-glucans (or their corresponding glucan derivatives). The advantage in the selection of glucose-oriented synthases is not known, but may have evolved from machinery already in use for producing the storage polymers of starch, which have a core backbone of α1,4-glucose. Production of a cell wall based on enzymes for the synthesis of starch would be convenient, but potentially impractical over time as cells possess corresponding α-glucanase activities and this may have driven the evolution of the β-linkage widely used in cell wall glucans and aminoglucans, though

59 in addition to a cell wall component algae use β1,3-glucan (laminaran) as an energy storage polymer, as well.

However, to maximize the chances of survival it would be advantageous for organisms that form a protective cyst or spore to withstand periods between hosts or nutrient deprivation, or for organisms that live in a niche with great competition and that experience enzymatic attack among rivals to evolve away from the glucan model with

(1→4)-linkages, to develop a more elaborate wall with greater modifications (e.g. acetylation, sulfation, cross-linking, etc.), and/or develop walls with alternate sugar compositions. Development of polymers based upon galactose instead of glucose including galacturonic acid, guluronic acid (the epimer of mannuronic acid), galactofuranose (5-carbon ring), and N-acetylgalactosamine (GalNAc) may have occurred in response to these selective pressures. An examination of the literature regarding polymers based upon galactose and GalNAc suggests this might be plausible.

In Aspergillus, Distler and Roseman (1960) partially characterized a polymer of

GalNAc (>60% deacetylated) from A. parasiticus QM 884 secreted into the culture medium, which produced only GalNH2 with an α-optical rotation (no other sugar or amino acid) upon complete acid hydrolysis. An alkali-extractable polymer composed solely of GalNAc was also identified from washed mycelia, and high concentrations of

GalNH2 was detected following hydrolysis of young mycelia of Aspergillus parasiticus

(4.0% of dry weight), A. oryzae (2.7%), A. niger (2.3%) and lesser amounts in

Helminthosporium sativum, Neurospora spp., and Rhizopus sp..

The GalNAc polymer secreted into the culture fluid was further characterized from Aspergillus parasiticus AHU 7165 by Araki et al. (1979), who generated

oligosaccharides of n=50 maximum chain length by partial acid hydrolysis, demonstrated 60 enzymatic de-N-acetylation of the polymer by a purified Aspergillus deacetylase with a preference for chain lengths >14 and that was inactive with chitin or peptidoglycan

(Araki and Ito, 1988), showed that ammonium tartrate added to cultures stimulated polymer synthesis and deacetylase activity up to 7-fold, and identified poly(GalNAc) deacetylase activity in multiple other species.

Takada et al. (1981) fully characterized the Aspergillus parasiticus polymer as a linear α1,4-N-acetylgalactan or poly(GalNAc) (55-65% random N-deacetylated) with an average degree of polymerization of 200 after partial acid hydrolysis and generation of partially methylated methyl-glycosides assessed by GLC-MS and 1H-NMR. More recently, Fontaine et al. (2000) identified a β-1,3-glucanase and chitinase-resistant insoluble poly(GalNAc) polymer from the related A. fumigatus cell walls and culture supernatants, with 1-4 linkages as determined by GLC-MS of the partially methylated methyl-glycosides, but did not focus further on this polymer.

Interestingly, an uncharacterized polymer of GalNAc/GalNH2 was also found from a virus-containing strain of Penicillium stoloniferum (18% of cell wall dry weight) and was minimally present (<1%) in five other virus-free strains tested (Buck et al.,

1969), suggesting a model similar to the viral-encoded hyaluronan and chitin synthases demonstrated to produce polymers on the surface of infected cells (Delaroque et al.,

2001;Kawasaki et al., 2002; Yamada et al., 2005, 2006). However, no further work pertaining to an α1,4-poly(GalNAc) polymer or the associated synthase activity required for its synthesis have appeared in the literature since. The function of the secreted polymer from Aspergillus parasiticus remains undetermined, but it was speculated to function as a bioflocculant (Araki and Ito, 1988).

61 Neisseria sicca is a gram-positive member of the β-proteobacteria and is considered a commensal of the vertebrate throat. Their environment suggests the necessity to evade the host immune response, salivary enzymes, and other microorganisms. Thus, it is of interest to note that a branched GalNAc polymer has been described with α1,4/α1,6-linkages isolated from sonicated Pronase- and SDS-treated cell walls (Adams and Chaudhari, 1972). However, no further work has appeared in the literature since.

Kottel et al., (1975) described an additional poly(GalNAc) polymer from

Myxococcus xanthus, a rod-shaped Gram-negative bacterium of the δ-proteobacteria with a complex lifecycle similar to cellular slime molds such as Dictyostelium and

Polysphondylium, and the plasmodial slime mold Physarum. Myxococcus can be induced to differentiate and form myxospores in vitro in response to the addition of glycerol or to nutrient depletion in liquid culture, or can be induced to form myxospores from fruiting bodies by transfer to a nutrient-free agar surface. Myxospores were collected free of cells by treatment with 1M KCl, and myxospore walls were isolated following ballistic disintegration with glass beads and subsequent extraction with 1% SDS. Myxospore walls were resistant to boiling in 2% SDS with β-mercaptoethanol, 10M urea, 45% phenol at 65oC, pronase, trypsin, cellulase, or Helix pomatia gut enzyme mixture (Kottel et al., 1975).

It was determined that myxospore walls contain over 75% carbohydrate and 50% of the dry weight is a galactosamine polymer (> 85% N-acetylated) (Filer et al., 1977).

The galactosamine polymer was considered separate from α1,3glucan after treatment with Aspergillus α1,3glucanase (Sutherland et al., 1977) or peptidoglycan after treatment

62 with lysozyme (White et al., 1968). The peptidoglycan content of Myxococcus walls decreased during sporulation (Bui et al., 2009).

In Myxococcus, Filer et al. (1977) described a pathway of inducible enzyme activities from liquid cultures induced to form myxospores. Six of seven enzymes required to convert fructose-1,6-bisphosphate to UDP-GalNAc were identified from crude homogenates and their activities were induced 4-8 fold (85-224 nmol min mg protein-1) including a 5.25-fold induction of UDP-GlcNAc 4'-epimerase activity (to 210 nmol min mg protein-1). Later, White et al., (1977) demonstrated incorporation of

[14C]GalNAc from UDP-[14C]GalNAc into an acid insoluble material using cell free extracts of Myxococcus, and upon hydrolysis the resistant material yielded only

[14C]galactosamine, and unlabeled glucose and acetate.

In the plasmodial slime mold Physarum polycephalum, a galactosamine polymer was identified as the major constituent of the walls of spherules (95.5%) and spore walls

(97.5% of carbohydrate, 82% including melanin) upon partial and complete hydrolysis

(McCormick et al., 1970) and UDP-GlcNAc 4'-epimerase activity was detected (Hiatt and Whiteley, 1974). However, determination of the extent of N-acetylation or further characterization of this polymer was not attempted.

Amoebidium parasiticum, is an ectocommensal of some arthropods and attach thalli externally to the exoskeleton of larval fresh water insects or crustaceans. Trotter and Whisler (1965) found neither chitin nor cellulose in cell walls as acid hydrolysates of the wall material showed no evidence of glucose or glucosamine. Carbohydrate compositional analysis revealed 70% galactosamine, 23% galactose, and 7% xylose, in addition to protein. The apparent lack of chitin, glucan, and cellulose raised the

possibility of a lack of a relationship of A. parasiticum with other groups of fungi, and 63 DNA evidence confirms that A. parasiticum is a protist (Benny and O'Donnell, 2000;

Ustinova et al., 2000).

A final galactosaminoglycan characterized in detail from the cellular slime mold

Dictyostelium is a repeating block copolymer of [β1,3Gal-α1,3GalNAc] with β1,6Gal- branching and this polymer is a component of the spore walls and stalk associated with cellulose deposition (Sakurai et al., 2002).

Giardia Cyst Wall Synthesis: Scope of Research

Giardia requires a protective cyst wall to survive outside its host for up to 30-60 days before parasites are no longer viable. The synthesis of a unique wall structure resistant to common glycan hydrolases would provide a distinct advantage during this time. Previous biochemical evidence from our laboratory demonstrated large amounts of the uncommonly associated wall monosaccharide, GalNAc (detected as GalNH2 during carbohydrate compositional analysis) from extremely resistant cysts and cyst walls.

Thus, it was likely that Giardia elaborates a unique carbohydrate polymer of GalNAc, and its synthesis may have been the result of selective pressures of cysts in the watershed waiting to be consumed and continue the life cycle.

Based on additional biochemical data demonstrating an inducible pathway of enzymes including UDP-GlcNAc 4'-epimerase responsible for the synthesis of UDP-

GalNAc, which is feed-forward regulated in the anabolic direction, then it was reasonable to ask if Giardia possessed an enzyme activity similar to other glycan synthases which consume nucleotide diphosphate-sugars as substrates in the production of extracellular polysaccharides. Initial attempts to assay homogenates of encysting trophozoites using

14 the radiolabeled tracer UDP-GalNAc resulted in incorporation of [1- C]GalNAc into a 64 precipitable or filterable product that was chemically and enzymatically resistant in a similar fashion to the filamentous walls of mature in vitro- or in vivo-derived cysts. No incorporation of [1-14C]GalNAc was detectable in three assay systems using homogenates of non-encysting trophozoites as enzyme source.

Thus, the scope of this work is to identify, purify, and characterize the novel polysaccharide synthase responsible for the synthesis of the unique Giardia cyst wall filaments from trophozoites induced to encyst in vitro. The synthase activity will be characterized with respect to induction of activity during the encystment process, sub- cellular distribution, potential requirement for an acceptor, specificity for nucleotide- sugar donors, inhibitors, assay kinetics, and general assay conditions.

However, because an enzyme of this nature has not been identified previously, the purification and biochemical characterization of the unique cyst wall filaments synthesized by the enzyme in question will also be studied in order to fully characterize this novel enzyme activity involved in cyst wall polysaccharide synthesis. The isolated cyst walls will be characterized with respect to carbohydrate and amino acid composition and an attempt will be made to produce antiserum to purified cyst walls, if immunogenic.

The isolated cyst wall filaments will serve as source material for compositional analysis, to determine the extent of polymer branching, for elucidating the inter-residue linkage(s) and linkage conformation(s), potential monosaccharide substitutions or modifications, the percent N-acetylation of the GalNAc monomers, and the degree of polymerization of isolated oligosaccharides following controlled partial acid hydrolysis.

Cyst walls will also serve as source material to determine susceptibility to known glycosidases and will be examined by electron microscopy. Further, physical data and

computer-aided modeling will be used to begin constructing a model of the filamentous 65 cyst wall and to better understand its extreme resistance to chemical treatments, enzymatic degradation, and to dissolution by most available means.

Results of these studies and implications for the process of encystment in Giardia will be presented and discussed. In addition, this investigation of parasite cytodifferentiation is likely to yield new insights into the basic understanding of pathways associated with the developmental regulation, protein sorting and secretion, and the biochemical synthesis of the Giardia cyst wall. Potentially these results may lay the ground work for future identification of the genes associated with cyst wall microfibril formation, their cellular regulation, potential targets for antiprotozoal treatments, and a deeper understanding of the process of cyst wall biogenesis in this early diverging and yet highly evolved eukaryote.

66 CHAPTER II

METHODS

Reagents

Radiochemicals used were: UDP-N-acetyl-D-[1-14C]galactosamine (54.7 mCi mmol-1; New England Nuclear, Boston, MA), UDP-N-acetyl-D-[1-14C]glucosamine

(282.8 mCi mmol-1; New England Nuclear), UDP-D-[U-14C]galactose (272.8 mCi mmol-1

, New England Nuclear), UDP-D-[1-14C]glucose (318.1 mCi mmol-1; New England

Nuclear), D-[1-14C]galactosamine (55.4 mCi mmol-1; Amersham, Arlington Heights, IL),

D-[1-14C]Glucosamine (45.0 mCi mmol-1; ICN, Costa Mesa, CA), UDP-N-acetyl-D-[6-

3H]galactosamine (15 Ci mmol-1; American Radiochemical Inc., St. Louis, MO), UDP-

N-acetyl-D-[1-3H]glucosamine (20 Ci mmol-1; Amersham, Arlington Heights, IL), where

1.0 mCi mmol-1 equals 37 MBq mmol-1. [14C]mannose was a gift from Dr. Carlos

Semino.

Maintenance of Parasites in Culture and Induction of Encystment

Giardia intestinalis (strain MR4) trophozoites were cultured axenically in the presence of Low Bile Culture Medium (LB; 10% Bovine serum, 1.0 mg ml-1 Bovine bile, pH 7.0) and induced to encyst asynchronously in the presence of High Bile Encystment

Medium (HB; 5% Bovine serum, 10.0 mg ml-1 Bovine bile, pH 7.6), modified from previously described methods (Schupp et al., 1988; Macechko et al., 1992). The following modifications to the original protocols were made: 1) the medium was reused for standard culture (up to 3 cycles of 72 h), 2) fetal bovine serum was substituted for bovine serum, and 3) cells were induced to differentiate in the presence of 5% instead of

67 10% serum. Percent encystment was quantified based on counting at least 200 Giardia forms in a hemocytometer using the following calculation:

(refractile complete cysts + rounded wall-positive cyst forms) x 100

(refractile complete cysts + rounded wall-positive cyst forms + trophozoites)

This encystment procedure consistently produced high levels of encysting forms and cysts (50 to 70 % after 48 h).

CWS / 3GaN-T Purification Procedure

Culture flasks of 24 to 36 hour encysting trophozoites were chilled to 4oC for 60 min to remove adherent trophozoites, collected by centrifugation of culture medium at

1,000 g for 10 min, washed in 0.25 M sucrose three times, and mechanically disrupted on ice with 30 to 50 strokes in a Potter-Elvehjem tissue homogenizer, as described previously (Lindmark et al., 1980; Macechko et al., 1992, Steimle et al., 1997). The level of homogenization was assessed using phase contrast microscopy by periodically sampling the process.

Giardia homogenates were subjected to differential centrifugation at 4oC as previously described (Macechko et al., 1992; Lindmark et al., 1980). Briefly, defined subcellular fractions were collected by differential centrifugation (2 x 5 min at 500 g) to remove unbroken cells, nuclei, ventral adhesive disks and other large debris and the clarified supernatant was further subjected to centrifugation at 44,000 g for 60 min to produce non-sedimentable (S-fraction) and particulate (P-fraction) fractions. The differential centrifugation fractions were monitored for the presence of marker enzymes

68 (Lindmark et al., 1980), protein concentration (Bradford, 1976), and CWS activity (Karr and Jarroll, 2004).

The P-fraction was then further sub-fractionated to separate microsomal vesicle populations, membrane fragments, and insoluble components using isopycnic centrifugation modified from the method of Lindmark et al. (1988). Briefly, the P- fraction (~30-40 mg protein) was adjusted to 6.0 ml with ice cold 0.25 M (8.5%) sucrose and the pellet was resuspended by 3 strokes in a 15-ml glass Dounce Homogenizer

(Bellco, Vineland, NJ) on ice without foaming. A 32-ml continuous 10-66% sucrose gradient was then formed within a 39-ml polypropylene tube over a 3-ml plug of 66% sucrose.

The suspended P-fraction was then divided equally (3.0 ml or ~15-20 mg protein) and layered by peristaltic pump onto two identically prepared gradients and the tubes were overlaid with 0.15 M (5%) sucrose to ensure a compact uniform loading of sample and proper rotor balance. The completely filled tubes were then heat sealed and subjected to ultracentrifugation at 189,000 g (39,000 rpm) for 3.25 h at 10oC within a

Beckman VTi50 vertical ultracentrifuge rotor and using a Beckman L8-70 ultracentrifuge without deceleration breaking. There was no difference in visible bands, protein, or the distribution of marker enzymes in fractions subjected to centrifugation for 1.5 or 3.25 h.

Fractions (3.0-ml each) were collected by gravity after piercing the tube bottom.

All fractions were monitored for marker enzymes, CWS activity, protein, and the optical density of sucrose used as a measure of average fraction density, as described below.

Peak fractions of CWS activity were pooled, diluted 1:3 with CWS buffer (10 mM Trizma pH 7.5, 10 mM MgSO4, 20% glycerol), subjected to 3 freeze-thaw cycles

over dry-ice, and sedimentable proteins, microsomes, and membrane fragments were 69 concentrated free of sucrose and non-sedimentable components by centrifugation at

105,000 g (15,000 rpm) for 45 min using a Beckman SW 40 Ti rotor at 10oC without deceleration breaking.

Sedimentable proteins (100% recovery of CWS activity) were resuspended and gently mixed using a 15-ml glass Dounce Homogenizer (Bellco, Vineland, NJ) in ice cold 1% deoxyBigCHAPS in CWS buffer and intermittently mixed while on ice for up to

4.0 h. Detergent-insoluble proteins were collected by centrifugation at 105,000 g for 45 min as described above and retreated with 1% deoxyBigCHAPS. The final deoxyBigCHAP-insoluble pellet (100-125% recovery) was resuspended in CWS buffer

(including 20% glycerol) and stored at –20oC. No loss of CWS activity was detected in samples tested after 3 years of storage.

Glycosyltransferase Assays

Initial transferase assays conducted were based on that of Cabib (1972) and Duran and Cabib (1978) for the assay of chitin synthases from yeast. Typically, 20 g of partially purified CWS subcellular fractions were assayed for 30 min in the presence of

14 10-100 M UDP-GalNAc (20-200 nCi UDP-[1- C]-GalNAc), 10 mM CaCl2 or MgSO4, and 10 mM Trizma buffer pH 7.5. Iodoacetamide (5.0 mM) was included in initial subcellular fractions as an irreversible alkylating agent useful as a cysteine proteinase inhibitor. Assays conducted in a 50 l volume were terminated by the addition of 1.0 ml of 67% ethanol. The reaction product(s) was separated from excess substrate either by filtration, chemical precipitation and centrifugation, or by reducing SDS-PAGE. The

70 radiolabeled reaction product was quantified by standard scintillation counting or by autoradiography.

Later, more highly purified fractions were assayed for CWS reaction product(s) by: 1) colorimetric assay of UDP release and 2) potential incorporation into defined biotinylated-acceptors monitored by streptavidin membrane capture and scintillation proximity assay of solution-phase biotinylated-acceptors or using glass microarrays of solid-phase acceptors with [1-14C]GalNAc incorporation monitored by phosphor imaging and [6-3H]GalNAc incorporation monitored by CCD imaging, as described below.

Separation of reaction product(s) by filtration

The ethanol- or TCA-precipitated CWS product(s) was collected by vacuum filtration over a 2.4 cm diameter 1.2 m pore size glass fiber filter (Whatman GF-C,

Polyfiltronics, Cat. 28497-641, MA). The filters were prewetted with and the filter residue washed once with 10 ml of 0.8 M acetic acid in 20% ethanol followed by a wash with 5.0 ml of 100% ethanol. Washed filters were counted in 5.0 ml of Scintiverse E scintillation cocktail (Fisher Scientific, IN) in a Beckman scintillation counter. The limit of detection for enzyme activity was determined to be 0.001 nmol min-1 mg-1 protein or

1.0 pmol incorporation (background of 100-150 cpm, <0.1% of available substrate) in a

60 min assay.

71 Separation of reaction product(s) by precipitation and centrifugation

Alternately, the CWS product(s) was precipitated with 1.0 ml of 67% ethanol and concentrated to pellet by centrifugation at 15,000 g for 2 min. The supernatant was carefully removed by pipette and the pellet resuspended and washed twice more with 0.4 ml of 100% ethanol. The final pellet was collected by resuspension in 1.0 ml of

Scintiverse E (Fisher Scientific, IN) and transferred by plastic pipette to 4 ml of

Scintiverse E for counting.

Separation of reaction product(s) by reducing 1D SDS-PAGE and autoradiography

For analysis of a potential glycoprotein product(s), complete reaction mixtures and/or ethanol precipitates assayed in the presence of the substrate UDP-[1-14C]-GalNAc were subjected to heating to 100oC in standard 2% SDS / 5% BME sample buffer for 5 minutes and were separated by 1D SDS-PAGE, as described. Gels were stained with

Coomassie® Brillant Blue R-250 and photographed. For autoradiograms, gels were either treated with En3hance™ (Perkin-Elmer, Waltham, MA) before drying and exposed to X-

Omat AR5 film (Perkin-Elmer) for 2 weeks at –80oC, or gels presoaked in 5% glycerol were dried and exposed to BioMax™ MS film (Perkin-Elmer, Waltham, MA) with

BioMax™ Transcreen® LE (Perkin-Elmer) between the gel and film at –80oC for up to 3 days.

Susceptibility of in vitro generated radiolabeled CWS reaction product to various proteinase treatments was performed on washed reaction products from peak CWS- containing isopycnic fractions prior to separation and autoradiography. All assays were conducted in duplicate with one set measured for radioactive incorporation by

scintillation counting. Typically, CWS-positive samples produced up to 45,000 d.p.m. 72 (~10% of total label) of incorporated [1-14C]GalNAc prior to separation by 1D SDS-

PAGE, with or without treatment.

Conditions for the assay of proteinase susceptibility were as described in Manning et al. (1992) except for the inclusion of Pronase E (Sigma, St. Louis, MO), which was assayed in 0.05 M sodium phosphate buffer pH 6.8 at 37oC. Briefly, all proteinase digestions were performed using 1.0 mg ml-1 proteinase (>4 to >10,000 U mg-1) in 0.25 ml for 2 hours. An equal volume of 2 X SDS-PAGE sample buffer was added and the samples were heated to 100oC, a sample was immediately loaded and separated on a 10%

1D-SDS-PAGE with a 5% stacking gel, and subjected to autoradiography as described.

Monitoring the release of the reaction product UDP

Incorporation of one mole of GalNAc from UDP-GalNAc or monosaccharide from other nucleotide-sugars into product is accompanied by the liberation of one mole of UDP. Simultaneous incubation with 1 unit per well of alkaline phosphatase (EC

3.1.3.1, Bovine intestinal, phosphodiesterase-free, Sigma, St. Louis, MO) results in the liberation of 2 moles of free PO4 per mole of sugar incorporated. The degradation of nucleotide-phosphates during the assay also relieves product inhibition of sensitive enzymes.

The liberated PO4 can be detected by a modification of Hoenig et al. (1989) with the addition of an equal volume of 2X Phosphate Detection Reagent (0.05% Synperonic®

PE/F68, 0.05% malachite green, 1.0% ammonium molybdate, 2.0 M HCl). The 2X

Phosphate Detection Reagent Stock is prepared in parts; Part 1 - the Malachite Green

-1 Stock (0.8 g dye L , adjusted for dye purity) is prepared in dH2O, mixed well, and

filtered through a PES 0.22 µm filter unit (#431098; Corning Life Sciences, Lowell, MA) 73 and Part 2 - the Ammonium Molybdate Stock (40 g ammonium molybdate added slowly with stirring to 0.666 L of 12 M HCl is then adjusted to 1 L with dH2O, which should remain clear). Then stock solutions Part 1 and Part 2 are mixed in a 3:1 ratio producing the 2X Phosphate Detection Reagent Stock, which is stable at room temperature for more

® than one year. On the day of use, Synperonic PE/F68 (CAS # 9003-11-6) in dH2O is added fresh (50 µl of a 1.0% stock ml-1) to an aliquot of the 2X Phosphate Detection

Reagent Stock, which remains stable for 24 hours.

The limit of detection of the assay is approximately 2.0 M free PO4 (1.0 µM

UDP from UDP-sugar) equivalent to 10.0 pmol UDP-sugar consumed. Detection in a 10

µl 384-well microtiter plate format was accomplished using an EnVision (Perkin-Elmer) or Saphire (Tecan, Durham, NC) microtiter plate reader at a wavelength of 630 nm (± 2.5 nm). The assay system is insensitive to buffers and EDTA, but alkaline phosphatase requires Zn2+ (which is tightly bound) and is activated by Ca2+ or Mg2+.

Separating products of lipid extraction by paper chromatography

CWS reactions were scaled up for immediate use and all fractions were kept on ice. The 15,000 g pellets from CWS reactions were washed 3 times to remove any free

14 [ C]substrate by resuspension in dH2O and centrifugation at 15,000 g for 10 min. The

CWS reaction pellets were then pooled as source material for potential extraction of lipid-linked intermediates following the methods of Semino and Dankurt, (1993). The

CWS pellet was extracted with 1 volume of chloroform:methanol:dH2O (1:2:0.3; 1203- solvent) and the 1203-extract was treated with 0.01 M HCl for 10 min at 100oC. After centrifugation at 15,000 g for 10 min, the supernatant was then treated with 0.1 M HCl

74 for 10 min at 100oC. Samples of each fraction were retained for liquid scintillation counting.

To distinguish between lipid-intermediates and residual mono or oligosaccharide components of the 1203- and the hydrolyzed 1203-extract, the remainder of each sample was reduced to near dryness using a stream of nitrogen and paper electrophoresis was performed according to Semino and Dankert (1993). The paper chromatogram lanes were sectioned (1 cm intervals) and counted in a liquid scintillation counter. As standards, charged UDP-[14C]GalNAc and neutral [14C]mannose were included in separate lanes during paper chromatography. The [14C]GalNAc standard was prepared by hydrolysis with 0.1 M HCl for 10 min at 100oC.

Separation of reaction product by streptavidin membrane capture

A library of linear and branched oligosaccharides (Lectinity Holdings, NY) conjugated at their reducing ends to a biotin molecule through a multi-carbon spacer was assayed at 100 µM by biotin capture on SAM2® Biotin Capture Membrane (Promega,

Madison, WI) and by scintillation proximity assay of biotinylated-acceptors using streptavidin-functionalized Yttrium oxide SPA™ imaging beads (# RPNQ0271) and a

LEADseeker™ imaging system (GE Healthcare, Piscataway, NJ).

Using SAM2® Biotin Capture Membranes, up to 250 pmoles of the biotinylated- neoglycoconjugate acceptors assayed were spotted in duplicate to a membrane (1,000 pmoles maximum binding per cm2), and after 10 min the membrane was washed inverted

1 time each for 5 min in 100-ml baths of: 1) 0.2 M NaCl, 2) 0.01% Triton X100, 3) dH2O, and then 4) 20% ethanol. After air drying, the membrane was placed against a

phosphor imaging film screen (BAS-TR2025, Fuji Film Medical Systems, USA) for 24- 75 72 hours. Detection of [14C]-labeled reaction product(s) was accomplished by phosphor imaging (Fuji Film Medical Systems, USA) of the exposed film and quantification was done using the manufacturer’s software.

For assays with SPA™ imaging beads, 10 µl assays in 384-well microtiter plates contained: 1) biotinylated-peptides (2 µM, 20 pmoles), UDP-[6-3H]-GalNAc (2 µM, 20 pmoles, 200 nCi), and appropriate cofactors (10 mM Ca2+ for CWS; 10 mM Mn2+ for the human ppGaN-T11, and gpGaN-T10), or 2) biotinylated-oligosaccharides (50 µM, 500 pmoles), UDP-[6-3H]-GalNAc or UDP-[1-3H]GlcNAc (100 µM, 1000 pmoles, 200 nCi), and appropriate cofactors (10 mM Ca2+ for CWS; 10 mM Mn2+ for the human β3Gn-T2).

For assays of biotinylated-peptides, reactions were terminated with the addition of 10 µl detection mixture containing 100 µg (25 pmoles binding capacity) streptavidin- functionalized yttrium oxide SPA™ imaging beads in 100 mM EDTA with 0.01% Triton

X-100-PC, pH 10. For assays of biotinylated-oligosaccharides, 2 µl aliquots (in duplicate) were transferred to wells of a 384-well microtiter plate containing 40 µl of the detection mixture (above) with 400 µg (100 pmoles binding capacity) streptavidin- functionalized yttrium oxide SPA™ imaging beads. Imaging was accomplished using a

LEADseeker™ imaging system with CCD camera and protocol was optimized for coincidence counting using the manufacturer's software.

Positive controls included: 1) endogenous CWS activity measured separately, 2) a UDP-GlcNAc : β-D-galactosaminide N-acetylglucosaminyltransferase activity (human

3Gn-T1 / GenBank™ AF092051 [synonym β3Gn-T2 / AB049584], Millennium

Pharmaceuticals), 3) a polypeptide GalNAc transferase (human ppGaN-T11 / GenBank™

EAL24518, Millennium Pharmaceuticals) active against the mucin repeat peptides such

76 as EA2 and Muc7 (New England Peptide, Fitchburg, MA), and 4) a glycopeptide

GalNAc transferase (human gpGaN-T10 / GenBank™ BAC56890, Millennium

Pharmaceuticals) active only with a GalNAc-α-O-modified peptides such as gp-EA2

(Sussex Research Laboratories, Ottawa, CA).

Characterization of CWS / 3GaN-T Activity

CWS activity partially purified from the detergent-insoluble fraction of microsomal ESV's was characterized for the effect of typical reaction conditions including enzyme and/or acceptor concentration, acceptor specificity, substrate specificity, cofactor requirement, temperature and pH optima, and enzyme kinetics.

The concentration of enzyme was varied initially from 0-1.0 mg protein ml-1 and over a range of incubation time (0-120 min) to determine a linear range within which to work. Assays were generally conducted for 30 min at 37oC in the presence of the typical assay mixture which included 10-20 g total protein, 10-100 M UDP-GalNAc (20-200

14 nCi UDP-[1- C]-GalNAc), 10 mM MgSO4, and 10 mM Trizma buffer pH 7.5, unless otherwise noted.

The potential acceptor specificity was addressed by assaying CWS (as described) in the presence of UDP-[1-14C]GalNAc or UDP-[6-3H]GalNAc and the following potential acceptors: 1.0 mg ml-1 ovalbumin (chicken egg white; Sigma), 1.0 mg ml-1 asialofetuin (fetal calf type II, Sigma), 1.0 mg ml-1 mucin (bovine submaxillary type I-S,

9-17% sialic acid; Sigma), 1.0 mg ml-1 asialomucin (porcine stomach type III, 0.5-1%

-1 -1 sialic acid; Sigma), 1.0 mg ml bovine serum albumin, 1.0 mg ml GalNH2-conjugated

-1 BSA (Sigma), 1.0 mg ml GlcNH2-conjugated BSA (Sigma), 2 M of 12 biotinylated

77 mucin repeat (glyco)peptides (custom synthesized by New England Peptide, Fitchburg,

MA; and Sussex Research Laboratories, Ottawa, CA) (Table II), 0.1 mg ml-1 GalNAc-

1,3-GalNAc-sp-biotin (Lectinity Holdings, Inc., NY), 100 M of 109 monovalent solution-phase biotinylated-oligosaccharides (custom library by Lectinity Holdings, Inc.,

NY) (Table III), a micro array of solid-phase polyvalent oligosaccharides (GlycoMinds

Ltd., Lod, Israel) (Table IV), and protein from encysting Giardia trophozoites. As an acceptor was not identified and likely not required, it was not possible to vary an acceptor independently.

Table II. Solution-phase biotinylated (glyco)peptide acceptors assayed.

Code Peptide Sequence EA2 -PTTDSTTPAPTTK-amide red-EA2 -GSTTPAPG-amide gp-EA2 -PTTDST(GalNAc-α-O-)TPAPTTL-amide Muc1a -PPAHGVTSAPDTRPA-amide Muc2 -PTTTPISTTTMVTPTPTPTC-amide r-Muc2 -SPTTSTPISSTPQPTS-amide Muc5ac -GTTPSPVPTTSTTSAP-amide gp-Muc5ac -GT(GalNAc-α-O-)TPSPVPTTST(GalNAc-α-O-)TSAP-amide mg-Muc -QTSSPNTGKTSTISTT-amide mod-HIV -RGPGRAFVTIGKIGNMR-amide Epo-T -PPDAATAAPLR-amide Zonadhesin -PTERTTTPTKRTTTPTIR-amide * All acceptors were N-terminal biotin modified through a 6-aminocaproic acid spacer. ** Purity was assessed by mass analysis using LC/MS and for glycosylation by NMR. All peptides were at least 95% pure except glycopeptides, which were >99% pure. ***Glycosylation sites indicated were verified by LC/MS and NMR and are underlined.

78 Table III. Solution-phase biotinylated neoglycoconjugate acceptors assayed.

Code Sequence GA-001 Neu5Acα-biot GA-002 Neu5Acα-biot GA-003 Neu5Acβ-biot GA-004 Rhaα-biot GA-005 Fucα-biot GA-006 Manα-biot

GA-007 6-PO4-Manα-biot GA-008 Glcα-biot GA-009 Glcβ-biot GA-010 Galα-biot GA-011 Galβ-biot GA-012 (Fucα1-2)Galβ-biot GA-013 GlcNAcβ-biot GA-014 (Fucα1-3)GlcNAcβ-biot GA-015 (Fucβ1-3)GlcNAcβ-biot GA-016 (Fucα1-4)GlcNAcβ-biot GA-017 GalNAcα-biot GA-018 GalNAcβ-biot

GA-019 3-O-SO3-Galβ-biot

GA-020 6-O-SO3-GlcNAcβ-biot

GA-021 6-SO3-GalNAcα-biot GA-022 Glcα1-4Glcβ-biot GA-023 GlcNAcβ1-4GlcNAcβ-biot GA-024 GlcNAcβ1-4GlcNAcβ-biot GA-025 GlcNAcβ1-3GalNAcα-biot GA-026 GlcNAcβ1-3(GlcNAcβ1-6)GalNAcα-biot GA-027 GlcNAcβ1-4(GlcNAcβ1-6)GalNAcα-biot GA-028 GlcNAcβ1-6GalNAcα-biot GA-029 Galα1-2Galβ-biot GA-030 Galβ1-2Galβ-biot GA-031 Galα1-3Galβ-biot GA-032 Galα1-3(Fucα1-2)Galβ-biot GA-033 Galα1-3(Fucα1-2)Galβ-biot GA-034 Galβ1-3Galβ-biot GA-035 Galα1-3GalNAcα-biot GA-036 Galα1-3GalNAcβ-biot GA-037 Galβ1-3GalNAcα-biot GA-038 3'-O-SO -Galβ1-3GalNAcα-biot 3 79 Code Sequence GA-039 Galβ1-3(GlcNAcβ1-6)GalNAcα-biot GA-040 (Fucα1-2)Galβ1-3GalNAcα-biot GA-041 Galβ1-3GalNAcβ-biot GA-042 (Blank)------GA-043 Galβ1-3GlcNAcβ-biot GA-044 (Fucα1-2)Galβ1-3GlcNAcβ-biot GA-045 Galβ1-3(Fucα1-4)GlcNAcβ-biot

GA-046 3'-SO3-Galβ1-3GlcNAcβ-biot

GA-047 3-O-SO3-Galβ1-3(Fucα1-4)GlcNAcβ-biot GA-048 Galβ1-4(Fucα1-3)GlcNAcβ-biot

GA-049 3-O-SO3-Galβ1-4(Fucα1-3)GlcNAcβ-biot GA-050 Galβ1-4Glcβ-biot GA-051 Galα1-4GlcNAc-biot GA-052 Galβ1-4GlcNAcβ-biot

GA-053 Galβ1-4(6-O-SO3)GlcNAcβ-biot GA-054 (Fucα1-2)Galβ1-4GlcNAcβ-biot

GA-055 3'-SO3-Galβ1-4GlcNAcβ-biot

GA-056 6'-SO3-Galβ1-4GlcNAcβ-biot GA-057 GalNAcα1-3(Fucα1-2)Galβ-biot GA-058 GalNAcα1-3(Fucα1-2)Galβ-biot GA-059 GalNAcα1-3GalNAcβ-biot GA-060 GalNAcβ1-3GalNAcβ-biot GA-061 GalNAcβ1-4GlcNAcβ-biot GA-062 Neu5Acα2-3GalNAcα-biot GA-063 (Blank)------GA-064 Neu5Gcα2-6GalNAcα-biot GA-065 Neu5Acα2-6GalNAcα-biot GA-066 Neu5Acβ2-6GalNAcα-biot GA-067 Neu5Acα2-8Neu5Acα2-biot GA-068 GlcNAcβ1-2Galβ1-3GalNAcα-biot GA-069 GlcNAcα1-3Galβ1-3GalNAcα-biot GA-070 GlcNAcβ1-3Galβ1-4GlcNAc-biot GA-071 GlcNAcβ1-4GlcNAcβ1-4GlcNAcβ-biot GA-072 Galα1-3Galβ1-4GlcNAcβ-biot GA-073 Galα1-4Galβ1-4GlcNAcβ-biot GA-074 Galβ1-4GlcNAcβ1-3GalNAcα-biot GA-075 Galβ1-4GlcNAcβ1-6GalNAcα-biot GA-076 (Blank)------GA-077 Manβ1-4GlcNAcβ1-4GlcNAcβ-biot

80 Code Sequence GA-078 Neu5Acα2-8Neu5Acα2-8Neu5Ac-biot GA-079 Neu5Acα2-3Galβ1-3GlcNAc-biot GA-080 Neu5Acα2-3Galβ1-4GlcNAcβ-biot GA-081 Neu5Acα2-3Galβ1-4Glcβ-biot GA-082 Neu5Acα2-6Galβ1-4Glcβ-biot GA-083 Neu5Acα2-3Galβ1-3GalNAcα-biot GA-084 Neu5Acα2-6Galβ1-4GlcNAcα-biot GA-085 Neu5Acα1-6(Galβ1-3)GalNAcα-biot GA-086 Neu5Acβ1-6(Galβ1-3)GalNAcα-biot

GA-087 (Galβ1-4GlcNAcβ)2-3,6-GalNAcα-biot GA-088 GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ-biot GA-089 Galα1-3(Fucα1-2)Galβ1-4GlcNAcβ-biot GA-090 Galα1-3Galβ1-4(Fucα1-3)GlcNAcβ-biot GA-091 Fucα1-2Galβ1-3(Fucα1-4)GlcNAcβ-biot GA-092 Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ-biot GA-093 Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ-biot GA-094 Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAcα-biot GA-095 GlcNAcβ1-4GlcNAcβ1-4GlcNAcβ1-4GlcNAcβ-biot GA-096 Galβ1-4GalNAcβ1-3(Fucα1-2)Galβ1-4GlcNAcβ-biot GA-097 Galα1-4(Galα1-3)Galβ1-4GlcNAcβ-biot GA-098 GalNAcα1-4(Fucα1-2)Galβ1-4GlcNAcβ-biot GA-099 Galβ1-4GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ-biot GA-100 GalNAcβ1-3(Fucα1-2)Galβ1-4GlcNAcβ-biot GA-101 Galα1-4(Fucα1-2)Galβ1-4GlcNAcβ-biot GA-102 GlcNAcβ1-3(GlcNAcβ1-6)GlcNAcβ1-4 GalNAcα-biot GA-103 Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ-biot GA-104 Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ-biot

GA-105 (Neu5Acα2)2-3,6-GalNAcα-biot

GA-106 (Neu5Acα2-6Galβ1-4GlcNAcβ1-2Man)2-Man-GlcNAcβ1-4GlcNAcβ-biot

GA-107 (Galβ1-4GlcNAcβ1-2Man)2-Man-GlcNAcβ1-4GlcNAcβ-biot GA-108 Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc-biot GA-109 GlcNAc-Manα1-3(GlcNAc-Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc-biot GA-110 GlcNAcβ1-4GlcNAcβ1-O-Asparagine-biot

GA-111 3-O-SO3-GalNacβ-biot

GA-112 Neu5Acα2-3Galβ1-4(Fucα1-3)-[6'-O-SO3-GlcNAc]β-biot

* Acceptors were biotinylated through a spacer at the reducing terminus. ** Purity was determined to be at least 95% pure as assessed by LC/MS and NMR.

81 Table IV. Solid-phase biotinylated neoglycoconjugate acceptors assayed.

Code Sequence Code Sequence C01 Glcβ1-4Glcβ1-4Glcβ1-4Glcβ C26 Galα C02 6-O-SO3-GlcNAcβ C27 Galα1-3(Fucα1-2)Galβ C03 GlcNAcα C28 Galα1-3Galβ1-4GlcNAcβ C04 GlcNAcβ C29 Galα1-4Galβ1-4Glcβ C05 GlcNAcβ1-3Galβ1-4Glcβ C30 Galβ C06 GlcNAcβ1-3GalNAcα C31 Galβ1-3(GlcNAcβ1-6)GalNAα C07 GlcNAcβ1-4GlcNAcβ C32 Galβ1-3GalNAcα C08 GlcNAcβ1-6GalNAcα C33 Galβ1-3GlcNAcβ C09 L-Rha-α C34 Galβ1-4Glcβ C10 GalA-β C35 Galβ1-4GlcNAcβ C11 Manα C36 Galβ1-6Galβ C12 Manα1-3(Manα1-6)Manβ C37 GalNAcα C13 Manα1-3Manα C38 GalNAcα1-3(Fucα1-2)Galβ C14 Manβ C39 GalNAcβ C15 Manβ1-4Glcβ C40 Biotin / Control C16 Neu5Acα C41 Fucα C17 Neu5Acα1-3Galβ1-4(Fucα1-3)GlcNAβ C42 Fucα1-2Galβ C18 Neu5Acα1-3Galβ1-4Glcβ C43 Fucβ C19 Neu5Acα1-3Galβ1-4GlcNAcβ C44 Glcα C20 Neu5Acα1-6Galβ1-4GlcNAcβ C45 Glcα1-4Glcα C21 Hydroxyl / Control C46 Glcα1-4Glcβ C22 L-Araf-α C47 Glcβ C23 GlcU-β C48 Glcβ1-3Glcβ C24 Xyl-α C49 Glcβ1-4Glcβ C25 Xyl-β C50 Glcβ1-4Glcβ1-4Glcβ * All acceptors were surface bound through a spacer at their reducing ends. ** Sample spots were separated by a Teflon® template which created micro wells of approximately 0.5-1.0 µl, each.

82 The substrate specificity of CWS was determined using the detergent-insoluble

ESV-fraction from encysting trophozoites. Potential substrates were assayed for 60 min in the presence of 10.0 mM MgCl2, 1.0 mM CaCl2, 1.0 mM MnCl2, and 100 M (200 nCi per reaction) UDP-[1-14C]GalNAc, UDP-[1-14C]GlcNAc, UDP-[U-14C]Gal, UDP-[1-

14 14 14 o C]Glc, [1- C]GalNH2, and [1- C]GlcNH2 at 37 C. In addition, both the P-fraction and

S-faction from both encysting cells were assayed for any detectable endogenous glycosyltransferase activity capable of incorporating any available radiolabeled nucleotide-sugar into an ethanol-precipitable product.

The effect of pH was determined using a 20 mM acetate-borate-cacodylate buffer system over the pH range of 4-10. Temperature optima was determined by varying temperature from 10-60oC during a 30 min assay.

Sub-cellular Organelle Marker Enzyme Assays

P-fraction (lysosome-like vesicles) / acid phosphatase

Acid phosphatase (EC 3.1.3.2) activity was determined colorimetrically according to the protocol of Müller (1973) measuring the release of -nitrophenol from - nitrophenolphosphate at 410 nm. Typical conditions included 75 g of Giardia homogenate or other crude fractions assayed in the presence of 12.5 mM - nitrophenylphosphate, 0.2% Triton X-100, and 100 mM acetate buffer pH 5.0 for 15 min at 30oC. Reactions were terminated with the addition of 3.0 volumes of ice cold stop mix

(100 mM NaCl, 70 mM sodium carbonate, 133 mM glycine pH 10.7).

83 S-fraction (non-sedimentable/cytosolic proteins) / malate dehydrogenase

Malate dehydrogenase (decarboxylating) activity (EC 1.1.1.37), found in the cytosol of amitochondriate eukaryotes, was determined using a continuous spectrophotometric assay following the reduction of NAD in a malate-dependent manner

(Lindmark, 1980). Assay conditions included 330 mM NAD, 0.66 mM CoCl2, 3.3 mM malate, and 0.1% Triton X-100 in 3.0 ml of 53 mM Trizma pH 7.3. Samples were assayed at 30oC and followed by the absorbance differential measured at 340 nm.

One Dimensional SDS-PAGE

Separation of reduced proteins was accomplished using 1.0 mm thick by 7.0 cm long (25 mA constant current) or 1.5 cm thick by 14.0 cm long (50 mA constant current)

1D SDS-PAGE using 10% separating gels, 5% stacking gel, and a discontinuous buffer system (Maniatis, 1984). Samples were prepared prior to electrophoresis by reduction in a 100oC water bath for 5 min with sample buffer containing 2% SDS, 5% - mercaptoethanol, 10% glycerol, 0.001% bromophenol blue, 0.01% pyronin-Y, and modified with or without 5.0 mM iodoacetamide (Lane, 1978). Molecular mass markers

(Sigma) included in each gel were: 1) myosin (205 kDa), 2) B-galactosidase (116 kDa),

3) phosphorylase B (97.4 kDa), 4) bovine albumin (66 kDa), 5) ovalbumin (45 kDa), 6) glyceraldehyde-3-phophate dehydrogenase (36 kDa), 7) carbonic anhydrase (29 kDa), 8) trypsinogen (24 kDa, 9) trypsin inhibitor (20.1 kDa), and 10) -lactalbumin (14.2 kDa).

Proteins were stained with either a modified Coomassie® Brillant Blue R-250 method

(0.02% Coomassie® Brillant Blue R-250, 5% acetic acid, 25% methanol) overnight, or gels were quick stained by rinsing in dH2O 3 x 30 sec followed by 5 min in standard

84 staining reagent (2.0% Coomassie® Brillant Blue R-250, 5% acetic acid, 45% methanol).

For semi-denaturing 1D SDS-PAGE, proteins were prepared in one tenth the reducing agent (0.5% -mercaptoethanol).

Preparative One Dimensional SDS-PAGE and Electroelution

CWS activity (2.0 mg of total protein) partially purified from ESV fractions by repeated deoxyBigCHAP treatments was mixed with 1 volume of 2X reducing sample buffer, layered onto a 1.5 x 14.0 cm 10% preparative acrylamide gel, and electrophoresed at 25 mA for 4.0 h at 4oC. Outer gel slices were removed and quick stained as described above to reveal the putative 30-33 kDa protein doublet. The region of the gel containing the doublet was excised and electroeluted from the gel slice into dialysis tubing using a makeshift horizontal electrophoresis chamber (horizontal agarose slab electrophoresis chamber) at 150 mA / 100 V for 3 h at 4oC. The protein sample was then dialyzed

o ® overnight against dH2O at 4 C and then concentrated using first a Centriprep and then a

Microcon® centrifugal filter unit (each with a 10 kDa nominal molecular mass cutoff;

Millipore, MA). All gel pieces except the excised band were restained together to verify correct protein excision had occurred. Recovery was confirmed by semi-reducing (0.5 mM BME) and reducing (50 mM BME) 1D SDS-PAGE, as described above. Recovery of protein excised from the preparative gel in the region of the protein doublet was 193

g or 9.65% of the total protein (2.0 mg) applied to the preparative gel.

85 Two Dimensional SDS-PAGE

Two-dimensional electrophoresis was performed according to the method of

O'Farrell (1975) by Kendrick Labs, Inc. (Madison, WI) as follows: isoelectric focusing was carried out in glass tubes of inner diameter 2.0 mm using 2% pH 4-8 ampholines for

9600 volt-hrs. Fifty nanograms of an IEF internal standard, tropomyosin, was added to each sample. This protein migrates as a doublet with lower polypeptide spot of 32.7 kDa and pI 5.2. The tube gel pH gradient was determined with a surface pH electrode. After equilibration for 10 min in buffer (10% glycerol, 50 mM dithiothreitol, 2.3% SDS and

62.5 mM Trizma, pH 6.8) the tube gel was sealed to the top of a stacking gel which is on top of a 10% acrylamide slab gel (0.75 mm thick). SDS slab gel electrophoresis was carried out for about 4.0 h at 12.5 mA per gel. The slab gels were fixed in a solution of

10% acetic acid/50% methanol overnight. The following proteins (Sigma Chemical Co.,

St. Louis, MO) were added as molecular weight standards to the agarose which sealed the tube gel to the slab gel for the reduced samples: myosin (220 kDa), phosphorylase A

(94.7 kDa), catalase (60 kDa), actin (43 kDa), carbonic anhydrase (29 kDa), and lysozyme (14 kDa). These standards appear as horizontal lines on the silver stained

(Oakley et al., 1980) 10% acrylamide slab gels. The silver-stained gel was dried between sheets of cellophane with the acid edge to the left of the recorded image. After slab gel electrophoresis, duplicate gels were transferred to transfer buffer (25 mM Trizma pH 8.8,

96 mM glycine, 10% methanol) and transblotted onto PVDF membrane overnight at 200 mA (~50 volts gel-1). The blot was stained with Coomassie® Brillant Blue R-250 for visualization of proteins.

86 Internal Polypeptide Digestion and Protein Microsequencing

Isoforms consistent with multiple protein phosphorylation states were identified from multiple spots of each band of the 1D SDS-PAGE protein doublet after separation by 2D SDS-PAGE. The presence of isoforms was confirmed after the major cleaved peptides of endoproteinase Asp-N hydrolysis were separated by HPLC and identified by protein microsequencing at the Protein Chemistry Core Facility (Howard Hughes

Medical Institute, Columbia University, NY) by Dr. Mary Ann Gawinowicz.

Autoradiography

After drying in the presence of 5% glycerol, gels were sprayed with Amplify™

(Amersham, Cardiff, Wales) and applied to X-Omat AR5 X-ray film or Amplify™ was omitted and the dried gel directly applied to BioMax™ MS autoradiography film (Perkin

Elmer, Waltham, MA) with a BioMax™ Transcreen® LE phosphor-coated intensifying screen (Perkin Elmer, Waltham, MA) between the film and gel. Exposure lasted up to 3 weeks at -80oC with no further bands appearing after 2 weeks when sprayed with

Amplify™ and applied to traditional autoradiography film. Development times were greatly reduced using the green-sensitive film and phosphor-screen requiring only 1 to 3 days. Green-sensitive film was developed using an X-Omat automatic film developer according to the manufacturer's instructions while the traditional film was developed by hand with reagents from Kodak.

87 Western Blot

Proteins were transferred from a 1.5 mm thick 14 x 14 cm acrylamide slab gel to nitrocellulose membrane using a semi-dry electro-blot apparatus and buffer kit (#ER35,

Owl Separation Systems, Waltham, MA) at 400 mA constant current (2.0 mA per cm2 of gel) for 90 min at room temperature.

Lectin Blot

Protein samples were prepared under reducing conditions, separated by 1D SDS-

PAGE, and transferred as described above onto PVDF membrane. Milk proteins (Stop-n-

Shop, non-fat dry milk) and chemically-defined reduced N-glycosylated ovalbumin

(Sigma; [GlcNac2-Man5-6]) served as positive and negative controls for lectin binding of

GalNAc-sensitive lectins, respectively. Following transblotting, the membrane was blocked in a solution of 3% BSA in Tris-buffered-saline (TBS) for 2.0 h at room temperature, washed 3 x 5 min in TBS, and probed with either 10 g ml-1 Vicia villosa-

HRP isolectin B4 (Sigma; affinity for terminal α- or β-GalNAc-O-serine or threonine of glycoproteins; the "Tn antigen"), 5.0 g ml-1 Arachis hypogaea-HRP lectin (Sigma; affinity for Gal--, Gal-1-3-GalNAc- [the T-antigen], and Gal-1-4-Glc-modified glycoproteins), or 20 g ml-1 Sophora japonica-biotin (Sigma; affinity for terminal

GalNAc- or Gal-modified glycoproteins) for 2.0 h at room temperature. Unbound lectin was removed and blots were washed 3 x 5 min in TBS. Blots probed with the primary lectin-biotin conjugate were washed, further treated with 2.0 g ml-1 of streptavidin-HRP

(Sigma) for 1.0 hour at room temperature, and then washed 3 x in TBS. Lectin binding was visualized after color development of the horse radish peroxidase (HRP)-conjugated

88 lectins or HRP-streptavidin-bound lectin with 0.67 mg ml-1 4-chloro-napthol and 0.02% hydrogen peroxide in TBS.

Production and Purification of Giardia Cyst Wall Material

Approximately 40 L of G. intestinalis (strain MR4) trophozoites were grown in axenic culture and encysted for 24-48 h as described previously (Macechko et al., 1992).

Cysts were collected by centrifugation of encystment medium at 750 g for 10 min at

25oC, and subjected to homogenization to disrupt trophozoites and partially encysted cells (Macechko et al., 1992). Cyst pellets were washed 5 x in 10 ml of distilled water and collected at 500 g.

Cyst wall filaments preparations were made by a modification of the procedure of

Manning et al. (1992) as described in Gerwig et al. (2002). All samples were stored at

–20oC when not in use and supernatants discarded as wash steps were monitored by phase contrast microscopy for the presence of intact cyst wall material prior to disposal.

The pooled sample was divided into two equal portions and processed separately.

Each cyst pellet was heated to 100oC in an equal volume of 10% SDS for 5 min. The cysts were concentrated by centrifugation at 12,000 g for 5 min (as in subsequent steps), and the supernatant was discarded. The cyst pellet was washed in 10 ml of distilled water

5 x until there were no visible signs of the detergent. The resulting cyst pellet was resuspended in 3 ml of amyloglucosidase buffer (20 mM acetate buffer, pH 4.5), transferred to an acid washed 20-ml glass, screw-capped scintillation vial with a flat bottom and a stir bar. One hundred U of amyloglucosidase (EC 3.2.1.3, Sigma A-3514,

St. Louis, MO) was added to the vial that was placed on a stir plate in a 55oC warm air

incubator for 60 min. This procedure was repeated once more and each time the 89 supernatant was discarded. After the two amyloglucosidase treatments, the SDS treatment described above was performed once again. The resulting cyst pellet was washed in 10 ml of freshly made papain buffer (100 mM PBS, pH 7.2, with 5 mM cysteine and 5 mM EDTA). The pellet was resuspended in 3 ml papain buffer and again placed in a glass scintillation vial with a stir bar. The sample was stirred with 50 U of papain (Carica papaya EC 3.4.22.2, Sigma P-4762, St. Louis, MO) for 3 h at 60oC. This procedure was repeated once more and each time the supernatant was discarded. Again the resulting cyst pellet was collected by centrifugation and washed, as in step one, with

10% SDS and water. The pellet resulting from the papain treatment was subjected to

DNase 1 (Bos taurus pancreas EC 3.1.21.1, Sigma DN-25, St. Louis, MO) and RNase A

(Bos taurus pancreas EC 3.1.27.5, Sigma R-4875, St. Louis, MO) treatments. The pellet was resuspended in 3 ml of XNase buffer (0.1 M PBS, pH 7.2, with 5 mM MgCl2) in a glass scintillation vial with a stir bar. The sample was incubated with 500 Kunitz units of

DNase and 5 Kunitz units of RNase stirring for 2 h at 37oC. This treatment was repeated once more and the pellet washed with amyloglucosidase buffer. At this point, all subsequent centrifugation steps were carried out at 44,000 g for 10 min at 10oC.

Following the DNase/RNase treatment, the pellet was subjected to another amyloglucosidase treatment as described above and then to a proteinase K (Tritirachium album, EC 3.4.21.64 syn. EC 3.4.21.14, P-0390, St. Louis, MO) treatment. The pellet was washed in proteinase K buffer 1 x (50 mM Tris, pH 8.0, 0.2% Triton X-100, 1.0 mM

CaCl2). The pellet was resuspended in the proteinase K buffer, placed in a glass scintillation vial with 50 U of proteinase K and stirred for 2 h at 60oC. This procedure was repeated once more and the pellet was washed with an equal volume of 10% SDS

o containing 100 mM DTT for 5 min at 100 C. The pellet was then washed 5 x in distilled 90 water to remove the detergent and DTT. The resulting cyst wall material (CWM) was lyophilized for 48 h yielding approximately 150 mg dry fine-powdered white material.

Complete Acid Hydrolysis

The chemical structural analysis of the cyst wall polysaccharide was performed at the Bijvoet Center (Utrecht University).

Ddried CWM (1.0 mg) and GalNAc (1.0 mg) were each treated with 1.0 ml of 2.0

M HCl for 3 h at 100oC, and with 1 ml of 4 M trifluoroacetic acid (TFA) for 5 h at 100oC.

After lyophilization, the residues were dissolved in 1.0 ml H2O. Aliquots of 25 l of the four samples were taken, lyophilized and trimethylsilylated with 25 l pyridine / hexamethyldisilazane / chlorotrimethylsilane 5:1:1 (v/v) for 30 min at room temperature.

Of each sample, 1.0 l was analyzed by GLC (Chrompack CP 9002 gas chromatograph with CP Sil 5CB column (25 m x 0.32 mm); temperature program: 140 - 240oC at 4oC min-1).

Controlled Partial Acid Hydrolysis

Dried CWM (8 mg) was treated with 0.5 ml of 0.5 M TFA for 15 min at 100oC.

After centrifugation, the supernatant was collected and the sediment was treated again with 0.5 ml of 0.5 M TFA for 15 min at 100oC. This procedure was repeated 15 times.

Then, the total supernatant was concentrated by a stream of N2 and lyophilized twice.

The residue was suspended in 2 ml H2O, filtered through a 0.22 m Millex-GS filter

(Millipore), and the filtrate was fractionated on a column (68 x 1.6 cm) of Bio-Gel P-2

91 -1 (Bio-Rad), using bidistilled H2O as eluent (22 ml min ) and UV monitoring at 206 nm.

Definite fractions were rechromatographed on the same column.

Monosaccharide and Amino Acid Compositional Analysis

Monosaccharide analyses after methanolysis, re-N-acetylation and trimethyl- silylation, including absolute configuration determination using (-)-2-butyl glycosides, was performed at the Bijvoet Center (Utrecht University) and the Complex Carbohydrate

Research Center (Athens, GA) by gas-liquid chromatography-mass spectrometry (GLC-

MS) as described (Gerwig et al., 1979; Kamerling and Vliegenthart, 1989). Quantitative amino acid analyses of the isolated cyst wall material were carried out as described in De

Baaij et al. (1986).

Amino sugar analysis was performed separately, as described (Risk et al, 1996).

Briefly, samples were suspended in distilled water (1 mg ml-1), and 10 pg of each sample was hydrolyzed in vucuo with gaseous 6M HCl at 105°C for 24 hours, using a Picotag work station (Millipore Corporation, Bedford, MA). Hydrolysates were derivatized with phenylisothiocyanate (Cohen et al. 1984) and the derivatized amino residues were separated by reverse-phase HPLC using a 15 cm Picotag Amino Acid Column.

Separation was achieved using a linear gradient shifting between 100% polar eluent and

100% non-polar eluent in 14.5 minutes at 42°C. Elution was monitored by absorption at

254 nm. Identification of the amino residues was by comparison with standards of known retention times. Quantification of amino residues was carried out by peak area comparison with identically processed external amino acid standards and the amino sugar standards D-glucosamine hydrochloride and D-galactosamine hydrochloride.

92 Polysaccharide Linkage Analysis (Methylation Analysis)

Permethylation was performed at the Bijvoet Center, Utrecht University, essentially as described (Ciucanu and Kerek, 1984). Dried sample (0.1 - 1.0 mg) was dissolved in 1.0 ml of dry dimethyl sulfoxide by ultrasonication for 30 min at 40oC.

Freshly powdered NaOH (~ 10 mg) was added under an inert atmosphere, and the mixture was sonicated for 20 min at room temperature. After cooling to 0oC, 400 l of methyl iodide was added, and sonication was continued for 30 min at a temperature not exceeding 20oC. The latter step was repeated with a second addition of 400 l methyl iodide. Then, 1.0 ml of dH2O containing a few crystals of sodium thiosulfate was added, and the methylated product was isolated by extraction with chloroform (3 x 0.5 ml). The organic phase was washed with water (3 x 0.5 ml) and concentrated under a stream of N2.

The residue was hydrolyzed with 1.0 ml of 4 M TFA (4 h, 100oC), and, after co- evaporation with methanol, reduced with NaBD4 in water (2 h, room temperature). The excess of reductant was destroyed by the addition of 2 M of acetic acid. Boric acid was removed by repeated evaporations with methanol. The sample was finally acetylated with 0.5 ml pyridine:acetic anhydride 1:1 (v/v) for 30 min at 100oC. The solvent was evaporated by a gentle stream of N2 with intermediate addition of 1 drop of toluene, and the residue was redissolved in dichloromethane. The partially methylated alditol acetates were analyzed by GLC-MS (Kamerling and Vliegenthart, 1989).

Proteinase Digestion of CWM (Pronase)

Samples (1.0 mg) were treated under stirring with Pronase in 1.0 ml of 0.1 M

Trizma buffer, pH 7.6, containing 5.0 mM CaCl2. Portions of 40 g Pronase (from

93 Streptomyces griseus EC 3.4.21.81, Boehringer Mannheim) were added at 0, 6, 24 and 32

o h, and the incubation was carried out for 48 h at 40 C under N2. After heat inactivation

(3 min at 100oC) of proteolytic activity, the solution was centrifuged, and the supernatant passed through a Sephadex G-25 column (HiTrap, 5 x 5 ml, Pharmacia) using dH2O as eluent (3 ml/h) and UV detection at 214 nm. Isolated peak fractions were investigated by monosaccharide and amino acid analysis.

Deglycosylation of CWM

N-linked enzymatic deglycosylation (PNGase F)

Native CWM and Pronase digested samples were treated with peptide-N- glycosidase (EC 3.5.1.52 PNGase F, Boehringer Mannheim) in 50 mM Tris-HCl, pH 7.4, containing 50 mM EDTA and 1% (v/v) 2-mercaptoethanol, for 24 h at room temperature.

Precipitation of protein material was induced by the addition of ice-cold methanol to the incubation mixture and centrifugation. The supernatant was fractionated on a Sephadex

-1 G-25 column (HiTrap, 5 x 5 ml, Pharmacia) using dH2O as eluent (3 ml min ) and UV detection at 214 nm. Isolated peak fractions were investigated by monosaccharide and amino acid analysis.

N- and O-linked chemical deglycosylation (alkaline borohydride)

CMW (3.0 mg) was treated with 1.0 ml of 0.1 M NaOH containing 1.0 M NaBD4.

o After stirring for 48 h at 40 C under N2 in the dark, the solution was acidified to pH 5 with 3.0 M acetic acid at 0oC. The solution was centrifuged (10 min, 3000 rpm), and the supernatant was repeatedly evaporated with methanol to remove boric acid. The residue

was dissolved in dH2O and passed through a Sephadex G-25 column (HiTrap, 5 x 5 ml, 94 Pharmacia) to remove residual salts. After lyophilization, the sample was investigated by

1H NMR spectroscopy and monosaccharide analysis. The sediment was dialyzed against

H2O to remove salts and investigated by monosaccharide analysis.

Deglycosylation of CWM by known glycosidases

CMW (50 g total weight, 30 g total GalNAc polymer) was assayed for 24 h in the presence of 0.25 to 70 U of 22 individual known glycosidases, under conditions and temperature recommended by the manufacturer. Enzymes obtained from Sigma-Aldrich

(St. Louis, MO) were assayed as follows: 0.25 U -N-acetylgalactosaminidase (EC

3.2.1.49), 0.25 U -Galactosidase (EC 3.2.1.22), 1.0 U -Galactosidase (EC 3.2.1.23), 10

U -Glucosidase (EC 3.2.1.20), 10 U Amyloglucosidase (EC 3.2.1.3), 20 U -Amylase

(EC 3.2.1.1), 20 U -Amylase (EC 3.2.1.2), 0.25 U -N-Acetylglucosaminidase A (EC

3.2.1.52, Bovine epididymis), 0.25 U -N-Acetylglucosaminidase B (EC 3.2.1.52, bovine epididymis), 1.0 U -N-Acetylglucosaminidase (EC 3.2.1.52, Canavalia ensiformis), 10

U Chitinase (EC 3.2.1.14, Serratia marcescens, C-1650), 10 U Chitinase (EC 3.2.1.14,

Streptomyces griseus, C-1525), 5.0 U -Galactosidase (EC 3.2.1.23), 10 U Dextranase

(EC 3.2.1.11), 100 g Lysing Enzyme (enzyme mixture, Rhizoctonia, L-8757), 100 g

Lysing Enzyme (enzyme mixture, Aspergillus, L-3768), 1.0 U Lyticase (poly-β1,3-

Glucan hydrolase, L-8137), 50 U Mutanolysin (N-acetylmuramidase, Streptococcus faecalis-acting, M-9901), 70 U Lysozyme (N-acetylmuramidase, Micrococcus-acting, L-

6876). Gross morphological or refractive changes of glycosidase-treated cysts were assessed by phase contrast microscopy compared to a sample of untreated cysts.

95 The release of free GalNAc monosaccharides was monitored by a modification of the Morgan-Elson assay for N-acetylated hexosamines (Chaplin and Kennedy, 1986). In brief, 150 μl of sample was incubated for 10 min at ambient temperature with the addition of 15 µl of 0.75% acetic anhydride in acetone to ensure complete acetylation and then 30 μl of 0.8 M H2BO3 (pH 9.1) was added and boiled for 3 min. Color was developed with the addition of 800 μl of (1% w/v) N,N-dimethyl--aminobenzaldehyde in acid (17.3 M acetic acid/1.0% 12 M HCl) following incubation at 37°C for 30 min.

Sample absorbance was read at 585 nm. After time for insoluble CWM to settle, samples were measured for absorbance at 585 nm using a Beckman DU-64 spectrophotometer.

Negative controls included the glycosidases in buffer without CWM added, CWM alone, and unacetylated galactosamine in buffer. A GalNAc-standard curve was prepared in the respective assay buffers and processed identically to the samples tested. The limit of detection was 4 g GalNAc per ml (1.0 g per assay or 3.3% of CWM polysaccharide tested). Chitin and chitinase were included as positive controls.

Matrix Assisted Laser Desorption-Time of Flight Mass Spectrometry

Positive-ion MALDI-TOF mass spectra were obtained on a Voyager-DE

(PerSeptive Biosystems) instrument operating at an accelerating voltage of 21 kV (grid voltage 95%, ion guide wire voltage 0.05%) and equipped with a VSL-337ND-N2 laser.

-1 The samples were dissolved in dH2O (1.0 mg ml ) and mixed with 2,5-dihydroxybenzoic acid (10 mg ml-1) in a ratio of 1:2. Recorded data were processed using GRAMS/386 software (v. 3.04, Galactic Industries Corporation).

96 1H NMR Spectroscopy

Samples were exchanged twice in D2O (99.9 atom% D, Isotec) with intermediate lyophilization. Finally, the material was dissolved in 0.5 ml of D2O (99.96 atom% D,

Isotec). One-dimensional resolution-enhanced 500-MHz 1H-NMR spectra were recorded on a Bruker AMX-500 spectrometer at a probe temperature of 300 K. Chemical shifts are expressed in ppm by reference to internal acetone ( 2.225) (Vliegenthart et al.,

1983). 2D TOCSY spectra with mixing times of 12, 25, 41, 50, and 125 ms were recorded at 500 MHz on a Bruker DRX-500 instrument. The spectra were processed using locally developed NMR software (Van Kuik, Bijvoet Center, Utrecht University).

2D ROESY experiments were carried out at 280 K with mixing times of 100, 200, 300, and 500 ms. The HOD signal was presaturated for 1 s during the relaxation delay.

Antibody Production

Polyclonal antiserum to CWM was generated in a New Zealand white rabbit.

After collection of a pre-immune serum, approximately 1.0 mg of CWM was mixed with

TiterMax® adjuvant (Sigma, St. Louis, MO) and injected intramuscularly. After 1 month, the rabbit was boosted with an additional 1.0 mg bolus in adjuvant. The animal was sacrificed after several months and approximately 80 ml of blood collected.

Electron Microscopy of CWM and CWS-containing Vesicles

Scanning electron microscopy (SEM) and transmission electron microscopy

(TEM) was performed by William Fowle and Rita Droste (Northeastern University,

Boston, MA) on the isolated CWM for gross comparison to intact mature cysts.

97 High resolution low voltage scanning electron microscopy (LVSEM) was performed by Drs. Stanley Erlandsen and Tim Macechko (University of Minnesota

Medical School, Minneapolis MN) on CWS-containing vesicle fractions assayed with and without UDP-GalNAc prior to fixation and samples of CWM collected at each step of the extraction and isolation procedure. Materials were fixed in 2% paraformaldehyde and 2% glutaraldehyde in 0.15 M sodium cacodylate buffer (pH 7.4), postfixed for 90–

120 min in 1% OsO4 in 0.15 M cacodylate buffer containing 1.5% potassium ferrocyanide, and dehydrated in an ascending ethanol series [50, 70, 80, 95, and 100%

(twice)] before critical point drying with CO2. Specimens were mounted on adhesive carbon films and then coated with 1 nm of platinum using an Ion Tech argon ion beam coater. Examination of samples was performed in a Hitachi S-4700 field emission SEM operated at 2 keV. Images were recorded on Polaroid type 55 film (Eastman Kodak;

Rochester, NY).

Field emission scanning electron microscopy (FESEM) was performed by Dr.

Stanley Erlandsen (University of Minnesota Medical School, Minneapolis MN). The primary mAbs used were 8C5.C11 and 5A4.G6, which recognize epitopes of CWP1 and

CWP2 (Campbell and Faubert, 1994; Lee and Faubert, 2003). Affinity-purified goat anti-mouse antibodies conjugated to 12-nm colloidal gold were obtained from Jackson

ImmunoResearch Laboratories (West Grove, PA). Cyst walls stained with immunogold were transferred to glass specimen carriers coated with 0.1% poly-L-lysine (Sigma; St

Louis, MO) and incubated at 4°C for 5-10 min. The cells were then fixed overnight at room temperature with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, containing 7.5% sucrose. Specimens were postfixed for 15-30 min in the same buffer

containing 1% OsO4 (Fisher Scientific; Fair Lawn, NJ), followed by dehydration in an 98 ascending alcohol series and critical point-drying with CO2 (Erlandsen et al., 1993), and then by platinum sputter coating. Detection was accomplished using a Hitachi S-900 field emission SEM using a YAG detector in the backscatter electron imaging mode with an acceleration voltage of 3.0-4.0 kV. Images were recorded on Polaroid type 55 film

(Eastman Kodak; Rochester, NY).

99 CHAPTER III

RESULTS

Identification of Induced CWS / 3GaN-T Activity

The initial assay of glycosyltransferase activity followed those of Cabib (1972) and Duran and Cabib (1978) for yeast chitin synthase. In the modification of these published assays, partially purified 3GaN-T fractions were incubated with UDP-[1-14C]-

o GalNAc and 0-50 mM CaCl2 or MgSO4 in 10 mM Trizma pH 7.5 for 30 min at 37 C.

This activity, tentatively named cyst wall synthase (CWS), was below the limits of detection (<0.001 nmol min-1 mg-1 protein) in non-encysting trophozoites, increased during the first 36 h of encystment (ca. 24-36 h), and declined as maximal encysment was reached at ca. 48 h (Figure 7).

CWS Sub-Cellular Localization and Purification

Figure 8 outlines the purification scheme used to partially purify CWS activity from encysting Giardia trophozoites. Based on the methods of Lindmark (1988), Figure 9 shows that CWS appears confined to the particulate fraction after differential velocity sedimentation of Giardia homogenates as determined by the biochemical markers malate dehydrogenase (non-sedimentable) and acid phosphatase (particulate).

To identify the particulate nature of the CWS activity isopycnic centrifugation was performed using a 10-66% continuous sucrose gradient and centrifuged from 1.25 to

3.25 h at 189,000 g max (Lindmark, 1988). CWS is found to localize within a particle population having a peak density of 1.16 g ml-1 in sucrose, direct physical measurement

100 Figure 7. Correlation of Time Course of Encystment and Induction of CWS Activity.

Representative graph depicting correlation of time course of encystment and the induction of CWS activity from in vitro encysting Giardia intestinalis. Incorporation of [1-14C]GalNAc by CWS was monitored quantitatively by liquid scintillation counting, as described in Methods. Specific activity on the left refers to the CWS activity in cell homogenates; maximal encystment equalled 68% at 36 h post-induction. The time-course plot of induction of CWS activity ( ) and encystment ( ) is superimposed on an autoradiograph corresponding to an SDS-PAGE gel in which the remaining assay mixture (100 mg protein per assay per lane) was loaded after scintillation counting aliquots were taken. Incorporation of [1-14C]GalNAc by CWS was monitored qualitatively by SDS-PAGE/autoradiography, as described in Methods. Radiolabelled CWS reaction products appear too large to enter the SDS-PAGE gel (e.g. the arrow points to the top of the stacking gel with radiolabeled product). For SDS-PAGE, all samples were mixed with sample buffer containing DTT, and electrophoresed through a 5% stacking and 10% running gel. The gel was stained with Coomassie® Brillant Blue R- 250, and the dried gel was sprayed with Enhance™ and exposed to autoradiographic film for 2 weeks at –70oC.

101 Figure 8. Diagram of CWS purification scheme from encysting Giardia trophozoites.

Encysting Giardia CWS 100% whole cell homogenate ME 100% AP 100%

N-fraction 2 x 500 g, 4 min (nuclei, unbroken cells, etc) 0.25 M sucrose

E-fraction (non-sedimentable)

44,000 g, 60 min S-fraction 0.25 M sucrose (non-sedimentable)

CWS 96% P-fraction ME 4% (organelles, insoluble, etc) AP 91% 189,000 g, 75-205 min 0-60% sucrose gradient *

* CWS ~50% in peak fraction

Fraction Density (g sucrose ml-1)

105,000 g, 45 min Supernatant CWS buffer (non-sedimentable)

Insoluble-fraction CWS ~100% [CWS] recovery

105,000 g, 45 min Supernatant 2 x 1% deoxyBigCHAP / CWS buffer (non-sedimentable)

Detergent insoluble-fraction CWS 100-125% [CWS] recovery

Figure 8. CWS was purified from encysting cells ~150-fold following: 1) homogenization in isotonic medium, 2) differential centrifugation, 3) isopycnic centrifugation, and 4) detergent extraction. Conditions were as described in Methods. Marker enzymes and CWS activity during the purification are given in percent and are representative of typical total recovery. CWS = cyst wall synthase activity. ME = malate dehydrogenase (decarboxylating; a marker enzyme for the non-sedimentable fraction - S-fraction). AP = acid phosphatase (a marker enzyme for the microsomal or particle fraction - P-fraction).

102 Figure 9. Sub-cellular localization of CWS activity within encysting Giardia.

A. B.

CWS 50 CWS 2

40

30

1 RSA

RSA 20

10

0 0 0 25 50 75 100 1.0 1.1 1.2 1.3

50 2 AP AP

40

30

1 RSA

RSA 20

10

0 0 0 25 50 75 100 1.0 1.1 1.2 1.3

50 2 ME

40

30

1

RSA 20

% Protein 10

0 0 0 25 50 75 100 1.0 1.1 1.2 1.3 Percent Equilibrium Protein Density

Figure 9. Distribution of cyst wall synthase (CWS) from encysting Giardia. The relative specific activities (RSA) of CWS and the marker enzymes alkaline phosphatase (AP; lysosome-like vesicles) and malate dehydrogenase (ME; cytosolic enzyme) are depicted in column A. The subcellular distribution of CWS and AP (from the P-fraction, shaded in column A) are depicted in column B. Equilibrium density was directly determined by optical refractometry. (from Karr and Jarroll, 2004)

103 determined by optical refractometry. Lysosome-like peripheral vacuoles of Giardia have a peak density in sucrose of 1.19 g ml-1, and are distinguished from ESVs based on buoyant density, biochemical marker enzyme comparison (acid phosphatase versus

CWS), induction pattern during encystment, and antibody localization. In addition, dramatic differences are evident in these vesicle populations including average vesicle diameter, intracellular location, and electron density as seen by TEM (Reiner et al., 1990;

McCaffery et al., 1994; Lanfredi-Rangel et al., 2003). Use of LVSEM to image the peak

CWS fraction from isopycnic centrifugation clearly shows an enriched population of large diameter vesicles (Figure 10). This CWS-containing microsomal population was strongly suggested to be the encystment specific vesicles (ESV’s) (Faubert et al., 1991) that appear during differentiation as CWS activity co-localizes with the binding of specific monoclonal antibodies generated using ESV-specific proteins, including the cyst wall specific proteins CWP1 and CWP2 (Table V).

CWS Localization

Using the peak CWS fraction from isopycnic centrifugation, 100% of the CWS activity was recovered in a 60 min 44,000 g pellet fraction after either 15 freeze-thaw cycles or after various detergent treatments and did not show latency or a requirement for reducing agents. Figure 11 shows a LVSEM of ESV's assayed in the presence of UDP-

GalNAc and collected by glutaraldehyde fixation, showing numerous random protrusions appearing to be outgrowths of GalNAc-containing cyst wall filaments. The filamentous outgrowths were not present in micrographs of the same fraction incubated without exogenous substrate. However, no direct biochemical or lectin-staining was pursued.

104 Figure 10. LVSEM photomicrographs: peak CWS-containing microsomal population

A. LVSEM of the peak CWS-containing microsomal fraction from 30-h encysting cells.

CWS and vesicles were enriched after isopycnic centrifugation showing a fairly uniform population of microsomal vesicles. The concave nature of the vesicles is likely the result of the hypertonic medium (1.16 g ml-1 sucrose) within which the vesicles were collected and subsequently processed with glutaraldehyde prior to electron microscopy.

B. Microsomal vesicles at higher magnification.

LVSEM at higher magnification of the same sample of CWS-containing microsomal vesicles, as described in A (above). Work performed at University of Minnesota, in collaboration with Dr. Stanley Erlandsen.

105 Figure 11. LVSEM of Giardia cyst wall filaments in vivo and CWS product in vitro.

A. Magnification of Figure 3 showing meshwork of cyst wall filaments.

(from Erlandsen et al., 1989. t = trophozoite * = filaments. bar = 0.5 µm.)

B. Likely In vitro-derived cyst wall synthase product from peak microsomal fraction.

(product of this work in collaboration with Dr. Stanley Erlandsen. bar = 0.25 µm.)

106 Table V. Co-localization of ESVs and CWS activity from the detergent insoluble fraction of Peak CWS-containing microsomes.

Partially Purified CWS Monoclonal Localization

NIH Immunogen Western Western Encysting Mature Monoclonal + 2BME – 2BME Trophozoites Cysts Antibody 5-3C a CWP1,2 30-33 60-65, 180-210 ESV CW *8G8 b rCWP2 30-33 60-65, 180-220 ESV CW *7D2 b rCWP2 31-63 60-65 ESV CW *5D2 ― 100 ESV CW *6F5 200 200 ESV CW 9C9 c BiP ― 100 ESV / ER CW *7E7 d gGSP 21-24 21-24 ESV / SV ― *9C3d gGSP ― ― ESV ― *3B5 36-40, 50 36-40, 50 ― CW 1G8 ― ― ESV CW 1C8 ― ― ESV CW 1F9 ― ― ESV CW 3H3 ― ― ESV CW 3F8 ― ― ESV CW

Table V. A peak CWS-containing microsomal population was subjected to repeated 1% deoxyBigCHAP treatments to remove detergent-soluble proteins, and the CWS activity was recovered in essentially 100% yield in the detergent-insoluble pellet. The partially pure CWS-containing fraction was separated by both reducing (+BME) and non-reducing (-BME) SDS-PAGE and challenged with a panel of monoclonal antibodies (mAb) after Western Blotting (in the laboratory of Dr. Ted Nash, NIH, by Dr. Hugo Luján). The mAb's recognized membrane-associated proteins corresponding to ESVs destined for the cyst wall. Ascites fluid from mAb's positive in this test (*'s) were used to challenge CWS, but none affected enzyme activity.

ESV = Encystment Specific Vesicles, CW = cyst wall, ER = endoplasmic reticulum, SV = small vesicles. a = mAb 5-3C (generated against CWP1, 23 kDa) was reported to cross-react with protein bands of 26 kDa (processed CWP2) and 39 kDa (CWP2) in reducing SDS-PAGE gels from ESVs, and CWP1 is considered a marker for ESVs prior to its incorporation into cyst walls (Mowatt et al., 1995; Gottig et al., 2006). b = mAb 7D2 and 8G8 generated against recombinant full length 39 kDa CWP2 from ESVs also cross- reacts with a protein band of 26 kDa (processed CWP2) in reducing SDS-PAGE gels. CWP2 is considered a marker for ESVs prior to its incorporation into cyst walls (Luján et al., 1995; Gottig et al., 2006). c = Monoclonal antibody 9C3 recognizes full length gGSP, while 7E7 binds only to the processed gGSP N- terminus. Induced gGSP is a soluble 54 kDa calcium-binding protein localized to the ESV population, and is thought to regulate degranulation of ESV contents during cyst wall formation (Touz et al., 2002). d = BiP (syn. GRP78) is a molecular chaperone of the ER also found in the ESVs (Gottig et al., 2006).

107 Further purification takes advantage of the stability and insolubility of CWS in the membranous state. Of the 24 different detergents tested, only a few non-ionic detergents did not inhibit enzyme activity above their respective critical micelle concentration

(CMC) including deoxyBigCHAP (N,N-Bis[3-(D-gluconamido)propyl]deoxycholamide) and MEGA-8 (n-octanoyl-N-methylglucamide), which possess a sterol-like and a fatty acid-like hydrophobic portions, respectively, but are similar in their hydrophilic side chains; and Synperonic PE/F68 (syn. Pluronic® F68, polyoxamer 188), a self-assembling tri-block copolymer of polyethylene oxide and polypropylene oxide with the repeating structure -[PEO78PPO30PEO78]-[PEO78PPO30PEO78]-. The detergent deoxyBigCHAP stimulated CWS activity consistently to 125% when present at 1% w/v (~10X the CMC) and inhibition was not found until the detergent concentration exceeded 3%.

Of those detergents that did inhibit CWS above their CMC, reconstitution studies failed to recover lost activity. Thus, selective solubilization and extraction of proteins not essential for CWS activity from the microsomal membranes using the non-ionic detergent deoxyBigCHAP was deemed as most useful for further purification.

Maximal CWS purification was obtained by subjecting microsomal particles from isopycnic centrifugation to 15 freeze-thaw cycles followed by multiple extractions with

1% deoxyBigCHAP for 1-4 hours at 4oC. CWS activity did not exhibit latency or activation following freeze-thaw treatment alone, it was recovered completely from the washed and resuspended pellet after centrifugation for 45 min at 105,000 g. CWS activity was not detected in any 105,000 g supernatant fraction for any of the 24 detergents tested. Since inclusion of deoxyBigCHAP routinely resulted in an increase in

CWS activity to 125% over controls prior to or after centrifugation, then the increase in

108 "recovery" could be the result of detergent stimulation, with 100% CWS recovery implied.

Purification of Cyst Wall Synthase (CWS)

CWS was partially purified from 30 h encysting Giardia trophozoites (Table VI).

Following homogenization in an isotonic medium and differential centrifugation, CWS was essentially confined to the sedimentable fraction

Table VI. Purification of cyst wall synthase from encysting Giardia. (from Karr and Jarroll, 2004).

Total Total Specific Purification Yield Purification Step Protein Activity Activity b Factor [mg] [mU] a [fold] [%]

Cell Homogenate 609.1 2.359 0.004 1 100

Particle Fraction c 193.6 2.294 0.012 3.1 97

Isopycnic Fraction d 10.5 2,183 0.208 53.6 93 deoxyBigCHAP Pellet e,f 2.0 1.208 0.604 155.9 51 a One milliunit (mU) of activity equals 1 nmol [1-14C]-GalNAc incorporated min-1. b Specific Activity equals mU mg protein-1. c 44,000 g Max, 45 min. d 189,000 g Max, 3 h 15 min, buoyant density 1.16 g sucrose ml-1. e 105,000 g Max, 45 min, after repeated detergent extraction with deoxyBigCHAP (N,N-Bis[3-(D-gluconamido)propyl]deoxycholamide). f represents only the protein from the peak isopycnic fraction (~50% of CWS) processed for detergent extraction.

109 Identification of cyst wall synthase (CWS) subunit(s)

Analysis of various purification steps by 1D SDS-PAGE revealed protein bands of ca. 30, 33, 55, and 81 kDa that co-localized with peak CWS activity (Figure 12A).

However, the 30-33 kDa protein doublet co-purified strongly with CWS activity as determined both by 1D SDS-PAGE and Western Blotting with antiserum to 20 hour encysting trophozoites preadsorbed with non-encysting cells (kindly provided by Drs.

Shields and van Keulen, Figure 12B).

In an attempt to identify the 30-33 kDa proteins, approximately 2 mg of partially purified CWS was subjected to 1D preparative SDS-PAGE. Outer portions of the gel were excised, quick stained with Coomassie® Brillant Blue R-250 as described in

Methods, and then repositioned next to the unstained portion of the gel to allow the correct bands to be excised. The excised protein bands were electroeluted into dialysis tubing, dialyzed, and then concentrated. To confirm that the correct protein bands had been recovered, the entire 1D SDS-PAGE except the excised gel slice was stained with

Coomassie® Brillant Blue R-250 overnight (Figure 13A) and an aliquot of the concentrated protein recovered after electroelution was analyzed for purity by denaturing

1D SDS-PAGE (Figure 13B) and semi-denaturing 1D SDS-PAGE. Under semi- denaturing conditions, additional Coomassie® Brillant Blue R-250-stained bands appeared with molecular masses equal to multiples of 30-33 kDa (data not shown), suggesting both of these proteins interacted with themselves to form dimers, trimers, and higher assemblages.

The 30-33 kDa doublet recovered after electroelution was then analyzed by 2D

SDS-PAGE and multiple isoforms of both species were found (Figure 13C).

110 Figure 12. Identification of protein bands enriched during CWS purification–I

A. A-I A-II B. B-I B-II

H 6 D6 H 6 D6

Figure 12A. 30-hour encysting Giardia trophozoites were homogenized Figure 12B. CWS was partially purified 155-fold from 30-hour

and subjected to differential and isopycnic fractionation. CWS activity encysting Giardia trophozoites, as described. Fractions corresponding

was monitored and proteins from peak isopycnic fractions (4-7) were to the whole cell homogenate (H), peak isopycnic microsomal fraction

separated by (A-I) SDS-PAGE and (A-II) probed with anti-20h (6), and the detergent extracted fraction 6 (D6). A prominent 30-32

trophozoite antiserum (pre-adsorbed with non-encysting trophozoite kDa protein doublet consistently tracks with purification of CWS

homogenate). Fraction 6 (followed by fraction 5) contained maximal activity as seen by (B-I) SDS-PAGE and by (B-II) immuno-reactivity

CWS activity while fraction 7 contained maximal acid phosphatase to anti-20h trophozoite antiserum (pre-adsorbed with non-encysting

activity (a marker for lysosome-like vesicles). Prominent bands trophozoite homogenate). Molecular mass is given in kDa.

consistent with CWS purification are indicated. Molecular mass is given

in kDa. 111 Figure 13. Identification of protein bands enriched during CWS purification–II

Figure 13. Detergent-extracted peak CWS-containing microsomal protein was prepared for microsequencing. The 30-32 kDa protein region, which tracked strongly with CWS activity and anti-20h encysting trophozoite antiserum was separated by preparative 1D-SDS-PAGE (B), the correct region was excised after quick staining the outer portions of the preparative gel, and the proteins were electroeluted into dialysis tubing from the gel slice. The recovery of the 30-32 kDa region proteins was assessed by 1D- SDS-PAGE (A). (C) 2D-PAGE demonstrated multiple spots for each protein band as determined by 1D- PAGE and were suggested to be isoforms likely with variable phosphorylation states. The 2D-PAGE was performed at Kendrick Labs (Madison, WI) and confirmation of the isoforms by protein microsequencing was performed at (Howard Hughes Medical Institute, Columbia University). Molecular mass markers are visible and are given in kDa.

112 Preliminary data suggested that the charge-related isoforms were the result of varying levels of phosphorylation.

Isoforms consistent with multiple protein phosphorylation states were identified from multiple spots of each band of the 1D SDS-PAGE protein doublet after separation by 2D SDS-PAGE. The presence of isoforms was confirmed after the major cleaved peptides of endoproteinase Asp-N hydrolysis were separated by HPLC and identified by protein microsequencing.

The pooled isoforms of the upper band were identified as β-giardin (GenBank™ accession: CAA59935.1) from 13 peptide sequences covering 54.4% of 272 amino acids possible. Using regions covered by overlapping peptide sequences

(149EALKSLNDLETGIATENAERKKMYDQLNEKVAEGFARISAAIEKETIAR197,

9TLTQTMDKPDDLTRSATETAVKLSNMNQR37), a BLAST analysis was performed on the Giardia genome confirming the identification as β-giardin; no other protein match was found. The molecular mass determined from reducing 1D and 2D PAGE is consistent with an unprocessed protein of 272 amino acids (~30.5 kDa). The sequence for β-giardin contains a DxD motif (55DDD57), found in some glycosyltransferase classes.

However, no relationship with other glycosyltransferases was detected by sequence similarity.

Pooled isoforms of the smaller mass band were identified as α1-giardin

(GenBank™ accession: ABU56103.1) from the identity of 2 larger peptide sequences covering 6.1% of 295 amino acids possible. The numerous remaining peptide fragments were small; there were no other peptide fragments larger than 1,117 Da. To ensure that protein still did not remain in the sample it was again digested, but with trypsin, and no

additional peptides were detected. 113 There are no other matches in the Giardia genome containing both of the identified sequences (52DDIKKALKGGSEE68, 185DFFGTVPS192), though multiple other sequences of the Giardia annexin family are present. When the complete α1-giardin sequence was used for BLAST analysis including all publically available databases a specific motif of D(I/L)KxxLxxGxxE (from α1-giardin; 52DDIKKALKGGSEE68) was revealed in the N-terminus for sequences of annexin VI from Mus, Rattus, and Homo sapiens, and annexin VII from Xenopus and Manduca sexta, even though the overall similarity is only 1.5 x 10-2 for the least similar (Manduca sexta) result. The molecular mass determined from reducing 1D and 2D SDS-PAGE is consistent with an unprocessed protein of 33 kDa, which corresponds with the 295 amino acid protein identified. The presence of α1-giardin in ESVs was subsequently confirmed by proteomics analysis with

32% peptide coverage by protein microsequencing (Stefanic et al., 2006).

Characterization of cyst wall synthase (CWS) activity

CWS activity was characterized with respect to optimal assay conditions, cofactors, substrate specificity, inhibitors, and apparent enzyme kinetics using partially purified CWS from detergent-extracted pellets of peak microsomal particle fractions as enzyme. The results are summarized in Table VII.

In brief, CWS activity is optimal at pH 7.5 and from 30-37oC in the presence of

25 mM calcium or magnesium (Figure 14). However, CWS activity was at least 50% as active over the ranges pH 6.0-10, 25-40oC, and 5-50 mM calcium or magnesium during a

30 min assay

114 Figure 14. Effect of pH, temperature, and divalent cations on Cyst Wall Synthase.

A. B.

Figure 14. Cyst wall synthase assay optima and range

(A) pH optima and range were determined using an C. overlapping buffer series, as described in Methods. Reactions were conducted at 37oC for 30 minutes in the presence of 10 mM Mg2+. Error bars are STDEV. (B) Temperature optimum and range were determined in the presence of 10 mM Mg2+, at pH 7.5, and for 30 minutes. Data are means of three independent trials. (C) Divalent cations found to stimulate CWS activity were assayed at 37oC for 30 minutes. Data are means of three independent trials.

*For all experiments, characterization was performed using peak isopycnic microsomal fractions as enzyme.

115

In contrast, CWS activity showed an absolute requirement for its substrate UDP-

GalNAc (Table VII) and a divalent metal cation cofactor (Figure 14). CWS activity levels were below the limits of detection (0.001 nmol min-1 mg-1 protein) with any other

UDP-sugar donor or free sugar tested. The divalent cation chelator, EDTA, inhibited

CWS activity (reversibly) to below the limits of detection indicating an absolute requirement for a suitable divalent cation.

Table VII. Substrate specificity of Giardia cyst wall synthase (CWS) activity. From Karr and Jarroll (2004).

Specific Activity a Sugar Substrate P-fraction S-fraction

UDP-N-acetyl- D-galactosamine 0.41 + 0.06 < 0.011 a b b UDP-N-acetyl-D-glucosamine < 0.001 < 0.001 UDP-D-Galactose < 0.001 b < 0.001 b UDP-D-Glucose < 0.001 b < 0.001 b D-Galactosamine < 0.001 b < 0.001 b D-Glucosamine < 0.001 b < 0.001 b a Defined as 1 nmol [14C]-labeled substrate incorporated min-1 mg protein-1. b Below the limits of detection.

Enzyme kinetics of the CWS reaction were studied using various fractions taken during purification including the most pure fraction from the peak microsomal population isolated after a repeated deoxyBigCHAP detergent enrichment step. At this stage, CWS was partially purified to ca. 155-fold (Table VI). CWS exhibited an apparent Kmapp of 48

+ 0.3 M and a Vmaxapp of 0.70 ± 0.085 nmol UDP-GalNAc incorporated min-1 mg protein-1 as determined by non-linear regression analysis (GraphPad Prism®, Figure 15).

116 Kinetic parameters obtained by linear regression were similar with an apparent Kmapp of

55 M and a Vmaxapp of 0.76 nmol UDP-GalNAc incorporated min-1 mg protein-1, as calculated (y = 0.0723x + 1.3193, R2 = 0.9873, Microsoft Excel®, Figure 15 inset).

While CWS was active without the addition of an exogenous acceptor and it does not appear that an acceptor is required, an acceptor may yet be found. If this is the case then the kinetic parameters determined would likely be different. Thus, we chose to

app represent these kinetic parameters as the apparent maximal velocity (Vmax ) and

app apparent Km (Km ).

Figure 15. Determination of enzyme kinetics by non-linear regression analysis.

Figure 15. Cyst wall synthase reaction kinetics for the substrate UDP-GalNAc as determined by non-linear regression analysis (GraphPad Prism™) and (inset) linear regression analysis (Excel®). Partially purified CWS from detergent-extracted peak microsomal fractions was used as enzyme. app Data are from three separate enzyme preparations assayed in duplicate. The Km (55 µm) and -1 -1 Vmax (0.76 nmol min mg protein ) as determined by linear regression analysis (y = 0.0723x + 1.3193, R2 = 0.9873, Microsoft Excel®) was similar to data with non-linear regression analysis (From Karr and Jarroll, 2004).

117 However, when CWS was assayed without exogenous acceptor and at a UDP-

app GalNAc concentration below its Km value the synthetic UDP-sugar substrate analogs nikkomycin Z and polyoxin D, the natural substrate analog UDP-GlcNAc, and the partial substrate analogs UMP, UDP, and UTP inhibited CWS activity in a concentration dependent manner (Table VIII). As an internal control, additional substrate (UDP-

GalNAc) increased CWS activity, as expected.

Table VIII. Identification of inhibitors of Giardia cyst wall synthase (CWS) activity.

a Concentration Relative to Relative Activity Inhibitor or Control the Substrate (UDP-GalNAc) (%) Control 100 b UDP-GalNAc 1X 149 c 10X 179 c 1X 87 Polyoxin D 10X 26

1X 84 Nikkomycin Z 10X 37

1X 83 UDP-GlcNAc d 10X 21 1X 49 UTP 5X 33 10X 22 1X 76 UDP 5X 42 10X 26 1X 89 UMP 5X 48 10X 29 a app The substrate concentration of UDP-GalNAc used (35 µM) was below the Km . b Control activity was 0.307 + 0.026 nmol min-1 mg protein-1 and was defined as 100%. c Additional UDP-GalNAc substrate increased activity and served as an internal control. d UDP-GlcNAc did not function as a substrate in the reaction (see Table VII)

118 Acceptor Profiling of CWS Activity

In an attempt to demonstrate a potential requirement for an acceptor for the in vitro CWS reaction two approaches were pursued; we assessed the potential for an endogenous acceptor(s) and examined exogenous acceptors known from other systems.

Endogenous Acceptors

To investigate potential endogenous acceptors, we pursued three lines of investigation including the potential for [1-14C]GalNAc to be incorporated into: 1) protein or glycoprotein, 2) lipid or glycolipid, and 3) high molecular weight polysaccharide(s).

Potential endogenous protein or glycoprotein acceptors were examined after separating radiolabeled in vitro-derived CWS reaction product(s) by reducing 1D SDS-

PAGE and assessing incorporation of [1-14C]GalNAc into components of the gel by autoradiography, as described in Methods. Figure 16 is a representative experiment that shows [1-14C]GalNAc is: 1) not incorporated above the level of detection by P-fractions from non-encysting (no bile) trophozoites using the complete reaction mixture, but 2) is incorporated into high molecular weight material(s) from P-fractions of 24-hour encysting cells using either the washed reaction precipitate or the entire assay mixture.

When entire reaction mixtures from encysting P-fraction assays were prepared in reducing SDS-PAGE sample buffer and loaded to the gel, radiolabeled products were present predominantly as a high molecular weight material excluded from entering the stacking gel. Minor incorporation was seen in a component retained at the interface between the 5% stacking and 10% running gel, but was only visible upon prolonged

119 Figure 16. Analysis of CWS Reaction Product by 1D-PAGE and Autoradiography-I.

A1 A2 A3 B1 B2 B3

Figure 16. Representative SDS-PAGE gel (A) and autoradiogram (B) of Giardia CWS reaction product. CWS was assayed with UDP-[1-14C]GalNAc as described in Methods. Lanes contained total assay mixture (1) and washed ethanol precipitate only (2) using 90 µg protein (P-fraction) from encysting cells as enzyme, and total assay mixture (3) using 90 µg protein (P-fraction) from non-encysting cells as enzyme. All samples were mixed with sample buffer containing DTT, and electrophoresed through a 5% stacking and 10% running gel. Gels were stained with Coomassie® Brillant Blue R-250 (left); the dried gel was sprayed with Enhance™ and exposed to autoradiographic film (right) for 2 weeks at –70oC. Molecular masses are given in kDa. Solid arrow = prominent radiolabeled component retained at the PAGE well. Open arrow = minor radiolabeled component from the interface between the 5% stacking and 10% running gels. (Karr and Jarroll, 2004)

120 exposure of the film (3 weeks), a time at which the film likely saturated in the spots of high molecular weight material. However, these differences were not quantified.

To ensure that an endogenous acceptor was not formed early during encystment as a priming step and subsequently extended later into high molecular weight material, a time course experiment was conducted. Using homogenates of non-encysting (low-bile cultured, time 0h) and cells induced to encyst for 4, 8, 12, 16, 24, 30, 36, 48, and 72h as enzyme source, in vitro CWS assays were performed on equivalent cell number (1 x 106 cells) samples. The complete radioassay mixtures were prepared in reducing SDS-PAGE buffer, separated by 1D SDS-PAGE, and dried gels were exposed to autoradiographic film, as described in Methods. Figure 7 is a representative time course experiment that correlates the percentage of cysts formed and CWS activity, using two assay formats.

Figure 7 shows the reducing 1D SDS-PAGE gel with nearly uniform protein loading

(color image) and the corresponding autoradiographic film (black spots of 14C- incorporation) aligned and overlaid at the time the image was scanned. Incorporation of

[1-14C]GalNAc is barely discernable from low-bile cultured trophozoites (time 0h) and trophozoites induced to encyst up to 4 hours by autoradiographic film, which could be remnants of the assay mixture containing excess UDP-[1-14C]GalNAc since incorporation into product as detected from precipitates in the CWS in vitro assay did not show detectable levels of incorporation. Incorporation into a high molecular weight product(s) is clearly shown to increase at 8h and continues to increase until 24h, which corresponds to nearly maximal encystment. No incorporation of [1-14C]GalNAc into a protein or glycoprotein which separates on reducing SDS-PAGE was detected at any time point.

121 Potential glycosylation of endogenous proteins or glycoproteins was further examined by assessing the sensitivity of the high molecular weight in vitro-derived CWS reaction product to hydrolysis by known proteinases. To determine the effects of proteinase treatment, radiolabeled reaction products were generated from samples of peak

CWS-containing isopycnic fractions (33.6 µg) as enzyme, and treated with proteinases individually, as described in Methods. The CWS reaction product was partly sensitive to proteinase K and papain (~50% reduction in filtered reaction product(s)), but insensitive to digestion by trypsin or Pronase (mixture of hydrolytic enzymes) (Table IX).

Table IX. Effects of various treatments on the cyst wall synthase reaction product. From Karr and Jarroll (2004).

a Control Precipitate Treated precipitate (range) Treatment (d.p.m.) b (% of control d.p.m)

1% SDS 21,715 88 (86–90) Pronase E 22,010 104 (99–109) Trypsin 22,010 102 (101–104) Proteinase K 22,010 54 (52–57) Papain 19,867 52 (43–57) 1 M KOH, (1h / 100oC) 22,942 46 (40–50) 2 M HCl, (3h / 100oC) 23,644 2 (1.6–2.2) a see Methods. b Control values represent [1-14C]GalNAc incorporation from UDP-[1-14C]GalNAc into the ethanol precipitate, which was subjected to the same conditions as treated precipitates (performed in triplicate) but without the active agent.

122 Confirmation of proteinase activity prior to each assay was accomplished by incubating each proteinase solution with a polyacrylamide gel slice (polymerized with

0.2% gelatin) for 30 min under conditions described in Methods. Gelatin gel slices were then washed in 1% Triton X-100, dH2O, and subsequently stained with Coomassie®

Brillant Blue R-250. Proteinase activity was indicated from the portion of the gel submerged in proteinase solution, which was unstained. As a positive control, the untreated portion of the gelatin gel stained positive with Coomassie® Brillant Blue R-250.

In an attempt to confirm the loss of [1-14C]GalNAc from proteinase-treated high molecular weight material and examine the release of potential radiolabled-polypeptides, the complete proteinase-treated (proteinase K, papain) reaction mixtures were reduced in

SDS-PAGE sample buffer, separated by reducing 1D SDS-PAGE, and dried gels exposed to autoradiographic film, as described. However, after treatment with proteinase K or papain there were no radiolabeled polypeptides detected in the gel (Figure 17).

Confirmation of proteolysis was visualized by Coomassie® Brillant Blue R-250-stained hydrolysis products at and below the dye front for proteinase-treated samples.

In addition, incorporation of [1-14C]GalNAc into the high molecular weight fraction excluded from entering the stacking gel (Figure 17, solid arrow) was not reduced versus controls. This suggested that the proteinase activity had produced some components still too large to enter the gel, but too small to precipitate under conditions of the CWS precipitation assay. In fact, when the 1 M KOH-treated CWS reaction products were precipitated in 67% ethanol at 15,000 g for 5 min the supernatant contained an additional 8.5% of the initial label that did not precipitate, but was retained in the CWS filter assay.

123 Figure 17. Analysis of CWS reaction product by 1D-PAGE and autoradiography-II.

Proteinase K: -- + ------+ :BSA Papain : ------+ -- -- + -- :BSA-GlcNAc -- + -- -- :BSA-GalNAc

Figure 17. Representative SDS-PAGE gel and autoradiogram (overlay) attempting to demonstrate a natural or introduced (glyco)protein acceptor of Giardia CWS activity.

(Left) Radiolabelled CWS reaction products were generated as washed pellets (22,239 d.p.m., average) and incubated mixed with or without 1.0 mg ml-1 papain or proteinase K for 2 h, as described in Methods. Each complete reaction mixture was heated to 100oC with 5% SDS and 100 mM DTT and electrophoresed on a 10% SDS-PAGE gel with a 5% stacker. Coomassie® Brillant Blue R-250-stained hydrolysis products are visible at and below the dye front for proteinase-treated samples. However, no change in total incorporation of the radiolabel or distribution of radiolabel was detectable (solid arrow).

(Right) CWS was incubated with or without bovine serum albumin (BSA) or glycoconjugated-BSA. However, no change in total incorporation into high molecular weight material(s) (solid arrow) or evidence of protein glycosylation could be detected within the gel.

Open arrow = interface between the 10% running and 5% stacking gel.

124 Endogenous lipid-linked acceptors or intermediates

In search of a potential lipid-linked intermediate or acceptor, CWS reactions were scaled up for immediate use and all fractions were kept on ice. The 15,000 g pellets from

CWS assays were washed 3 times to remove any unbound [14C]substrate by resuspension in dH2O and centrifugation at 15,000 g for 10 min. The CWS reaction pellets were then pooled (290,869 d.p.m) as source material for potential extraction of lipid-linked intermediates, as described in Methods. Following extraction with 1203-solvent 6.8%

(19,891 d.p.m) of the radiolabel from the pooled CWS assay pellets was recovered. The

1203-extract was then treated with 0.01 M HCl (pH 2.5) for 10 min at 100oC, which released 2.9% (8,478 d.p.m.) of the total starting label resulting from contaminant UDP-

[1-14C]GalNAc. The 0.01 M HCl-treated 1203-extracted pellet was then treated with 0.1

M HCl (pH 1.0) for 10 min at 100oC, but no further radiolabel was released.

To distinguish between lipid-intermediates and residual mono or oligosaccharide, contaminants, the remainder of each sample not used for liquid scintillation counting was reduced to near dryness using a stream of nitrogen and paper electrophoresis was performed according to Semino and Dankert (1993). The paper chromatogram lanes were sectioned (1 cm intervals) and counted in a liquid scintillation counter. The position of radiolabel was inconsistent with incorporation into lipid-linked intermediates. The

UDP-[14C]GalNAc standard remained at the origin, while all other experimental samples migrated in overlapping fractions with the commercial standard [14C]mannose and [1-

14C]GalNAc (fractions 8-10) prepared by hydrolyzing UDP-[14C]GalNAc with 0.01 M

HCl for 10 min at 100oC. Fractions beyond this (through fraction 30) which should contain progressively more hydrophobic components were not labeled.

125 Endogenous glycoprotein acceptors

To determine if GalNAc-terminated glycoproteins were present in Giardia which might serve as endogenous acceptors of [14C]GalNAc, protein samples from encysting trophozoites homogenates were probed with lectins, as described in Methods. The lectins

Vicia villosa isolectin B4 (affinity for terminal α- or β-GalNAc-O-Ser or O-Thr), Arachis hypogaea lectin (affinity for Gal--, Gal-1-3-GalNAc-, and Gal-1-4-Glc-), or Sophora japonica lectin (affinity for terminal GalNAc- or Gal-modified glycoproteins) did not bind to polypeptides from 24 and 30 hour encysting cells even after incubation of the blots overnight. Milk proteins (Stop-n-Shop, non-fat dry milk) and chemically-defined reduced N-glycosylated ovalbumin (GlcNac2-Man5-6) served as positive and negative controls for lectin binding, respectively.

Exogenous (glyco)protein and (glyco)peptide acceptors

While an endogenous protein or glycoprotein acceptor was not detected using

SDS-PAGE and proteinase treatment, the possibility that the CWS activity was a polypeptide or glycopeptide glycosyltransferase remained.

A first approach was to assay CWS in the presence of exogenous glycoproteins and glycoconjugates of BSA (BSA-GalNH2 and BSA-GlcNH2) as potential substrates.

Incorporation of unlabelled UDP-GalNAc was measured in the presence of phosphodiesterase-free alkaline phosphatase, which catalyzes the release of phosphate from the reaction product UDP. Released phosphate was detected by spectrophotmetric assay at 635 nm following the addition of Phosphate Detection Reagent, as described in

Methods. No activity above the endogenous CWS activity was detected when exogenous

126 glycoprotein acceptors (ovalbumin, asialofetuin, mucin, asialomucin, BSA, BSA-

-1 GalNH2, BSA-GlcNH2) were assayed at 1.0 mg ml (20 µg per reaction).

In a second approach CWS was assayed by scintillation proximity assay in the presence of UDP-[6-3H]GalNAc using a panel of biotinylated (glyco)peptides (Table II) known to function as acceptors (Elhammer et al, 1999; Ten Hagen et al,. 2003) for polypeptide- and glycopeptide-GalNAc transferases. Incorporation of [6-3H]GalNAc into biotinylated peptides or glycopeptides brings the 3H-label into proximity of scintillant-doped SPA™ beads functionalized by streptavidin. The weak decay of UDP-

[6-3H]GalNAc free in solution is too far from the scintillant within the bead to produce a signal; thus, creating a homogeneous assay. However, there was no incorporation of

[6-3H]GalNAc into any of the peptides by CWS. The human ppGaN-T11 and gpGaN-

T10 served as positive controls in these assays (Figure 18).

127 Figure 18. (Glyco)peptide acceptor profiling.

(A) Identification of the acceptor specificity of human ppGaN-T11.

(B) Identification of the acceptor specificity of human gpGaN-T10.

Figure 18. (Glyco)peptide acceptor profiling; ppGaN-T11 and gpGaN-10 controls. Assays were performed as described in Methods. Control enzymes were tested at 100- fold less protein (0.1 µg well-1) over a time course of 2h in the presence of UDP-[6- 3H]GalNAc (200 nCi, 2 µM, 20 pmoles) and biotinylated peptides (2 µM, 20 pmoles). Assays were terminated with the addition of 100 µg SPA™ imaging beads (25 pmole binding capacity) in EDTA. 384-well microtiter plates were imaged after bead settling (15 min) using a LEADseeker imaging plate reader. Response values are arbitrary units. The graph insets are images of the plates; relative light units are color coded in each inset legend. Data points are the means and error bars are STDEV (n=3).

128 Exogenous carbohydrate-based acceptors

Glycosyltransferases can transfer monosaccharide from a nucleotide-donor to the growing polysaccharide chain in a processive fashion or through the specific terminal addition a single residue. To test if CWS can perform the function of specific or non- specific terminal addition, several panels of oligomers were accessed.

The first attempt at deorphaning CWS utilized a solid-phase array, which can create a microenvironment consistent with the physiological presentation of terminal glycan chains. CWS was tested at 1.0 mg ml-1 against a panel of 48 oligosaccharides in the presence of UDP-[1-14C]GalNAc, on a chip sealed by a Teflon ring and covered with an inert coverslip. After incubation at 30oC for 19 hours the array was washed, rinsed with ethanol and the dry chip was placed against phosphor imaging screen for 4 hours and then 72 hours. No incorporation into any acceptor was detected by phosphor imaging.

Controls for the solid phase array included the enzymes: 1) human β3Gn-T2, 2) bovine β4Gal-T (without lactalbumin), and 3) porcine α3Gal-T. These enzymes were tested at 100-fold less protein than was CWS and for only 2 hours. Reaction conditions for these control enzymes were as described in Methods. Figure 19 presents the data for the arrays tested, while Table X contains the corresponding quantitative data. The results for the controls were consistent with expected acceptor profiles.

A variant approach to acceptor profiling uses a solution-phase array of substrates.

The 109 acceptors accessed were conjugated via a hydrophilic spacer to a biotin through their reducing end. Thus, they are only acceptors for non-reducing end transfer. Assay of the solution phase array was accomplished by scintillation proximity assay, as described

in Methods, using SPA™ imaging beads functionalized with streptavidin. Transfer of 129 radiolabeled substrate onto an acceptor, subsequently captured to the bead brings the

[3H]-sugars into proximity with the internally scintillant-doped bead producing the signal.

CWS was assayed at 0.76 mg ml-1 protein and for 20 hours in the presence of 100

µM UDP-GalNAc (10 nCi, UDP-[6-3H]GalNAc), while the control enzyme β3Gn-T2 was assayed at 10 µg ml-1 (10-fold less) for 2 hours (10-fold less) with 100 µM UDP-

GlcNAc (10 nCi UDP-[1-3H]GlcNAc). No incorporation was detected into any of the

109 potential acceptors using CWS (Table XI). However, the results for the control enzyme, human β3Gn-T2, matched the major known acceptor motif of terminal poly[Gal(β1-4)GlcNAc] (LacNAc), as expected (Zhou et al., 1999; Shiraishi et al. 2001;

Seko and Yamashita 2005).

130 Figure 19. Solid-phase acceptor profiling of CWS and control enzymes.

A. Bovine Gal-T B. Human β3Gn-T2 C. Porcine α3Gal-T D. Giardia CWS (0.01 mg ml-1) (0.01 mg ml-1) (0.01 mg ml-1) (0.76 mg ml-1)

1 26 2 27 3 28 4 29 5 30 6 31 7 32 8 33 9 34 10 35 11 36 12 37 13 38 14 39 15 40 16 41 17 42 18 43 19 44 20 45 21 46 22 47 23 48 24 49 25 50

*Key to acceptor microarray layout. Neoglycoconjugate acceptors represented as n=4. C01 Glcβ1-4Glcβ1-4Glcβ1-4Glcβ1-4Glcβ C26 Galα C02 6-O-SO3-GlcNAcβ C27 Galα1-3(Fucα1-2)Galβ C03 GlcNAcα C28 Galα1-3Galβ1-4GlcNAcβ C04 GlcNAcβ C29 Galα1-4Galβ1-4Glcβ C05 GlcNAcβ1-3Galβ1-4Glcβ C30 Galβ C06 GlcNAcβ1-3GalNAcα C31 Galβ1-3(GlcNAcβ1-6)GalNAcα C07 GlcNAcβ1-4GlcNAcβ C32 Galβ1-3GalNAcα C08 GlcNAcβ1-6GalNAcα C33 Galβ1-3GlcNAcβ C09 L-Rha-α C34 Galβ1-4Glcβ C10 GalA-β C35 Galβ1-4GlcNAcβ C11 Manα C36 Galβ1-6Galβ C12 Manα1-3(Manα1-6)Manβ C37 GalNAcα C13 Manα1-3Manα C38 GalNAcα1-3(Fucα1-2)Galβ C14 Manβ C39 GalNAcβ C15 Manβ1-4Glcβ C40 Biotin / Control C16 Neu5Acα C41 Fucα C17 Neu5Acα1-3Galβ1-4(Fucα1-3)GlcNAcβ C42 Fucα1-2Galβ C18 Neu5Acα1-3Galβ1-4Glcβ C43 Fucβ C19 Neu5Acα1-3Galβ1-4GlcNAcβ C44 Glcα C20 Neu5Acα1-6Galβ1-4GlcNAcβ C45 Glcα1-4Glcα C21 Hydroxyl-blank C46 Glcα1-4Glcβ C22 L-Araf-α C47 Glcβ C23 GlcU-β C48 Glcβ1-3Glcβ C24 Xyl-α C49 Glcβ1-4Glcβ C25 Xyl-β C50 Glcβ1-4Glcβ1-4Glcβ

131 Table X. Solid-phase acceptor profiling of CWS and control enzymes.

Bovine Human Porcine Giardia Code Solid-phase Neoglycoconjugate β4GalT β3GnT2 α3GalT CWS C01 Glcβ1-4Glcβ1-4Glcβ1-4Glcβ1-4Glcβ 56 5 -1 6 C02 6-SO3-GlcNAcβ 312 0 0 3 C03 GlcNAcα 374 0 0 0 C04 GlcNAcβ 630 0 0 1 C05 GlcNAcβ1-3Galβ1,4Glcβ 1053 13 1 2 C06 GlcNAcβ1-3GalNAcα 733 2 0 0 C07 GlcNAcβ1-4GlcNAcβ 737 4 0 0 C08 GlcNAcβ1-6GalNAcα 498 1 0 -1 C09 L-Rha-α 3 1 2 -1 C10 GalA-β 1 11 5 3 C11 Manα 5 28 3 4 C12 Manα1-3(Manα1-6)Manβ 3 7 1 1 C13 Manα1-3Manα -1 4 0 -1 C14 Manβ 2 44 -1 1 C15 Manβ1-4Glcβ 4 245 2 0 C16 Neu5Acα 6 3 1 0 C17 Neu5Acα1-3Galβ1-4(Fucα1-3)GlcNAcβ 4 2 0 0 C18 Neu5Acα1-3Galβ1-4Glcβ -13 111 12 0 C19 Neu5Acα1-3Galβ1-4GlcNAcβ 8 39 3 -1 C20 Neu5Acα1-6Galβ1-4GlcNAcβ 1 83 9 0 C21 Hydroxyl / Control 0 2 0 -1 C22 L-Araf-α 2 3 1 1 C23 GlcU-β -1 5 1 1 C24 Xyl-α 3 3 0 0 C25 Xyl-β 5 7 0 3 C26 Galα 39 347 7 0 C27 Galα1-3(Fucα1-2)Galβ 18 1 0 1 C28 Galα1-3Galβ1-4GlcNAcβ 14 28 1 -1 C29 Galα1-4Galβ1-4Glcβ 4 7 5 -1 C30 Galβ 18 481 45 0 C31 Galβ1-3(GlcNAcβ1-6)GalNAcα 491 81 5 0 C32 Galβ1-3GalNAcα 17 198 9 -2 C33 Galβ1-3GlcNAcβ 7 832 57 2 C34 Galβ1-4Glcβ -2 1938 86 2 C35 Galβ1-4GlcNAcβ 12 2538 88 2 C36 Galβ1-6Galβ 31 336 23 3 C37 GalNAcα 20 70 10 3 C38 GalNAcα1-3(Fucα1-2)Galβ 12 4 1 1 C39 GalNAcβ 14 233 3 1 C40 Biotin / Control 5 2 0 -2 C41 Fucα 9 3 1 -1 C42 Fucα1-2Galβ 7 21 -1 -1 C43 Fucβ 12 2 0 -2 C44 Glcα 6 3 1 -1 C45 Glcα1-4Glcα 8 5 1 -1 C46 Glcα1-4Glcβ 12 6 -1 -1 C47 Glcβ 141 8 0 -2 C48 Glcβ1-3Glcβ 90 7 0 1 C49 Glcβ1-4Glcβ 118 3 0 1 C50 Glcβ1-4Glcβ1-4Glcβ 45 6 0 -3 *Data are the means of 4 replicates, are expressed as relative pixel units, and are not background corrected. Controls include biotin- & hydroxyl-blanks. CWS was tested at 76X protein and 10X time vs. controls.

132

Table XI. Solution-phase acceptor profiling of CWS and human β3Gn-T2.

Code Response Sequence GA-075 1851.9 Gal1-4GlcNAc1-6GalNAc-sp-biot GA-052 1756.8 Gal1-4GlcNAc-sp-biot GA-074 1659.3 Gal1-4GlcNAc1-3GalNAc-sp-biot GA-050 1435.4 Gal1-4Glc-sp-biot GA-061 1381.7 GalNAc1-4GlcNAc-sp-biot GA-094 1111.8 Gal1-4GlcNAc1-6(Gal1-3)GalNAc-sp-biot GA-087 928.0 (Gal1-4GlcNAc)2-3-6-GalNAc-sp-biot GA-053 860.3 Gal1-4(6-O-SO3)GlcNAc-sp-biot GA-054 343.0 (Fuc1-2)Gal1-4GlcNAc-sp-biot GA-043 189.7 Gal1-3GlcNAc-sp-biot GA-093 184.6 Gal1-3GlcNAc1-3Gal1-4Glc-sp-biot GA-081 131.6 Neu5Ac2-3Gal1-4Glc-sp-biot GA-077 128.1 Man1-4GlcNAc1-4GlcNAc-sp-biot GA-080 103.4 Neu5Ac2-3Gal1-4GlcNAc-sp-biot GA-011 75.0 Gal-sp-biot GA-010 66.2 Gal-sp-biot GA-082 58.1 Neu5Ac2-6Gal1-4Glc-sp-biot GA-051 52.4 Gal1-4GlcNAc-sp-biot a For CWS, the background control value (red bars) was 28.6±4.1 (mean±STDEV; n=7) pixel units. Acceptor activity was defined as incorporation > background+3*STDEV (41.0 pixel units). b For β3Gn-T2, the background control value (red bars) was 33.5± 6.0 (mean±STDEV; n=7) pixel units. Acceptor activity (blue bars) was defined as incorporation > background+3*STDEV (51.4 pixel units). c Positive control wells (green bars, n=8) were spiked with [3H]biotin. d CWS was assayed at 0.76 mg ml-1 protein and for 20 hours with 100 µM UDP-GalNAc (200 nCi UDP- [6-3H]GalNAc). The control enzyme β3Gn-T2 was assayed at 10 µg ml-1 for 2 hours with 100 µM UDP-GlcNAc (200 nCi UDP-[1-3H]GlcNAc). Biotinylated-acceptors were used at 100 µM, each. e Detection was accomplished by scintillation proximity assay of biotinylated-acceptors using streptavidin-functionalized SPA™ beads and a LEADseeker™ imaging system. Control results obtained match the major known acceptor motif of terminal poly[Gal(β1-4)GlcNAc] (LacNAc), as expected (Zhou et al., 1999; Shiraishi et al. 2001; Seko and Yamashita 2005).

133 Characterization of the CWS Product

Because an enzyme of this nature has not yet been described in the literature, it was necessary to analyze the CWS product in order to fully characterize this novel enzyme and its likely polysaccharide product. Early experiments were designed to distinguish between transferase activity to a glycoprotein, glycolipid, or a carbohydrate polymer acceptor. Since the labeled ethanol-precipitable product was excluded from entering the 5% stacking gel during reducing SDS-PAGE even after proteinase treatments, then this suggested the labeled component(s) is either very large or lacks the charge necessary to migrate in the electric field.

The possibility that a fraction of the radiolabeled product was either soluble, neutral in charge, and/or too small to be detected by 1D SDS-PAGE and autoradiography led to attempts to fractionate the radiolabeled product by size exclusion over a 10 cm

Biogel P-2 column (120 mM Pyridine/Acetic acid pH 5). However, the radiolabeled product remained trapped at the top of the column even after mild acid hydrolysis (0.1 M

HCl, 10 min, 100oC) suggesting that size was the predominant factor affecting its mobility. Only monosaccharides were recovered from column fractions after mild acid hydrolysis as confirmed by the authentic GalNAc standard generated by hydrolysis of

UDP-[1-14C]GalNAc; the remaining radiolabel was recovered from the top of the column and counted in a liquid scintillation counter.

CWM Compositional Analysis

Once it was established that the acceptor was likely a carbohydrate polymer, further studies to identify the composition and the linkage(s) of the polysaccharide

acceptor were necessary to confirm the above hypothesis and to characterize the CWS 134 product (and therefore the CWS biochemical activity). Preliminary analysis of CWM hydrolysates was performed by HPLC using a Waters PicoTag Amino Acid column and a protocol developed to identify and quantify amino sugars and amino acids, simultaneously (Risk et al., 1996). These experiments demonstrated the presence of

GalNAc and amino acids, and confirmed that GlcNAc was not present above the levels of detection.

Based on these data, approximately 150 mg of isolated CWM was generated from approximately 40 L of 24-48 h encysting cells. Samples of CWM were analyzed by

GC/MS for carbohydrate composition and linkage analysis in collaboration with the

Complex Carbohydrate Research Center (Athens, GA). Initial data generated revealed that GalNAc was the only sugar detected, but failed to conclusively identify the polymer linkage(s) likely due to undermethylation of the extremely insoluble CWM.

Further carbohydrate analysis was performed at the Bijvoet Center for

Biomolecular Research (Utrecht University, The Netherlands) using samples from the same source of purified CWM. To overcome the extreme insolubility of the polymer, the

CWM was subjected to controlled partial acid hydrolysis. The results of compositional analysis on intact- and partial acid hydrolysis solubilized-CWM confirmed previous data that GalNAc composes 63% of the resistant carbohydrate filaments and that the remainder is composed of amino acids rich in leucine (Gerwig et al., 2002). No other sugars, lipids, or components were detected in the CWM analyzed (Gerwig et al., 2002).

Figure 20 shows representative electron micrographs of typical cysts and cyst walls before and after the cyst wall isolation procedure, described in Methods. The gross morphology of the cyst wall was unchanged following chemical and hydrolytic treatment.

135 Figure 20. Electron micrographs of Giardia cyst walls before and after purification.

Figure 20. Transmission (A) and Scanning (C) electron micrographs of typical Giardia cyst. (A = from Erlandsen et al., 1989. bar = 0.25 um; B is courtesy of Dr. Stanley Erlandsen, University of Minnesota Medical School, Minneapolis, MN. bar = 1.0 um).

Transmission (B) and Scanning (D) electron micrographs of isolated Giardia cyst walls (B and D courtesy of William Fowle and Rita Droste, Northeastern University, Boston, MA. bars are 0.25 µm in B and 1.0 µm in D).

136 Identification of the Protein Component of CWM

Upon further analysis it was determined that: 1) the amino acid content of the

CWM was similar to published sequence data for the related mature cyst wall specific proteins - CWP1, CWP2, though it differed from CWP3 (Table XII), 2) the mAbs

8C5.C11 and 5A4.G6, which have been demonstrated to bind CWP1 and CWP2

(Campbell and Faubert, 1994; Lee and Faubert, 2006) still recognized epitopes evenly distributed over the CWM as determined by indirect immuno-gold labeling and FE-SEM

(Figure 21B, similar data for mAb 5A4.G6 not shown) and 3) an anti-CWM polyclonal antiserum generated from the isolated CWM recognized three major bands matching molecular masses of the mature and processed cyst wall proteins by western blot analysis

(Figure 22). Based on these findings, it is likely the majority of the structural protein component of the CWM is composed of CWP1 and CWP2, which have been described previously (Luján et al., 1995, Mowatt et al., 1995). The relatively larger difference in amino acid composition of CWP3, suggests a minor structural role for this protein.

These data also suggested that some CWM protein may be covalently attached to or protected from proteolytic and chemical degradation within crystalline cyst wall filaments. However, no covalent association could be demonstrated between any amino acid residue and GalNAc-containing filaments solubilized by partial acid hydrolysis

(Gerwig et al., 2002). Further studies to elucidate the protein-carbohydrate association will be necessary to understand the intractable nature of the cyst wall.

137 Table XII. Amino acid composition of the cyst wall protein component.

Amino Acid Relative Amount (mole %) aCWM bCWM PH1 cCWP1 cCWP2 cCWP3 0X 1X 2X 3X d Asx 18.8 19.4 18.9 14.7 14.1 15.5 17.1 18.5 Thr 11.4 11.5 10.3 12.7 14.8 10.8 11.6 5.3 Ser 4.5 4.5 11.4 5.9 5.9 4.7 3.7 9.3 d Glx 9.0 9.3 13.4 10.8 10.4 8.5 6.5 5.7 Pro 5.9 7.7 9.1 11.8 12.6 6.6 6.9 3.1 Gly 8.4 9.2 13.1 12.7 12.6 8.0 6.5 8.4 Ala 7.8 8.0 6.6 7.5 6.7 5.2 7.4 4.0 Val n.d. n.d. n.d. n.d. n.d. [4.4] [3.1] [6.9] Cys 3.8 3.6 0.1 - - 8.5 7.4 6.2 Met 2.1 0.9 0.3 1.0 1.5 2.8 3.2 2.2 Ile 4.8 4.5 2.2 2.5 2.2 4.7 5.6 2.6 Leu 14.3 14.4 6.3 8.8 8.9 15.0 13.4 17.6 Tyr 3.8 1.7 2.0 2.9 3.7 4.2 5.1 7.0 Phe 1.9 2.2 1.7 2.0 2.2 2.3 1.9 2.2 Lys 1.9 1.4 1.4 2.0 1.5 1.9 0.9 2.6 His 0.7 0.6 0.9 1.0 0.7 0.9 1.4 2.6 Arg 1.0 1.2 2.2 2.9 2.2 0.5 1.4 2.6 Trp n.d. n.d. n.d. n.d. n.d. [1.8] [1.8] [1.2] Total 100.1 100.1 99.9 99.2 100.0 100.0 100.0 100.0

GalNAc 100.0 100.0 100.0 100.0 100.0 - n.d. n.d. GlcNAc ------n.d. n.d. Neutral ------n.d. n.d. sugars a Isolated cyst wall material (CWM) generated from 24-48h encysting Giardia, as described in Methods. b CWM PH1 represents the large insoluble size exclusion fraction generated by repeated partial acid hydrolysis. The samples 0X, 1X, 2X, and 3X refer to either untreated or successive pronase-treated CWM. c Amino acid composition data for CWP1, CWP2, and CWP3 were calculated from amino acid sequence data as supplied (Mowatt et al., 1995/GenBank™ EDO77216; Luján et al., 1995/GenBank™ EDO82568; Sun et al., 2003/GenBank™ EDO79967). As indicated in the publications, the N-terminal 14 amino acids of both CWP1 and CWP2, and the C-terminal 121 amino acids of CWP2 are processed from the premature proteins prior to incorporation into the cyst wall and are thus excluded from this compositional analysis. CWP3 migrates as a 28 kDa band under reducing conditions (Sun et al., 2003), consistent with a predicted molecular weight of 27.3 kDa based on the complete 247 amino acid sequence. Thus, the calculated amino acid composition includes the potential signal sequence of 17 amino acids. Actual data from sequences supplied were: (CWP1; Thr=10.1, Ser=4.4, Cys=7.9, and Leu=14.1), (CWP2; Thr=11.6, Ser=3.7, Cys=7.4, and Leu=13.4), and (CWP3; Thr=4.9, Ser=8.5, Cys=5.7, and Leu=16.2) where data for Val and Trp is available. The calculated data in the table were normalized for Val and Trp content, shown in brackets, which are not resolved in our experimental data. d During sample processing, asparagine and glutamine are hydrolyzed to aspartic acid and glutamic acid, respectfully. Thus, Asx = aspartic acid + asparagine, and Glx = glutamic acid + glutamine. n.d. = not detected

138 Figure 21. Isolated CWM as seen by LVSEM and indirect immuno-gold staining.

Cyst wall material (CWM) was isolated for analysis after treatment with SDS/DTT, amyloglucosidase, RNAse, DNAse, Papain, and Proteinase K, as described.

A. LVSEM of the Treated CWM demonstrating typical filamentous appearance.

Courtesy of Drs. Stanley Erlandsen and Timothy Macechko, University of Minnesota Medical School. Bar = 0.3 µm.

B. FESEM of CWM probed with monoclonal antibody to CWP2 by indirect immuno- gold labeling and backscatter mode showing uniform staining.

The electron micrograph under the same magnification as in A (above) was taken in backscatter mode using indirect immuno-gold labeling. The primary mAb 8C5.C11 used recognizes CWP2 (Campbell and Faubert, 1994; Lee and Faubert, 2006). Courtesy of Drs. Stanley Erlandsen and Timothy Macechko, University of Minnesota Medical School. Bar = 0.3 µm.

139 Figure 22. Protein epitopes recognized by polyclonal antibodies to isolated CWM.

Figure 22. Giardia trophozoites were induced to encyst and samples were collected at 0, 4, 8, 12, 16, 20, 24, 30, 36, 48, and 72 hours. The percent encystment was determined for each time point (listed across the top of the figure). Time course sample were separated by 1D-SDS-PAGE (5% stacker, 10% running gel) under reducing conditions, transferred to nitrocellulose, and probed for cyst wall antigen positive proteins. Cyst wall material (CWM) isolated as described in Methods was used to produce the polyclonal antiserum. The stacking gel was retained following electrophoresis and included in the western blot probed by anti-CWM antiserum. Molecular masses are given in kDa. The solvent front is stained by pyronin-Y at the bottom of the blot.

140 The information obtainable from insoluble wall material is limited by analytical techniques such as MS and 1H-NMR that require soluble structures. A common approach to the study of insoluble cell wall material from other systems is to digest the isolated walls with glycan hydrolases, such as cellulase, chitinase, glucanase, or lysozyme, which results in small soluble oligomers or uncross-linked polysaccharides. Since a glycosidase that cleaves GalNAc-β1,3-GalNAc bonds has not previously been identified, an effort was made to test known glycan hydrolases against the isolated CWM to facilitate structural analysis of the wall components.

Sufficient CWM (50 µg total / 30 µg GalNAc polymer) was incubated overnight in the presence of 15 separate glycan hydrolytic activities from 22 sources. At 24 hours a sample was observed by phase contrast microscopy for alterations in gross morphology, cyst number, and changes in the refractile property of the cyst wall. The remaining assay mixture was assessed for free GalNAc by a modification of the Morgan-Elson assay for

N-acetylhexosamines, as described in Methods The limit of detection with the assay, based on standard curves generated in the assay buffers used, was 3.3% of the total

GalNAc in the sample. No glycosidase tested produced free GalNAc under the conditions of these assays (Table XIII) and thus it was not possible to produce soluble oligosaccharides for further study using this approach. The control for the assay was the digestion of insoluble chitin by chitinase, releasing monosaccharides of the N- acetylhexosamine GlcNAc.

141 Table XIII. Resistance of CWM to known glycosidases.

Effect on Glycosidase Known Substrate(s) CWM a 1-4 Hydrolytic Activities Reported -N-acetylgalactosaminidase, A9763 1,4(GalNAc-HexNAc) — -Galactosidase, G4408 1,4(Gal-hexose) — -Glucosidase, G4634 1,4(Glc-hexose) — Amyloglucosidase, A3042 1,4(Glc-Glc) — Amyloglucosidase, A7420 1,4(Glc-Glc) — -Amylase, A6255 1,4(Glc-Glc) — -Amylase, A3176 1,4(Glc-Glc) — Lysing Enzyme, Aspergillus, L3768 1,4(GalU-GalU) — Lysing Enzyme, Rhizoctonia, L8757 1,4(GalU-GalU) —

 1-4 Hydrolytic Activities Reported -N-Acetylglucosaminidase A, A3391 1,4(GlcNAc-HexNAc) — -N-Acetylglucosaminidase, A2264 1,4(GlcNAc-HexNAc) — -N-Acetylglucosaminidase B, A7640 1,4(GlcNAc-HexNAc) — -Galactosidase, G6008 1,4(Gal-Gal/Xyl) — Chitinase, Serratia marcescens, C1650 1,4(GlcNAc-GlcNAc) — Chitinase, Streptomyces griseus, C1525 1,4(GlcNAc-GlcNAc) — Lysozyme, L6876 1,4(GlcNAc-MurNAc) — Mutanolysin, M9901 1,4(GlcNAc-MurNAc) —

1-3 Hydrolytic Activities Reported -Galactosidase, G6008 1,3(Gal-Gal/Xyl) — Lysing Enzyme, Rhizoctonia, L8757 1,3(Glc-Glc) — Lysing Enzyme, Aspergillus, L3768 1,3(Glc-Glc) — Lyticase, L8137 1,3(Glc-Glc) —

1-6 Hydrolytic Activities Reported Dextranase, D8144 1,6(Glc-Glc) — a Effect on CWM was determined after 24 h attempted digestion with glycohydrolases (as described in Methods). CWM was monitored post-treatment by phase contrast microscopy to determine gross morphologic effects, and by chemical detection of released N-acetylgalactosamine, as described.

142 Characterization of the Cyst Wall Material

The insoluble cyst walls were isolated from homogenates of encysting cells, and subsequently treated with SDS/DTT, amyloglucosidase, papain, DNase, RNase, and proteinase K, as described in Methods. Absence of internal cyst structures were confirmed by TEM (Figure 20) and referred to as cyst wall material (CWM). A sample of CWM was subjected to methanolysis followed by re-N-acetylation and analyzed for carbohydrate composition by GLC-MS (see Methods) revealed D-GalNAc as the only monosaccharide composing 63% (w/w) of the CWM (Gerwig et al., 2002). GlcNAc, neutral sugars, and fatty acids were below the level of detection. These results were confirmed by complete acid hydrolysis and analysis of the trimethylsilylated-residues by

GLC (Gerwig et al., 2002). The identity of the N-acetyl group of GalNAc from samples solubilized by partial acid hydrolyses (not re-N-acetylated) was confirmed by 1H-NMR and inferred from mass data, supporting a polymer composed essentially completely of

GalNAc, not GalNH2 (Gerwig et al., 2002).

The remainder of the cyst wall was identified as a protein component (36%) based on the presence of 16 of 16 amino acids that could be resolved during analysis (Table

XII); Trp is labile, Val co-eluted with GalNH2, and Asn and Gln are converted to Asp and

Glu, respectively. Expectations for a peptide component would suggest fewer amino acids and/or enrichment for a select few, (e.g. bacterial peptidoglycan). Amino acid analysis was performed at multiple points (Table XII) prior to and during subsequent size exclusion chromatography, but at no time were amino acids found in fractions that were soluble or in direct associated with carbohydrate. Pronase treatment of the large insoluble fraction after partial acid hydrolysis resulted in the loss of detectable cysteine

(from 3.6 mole % undetectable) and reduction of Leu (14.3 to 6.3-9.0 mole percent). All 143 other amino acids remained at the same levels. CWM is relatively rich in Leu (14.3%) and Thr (11.4%), and this is similar to the calculated amino acid compositions for the known cyst wall proteins CWP1 (Leu 15.0, Thr 10.8) and CWP2 (Leu 13.4, Thr 11.6).

Overall the amino acid compositions for the CWM and CWP1-2 are closely matched, and combined with data of indirect immunogold labeling (Figure 21) of CWM by the mAbs that recognize CWP1-2 and data by Sun et al. (2003) who identified CWP3 by sequence similarity, but no other wall associated proteins of similar sequence, it is likely that these proteins compose a majority of the CWM protein component.

Structural Characterization of the CWM Filaments

In an attempt to study the carbohydrate polymer component of the intractable

CWM it was necessary to produce soluble fragments of the cyst wall filaments. CWM was subjected to controlled partial acid hydrolysis (PH) by repeated treatment of the insoluble material with 0.5 M trifluoroacetic acid (see Methods). After each treatment the soluble components were collected, neutralized, and pooled. The concentrated soluble pool was then separated by size exclusion chromatography. Distinct peaks were re-chromatographed over the same column to produce clearly defined peaks (Figure 23).

Following size exclusion chromatography of the soluble pool, all fractions of the

CWM filaments were confirmed by GC/MS to be composed of a GalNAc homopolymer with a chain length of at least 23 residues (Gerwig et al., 2002). Figure 24 shows the results of these analyses corresponding to fractions CWM PH1–4 from Figure 23; fraction CWM PH5 was identified as the monosaccharide GalNAc (not shown).

144 Figure 23. Elution profile of solubilized cyst wall material from partial hydrolysis.

Figure 23. Isolation of soluble fractions of Giardia cyst walls following controlled partial acid hydrolysis. Giardia cyst walls were prepared as described in Methods, and subjected to repeated brief and mild acid hydrolysis with 0.5 M TFA. Neutralized and desalted, the soluble pool was (A) separated over Bio-Gel P-2 with H2O elution and (B) rechromatographed on the same column to obtain discreet fractions. Oligosaccharides were monitored in the UV at 206 nm. Vo = void. (Gerwig et al., 2002).

Determination of linkage, conformation, chain length, and branching was accomplished by methylation analysis of the partially methylated alditol acetates of the soluble PH CWM fractions, 1H-NMR, and by MALDI-TOF mass analysis (Gerwig et al.,

2002). These analysis revealed: 1) the presence of GalNAc-ol (CWM PH5, GalNAc), 2) terminal GalNAc and 3-substituted GalNAc-ol (CWM PH4, GalNAc-β1,3-GalNAc), 3) primarily terminal GalNAc, 3-substituted GalNAc, and 3-substituted GalNAc-ol (CWM

PH3, GalNAc-β1,3-GalNAc-β1,3-GalNAc), 4) terminal GalNAc and 3-substituted

GalNAc (CWM PH2, [β-3-GalNAc-1]4-9), and 5) terminal GalNAc and 3-substituted

GalNAc (CWM PH1, [β-GalNAc-1]5-23), Branching, which would be demonstrated by multiply-substituted-monosaccharides, was not detected in any sample analyzed.

145 Figure 24. Structural analysis of the Giardia cyst wall filaments.

Figure 24. Purified Giardia cyst walls were subjected to controlled, repeated partial acid hydrolysis and soluble oligosaccharides were pooled and separated as + described in Figure 23 resulting in fractions CWM PH 1-4. Compositional analysis by MALDI-TOF-MS (A & B) revealed only pseudomolecular ions [M+Na] of HexNAc polymers of 5 to 23 residues (CWM PH1) while other fractions corresponded to smaller chain lengths (shown on X axis). Spectral analysis by 1D 1H NMR of CWM PH1 (C) and PH4 (D) revealed only terminal GalNAc, and 3-substituted GalNAc and/or reducing end 3-substituted GalNAc-ol after reduction and methylation. The 1H NMR proton spectrum was completely assigned by TOCSY and ROESY measurements including assignment of N-acetyl substitution. (From Gerwig et al., 2002)

146 CHAPTER IV

DISCUSSION

During Giardia's life cycle, this parasite must differentiate into an infectious cyst and elaborate a resistant cyst wall prior to exiting its host. The mature cyst is surrounded by a thick filamentous layer that had been suggested to be composed of chitin. Early support for the presence of chitin was limited to indirect studies of lectin-binding (Ward et al., 1985; 1988) and biochemical analysis of lectin-affinity-selected GlcNAc- containing glycoproteins (Ortega-Barria et al., 1990). Table I and Figure 6 present data for the biochemically characterized polymers currently known; most of which are composed of glucose or glucose-derivatives such as N-acetylglucosamine (GlcNAc).

Thus, because of the cosmopolitan nature of chitin, and because of the early lectin data, the view that the Giardia filamentous cyst wall was composed of chitin was not immediately challenged.

To directly address the biochemical synthesis of the Giardia cyst wall, this lab demonstrated previously: 1) the exclusive presence of large amounts of the cyst-specific sugar N-acetylgalactosamine (GalNAc) in cyst walls with only trace amounts of the chitin constituent GlcNAc (Jarroll et al., 1989) and 2) the presence of a pathway of regulated carbohydrate synthesizing enzymes (Figure 5) (Steimle et al., 1997; Bulik et al., 1998; van Keulen et al., 1998; Steimle et al., 1998; Bulik et al., 2000; Jarroll et al., 2001; Karr et al., 2004) including an inducible UDP-N-acetylglucosamine 4'-epimerase (Macechko et al., 1992) responsible for the generation of the penultimate product in cyst wall polysaccharide synthesis - UDP-GalNAc.

147 Now with this work we provide conclusive data refuting the presence of detectable levels of chitin in the Giardia filamentous cyst wall and establish its true biosynthesis by: 1) demonstrating the presence of the novel 1,3-N-acetylgalactosaminyl transferase activity (CWS) which is 2) responsible for the synthesis of the unique cyst- specific (1→3)--D-N-acetylgalactosamine homopolymer that constitutes essentially

100% of the carbohydrate portion of the Giardia cyst wall filaments and 63% of its dry weight. We propose the name giardan as the common name for this unique polymer.

The novel glycan synthase activity identified catalyzes the following reaction:

( UDP-GalNAc + [3)-β-D-GalNAc-(1→]n → [3)-β-D-GalNAc-(1→]n+1 + UDP ) and has been given the common name of cyst wall synthase (CWS) for its unique action in synthesizing the cyst wall filaments. According to IUBMB convention, similar to chitin synthase (EC 2.4.1.16), the systematic nomenclature we propose for CWS is:

UDP-N-acetyl-D-galactosamine:(1→3)giardan–3--N-acetyl-D-galactosaminyltransferase.

However, like other glycan synthases that have never been demonstrated to use an acceptor (chitin synthase, β1,3glucan synthase, and cellulose synthase), this systematic nomenclature is imprecise, since processive enzymes are considered to extend only nascent chains and once released they are no longer acceptors for these enzymes.

The inducible CWS activity was identified from homogenates of encysting

Giardia intestinalis (MR4) cultured in vitro and was not found in bile-free cultures of non-encysting trophozoites. The induction of CWS activity (Figure 7) corresponds to the production of in vitro-derived cysts (Figure 7) and thus, to the need for cyst wall polysaccharide filaments, with detection of GalNAc in encysting cells from below the limits of detection in trophozoites (Jarroll et al., 1989), with morphologic studies of

148 encysting cells using cyst wall-specific antibodies (Erlandsen et al., 1996; Erlandsen,

1989; Reiner et al., 1990; Erlandsen et al.; 1990; McCaffery et al., 1994), with the synthesis of cyst wall-specific proteins and encystment specific vesicles (ESVs) (Faubert,

1991; Luján et al., 1995; Mowatt et al., 1995; Touz et al., 2002; Sun et al., 2003;

Lanfredi-Rengel et al., 2003), and with the time course induction of the complete pathway of enzymes involved in UDP-GalNAc synthesis during Giardia encystment

(Figure 5, reviewed in Jarroll et al., 2001; Karr et al., 2004). Induction of activity increased from 8h through 30h (24-36h range) (Figure 7) following the percent encystment until maximal encystment had been attained and CWS activity decreased thereafter. These results are consistent with the inductions observed by the other pathway specific enzymes of this system (Figure 5).

By 1D SDS-PAGE, the in vitro generated CWS product was of large size as it was excluded from entering the 5% stacking gel in native form (Figure 7, Figure 16) or following proteinase digestion (Figure 17). The CWS reaction product also did not migrate past the top of a size exclusion column, suggesting size rather than charge was the predominant reason for exclusion from enterting the SDS-PAGE gel. The product formed could be precipitated in 67% ethanol by centrifugation, in 10% TCA by centrifugation, or filtered over glass fiber filters and retained. Since these assays are modification of those used for chitin synthase (Cabib, 1972; Duran and Cabib, 1978) and are now generally used in the literature to demonstrate glycan synthesis, then these results with CWS activity are consistent with the formation of a glycan product.

The in vitro-derived [14C]-labeled product was also resistent to extraction in boiling 2% SDS and resistant to treatments with 1 mg ml-1 pronase and trypsin

completely, but was partially susceptible to papain, proteinase K, and 1 M KOH (1 h at 149 100oC), and was completely hydrolyzed by 2 M HCl (3 h at 100oC) (Table IX).

Interestingly, it has been shown by multiple methods including HPLC and circular dichroism that pronase (electrophoretically pure as assessed by capillary electrophoresis) and Aspergillus pectinase non-specifically depolymerize chitosan oligosaccharides

(Sukwattanasinitt et al., 2002; Kumar et al., 2004) and these references suggest that the proteinase digestions may not fully imply protein degradation.

However, given the partial susceptibility to proteinase K and papain in the ethanol precipitation assay, similarly prepared reaction mixtures (not pellets) were proteinase- treated and analyzed by reducing SDS-PAGE / autoradiography (Figure 17) and there were no detectable [14C]-labeled proteins or peptides in the stacking or running gel, at the dye front or below. Given that the amount of radioactivity in the product(s) excluded from entering the 5% stacking gel appeared the same as untreated controls, it was concluded that the proteinase treatments may have produced fragments too small to have precipitated under the conditions of the ethanol precipitation assay, but still too large to enter the gel.

In general, the CWS reaction product was intractable to work with unless hydrolyzed to monomers, which was of little value. Jarroll et al. (1989) and Manning et al. (1992) extracted in vivo-derived cyst walls with SDS without biochemical or morphologically detectable effect. Manning et al. (1992) also attempted proteinase digestion of in vivo-derived Giardia muris cyst walls without complete effect reducing total amino acids by ~60%, however, the simultaneous effect on monosaccharide content was not determined during these experiments. Thus, it is likely that some portion of the carbohydrate polymer filaments would have been liberated even though not digested by

proteinase treatment. 150 Thus, the incorporated radiotracer could not be reduced from the reaction product by treatment with hydrolytic enzymes or chemical degradation that also did not affect the morphology of the in vivo-derived cysts or the thickness of the filamentous wall as demonstrated by scanning or transmission electron microscopy (Manning et al., 1992;

Jarroll et al., 1992). However, both the CWS reaction product and mature cysts were susceptible to acid hydrolysis. These data suggest that the nature of the in vitro CWS reaction product is resistant in a similar fashion to the cyst wall filaments derived in vivo and is consistent with the synthesis of a polysaccharide, which is composed of GalNAc.

CWS was partially purified from homogenates of 30h encysting trophozoites

(Table VI, Figure 8). Unlike the five enzymes of the inducible UDP-GalNAc synthesis pathway characterized to date that are localized to the non-sedimentable fraction (S- fraction), CWS activity was confined to the particle fraction (P-fraction) (Figure 9A).

When the P-fraction was separated by isopycnic centrifugation and monitored for appropriate marker enzymes, CWS activity was confined to a microsomal vesicle population (Figure 9B) with a density (1.16 g ml-1) distinct from the lysosome-like peripheral vesicles (1.18 g ml-1).

The CWS-containing vesicles appeared morphologically uniform in size by

LVSEM (Figure 10) and when incubated with the substrate UDP-GalNAc appeared with fibril-like protrusions from vesicle surfaces (Figure 11) that were not seen without added substrate. The activity of CWS did not exhibit latency after freshly prepared vesicles were tested prior to and after multiple freeze-thaw cycles suggesting a cytosolic active site for the enzyme and potentially a transmembrane pore complex as is the case in other glycan synthase systems.

151 Identification of the peak CWS-containing microsomal population as the previously described encystment specific vesicles (ESVs) was confirmed using a panel of mAb's from the National Institutes of Health (Drs. Nash and Luján) to known cyst wall proteins (CWP) (Table V). Of particular interest is the colocalization with the well- characterized CWP1 and CWP2 considered marker proteins for the ESV population and the cyst wall (Luján et al., 1995; Mowatt et al., 1995; Gottig et al., 2006; Stefanic et al.,

2006).

Further purification of CWS from ESVs involved repeated extraction with detergent. Of 19 detergents with differing properties all but three inhibited CWS below their CMC value. Reconstitution studies failed to recover active enzyme. The remaining three detergents deoxyBigCHAP, CHAPS, and Synperonic® PE/F68 (a poly(ethyleneoxide)78-poly(propyleneoxide)30-poly(ethyleneoxide)78 tri-block copolymer) did not inhibit activity more than 25% until 18.5X, 1.0X, and 60X their respective CMC values. However, no CWS activity was recovered in a soluble fraction when CWS was tested with these detergents. Thus, CWS activity was deemed detergent-resistant and detergent extraction of non-essential proteins from the ESV membrane fraction was selected for further purification. CWS activity was stimulated to ~125% by the addition of 1% deoxyBigCHAP as assessed in direct assay and was tolerant to 3% deoxyBigCHAP (49% of control), which is 25x the detergent's CMC. Based on enzyme and protein recovery no further purification occurred beyond two cycles of detergent extraction resulting in a final purification of ~155-fold, not including enzyme stimulation.

Stimulation (to ~116%) was also found with Synperonic® PE/F68 at 30X its CMC.

Interestingly, the association of other glycan synthase complexes with detergent-

resistant lipid microdomains (DRM) has recently been published. The following two 152 publications appear to represent the first reports of the association of DRMs with active glycan synthases. Bessueille et al., (2009) demonstrated that 70% of β1,3glucan synthase and cellulose synthase activity from three plants were localized to DRMs, which are enriched in sterols and glycosylsphingolipids. Briolay et al. (2009) also localized chitin synthase and β1,3glucan synthase to DRMs from the oomycete Saprolegnia, which contained a similar composition of sterols and glycosylsphingolipids. Given that CWS resists detergent extraction as well, it may be that DRMs play a role in vesicle sorting and

Giardia cyst wall synthesis.

Since CWS is a novel system, no antibodies or molecular probes from other systems were available to attempt to identify the polypeptide components of this synthase in Giardia. During purification, several bands correlating with both increased stain intensity by SDS-PAGE and immuno-reactivity with an anti-20h antiserum to encysting trophozoites were identified (Figure 12A) that specifically tracked purification from P- fraction through peak CWS-containing isopycnic fractions (30, 32, 55, 81 kDa). After deoxyBigCHAP extraction the 30 and 32 kDa proteins were the dominant detergent- resistant proteins and immuno-dominant component that tracked CWS activity (Figure

12B).

The 30 and 32 kDa proteins were separated in 2D (Figure 13C), were identified by protein microsequencing, and confirmed by BLAST analysis of the Giardia genome to be α1-giardin and β-giardin, which are members of the annexin family. Annexins are abundant proteins in cells both in number and by total protein content, and they are generally considered to function in vesicle transport, sorting, membrane fusion, and exocytosis (Futter and White, 2007; Gerke et al., 1996).

153 It is known that β-giardin forms 2.5 nm filaments associated with the cytoskeleton in Giardia and reversibly self-assembles at neutral pH and moderate salt conditions

(Crossley and Holberton, 1985).

α1-giardin has been better characterized and is a functional annexin with Ca2+- dependent phospholipid binding properties, was extracted from a detergent-insoluble fraction of Giardia (Bauer et al., 1999), is an immuno-dominant antigen found on the surface of recently excysted trophozoites, and binds glycosaminoglycans in a Ca2+- dependent manner (Weiland et al., 2003).

Functional roles for annexins in glycan synthase complexes may be relevant.

Bulone and Fèvre (1996) found that a 34 kDa protein strongly co-purified with

β1,3glucan synthase from Saprolegnia after enrichment by product entrapment and

CHAPS solubilization. Later, Bouzenzana et al. (2006) established the identity of the 34 kDa protein by proteomics analysis of the fractions enriched in β1,3glucan synthase and characterized the 34 kDa protein as a functional annexin that activated β1,3glucan synthase in vitro. In an unrelated system, Andrawis et al. (1993) identified a 34 kDa annexin that upon association with partially purified cotton fiber β1,3glucan synthase in vitro inhibited activity. Multiple annexins possess intrinsic phosphodiesterase activity functional with GTP and ATP and their nucleside diphosphates that are stimulated by

Ca2+-dependent binding to membranes including a p34 and p35 annexin from tomato

(Bandorowicz-Pikula et al., 2001). Thus, annexins may participate with GTP/GDP exchange factors such as Rho1p, which is the major regulatory subunit of β1,3glucan synthases.

What role these giardins have in encystment is unclear and will require additional

study. However, the association of annexins to the plasma membrane and vesicles 154 appears to involve more than just phospholipid binding. The presence of annexins in detergent-resistant fractions and the Ca2+-dependent association to lipid raft microdomains is clear (Babiychuk and Draeger, 2000; 2006). However, it is also possible that during enrichment for membrane-related complexes that these annexins become artifacts of the in vitro purification scheme.

Currently, attempts to further purify CWS have proven challenging. Use of detergent solubilization or other means to make various forms of column chromatography useful have failed due to an inability to reconstitute the activity after treatment. Without an ability to quantify the activity during further purification steps, it remains unclear if the activity associated with CWS is the result of multiple subunits or to determine if there are regulatory components, as with β1,3glucan synthase. Purification of CWS to a detergent-resistant fraction of microsomal membranes is consistent with the purification schemes for processive glycan synthases - chitin synthase, β3glucan synthase, and cellulose synthase - for the past 30 years.

Giardia CWS may be a processive glycan synthase as well, which is a polymer producing enzyme that does not require an acceptor and cannot elongate polymer chains once terminated. Examples of enzymes of this class include chitin synthase, β1,3glucan

(callose) synthase, vertebrate and Class I streptococcal hyaluronan synthase, and cellulose synthase. If this is the case, then an acceptor for the CWS reaction may not be required. However, because there was no information in the literature on an enzyme activity or polymer of this nature until publications from this lab (Gerwig et al., 2002;

Karr and Jarroll, 2004), then it was appropriate to search for an acceptor to better characterize the enzyme.

155 In an attempt to demonstrate a potential requirement for an acceptor for the in vitro CWS reaction two approaches were pursued; I assessed the potential for an endogenous acceptor(s) and examined exogenous acceptors known from other systems.

Potential endogenous proteins and glycoproteins were investigated. Also, potential exogenous oligosaccharide and (glyco)peptide acceptors (blocked at their reducing-end by a functional group required for detection) and exogenous glycoproteins were examined by following the tracer [14C]GalNAc or [3H]GalNAc in assays of CWS activity. No incorporation was found into any endogenous or exogenous (glyco)protein,

(glyco)peptide, or oligosaccharide acceptor was detected as monitored by SDS-PAGE / autoradiography, by scintillation proximity assay of functionalized oligosaccharide, glycopeptide, and peptide acceptors, or by increased activity as measured in multiple assay formats. Based upon the purity of the CWS assayed from the detergent-resistant

ESV fraction, as compared to other processive glycan synthase systems, then these results are consistent with a processive glycan synthase where an acceptor is not required.

To ensure that an endogenous acceptor was not formed early during encystment as a priming step and subsequently extended later into high molecular weight material, a time course experiment was conducted. However, Figure 7 clearly shows that the radiotracer was confined to the high molecular mass fraction excluded from entering the

5% gel.

Potential endogenous glycoprotein acceptors were also assessed by lectin blotting samples of 30h encysting trophozoites probed with lectins reported to detect the terminal sugar structures GalNAc/- (Helix pomatia lectin), GalNAc1,3GalNAc- / GalNAc-

(Dolichos bifloris), and Gal1,3-GalNAc- / Gal- (Arachis hypogaea lectin). However,

156 Gal/GalNAc-modified proteins were not detected even upon prolonged incubations with the lectin-conjugates. Subsequently, it has been shown that glycosylation in Giardia has evolved a minimalist glycosylation pathway dominated by truncated N-linked GlcNAc2,

GlcNAc-Glc-, and variants thereof (Samuelson et al., 2005; Ratner et al., 2008;

Papanastasiou et al., 1997) and by O-GlcNAc modifications (Banerjee et al., 2009).

These results regarding GlcNAc-modified glycoproteins plus the negative results from the Gal/GalNAc-lectin probes supports the position that CWS does not incorporate

[14C]GalNAc into endogenous (glyco)proteins, and thus would not serve as primers in the reaction.

Based on the localization of CWS to ESVs likely destined for the outer cyst membrane, it was alternately postulated that a glycolipid may serve as a primer in the

CWS reaction similar to a report by Peng et al. (2002) who suggested that β-sitosterol- glucans might function as a primer for a cellulose synthase. Alternately, Giardia cyst wall filaments might have been produced from lipid-linked intermediates similar to some other microbial polymers such as murein, pseudomurein, methanochondroitin, and yeast wall mannoprotein. While a glycolipid was not likely to be retained in the CWM or to play a significant structural role in the cyst wall filaments as lipids were below the limits of detection in hydrolysates of CWM analyzed by GC/MS, however, a glycolipid might serve as an intermediate in the reaction or function as a primer.

As a first approach, the effect of various antibiotics which target the enzymatic synthesis of glycolipid intermediates in both Prokaryotic and Eurkaryotic systems was investigated. In vitro cultures of parasites were induced to encyst for 24 h in the presence of sub-lethal doses of tunicamycin (250 µM) or bacitracin (1.0 mM) without a reduction

in trophozoite growth rate, motility, adherence, or deviation in gross morphology as 157 assessed by phase contrast microscopy. At these concentrations both tunicamycin and bacitracin reduced the percent encystment by 65% and 38%, respectively. Slightly higher concentrations were cidal. Since it was possible that secondary effects halted encystment and incubation with inhibitors for >24h would have resulted in trophozoite death as well, then an alternate approach was sought to confirm these results.

To determine if a lipid-linked intermediate was involved in a more direct fashion, the presence of potential intermediates was assessed by paper chromatography after lipid extraction of in vitro radiolabeled CWS reaction products. However, while ~6% of the incorporated radiolabel could be extracted no lipid-linked oligosaccharide could be detected. Mild acid hydrolysis of the lipid extract with: 1) 0.01 N, or 2) 0.01 N and then

0.1 N HCl for 10 min at 100oC revealed only the presence of free radiolabeled GalNAc.

These data indicated that a lipid-linked intermediate was not involved in CWS activity in vitro and suggested only the presence of small non-sedimentable radiolabeled oligosaccharides.

Thus, at 155-fold enriched the CWS activity was characterized with respect to assay conditions, nucleotide-donor specificity, reaction kinetics, and inhibitors

(summarized in Table XIIII). Standard assays were performed for 30 min, but linear for at least 60. CWS was optimally active in the presence of 25 mM Ca2+ or Mg2+, at 37oC, and at pH 7.5 (Figure 14). To test the donor specificity, CWS was incubated in the presence of 10 mM Ca2+, 1 mM Mg2+, and 1 mM Mn2+ to ensure that other activities were not missed in the reactions. CWS demonstrated an absolute requirement for UDP-

GalNAc (Table VI); activities with other UDP-sugars were below the limits of detection in these assays (0.001 nmol min-1 mg-1 protein).

158 Enzyme kinetics were established for CWS from the peak isopycnic fraction after a repeated deoxyBigCHAP detergent enrichment step. At this stage, CWS was partially

app app purified to ca. 155-fold (Table VI). CWS exhibited a Km of 48 + 15 M and a Vmax of 0.70 + 0.085 nmol min-1 mg protein-1 for UDP-GalNAc incorporated into a filterable product as determined by non-linear regression analysis using the software package

GraphPad Prism™. Similar results were obtained using linear regression analysis (Figure

15 inset) and under the conditions of the assay at the highest substrate concentration tested (0.8 mM UDP-GalNAc) substrate feedback inhibition and end product (UDP) inhibition were not significant.

Because it remains unconfirmed if an endogenous high molecular-mass polymer acceptor is required, then it would not be possible to add it to the reaction in a saturating concentration. This would affect the apparent kinetic parameters determined likely

app app underestimating the true values. Thus, we chose to use Km and Vmax to distinguish this possibility. However, since chitin synthase, β1,3glucan synthase, cellulose synthase, and Class I hyaluronan synthase from vertebrates and Streptococcus (all processive enzymes) have not been shown to use an acceptor (Duran and Cabib, 1978; Cabib, 1987;

Kang et al., 1984; DeAngelis, 1999), then it is likely that the kinetic data generated will stand.

app Under optimal conditions and in the presence of substrate below the Km of the reaction, CWS was profiled for inhibitors of the assay including substrate analogs and reaction products (Table VIII). As suspected, high concentrations of the reaction product

UDP would inhibit the reaction if conducted at much higher substrate concentrations and/or greater substrate conversion. This was not an issue in the assay conducted at low

159 substrate with low conversion. However, inclusion of the coupling enzyme alkaline phosphatase (phospodiesterase-free) would alleviate feedback inhibition, if assays resulting in a higher concentration of product (UDP) were required. Also, the substrate analog of UDP-GalNAc - UDP-GlcNAc (which did not serve as a donor in this assay,

Table VII), and the chitin synthase inhibitors (membrane-impermeant UDP-GlcNAc analogs) Polyoxin D and Nikkomycin Z demonstrated similar levels of inhibition as compared to UDP-GlcNAc when tested against CWS. None of these compounds were potent inhibitors of CWS and thus were not pursued.

However, one lab has made progress on synthesizing compounds to target CWS and or the UDP-GlcNAc 4'-epimerase necessary for production of UDP-GalNAc by synthesizing UDP-GlcNAc analogs with a stable membrane-permeant alkylphosphonate linkage between N-acetylglucosamine and the uridine moiety in place of the natural pyrophosphate linkage (Suk et al., 2007a; 2007b; 2007c). The best of multiple compounds produced had a minimal inhibitory concentration of 0.48 µM (~10-fold lower than metronidazole), a therapeutic index >250 as measured against Madin-Darby Bovine

Kidney cells, and reduced cyst formation in vitro to <6% of controls (Suk et al., 2007a;

2007b; 2007c). It would be of interest to test compounds that have poor MIC values, but inhibit encystment, for their effect on CWS, the UDP-GlcNAc 4'-epimerase, and the other pathway enzymes responsible for UDP-GalNAc synthesis. It would also be of interest to determine if blocking encystment, specifically, leads to cell death or if the encysting trophozoite can persist. These compounds could facilitate novel approaches to cell wall proteomics in Giardia, or provide opportunities to study the cyst wall inner membrane during encystment.

160 Table XIIII. Characteristics of partially purified CWS from encysting Giardia.

Assay Characteristics Optimum Range a

pH 7.5 6.0-10.0

Temperature 37oC 25-40oC

Reaction Time -- 0-60 min b

Acceptor Specificity None detected c --

Substrate Specificity UDP-GalNAc -- d

app e Substrate Km 48 µM, UDP-GalNAc

Maximal Velocity 0.7 mU mg-1 protein f

Divalent Metal Cofactor 25 mM Ca2+ or Mg2+ 5-50 mM g

deoxyBigCHAP h 1.0 % h 0.2-3.0%

EDTA, UTP, UDP, UMP, UDP-GlcNAc, Inhibitors i Nikkomycin Z, Polyoxin D a Required for at least 50% of maximal activity when assayed for 30 minutes. b Beyond 60 min, the activity was not linear. c No activity detected with any endogenous or exogenous acceptor tested. d No detectable activity with UDP-GlcNAc, UDP-Glc, UDP-Gal, GalNH2, or GlcNH2. e app The apparent Km (Km ) was determined, since inclusion of a saturating concentration of an acceptor (if any is required) in the reaction was not possible at this time. f Activity (mU) is defined as nmol of substrate incorporated per min. g No other divalent cation provided activity above 50% of maximal vs. Ca2+ or Mg2+. h Represents stimulation of activity when the detergent deoxyBigCHAP (N,N-Bis[3-(D-gluconamido)propyl]deoxycholamide) is included in the assay.. i EDTA (ethylenediaminetetraacetic acid) is a chelator of divalent and trivalent cations.

161 In order to address the overall reaction catalyzed by cyst wall synthase the enzyme's product needed to be characterized. However, preliminary data from the

Complex Carbohydrate Research Center (Athens, GA) suggested that analysis of the material was not possible at that time, since the amount of product obtained from the

CWS reaction was small, extremely insoluble all solvents tested, and only monosaccharide analysis could be obtained after complete acid hydrolysis. Structural analysis would require far more material in order to produce soluble components from the intractable material by controlled partial acid hydrolysis.

Thus, since GalNAc is a cyst-specific sugar (undetectable by GC/MS in non- encysting, no-bile, trophozoites) found essentially completely within the cyst wall (Jarroll et al., 1989; Manning et al., 1992), since the product of the CWS reaction behaved biochemically identically to the mature cyst wall filaments (Karr and Jarroll, 2004), and since the only GalNAc-incorporating activity that was found localized to the ESV fraction, then analysis of isolated mature cyst wall filaments was chosen to identify the product of the CWS reaction.

Approximately 150 mg dry weight of cyst wall filaments from 40 L of 24-36h encysting trophozoites were isolated by sequential mechanical, chemical, and hydrolytic means and the process was monitored by phase-contrast, scanning, and transmission electron microscopy (Figure 20). No detectable gross morphologic differences were identified in CWM after the isolation procedure, which attests the intractable nature of the Giardia cyst wall.

In an effort to produce soluble components for structural analysis, CWM was challenged with 15 different known glycosidases from 22 sources individually for 24

hours (Table XIII). None of the glycosidases altered the gross morphology of the cysts as 162 determined by phase contrast microscopy, and assays for released GalNAc confirmed this. While unsuccessful in demonstrating that a known glycosidase is "sloppy" in its substrate range, it did highlight the uniqueness of Giardia's cyst wall polysaccharide in light of the diversity of polymers that are currently known. This data also supports the hypothesis that Giardia may have evolved a unique cyst wall polysaccharide because of selective pressures while outside its host for 30-60 days waiting to be consumed and infect another host. It is likely that a glycosidase exists that can affect degradation of this polysaccharide, perhaps from Giardia, and it will be interesting to see this tested.

Monosaccharide and amino acid compositional analysis were performed on intact cyst wall material (CWM), separately. Only GalNAc (100% of total sugars) and amino acids were detectable in the ratio of 63:36 in the CWM. These results confirmed that

GalNAc composes 100% of the carbohydrate component of resistant filaments (100% fully acetylated N-acetylgalactopyranosamine) and that the remainder is composed of amino acids rich in leucine (Gerwig et al., 2002). No other sugars, lipids, or other components were detected in the CWM analyzed (Gerwig et al., 2002). The data from in vitro-derived Giardia intestinalis (MR4) cyst walls (63% GalNAc) was similar to data from in vivo-derived G. muris cyst walls (66% GalNAc, 42.2 nmol/63.7 nmol total per

106 cysts) (Manning et al., 1992), which suggests that the in vitro model of the filamentous wall is relevant and mature by 24-36 hours.

While the composition of the cyst wall material was determined to be 63% carbohydrate the remaining 37% was found to be protein and/or peptide high in leucine

(14.3%) and threonine/serine content (15.9%). Upon analysis it was determined that: 1) the amino acid content of the CWM was similar to published sequence data for the

related mature cyst wall specific proteins - CWP1 and CWP2, though it differed from 163 CWP3 (Table XII), 2) the mAbs 8C5.C11 and 5A4.G6, which have been demonstrated to bind CWP1 and CWP2 (Campbell and Faubert, 1994; Lee and Faubert, 2006) still recognized epitopes evenly distributed over the CWM as determined by indirect immuno- gold labeling and FESEM (Figure 21B, similar data for mAb 5A4.G6 not shown) and 3) an anti-CWM polyclonal antiserum generated from the isolated CWM recognized three major bands matching molecular masses of the mature and processed cyst wall proteins by western blot analysis (Figure 22). Based on these findings, it is likely the majority of the structural protein component of the CWM is composed of CWP1 and CWP2, which have been described previously (Luján et al., 1995, Mowatt et al., 1995). The relatively larger difference in amino acid composition of CWP3, suggests a minor structural role for this protein.

These results together confirm the presence of intact epitopes consistent with

CWP1 and CWP2 in the isolated CWM consistent with published reports. However, it was reported that CWP1 and CWP2 can be solublized from encysting trophozoites in the presence of reducing agents (Luján et al., 1995), which was not the case from intact cyst walls. This suggests that wall materials may be cross-linked. However, no covalent association could be demonstrated between any amino acid residue and GalNAc- containing filaments solubilized by partial acid hydrolysis (Gerwig et al., 2002).

The mature CWP1 and CWP2 protein sequences reveal a large number of available serine and threonine residues (14.5 % each), and computer prediction modeling of these sites (NetOGly modeling) suggest that some of these hydroxylated-amino acid motifs are sufficiently similar to biochemically demonstrated O-glycosylation sites typically found in Eukaryotic proteins. Since the anti-CWM antiserum recognized bands

of increasing molecular weight late during encystment (Figure 22), then it was reasonable 164 to suspect that CWPs may be glycosylated later during encystment. However, lectin blot analysis with probes for terminal-Gal/GalNAc motifs sufficient to detect GalNAc- modified glycoproteins failed to demonstrate any GalNAc-O-glycosylated proteins using encysting or non-encysting trophozoite homogenates. An O-GlcNAc transferase has been demonstrated in Giardia recently (Banerjee et al, 2009) and also a report of WGA- affinity selection for the N-glycome of encysting Giardia (Ratner et al., 2008), however, these authors noted that CWP1 and CWP2 were not detected. Thus, the nature of polysaccharide-protein association of the cyst wall filaments remains unknown and will require solubilization of fragments of the wall material that may reveal covalent cross- linking.

Structural analyses were performed at the Bijvoet Center for Biomolecular

Research (Utrecht University, The Netherlands) on purified CWM. To overcome the extreme insolubility of the polymer, the CWM was subjected to controlled partial acid hydrolysis and soluble oligosaccharides were separated by size exclusion chromatography for further analysis (Figure 23).

Methylation analysis was performed on soluble oligosaccharides of up to 23 residues isolated after partial acid hydrolysis. However, the chain lengths are likely significantly longer, cross-linked, and/or strongly interacting and insoluble when not subjected to partial acid hydrolysis. Only a (1→3) linkage was demonstrated implying an unbranched polysaccharide. In addition, subsequent analysis revealed that the linkage was only present in the β configuration. The monosaccharide GalNAc present in the

CWM was determined to be in the pyranose form, as in nearly all other microbial polymers. However, in contrast to the homopolymers of fungal chitin and Aspergillus

165 parasiticus polygalactosamine, the CWS monosaccharides are essentially completely N- acetylated.

In summary, this work establishes the true biosynthesis of the Giardia intestinalis cyst wall polysaccharide; a unique, linear, unbranched, essentially completely N- acetylated (1→3)--D-N-acetylgalactosamine homopolymer that comprises 100% of the cyst wall polysaccharide and 63% of the cyst wall by dry weight. We propose the common name of "giardan" and the descriptive name polyβ1,3(GalNAc) to reflect current literature convention. Giardan is synthesized by an inducible enzyme for which we have previously named cyst wall synthase (CWS) to reflect its unique function. CWS has been purified 155-fold from encystment specific vesicles of 30h encysting trophozoites and is undetectable in non-encysting cells. Cyst wall synthase catalyzes the following reaction:

(UDP-GalNAc + [3)--D-GalNAc-(1→]n → [3)--D-GalNAc-(1→]n+1 + UDP)

with an absolute specificity for its substrate (UDP-GalNAc) and requirement for a divalent cation cofactor (Ca2+/Mg2+). According to IUBMB convention, similar to chitin synthase (EC 2.4.1.16), the systematic nomenclature we propose for CWS is as follows:

UDP-N-acetyl-D-galactosamine:(1→3)giardan–3--N-acetyl-D-galactosaminyltransferase

Based upon physical data, an energy minimized model of the giardan structure has been proposed: Figure 25.

166 Figure 25. Model of the Giardia cyst wall polymer Giardan.

A. Giardan; modeled from 6 β1,3-GalNAc units in a minimum energy conformation

The model was built using SWEET-DB program (Bohne et al., 1999) for in silico energy minimization of polysaccharides and represents one stable conformation, a right hand turn, when free in aqueous solution. For reference the non-reducing end is on the right of the model and colors represent a measure of hydrophilic character (red=least hydrophilic and white=most hydrophilic character).

B. Giardan; modeled from 23 β1,3-GalNAc units in a minimum energy conformation.

The model was built using SWEET-DB program (Bohne et al., 1999) for in silico energy minimization of polysaccharides and represents one stable conformation, a right hand turn, when free in aqueous solution. For reference, the non-reducing end is on the left of the model. Colors represent the atoms nitrogen (blue), oxygen (white), and carbon (red).

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