Structural and Biochemical Characterization of UDP-Glucose: Glycoprotein Glucosyltransferase

Structural and Biochemical Characterization of UDP- glucose: Glycoprotein Glucosyltransferase

Xuyao Li

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Biochemistry University of Toronto

Structural and Biochemical Characterization of UDP-glucose: Glycoprotein Glucosyltransferase

Xuyao Li

Master of Science

Department of Biochemistry University of Toronto

2016 Abstract

Endoplasmic reticulum quality control is a crucial process in eukaryotic cells to ensure glycoprotein folding. It utilizes N-glycan modification as an indicator of glycoprotein folding status. Monoglucosylated glycoproteins are recognized by calnexin and calreticulin that recruit

ERp57 to assist folding. The glycoproteins are released from calnexin and calreticulin upon removal of the glucose residue by glucosidase II. UDP-glucose: glycoprotein glucosyltransferase

(UGGT) recognizes incompletely folded glycoprotein and adds a glucose residue to the glycoprotein. Addition of the glucose residue leads to re-association of the glycoprotein with calnexin and calreticulin for further folding. In this study, we focus on characterizing UGGT and its substrate interaction. We expressed Fabs against UGGT as a tool for crystallization. The binding of Fabs to UGGT was assayed by the surface plasmon resonance technique. We also assayed UGGT activity using a radioactive UDP-Glc-based assay. The results provided a basis for understanding UGGT and its substrate interaction.

Acknowledgments

It has been a great journey since I began the master program. I would like to thank everyone that helped me in this project.

I would like to express my immense gratitude to my supervisor, Dr. James M. Rini. This thesis would not have been possible without his guidance and support. His inspiration has opened my eyes to the wonders of science. I would also like to acknowledge both members of my supervisory committee, Dr. David B. Williams and Dr. Gil Privé for their precious advice and input throughout the project.

I would like to thank the Department of Biochemistry and all my colleagues in the Rini lab for their help and support. They have made such a friendly and peaceful environment for research and learning. I especially thank Dr. Sachdev Sidhu for providing the phage display Fab constructs, Dr. Cordula Enenkel for instructing me in yeast culture, Jesse Hackett for providing the original UGGT construct, Malathy Satkunarajah and Dongxia Zhou for their technical support, Alan Wong and Zhijie Li for their support and comments on the thesis, and Kristina Han for supporting me and providing the Her2 antibody expressed with kifunensine.

Last but not least, I would like to thank my family and friends for always being there for me.

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Table of Contents

Acknowledgments ...... iii

Table of Contents ...... iv

List of Tables ...... vii

List of Figures ...... viii

List of Abbreviations ...... ix

Chapter 1 Introduction ...... 1

1.1 Protein folding and quality control in the ER ...... 1

1.1.1 N-linked glycosylation ...... 1

1.1.2 Glucose processing ...... 3

1.1.3 Glucosidase II ...... 3

1.1.4 The calnexin/calreticulin cycle ...... 4

1.1.5 UDP-glucose glycoprotein glucosyltransferase ...... 7

1.2 Phage display Fabs ...... 15

1.3 Thesis objectives ...... 16

Chapter 2 Materials and Methods ...... 17

2.1 Chemicals, plasmids and cell culture ...... 17

2.2 Plasmid constructions ...... 17

2.3 Mammalian expression and purification of hUGGT1 ...... 18

2.4 Bacterial expression and purification of Fabs ...... 20

2.5 Mammalian expression and purification of Fabs and antibodies ...... 22

2.6 Papain digestion of antibodies ...... 24

2.7 Fab UGGT affinity assay ...... 24

2.8 UGGT glucosylation assay ...... 24

Chapter 3 Results ...... 25

3.1 Expression, purification and crystallization of UGGT ...... 25

3.1.1 Expression of UGGT ...... 25

3.1.2 Purification of UGGT ...... 25

3.1.3 Proteolysis of UGGT ...... 25

3.1.4 Crystallization of UGGT ...... 26

3.2 Preparation of Fabs as a tool for crystallization ...... 28

3.2.1 Bacterial expression and purification of Fabs ...... 28

3.2.2 Mammalian expression and purification of antibodies ...... 31

3.2.3 Papain digestion of antibodies ...... 31

3.2.4 Mammalian expression and purification of Fabs ...... 31

3.2.5 Fab UGGT affinity assay ...... 37

3.2.6 Co-crystallization of UGGT with Fabs ...... 37

3.3 UGGT enzyme assay ...... 37

3.3.1 Preparation of UGGT acceptor substrates ...... 37

3.3.2 UGGT glucosylation assay ...... 43

Chapter 4 Discussion ...... 45

4.1 Expression, purification and crystallization of UGGT ...... 45

4.1.1 Mammalian expression of UGGT ...... 45

4.1.2 Proteolysis of UGGT ...... 45

4.1.3 Crystallization of UGGT ...... 46

4.2 Generation of Fabs ...... 46

4.2.1 Bacterial expression of Fabs ...... 47

4.2.2 Mammalian expression of Fabs/antibodies ...... 47 v

4.2.3 Fab UGGT affinity assay ...... 48

4.3 Design of RiboB constructs ...... 49

4.4 Preparing M9 glycan containing protein ...... 50

4.5 UGGT glucosylation assay ...... 51

Chapter 5 Future Directions ...... 53

5.1 Fragmentation of UGGT ...... 53

5.2 Fabs as a tool for characterization ...... 53

5.3 Preparation of Man9 containing substrates ...... 54

5.4 RiboB substrates ...... 54

5.5 UGGT Sep15 interaction ...... 55

Conclusions ...... 56

References ...... 57

List of Tables

Table 1. Some UGGT acceptor substrates reported in the literature ...... 13

Table 2. Complementarity-determining regions of the hUUGT1 phage display Fabs ...... 22

Table 3. Summary of yields and solubility of Fabs/antibodies ...... 36

Table 4. Summary of Fab binding affinity to hUGGT1...... 40

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List of Figures

Figure 1. The N-glycans ...... 2

Figure 2. The calnexin/calreticulin (CNX/CRT) cycle ...... 6

Figure 3. Predicted UGGT domain structure and the X-ray structure of the Trx3 domain ...... 10

Figure 4. The vector map of PBPA expressing hUGGT1 ...... 19

Figure 5. The vector map of RH2.2 for bacterial expression of Fabs ...... 21

Figure 6. The PBH Vector Map ...... 23

Figure 7. Purification of hUGGT1 ...... 26

Figure 8. Proteolysis of hUGGT1 ...... 27

Figure 9. Purification of FabF1 by ion exchange chromatography...... 29

Figure 10. Purification of 6 Fabs from bacterial lysate by ion exchange chromatography ...... 30

Figure 11. Purification of 3 selected antibodies by ion exchange chromatography...... 32

Figure 12. Papain digestion of antibody F7...... 33

Figure 13. Purification of mammalian cell expressed Fabs...... 34

Figure 14. Purification of 6 mammalian expressed Fabs by ion exchange chromatography ...... 35

Figure 15. Fab binding to hUGGT1 ...... 38

Figure 16. Purification of RB constructs and enterokinase cleavage ...... 42

Figure 17. UGGT glucosylation assay...... 44

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List of Abbreviations

Ab antibody

BSA bovine serum albumin

CD circular dichroism

CNX calnexin

CRT calreticulin

EDTA ethylenediamine tetraacetic acid

EK enterokinase

ELISA enzyme linked immunosorbent assay

ER endoplasmic reticulum

Fab fragment antigen-binding

Fc fragment crystallizable

GII glucosidase II

GnTI N-acetylglucosaminyltransferase I hUGGT1 human UDP-glucose: glycoprotein glucosyltransferase 1

LB Lysogeny broth

M9 Man9GlcNAc2

MHC major histocompatibility complex

MRH mannose-6-phosphate receptor homology

NMR nuclear magnetic resonance

OST oligosaccharyltransferase

PAGE polyacrylamide gel electrophoresis

PB PiggyBac

PMSF phenylmethylsulfonyl fluoride

RiboB ribonuclease B

TEV tobacco etch virus

UDP uridine diphosphate

UGGT UDP-glucose: glycoprotein glucosyltransferase

x 1

Chapter 1 Introduction 1.1 Protein folding and quality control in the ER

In eukaryotic cells, the endoplasmic reticulum (ER) is the major site of protein folding and quality control for membrane and secreted proteins. Almost one-third of the proteins expressed in a eukaryotic cell are translocated into the ER (1). The ER provides an oxidative environment that supports disulfide bond formation during protein folding (2). The lumen of the ER contains molecular chaperones and folding enzymes that assist folding and prevent aggregation. Newly synthesized secreted and membrane proteins are transported out of the ER upon acquiring their native tertiary and quaternary structures (3–5). The stringent distinction between properly folded proteins and misfolded proteins ensures the fidelity and functionality of the expressed proteins.

1.1.1 N-linked glycosylation

N-linked glycosylation is a co-translational modification event that occurs on the luminal side of the rough ER in eukaryotic cells. Ribosomes attached on the cytosolic face of the rough ER synthesize peptides that thread through the translocon into the lumen. Nascent polypeptides emerging from translocon are scanned by oligosaccharyltransferase (OST), a membrane-bound multi-subunit enzyme (6). In most eukaryotic cells, OST transfers a pre-assembled, branched oligosaccharide (Glc3Man9GlcNAc2) (Figure 1) from a dolichol pyrophosphate anchor to the asparagine residue in the consensus sequence Asn-X-Ser/Thr (X cannot be Pro) on the nascent polypeptide chain (7–10). The pre-assembled, branched oligosaccharide (Glc3Man9GlcNAc2) transferred by OST is known as the N-glycan or N-linked glycan since the oligosaccharide is transferred to the nitrogen atom of the Asn side chain. The process of transferring N-glycans to nascent polypeptides is known as N-linked glycosylation.

Arm A Arm B α-1,2 Arm C

α-1,3

α-1,3 Glc α-1,2 α-1,2 α-1,2

α-1,2 α-1,3 α-1,6 Man

α-1,3 α-1,6 GlcNAc β-1,4

β-1,4

-Asn-X-Ser/Thr- A

GI & GII α-ManI GnTI α-ManII GnTII β4GalT SiaT

Asn Asn Asn Asn Asn Asn Asn Asn

Glc kifunensine Fut8 GnTIII GnTV Man GlcNAc Gal Sia Asn Asn Asn Fuc B

Figure 1. The N-glycans

(A) The structure of the full-length N-glycan in most eukaryotic cells transferred to the Asn residue of the consensus sequence Asn-X-Ser/Thr (X ≠ Pro).

(B) N-glycan processing in mammalian cells. The enzymes and the intermediate structures are shown.

1.1.2 Glucose processing

N-glycans are processed almost immediately after transfer of Glc3Man9GlcNAc2 to the glycoprotein by OST. As shown in Figure 1B, the initial processing involves hydrolysis of the outer glucose residues from the Glc3Man9GlcNAc2 glycan. The outermost α-1, 2 linked glucose residue is cleaved by an integral membrane enzyme, glucosidase I to generate Glc2Man9GlcNAc2 (11). The remaining inner two α-1, 3 linked glucose residues are sequentially removed by a luminal soluble heterodimeric enzyme, glucosidase II (12). Proteins with the monoglucosylated glycan, GlcMan9GlcNAc2, are recognized by the calnexin and calreticulin system that promotes folding (13).

1.1.3 Glucosidase II

Glucosidase II (GII) is a soluble enzyme comprised of a catalytic α subunit and a non-catalytic β subunit (14). The purified enzyme was found in a α2β2 state (12, 15, 16). The enzyme cleaves glucosidic α-1,3 and α-1,4 bonds with a pH optimum between 6.0 and 7.5 and is often assayed with p-nitrophenyl-alpha-D-glucopyranoside (12, 15).

The alpha subunit is approximately 100 kDa and is homologous with several other glucosidases (14). The catalytic domain of the alpha subunit contains a consensus sequence found in other glycohydrolase family 31 domains that hydrolyzes the glucose residue (17, 18). Sequence analysis showed that the alpha subunit lacks any known ER-retention motifs (14).

The beta subunit is approximately 60 kDa and contains a C-terminal HDEL ER retention signal, two EF-hand motifs that bind to Ca2+, a long stretch of consecutive Glu residues and a lectin domain homologous to the mannose-6-phosphate receptor homology (MRH) domain (14, 19).

The beta subunit has been proposed to be responsible for localization of GII in the ER (14). Removal of the HDEL ER retention signal from the beta subunit was shown to increase secretion of the alpha subunit (20). In the absence of the beta subunit, the ER levels of the alpha subunit could be restored by the addition of the ER retention signal to the alpha subunit (21).

It has been proposed that the beta subunit is responsible for N-glycan recognition by GII. It was reported that the GIIβ MRH domain bound to high-mannose-type glycans and when binding was abolished by mutagenesis the rate of glucose trimming of N-glycans by glucosidase II was

4 significantly decreased (22). Experiments showed that glucose trimming rates of N-glycans by glucosidase II were reduced upon hydrolysis of mannose residues from the B and C arms in the N-glycan (as shown in Figure 1), suggesting that the MRH domain may bind to mannose residues in the B and C arms (21, 23, 24).

A model has been proposed in which binding of the MRH domain to mannose residues on the B and C arms of the N-glycan increases the local substrate concentration for the alpha subunit (21). The 3D structure of GIIβ MRH domain was solved by NMR spectroscopy and amino acids in the binding pocket were proposed to bind to the C arm of the N glycan (25).

1.1.4 The calnexin/calreticulin cycle

The calnexin/calreticulin (CNX/CRT) cycle (Figure 2) plays a key role in N-glycan-dependent ER quality control (26). After removal of the two outermost glucose residues by GI and GII, monoglucosylated glycoproteins with the GlcMan9GlcNAc2 glycan are recognized by CNX/CRT (27). Binding of monoglucosylated N-glycan by CNX/CRT retains the glycoprotein in the ER and recruits ERp57, a protein disulfide isomerase, to assist folding (28). The removal of the last glucose residue by GII releases the glycoprotein from CNX/CRT. The glycoprotein is re- glucosylated by UDP-glucose: glycoprotein glucosyltransferase (UGGT) if it displays a non- native conformation. The re-glucosylation by UGGT leads to re-association with CNX/CRT. The cycle of de-glucosylation and re-glucosylation by the opposing activities of GII and UGGT continues until the glycoprotein acquires its native fold or is targeted for degradation by the ER associated degradation pathway.

1.1.4.1 Structures of CNX and CRT

CNX is a 90 kDa type I ER membrane protein (29) and CRT is its 60 kDa soluble paralog with a C-terminal ER retention signal (30). The X-ray crystal structure of the luminal portion of CNX revealed a globular β-sandwich legume-lectin-like domain and a P domain that consists of a non- globular proline-rich hairpin fold (31). Legume lectins form one of the largest carbohydrate- binding protein families and bind to either glucose/mannose or galactose with activity depending on presence of Ca2+ (32, 33). The X-ray crystal structure of the CRT lectin domain revealed a concave β-sheet structure responsible for saccharide binding (34). The P domain is highly conserved and forms a protrusion or arm extending from the core protein (31, 35). The NMR

5 structure of the CRT P domain was solved and the protrusion was similar but shorter compared to that of CNX (35).

The crystal structure and mutagenesis studies demonstrated that the carbohydrate-binding site is located in a cleft on the surface of the globular domain (31, 36). The site recognizes not only the terminal glucose residue but also three mannose residues in the A arm (Figure 1) (27, 37). Recognition of the terminal glucose residue is achieved by coordination with six critical residues in the binding site (38–40).

Association of CNX and CRT with the ERp57 thiol oxidoreductase is through the tip of the P domain (36, 41, 42). Impairment in ERp57 binding was observed with mutants lacking the most distal region of the P domain (36).

1.1.4.2 Functions of CNX and CRT

Newly synthesized glycoproteins may interact with one of CNX/CRT or bind to both either simultaneously or sequentially (43–45). The initial interaction can be co-translational and can be maintained during subsequent steps (46). Release from CNX/CRT and export out of the ER are often coupled (47–50).

Studies using glucosidase inhibitors showed that the folding and assembly of many, but not all, glycoproteins was affected by CNX and CRT (51, 52). In CRT-deficient cells, rapid export of non-native glycoproteins out of the ER, and increased degradation of non-native glycoproteins were observed, suggesting that CNX/CRT stabilizes non-native species and retains them in the ER (53–57).

G3M9 G1M9 GII Exit ER

GI GII CNX/CRT folded G1M9 protein

misfolded protein M9 UGGT + UDP- GII

Figure 2. The calnexin/calreticulin (CNX/CRT) cycle

Nascent polypeptides entering the ER are N-glycosylated with the Glc3Man9GlcNAc2 (G3M9) oligosaccharide. Immediately after transfer, the two outermost glucose residues are removed by the sequential action of glucosidase I and glucosidase II (GI and GII). The remaining monoglucosylated (G1M9) protein is recognized by the CNX/CRT chaperone system which retains the protein and recruits folding enzymes to assist folding. Upon removal of the last glucose residue by GII, the protein is released from CNX/CRT. Properly folded protein proceeds along the secretion pathway. UGGT recognizes misfolded glycoproteins and adds back the glucose residue to the M9 structure, reintroducing the protein to the CNX/CRT cycle.

1.1.5 UDP-glucose glycoprotein glucosyltransferase

1.1.5.1 Discovery of UGGT glucosylation activity

The glucosylation activity of UGGT was discovered by Parodi and his colleagues (58). They incubated microsomes with UDP-[14C]Glc and monitored the formation of radioactive

Glc1Man9GlcNac2 on glycoproteins (59). In this study, the glucosylation activity was found to be conserved in mammals, plants, fungi and protozoa (59). In another report, glucosylation activity was located in the lumen of the ER and both soluble and membrane-bound endogenous glycoproteins were found to be glucosylated (60).

1.1.5.2 Biological aspects of UGGT

UGGT is present in most eukaryotic cells except organisms that transfer aberrantly short glycans (i.e. Giardia lamblia and Plasmodium falciparum) (61). Up-regulation of UGGT occurs under conditions of ER stress such as elevated temperature or the addition of dithiothreitol or tunicamycin (62). UGGT knockout triggers the unfolded protein response, leading to the up- regulation of several ER chaperones (i.e. BiP) and folding enzymes (i.e. protein disulfide isomerases) (62). Plants (i.e. Arabidopsis thaliana) lacking UGGT expression exhibit delayed growth and are susceptible to stressors such as pathogens, heat and salt (63). Schizosaccharomyces pombe and Trypanosoma cruzi cells lacking UGGT expression have impaired growth under ER stress conditions (64, 65). The two UGGT homologues in Caenorhabditis elegans are reported to have distinct biological functions (66). The CeUGGT1 possesses the canonical UGGT activity and RNAi-mediated depletion of CeUGGT1 leads to a reduced lifespan (66). The inactive CeUGGT2 is required for the viability of mutants lacking calnexin and calreticulin and is significant in relieving ER stress in the absence of the unfolded protein response signaling pathway (66). The yeast Saccharomyces cerevisiae has only one gene coding for UGGT and the protein is reported to be inactive (67). Knockout of UGGT in mice is embryonically lethal (68).

1.1.5.3 UGGT function

UGGT is a unique protein that displays the activity of a glycosyltransferase and the specificity of a classic chaperone. It transfers a single Glc residue from UDP-Glc to the terminal Man of arm A in N-glycans (Figure 2). Unlike BiP, a chaperone of the Hsp70 family, that associates with

8 nascent peptides entering the ER lumen, UGGT recognizes proteins at late stages of folding, or in a molten globule state, where most of the disulfide bonds in the glycoproteins are already formed (69, 70). The ability of UGGT to distinguish between misfolded and native proteins makes it a gatekeeper that regulates the export of glycoproteins out of the ER. It is worth noting that UGGT re-glucosylation delays secretion but not degradation (71).

UGGT is implicated not only in the folding process of monomeric glycoproteins but also in the formation of multimeric glycoproteins (72). Incompletely formed complexes with correctly folded monomers may still be recognized by UGGT through the exposed hydrophobic protein interphases that are buried in the complete complexes.

It has been suggested that UGGT-dependent monoglucosylation of N-linked glycoproteins promotes substrate solubility in the ER (73). Investigation of the soluble/insoluble distribution of two misfolded α1-antitrypsin variants that are responsible for α1-antitrypsin deficiency diseases indicates that additional substrate solubility is conferred by the action of UGGT.

UGGT also plays a key role in major histocompatibility complex (MHC) class I-mediated antigen presentation. MHC class I antigen presentation is critical for adaptive immune responses. In the absence of UGGT, the formation of the peptide loading complex is not affected but maturation of the MHC class I molecule is delayed, peptide selection is impaired, and the cell surface levels of the MHC class I molecule are reduced (74). It has also demonstrated that UGGT preferentially reglucosylates MHC class I molecules that contain a suboptimal peptide (74).

It has been reported that UGGT may collaborate with a cytosolic AAA-ATPase, p97, to form a checkpoint for native ectodomains with ionizable intramembrane residues (75). This checkpoint prevents Golgi transport of a native ectodomains that pass the luminal quality control scrutiny but display an intramembrane defect. The cytosolic AAA-ATPase p97 is a multifunctional protein that participates in a vast number of supramolecular complexes implicated in ER- associated protein degradation, quality control and vesicular transport (76). This study suggests that UGGT may play an important role in the quality control of transmembrane proteins.

1.1.5.4 UGGT structure

The cDNA of rat UGGT predicts that the protein contains 1527 amino acids with an N-terminal signal peptide (77). UGGT contains a C-terminal HEEL sequence, a variant of the HDEL ER retention signal found in other ER luminal proteins. Four N-X-S/T sites (N269, N536, N1015 and N1228) were found in the UGGT sequence from Rattus norvegicus while three N-X-S/T sites (N269, N536 and N1228) were found in the UGGT sequences from Sus scrofa, Bos taurus and Homo sapiens (78). Only one site (N269) was reported to be glycosylated across the four species (78).

UGGT is composed of an N-terminal region and a C-terminal catalytic domain based on bioinformatic analysis and biochemical studies (Figure 3). Studies of Rattus norvegicus, Schizosaccharomyces pombe and Drosophila melanogaster UGGTs suggested that the linkage between the two regions is extremely sensitive to proteolysis (79). The C-terminal region displays high sequence similarity to members of CAZy (carbohydrate-active enzymes) glycosyltransferase family 8 that utilize nucleotide diphospho-sugar donors (79) and contain a “DXD” motif critical for enzymatic activity (77). Several studies suggested that the N-terminal region of UGGT is involved in substrate binding and serves as a folding sensor that distinguishes non-native conformations from native conformations (80–82). Studies using a synthetic tripartite oligosaccharide probe suggested that the C-terminal domain of UGGT participates in glycan recognition and recognition of the protein-carbohydrate linkage (83).

Zhu et al. predicted that the N-terminal region of UGGT harbors three tandem thioredoxin-like (Trx) domains as well as a β-strand-rich region (β-domain) (Figure 3A) and they solved the crystal structure of the third Trx domain (84). The crystal structure of the third Trx domain displayed a conventional Trx-like fold, a five-stranded β-sheet surrounded by six α-helices (84). The sixth helix was unstable and became completely disordered when a detergent was present, leading to exposure of hydrophobic patches that may serve as substrate binding sites (Figure 3B) (84). This is consistent with the reported decrease in the α-helical content of UGGT upon increasing substrate concentration (85).

Thioredoxin-like domains

catalytic Trx1 Trx2 Trx3 β-domain domain

N C 28 168 379 467 624 671 831 940 1140 1199 1476 1505

A the N-terminal region

Figure 3. Predicted UGGT domain structure and the X-ray structure of the Trx3 domain

(A) The predicted domain structure of Chaetomium thermophilum UGGT (84). Chaetomium thermophilum is a thermophilic fungus that survives at temperatures of up to 60 °C.

(B) Overlay of the X-ray crystal structures of the Trx3 domain solved in the presence (PDB: 3WZS, shown in blue) and absence of detergent (PDB: 3WZT, shown in yellow). The sixth helix (circled in red) becomes disordered when a detergent is present.

1.1.5.4.1 UGGT Sep15 interaction

A 15-kDa selenoprotein Sep15 has been proposed to serve as a structural extension of UGGT with complementary function (84). Studies showed that Sep15 formed a 1:1 tight complex with UGGT with an apparent Kd of 20 nM (86). The association of Sep15 with UGGT has been shown to increase the enzymatic activity of UGGT (87). The NMR structure revealed that Sep15 is an oxidoreductase that possesses a single thioredoxin-like domain (88). It is reported that Sep15 assists in oxidative folding of glycoproteins and is implicated in the unfolded protein response (89). As Sep15 is implicated in protein disulfide bond formation, the interaction of Sep15 with UGGT is proposed to contribute to ER quality control (84, 88, 90–92).

1.1.5.5 Endogenous proteins recognized by UGGT

Only a few of the endogenous proteins recognized by UGGT have been studied so far (Table 1).

Cruzipain, an abundant Trypanosoma cruzi lysosomal protease was the first identified endogenous protein recognized by UGGT (93). The N-glycan transferred from dolichol-P-P in trypanosomatids lacks Glc residues so that UGGT-mediated glucosylation is required before glycoproteins can associate with calreticulin (Trypanosoma cruzi lacks calnexin). Evidence showed that cruzipain interacts with calreticulin only after nearly all disulfide bridges have been formed, indicating that UGGT operates during the last folding stages in vivo (93). Reduced forms of cruzipain were mainly recognized by BiP, suggesting that different ER folding systems cooperate in the quality control mechanism from highly unstructured polypeptides recognized by BiP to late-folding, molten globule-like conformations recognized by UGGT- calnexin/calreticulin (69).

Prosaposin, a lysosomal protease rich in disulfide bridges, was identified as a prominent endogenous UGGT substrate (94). It was labeled in a [35S]Met/Cys pulse-chase labeling experiment and remained bound to GST-calreticulin after a 2-h chase (94). The hydropathy profiles of the amino acids C-terminal to the N-glycans of prosaposin revealed an oscillating hydrophobicity profile associated with UGGT recognition (94). This result is consistent with the finding that UGGT preferentially re-glucosylates glycopeptides possessing dual hydrophobic patches located C-terminal to the N-glycan (95).

1.1.5.6 Substrate studies in vitro

Ribonuclease B (RiboB), a small protein that has a single exposed N-glycan site (96), is widely used for UGGT substrate studies in vitro. Subtilisin cleavage of RiboB generates an S-peptide and an S-protein with an exposed hydrophobic inner core (97, 98). The efficiency of cleavage can be improved by using an engineered enterokinase site (99). It was reported that UGGT had a greater activity towards reduced and unfolded RiboB than native RiboB (77). The screen of UGGT activity towards a series of RiboB constructs (i.e. native, subtilisin-treated and alkylated) further supports the idea that UGGT preferentially recognizes partially structured nonnative forms (100). Denatured EndoH-treated RiboB (only the Asn-linked GlcNAc unit remains) was found to be a potent inhibitor of UGGT glucosylation while denatured RiboA (non glycosylated) did not inhibit the activity, suggesting that UGGT recognizes the Asn-linked GlcNAc unit, a moiety expected to be more accessible when glycoproteins are in molten globule-like conformation (81, 82). Studies using a RiboB S-protein associated with a denatured full-length RiboB through the S-peptide of the denatured protein revealed that only glycans linked to the misfolded domain were glucosylated, suggesting that UGGT only recognizes local folding defects and reglucosylates glycans attached to a misfolded domain (101). Another study using a variety of RiboB mutants further supported the suggestion that only glycans within the misfolded domain were glucosylated (102).

Studies using a single domain protein, β-glucanase, also provided insight into the spatial relationship between misfolding and the N-glycan glucosylated by UGGT (103). The studies revealed that both N-glycans (N165 and N325) within the domain were glucosylated by UGGT upon introducing a mutation (F280S) located in close proximity to the N165 site (103).

Studies using fragments of chymotrypsin inhibitor 2 suggested that UGGT recognizes a molten globule-like, non-native conformation with exposed hydrophobic patches (104). Analysis of polypeptide fragments of chymotrypsin inhibitor 2 showed that they lacked side chain packing and folding cooperativity, characteristics of molten globules (105). It was shown that UGGT preferentially recognized these fragments (104).

Yeast acid phosphatase is also used as a UGGT substrate for in vitro studies (77). It contains 12 potential sites for N-glycosylation and loses its activity at neutral pH, suggesting that the protein may unfold at neutral pH (106). In the presence of 0.5 µM acid phosphatase, a KM value of 44

µM was determined for the donor substrate, UDP-glucose (77). The KM value for the acceptor substrate was not obtained as the rate of glucosylation dropped when the acid phosphatase concentration was increased to 2 µM and higher, likely due to aggregation of acid phosphatase (77).

Synthetic substrates are also used for UGGT substrate studies. Inhibition experiments with methotrexate conjugated glycans revealed that UGGT recognizes the core pentasaccharide

Man3GlcNAc2 structure (107). Circular dichroism (CD) spectrum studies using fluorophore- conjugated glycans revealed that the α-helical content of UGGT diminished as the acceptor substrate concentration increased (85), an observation consistent with the disordering of the α- helix observed in the presence of detergent revealed by the crystal structure of the Trx-3 domain

(Figure 3B) (84). A Kd value of 4 µM was estimated based on CD spectral changes (85). A KM value for UDP-Glc was determined as 69 µM in the glucosyltransferase reaction (85).

Table 1. Some UGGT acceptor substrates reported in the literature

Substrates Summary of findings

UGGT recognizes incompletely formed complexes with correctly folded soybean agglutinin monomers (72)

α1-antitrypsin UGGT-dependent monoglucosylation promotes substrate solubility in the variants ER (73)

MHC class I UGGT preferentially reglucosylates MHC class I molecules that contain a complex suboptimal bound peptide (74)

UGGT operates at late stages of folding (93)

cruzipain Different ER folding systems cooperate in the quality control mechanism (69)

prosaposin UGGT recognizes hydrophobic patches with oscillating hydrophobicity

profiles (94)

UGGT preferentially recognizes partially structured nonnative forms

(77, 100) RiboB UGGT recognizes the exposed innermost GlcNAc unit (81, 82)

UGGT recognizes local folding defects (101, 102)

chymotrypsin UGGT preferentially recognizes molten globule-like conformers that lack inhibitor 2 side chain packing and folding cooperativity (104)

UGGT recognizes the core pentasaccharide, Man3GlcNAc2 (107) synthetic substrates The acceptor substrate causes a decrease in the α-helical content of UGGT (85)

β-glucanase UGGT can modify glycans distant from the misfolded domain (103)

acid phosphatase A KM value of 44 µM is determined for UDP-glucose (77)

1.1.5.7 Preparation of Man9GlcNAc2 containing substrates

The study of UGGT activity depends on substrates that are both misfolded and possess

Man9GlcNAc2 N-glycan(s). Several approaches have been reported to facilitate the generation of M9 glycan containing substrates.

Thomas and his colleagues constructed a special yeast strain, DT111, to produce Man9GlcNAc2 containing glycoproteins (77). The DT111 strain has triple deletions in the genes MNN1, OCH1 and MNS1. In yeast, additional mannose residues are attached to the terminal mannose units of the N-glycan by a Golgi α-1,3-mannosyltransferase encoded by the MNN1 gene (108) or an α- 1,6-mannosyltransferase encoded by the OCH1 gene (109); these enzymes lead to the production of hyperglycosylated yeast proteins. Deletions in the MNN1 and OCH1 genes were reported to produce Man8GlcNAc2 containing glycoproteins (109). The trimming of the M9 glycan to the

M8 glycan is catalyzed by an ER α-mannosidase which is the product of the MNS1 gene (110). The DT111 strain, lacking three genes (mns1, mnn1 and och1), has been used in several studies to produce Man9GlcNAc2 containing substrates for the study of UGGT (77, 95, 103).

Ito and his colleagues utilized chemical approaches to synthesize M9 glycans (84, 85, 107, 111, 112). Chemical synthesis of M9 glycans is technically demanding. The major difficulty in synthesis of M9 glycans is the stereoselective formation of the glycosidic linkages connecting sugar residues. To selectively synthesize the stereotypical glycosidic linkages, they adopted an intramolecular aglycan delivery technique that utilized an aglyconic group to cross-link sugar residues and yield stereo-specificity by quenching with nucleophiles (113).This method builds the M9 glycan on the synthetic compounds such as methotrexate or BODIPY instead of endogenous proteins.

Synthesis of M9 glycan containing protein can be achieved by chemical synthesis of an N-glycan oxazoline and subsequent enzymatic transglycosylation to transfer it to the protein (114). The transglycosylation reaction requires a glycosynthase mutant, EndoA-N171A, that transfers the M9 glycan to an EndoA (or EndoH) cleaved glycoprotein (114).

Kifunensine, a potent inhibitor of ER α-mannosidase I, can also be used to produce M9 glycan containing glycoproteins in a mammalian cell expression system (115). Kifunensine is a derivative of 1-amino mannojirimycin. It was reported to be a weak inhibitor of jack bean α- -4 mannosidase (IC50 of 1.2 × 10 M) (116) but a very potent inhibitor of the plant mannosidase I -8 (IC50 of 2-5 × 10 M) (115). Kifunensine showed no effect on mannosidase II or other aryl-α- mannosidases (115). Inhibition of α-mannosidase I by kifunensine is used to produce glycoproteins with the M9 structure that can be cleaved by EndoH to remove the sugar and improve crystallizability (117).

1.2 Phage display Fabs

Fabs have been widely used in protein crystallization (118), as binding of Fabs to antigens provides additional opportunities for crystallization. In this study, we used phage display Fabs as a tool for the crystallization of UGGT. Phage-displayed Fab constructs against UGGT1 were obtained from Dr. Sachdev Sidhu (collaboration with the Rini lab). To select phage display antibodies against hUGGT1, purified hUGGT1 was sent to Dr. Sachdev Sidhu by Jesse Hackett.

The phage display antibody libraries were built on a human IgG1 framework with diversified complementarity-determining regions (CDRs) (119). Nine amino acids (Y, S, G, A, F, W, H, P and V) were synthetically incorporated into four of the six CDRs (L3, H1, H2 and H3) (120).

Fabs are selected from the synthetic phage display antibody libraries by affinity to antigens (119). The Fab cDNA is fused to the phage coat protein p3 so that expressed Fabs are displayed on the surface of phage particles. Antigens are immobilized on a surface presented to phage particles with displayed Fabs. High-affinity binding Fabs are selected and amplified while low- affinity binding Fabs are washed off. After several cycles, high-affinity Fabs are sequenced from the phage DNA.

1.3 Thesis objectives

Although the X-ray crystal structure of the Trx3 domain has been solved (84), the 3-dimensional structure of full-length UGGT remains unknown. It is known that UGGT recognizes M9 glycan containing glycoproteins that are in the molten globule state (section 1.1.5), but which residues/motifs/domains/domain-domain interactions are implicated in the UGGT substrate interaction and how they mediate substrate recognition are not fully understood. Exploring how UGGT distinguishes glycoproteins in a molten globule state from native glycoproteins and how UGGT recognizes a variety of glycoproteins in a molten globule state will greatly enhance our understanding of the ER quality control system.

We used structural and biochemical approaches to characterize UGGT and as part of the effort, we expressed Fabs as a tool for the crystallization of UGGT. To ensure that the expressed and purified UGGT was enzymatically active, we performed a radioactive UDP-Glc-based assay. This assay will provide the basis for a detailed characterization of UGGT substrate interactions in the future, including mutagenesis and truncation studies.

Chapter 2 Materials and Methods

Chapter 2 describes the methods used to structurally and biochemically characterize human UGGT1. Jesse Hackett provided the original construct of hUGGT1 (residues 43-1554). Dr. Sachdev Sidhu (University of Toronto) provided the phage display Fab constructs against hUGGT1 (developed in collaboration with the Rini lab). Kristina Han provided purified Her2 antibodies expressed from the N-acetylglucosaminyltransferase I (GnTI)-deficient HEK 293S cell line with (Her2-kif) and without kifunensine treatment (Her2-S).

2.1 Chemicals, plasmids and cell culture

IgG Sepharose 6 Fast Flow and rProtein A Sepharose Fast Flow affinity resins were obtained from GE Healthcare. Enterokinase from bovine intestine and papain from papaya latex was supplied by Sigma-Aldrich Canada Ltd. (Oakville, ON). Kifunensine was obtained from GlycoSyn. DNA manipulations were performed using In-Fusion® HD Cloning Kits including the In-Fusion Enzyme and Stellar™ Competent Cells (Clontech Laboratories, Inc.). FreeStyle™ 293-F cells (Life Technologies) and N-acetylglucosaminyltransferase I (GnTI)-deficient 293S cells (121) were used for mammalian expression. Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) and FreeStyle™ 293 Expression Medium were obtained from Life Technologies.

2.2 Plasmid constructions

The nucleotide sequence of Ribonuclease B (RiboB) was synthesized by GenScript (Piscataway, NJ, USA). The DDDDK enterokinase recognition sequence was inserted between RiboB S- peptide and S-protein to make the RBEK construct. The RiboB sequences were cloned into a Protein A-tagged version of the PiggyBac (PB) transposon-based mammalian expression vector.

Fab sequences from phage display constructs were re-cloned into a non-tagged version of the PB mammalian expression vector for mammalian expression of Fabs and antibodies. To express Fabs, MreI and NotI restriction sites were used to clone the Fab light chain and heavy chain. To express antibodies, MreI and NotI restriction sites were used to clone the antibody light chain

18 and MreI and ApaI restriction sites were used to clone the antibody heavy chain that is in frame with the human IgG1 hinge region and Fc domain.

2.3 Mammalian expression and purification of hUGGT1

The expression of hUGGT1 followed the PB expression protocol (122). The Protein A-tagged hUGGT1 plasmid (Figure 4) along with helper plasmids were transfected into 293S GnTI- cells. Transiently expressed PiggyBac transposase mediates integration of the target transposons into the genome. The integrated 293S GnTI- cells were selected by dual drug resistance under 10 µg/ml puromycin and 5 µg/ml blasticidin. After selection, cells were expanded and grown in DMEM/F12 medium at 37 ºC in a roller bottle. The Protein A-tagged hUGGT1 was expressed under 1 mg/L doxycycline induction. 1mg/L aprotinin was supplemented in the medium to inhibit proteolytic degradation. Medium was harvested every 3 to 5 d when glucose levels dropped to 1 mM.

The harvested medium containing the secreted hUGGT1 was centrifuged at 4000 g for 20 min at 277 K to remove cell debris. The supernatant was concentrated 20-fold on a Prep/Scale Ultrafiltration Module (Millipore). The concentrated medium was incubated with IgG Sepharose 6 Fast Flow affinity resin at 277 K overnight. The resin was washed with 50 column volumes of 10 mM Tris-HCl, pH 7.5 and 150 mM NaCl. Tobacco Etch Virus (TEV) protease was added to the resin at a final concentration of 0.1 mg/ml. The target protein hUGGT1 was released from the resin upon TEV protease cleavage of the Protein A tag. The protein was then dialyzed in the low- salt buffer containing 20mM HEPES pH 7, 50 mM NaCl and purified by ion exchange chromatography on a HiTrap Q HP column (GE Healthcare) with a salt gradient from 50 mM to 500 mM. For the purpose of crystallization, hUGGT1 was concentrated to a volume of 0.5 ml using an Amicon Ultra centrifugation filter device with a 30 kDa cutoff (Millipore). The concentrated protein was further purified via size exclusion chromatography on a Superdex 200 10/300 GL gel filtration column (GE Healthcare).

Figure 4. The vector map of PBPA expressing hUGGT1

The map shows the features of the N-terminal Protein A tagged PBPA vector. The green arrow indicates the start codon. The AscI and NotI restriction sites are used for cloning. The N-terminal Protein A tag can be removed by TEV proteases. PB5’ LTR and 3’LTR were used for integration. Detailed features are available in the literature (122).

2.4 Bacterial expression and purification of Fabs

Fabs (Table 2) in the phage display bacterial expression vector, RH2.2 (Figure 5), were transformed into E. coli BL21 (DE3) cells. Starter cultures were grown overnight in 50 ml LB broth containing 50 µg/ml ampicillin at 37 ºC in a shaking incubator. The cells were expanded in

1 L LB containing 50 µg/ml ampicillin until the culture density reached an OD600 value of 0.5. Expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. The induced cells were grown at 37 ºC for 4h. The cells were harvested via centrifugation at 5000 x g for 20 min at 4 ºC. The cell pellet was stored at -20 ºC.

To purify the Fabs from the bacterial lysate, frozen cell pellet was thawed on ice and re- suspended in 50 ml cold lysis buffer (20 mM Tris pH 7.5, 50 mM NaCl, 1 mM PMSF and 1 mg/ml lysozyme). The supernatant was isolated by centrifugation at 15,000 g for 20 min at 4 ºC.

For periplasmic extraction, the pellet was warmed to room temperature and re-suspended in 50 ml of 0.5 M sucrose, 20 mM Tris pH 8, and 1 mM EDTA per gram of cells. Cells were centrifuged at 3,500 g for 10 min at 4 ºC. The pellet was re-suspended in 25 ml of cold distilled water per gram of cells and centrifuged at 3,500 g for 10 min at 4 ºC. The supernatant was collected.

Protein A Sepharose Fast Flow affinity resin was added to the supernatant and the mixture was incubated at 4 ºC overnight. The resin was washed with 10 mM Tris-HCl pH 7.5 and 150 mM NaCl. Fabs were eluted with 100 mM glycine pH 2.5 and neutralized with 0.5 M Tris pH 8, 250 mM NaCl. The protein was dialyzed in the buffer containing 20 mM NaOAc pH5, 50 mM NaCl and purified by ion exchange chromatography on a HiTrap SP HP column (GE Healthcare) with a salt gradient from 50 mM to 500 mM.

Figure 5. The vector map of RH2.2 for bacterial expression of Fabs

The map shows the phage display bacterial expression vector RH2.2 generated by Dr. Sachdev Sidhu (University of Toronto) for expressing the Fab heavy chain and light chain in bacteria (120). The Fabs are expressed as individual heavy chains and light chains. The heavy chain has a C-terminal His tag while the light chain has a C-terminal FLAG tag.

Table 2. Complementarity-determining regions of the hUUGT1 phage display Fabs

Fabs L1 L2 L3 H1 H2 H3

F1 SVSSA SASSLYS GYGYASGLI LSSSSM YISSYSGSTY SVSWHVWFSSSYYFYAM

F3 SVSSA SASSLYS GWALI LSYYYI SISPYYSSTS YFYAM

E4 SVSSA SASSLYS SYFVGPF LSSYSM SISSYSSYTS SWWYYSGI

E5 SVSSA SASSLYS HVSLI LSSYSM SISSSYGYTS GSGYFWGAI

F7 SVSSA SASSLYS GYAPI ISYYYM SISPYYGYTY GYVYGL

F11 SVSSA SASSLYS GYAPI LSSYYI YISSSSGSTY PYYAM

E12 SVSSA SASSLYS SGYFLI LSYYYI YISSYYGYTS SVWYAGAL

L1, L2 and L3 are complementarity determining regions on the Fab light chain while H1, H2 and H3 are complementarity determining regions on the Fab heavy chain.

2.5 Mammalian expression and purification of Fabs and antibodies

A non-tagged version of PB vector was used to express Fabs and antibodies in mammalian cells (Figure 6). The stably-transfected 293F cell lines expressing Fabs and antibodies were generated in the same way as described for hUGGT1. Cells were grown in FreeStyle 293 expression media at 37 ºC in a shake flask. 1 mg/L doxycycline was used to induce protein expression and 1mg/L aprotinin was added to prevent proteolysis. Medium was harvested once every week.

The medium with secreted Fab or antibody was centrifuged to remove cells and then concentrated 20-fold. Protein A Sepharose Fast Flow affinity resin was added to the concentrated medium and the mixture was incubated at 4 ºC overnight. The resin was washed with 10 mM Tris-HCl, pH 7.5 and 150 mM NaCl. Fabs and antibodies were eluted with 100 mM glycine pH 2.5 and neutralized with 0.5 M Tris pH 8, 250 mM NaCl. The protein was dialyzed in a buffer

23 containing 20 mM NaOAc pH5, 50 mM NaCl and purified via ion exchange chromatography on a HiTrap SP HP column (GE Healthcare) with a salt gradient from 50 mM to 500 mM.

Figure 6. The PBH Vector Map

The map shows the PB vector used for mammalian expression of Fabs or antibodies. To express Fabs, Fab heavy chain and light chain sequences are cloned using the MreI and NotI restriction sites. To express full-length antibodies, light chain sequences are cloned using MreI and NotI restriction sites while heavy chain sequences are cloned using MreI and ApaI restriction sites so that the Fab heavy chain sequence is in frame with an intron-containing human IgG1 Fc sequence.

2.6 Papain digestion of antibodies

In an attempt to generate Fabs from antibodies, papain digestion was performed on purified antibodies. 2 mg/ml papain was activated with 0.5 mM L-cysteine at 4 ºC for 1 hr. The reaction mixture contained 0.6 mg/ml antibody, 30 µg/ml papain, 100 mM HEPES, pH 7 and 2.5 mM EDTA. Samples were taken at time 0, 1, 2, 3 and 5 hr. The reaction was stopped by incubating with 10 µM E64 at room temperature for 15 min.

2.7 Fab UGGT affinity assay

The binding of Fabs to UGGT was analyzed using surface plasmon resonance (Biacore). CM5 sensor chips were used in this assay (GE Healthcare). The carboxymethyl group on the chip was activated by injecting a 70 µL freshly prepared mixture of 50 mM N-hydroxysuccinimide and 0.2 M 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride. The coupling reaction was performed by injecting 50 µg/ml hUGGT1 in 100 mM sodium acetate buffer pH 5. After coupling, the chip was blocked with 1 M ethanolamine. Six Fabs (F1, F3, E4, E5, F7 and F11) were prepared from the solubility limit to a concentration of 156 nM using 2-fold dilutions. Experiments on each Fab were performed once.

2.8 UGGT glucosylation assay

A radioactive glucosylation assay was performed to measure the enzymatic activity of UGGT. Frozen UGGT stock was used for the assay. UDP-[3H]-glucose with a specific activity 11.5 Ci/mmol (Amersham Biosciences) was used.

To prepare alkylated RiboB as a substrate for UGGT, 60 mg/ml commercial bovine pancreatic RiboB (Sigma) was incubated in 8 M urea, 10 mM DTT, 0.1 M Tris pH8. After 30 min incubation at 37 °C, the alkylating agent, 4-vinylpyridine (Sigma), was added to a final concentration of 80 mM and the mixture was incubated for an additional 30 min at 37 °C.

The reaction was initiated by adding substrates to a mixture containing 7 µM UGGT, 174 nM 3 UDP-[ H]-glucose (Sigma), 10 mM CaCl2 and 10 mM HEPES, pH 7. The reaction mixture was incubated at 310 K for 3 d. The mixture was loaded onto Sep-Pak C18 cartridge (Waters,

Milford, MA, USA) and washed with ddH2O. The column was eluted with 70% isopropanol and radioactivity was measured by liquid scintillation counting.

Chapter 3 Results 3.1 Expression, purification and crystallization of UGGT

3.1.1 Expression of UGGT

Human UGGT1 (hUGGT1), the major form of UGGT in humans, was expressed using the PiggyBac (PB) transposon-based mammalian expression system (122). The PB expression system is designed to secrete the target protein into the media, which allows purification of protein from the media. Protein A-tagged hUGGT1 was expressed as a secreted protein on a liter scale. The Protein A tag allows purification by IgG affinity chromatography. The N- acetylglucosaminyltransferase I (GnTI)-deficient HEK 293S cell line (121) was used to express glycoproteins with Man5GlcNAc2 N-glycans. This cell line minimizes heterogeneity due to glycoforms and has been routinely used in our laboratory to express glycoproteins for crystallization.

3.1.2 Purification of UGGT

Protein A-tagged hUGGT1 was purified through IgG affinity chromatography followed by on- column tag cleavage using TEV protease. The protein was further purified through ion-exchange chromatography. hUGGT1 is eluted at a conductivity value of approximately 27 mS/cm (Figure 7B). The average yield of hUGGT1 was approximately 0.7 mg per liter of culture, which is close to the reported yield (1.25 mg/L) of rat UGGT expressed from insect cells (77). Purified hUGGT1 could be concentrated up to 2 mg/ml in 20 mM HEPES, 200 mM NaCl, pH 7 without noticeable aggregation or precipitation.

3.1.3 Proteolysis of UGGT hUGGT1 was found proteolytically clipped upon storage at 4ºC. It was observed that the degradation of hUGGT1 followed a certain pattern as shown in Figure 8A. SDS-PAGE gel results showed the molecular weight bands at 175, 150, 135, 90, 80, 55, 40, 35 and 25 kDa, which is consistent with the literature (77). Upon storage at 4 ºC over a month, the majority of the protein appeared at 80 kDa. Bands at 55, 40 and 35 kDa were observed upon storage at 4 ºC over a year.

A screen for molecules that potentially inhibit proteolysis of UGGT showed that cOmplete™ protease inhibitor cocktail (Roche) was the most effective against proteolysis; EDTA and E64 were the next most effective (Figure 8B). No proteolytic bands below 80 kDa were observed under protease inhibitor cocktail treatment. No proteolytic bands at 55 and 40 kDa were observed under EDTA and E64 treatment. PMSF and aprotinin stocks used in the test may be contaminated with proteases as accelerated proteolysis was observed. The addition of calcium accelerated proteolysis, possibly by activating proteases.

3.1.4 Crystallization of UGGT

Crystallization of UGGT on its own was set up at 1 mg/ml using NeXtal Classics suite (Qiagen). No crystal formation was observed. 6

A B

Figure 7. Purification of hUGGT1

(A)63 Purification of hUGGT1 by IgG affinity chromatography. Lanes are: (1) molecular weight markers, (2) IgG beads, (3-5) wash fractions, (6) PA-hUGGT1 bound IgG beads, (7-9) elution fractions after incubating with TEV protease, (10) IgG beads after elution.

(B) Purification of hUGGT1 by ion exchange chromatography. Conductivity is shown by the A dashed line. Protein is eluted at a conductivity value of approximately 27 mS/cm.

A 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

Figure 8. Proteolysis of hUGGT1

(A) Lanes are: (1) molecular weight markers, (2) precipitate during dialysis, (3) ion exchange chromatography loading sample, (4-8) elution fractions from hUGGT1 ion exchange chromatography, (9) hUGGT1 sample stored at 4 ºC over a month and (10-14) hUGGT1 samples stored at 4 ºC over a year.

(B) Lanes are: (1) molecular weight markers, (2) non-treated UGGT, (3) UGGT with cOmplete™ protease inhibitor cocktail (EDTA free) (Roche), (4) UGGT with 1 mM PMSF, (5) UGGT with 2 µg/ml (0.3 µM) aprotinin, (6) UGGT with 10 µM EDTA, (7) UGGT with 10 mM

CaCl2 and (8) UGGT with 10 µM E64. All treatment groups were tested at room temperature for a week.

3.2 Preparation of Fabs as a tool for crystallization

3.2.1 Bacterial expression and purification of Fabs

To use Fabs as a tool for crystallization, we obtained phage-displayed Fab constructs against UGGT1 from Dr. Sachdev Sidhu (collaboration with the Rini lab). Purified hUGGT1 was sent to Dr. Sachdev Sidhu by Jesse Hackett to select phage display antibodies against hUGGT1. Seven Fabs against hUGGT1 (F1, F3, E4, E5, F7, F11 and E12) were identified in phage display screens (Table 2).

Fab constructs were obtained in the bacterial expression vector, RH2.2, made by Dr. Sachdev Sidhu (Figure 5). The phage-displayed Fabs consist of a C-terminal FLAG-tagged light chain and a C-terminal His-tagged heavy chain. The Fabs used in this system bind to protein-A (123). Fabs were expressed in liter scale in E.coli and purified from the bacterial lysate by Protein A affinity chromatography. Further purification was performed using ion exchange chromatography. E4 did not express. The yields of F1, F3 and F7 were approximately 0.6 mg/L (Table 3). E5, F11 and E12 had limited yields (Table 3).

As Fabs purified from bacterial lysate had low yields and showed non-symmetric peak shape on ion exchange chromatography (Figure 10), periplasmic expression and expression from SHuffle® T7 cells (New England Biolabs) with engineered protein disulfide bond isomerase were attempted for all Fabs. The periplasm is an oxidative environment between the cytoplasmic membrane and the bacterial outer membrane in gram-negative bacteria (i.e. E. coli). The periplasm contains chaperones (124) and other folding enzymes (125) that may improve the folding of Fabs. No significant improvement of Fab expression in terms of yields and purity was observed in both cases.

Figure 9. Purification of FabF1 by ion exchange chromatography.

Lanes are (1) molecular weight markers, (2) ion exchange chromatography loading sample, (3) flowing through and (4-12) elution fractions.

F1 F7

F3 F11

E5 E12

Figure 10. Purification of 6 Fabs from bacterial lysate by ion exchange chromatography

3.2.2 Mammalian expression and purification of antibodies

As mammalian expression was shown to produce high quality antibodies with high yields (126), we attempted to express antibodies and generate Fabs by papain digest. All seven Fabs were cloned into a version of our PB vector designed to express full-length antibodies (Figure 6). Antibodies were purified through Protein A chromatography and ion exchange chromatography as described. Three antibodies (F1, E4 and F7) with relatively high expression levels were selected for optimization with papain digest (Figure 11). The yields of antibodies F1, E4 and F7 were 8 mg/L, 5 mg/L and 2 mg/L (Table 3).

3.2.3 Papain digestion of antibodies

Purified antibodies were digested with papain to generate Fabs. The procedure was optimized to minimize over digestion (Figure 12). The optimal condition in which antibodies were mostly digested into Fabs and Fcs with minimal over-digestion was achieved at neutral pH, room temperature for 5 hours at a papain-to-antibody mass ratio of 1:20.

3.2.4 Mammalian expression and purification of Fabs

Parallel to the expression of antibodies, direct mammalian expression of Fabs was also attempted. Stably-transfected HEK 293F cell lines expressing 6 Fab constructs (F1, F3, E4, E5, F7 and F11) were generated. Fabs were expressed in liter scale and purified by Protein A chromatography followed by ion exchange chromatography as shown in Figure 13 and Figure 14. The yields are summarized in Table 3. Solubility values of Fabs were measured in 20 mM NaOAc pH 5 and 50 mM NaCl (Table 3).

Figure 11. Purification of 3 selected antibodies by ion exchange chromatography.

Gel lanes are (1) molecular weight markers and (2) ion exchange chromatography loading samples. Lanes 3-9, 3-8 and 3-5 in the various panels represent column fractions across the major eluted protein peak.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Ab 75 48 Fc Fab_H 25 Fab_L

non-reducing reducing

Figure 12. Papain digestion of antibody F7.

Lanes are (1) molecular weight markers, (2) non-reduced papain, (3) non-reduced antibodies, (4) digestion mixture at time 0, (5) time = 1 hr, (6) time = 2 hr, (7) time = 3 hr and (8) time = 4 hr. Lanes 9 to 15 are DTT reduced samples of lanes 2-8. Digestion was performed at an antibody-to- papain ratio of 1: 20 at room temperature.

1 2 3 4 5 6 7 8 9

Figure 13. Purification of mammalian cell expressed Fabs.

The 6 lanes (1-6) are Fabs F1, F3, E4, E5, F7 and F11.

F1 E5

F 3 F7

E4 F11

Figure 14. Purification of 6 mammalian expressed Fabs by ion exchange chromatography

Table 3. Summary of yields and solubility of Fabs/antibodies

Yields (mg/L) Solubility (mg/ml) Bacterial Fabs Mammalian antibodies Mammalian Fabs

F1 0.7 8 10 8

F3 0.7 * 6 0.5

E4 * 5 24 0.25

E5 <0.1 * 19 8

F7 0.6 2 21 2

F11 <0.1 * 6 4

E12 <0.1 * * No data

* was not tested in liter-scale production

Solubility was measured using mammalian expressed Fabs

3.2.5 Fab UGGT affinity assay

We chose to use mammalian expressed Fabs to examine the binding of Fabs to UGGT as the mammalian expression system is the natural expression system for human Fabs and it is easier to use directly expressed Fabs instead of purifying Fabs from papain-digested antibodies. The binding of mammalian expressed Fabs to UGGT was assayed using the Biacore system. Purified hUGGT1 was immobilized on a sensor chip (CM5). Purified Fabs with concentrations ranging from nM to µM were flowed over the surface in HBS buffer (50 mM HEPES, 140 mM NaCl, 1.5 mM Na2HPO4, pH 7.12) and the binding was monitored. Binding of most Fabs except Fab E5 did not achieve saturation as most Fabs have limited solubility that cannot be concentrated to 10× Kd value (Figure 15). Fab E5 achieved saturation in the binding curve and had a Kd value of 14.4 µM (Table 4). Binding experiment of each Fab on hUGGT1 was performed once.

3.2.6 Co-crystallization of UGGT with Fabs

Crystal trials of UGGT in equal molar mixture with Fabs F1, E4, E5 and F7 were set up using the NeXtal ProComplex suite (Qiagen). No6 crystal formation8 was observed.

3.3 UGGT enzyme assay

As we failed to obtain UGGT crystals, we performed a radioactive UDP-Glc-based assay to check whether the purified UGGT was in its native conformation. The enzyme assay can also be used to identify potential substrates of UGGT and to explore residues or motifs implicated in UGGT substrate interactions (i.e. mutagenesis studies or truncation studies).

3.3.1 Preparation of UGGT acceptor substrates

3.3.1.1 RiboB

Bovine pancreatic RiboB has been previously used by several groups to derive substrates for UGGT substrates (Table 1) and was used in this study to prepare substrates for UGGT. Bovine pancreatic RiboB contains a small portion of the M9 glycoform that is required for UGGT activity. Urea denaturation, subtilisin digestion and alkylation have been used to prepare partially denatured RiboB samples that are recognized by UGGT.

We decided to express RiboB as we can manipulate the sequence of RiboB. For example, an enterokinase (EK) cleavage site was engineered in (i.e. the RBEK construct) at the subtilisin cleavage site to avoid non-specific digestion by subtilisin. In addition, a C-terminal transglutaminase recognition sequence was engineered in (i.e. the construct RBTG) to allow modification of RiboB by transglutaminase (TG) for the purpose of introducing a fluorescent label for binding studies. The RBEKTG construct containing both the EK site and the TG recognition sequence was also made.

Fab UGGT Empty

4000 3000 2000 Kd=105.6M F1 1000 R2=1.00 0

Response (RU) Response 50 100 150 200

 -1000 concentration (M)

2000 1500 1000 Kd=2.0M F3 500 2

R =0.97 Response (RU) Response

 0 0 5 10 15 concentration (M)

300

200 Kd=2.4M E4 100

R2=0.97 Response (RU) Response

 0 0 2 4 6 concentration (M)

Figure 15. Fab binding to hUGGT1

Fab UGGT Empty

2500 2000 1500 E5 1000 Kd=14.4M 500 R2=0.99

0 Response (RU) Response

 -500 50 100 150 200 concentration (M)

3000

2000 Kd=32.0M F7 1000

R2=0.99 Response (RU) Response

 0 0 10 20 30 40 50 concentration (M)

1500

1000 Kd=62.8M F11 500

R2=1.00 Response (RU) Response

 0 0 20 40 60 80 100 concentration (M)

Figure 15. Fab binding to hUGGT1 (continued)

The left panel shows sensorgrams of 6 Fabs binding to hUGGT1 with the start of binding normalized at 0. The right panel shows the binding curves for the 6 Fabs. The reference channel did not contain a covalently attached protein. The binding experiment was performed once for each Fab.

Table 4. Summary of Fab binding affinity to hUGGT1.

rank Fab Kd R2

1 F3 2.0 µM 0.97

2 E4 2.4 µM 0.97

3 E5 14.4 µM 0.99

4 F7 32.0 μM 0.99

5 F11 62.8 μM 1.00

6 F1 105.6 μM 1.00

3.3.1.1.1 Expression and purification of RiboB

A synthesized RiboB cDNA was cloned into the PB vector with an N-terminal Protein A tag and modified to generate the RBEK, RBTG and RBEKTG constructs. Stably-transfected HEK 293S GnTI- cell lines expressing Protein A-tagged RB, RBEK, RBTG and RBEKTG were made and transient expression was tested. Protein A-tagged RB proteins were expressed in liter scale and purified by IgG affinity chromatography, followed by on-column tag cleavage using TEV protease. The protein was further purified through ion-exchange chromatography (Figure 16A). The yields of RB constructs were approximately 1 mg/L with over 60% of the protein unglycosylated (Figure 16B) as determined by deglycosylation experiments using EndoH and PNGase (data not shown). Purified RBEK was cleaved by enterokinase (EK) to validate the inserted EK cleavage site.

3.3.1.1.2 Expression of RiboB under kifunensine

We used the ER alpha-mannosidase inhibitor, kifunensine, to generate M9 glycan containing substrates. Kifunensine inhibits trimming of terminal mannose residues in the M9 glycan as shown in Figure 1. Kifunensine was added to 30-ml culture flasks expressing Protein A tagged RiboB to a final concentration of 2 mg/L. The culture was scaled up to one liter in the presence of 2 mg/L kifunensine. Limited protein expression was observed (<0.05 mg/L) (Figure 16D).

1 2 3 4 5 6 7

RiboB 25 RiboB 17 (unglycosylated) 11

A B

1 2 3 4 5 6 7 8 9

25 RBEK 17 S-protein RBEK S-protein 11 (unglycosylated) (unglycosylated)

C D

Figure 16. Purification of RB constructs and enterokinase cleavage

(A) Purification of RBEK by ion exchange chromatography.

(B) The SDS-PAGE gel of RBEK purified from ion exchange chromatography. Lanes are: (1) molecular weight markers, (2) TEV sample and (3-7) RBEK elution fractions.

(C) Enterokinase cleavage of RBEK. Lanes are (1) molecular weight markers, (2) EK at time 0, (3) EK at time 24hr, (4) RBEK at time 0, (5) RBEK at time 24hr, (6) RBEK + EK at time 0, (7) RBEK + EK at time 4hr, (8) RBEK + EK at time 20 hr, (9) RBEK + EK at time 24 hr.

(D) Purification of RBTG expressed under kifunensine by ion exchange chromatography.

3.3.1.2 Her2 antibody

The Her2 antibody expressed (by Kristina Han) in the presence of kifunensine was used as a potential UGGT acceptor substrate. The Her2 antibody has an N-glycan site in the Fc region. Expression of Her2 antibody under kifunensine in 30 ml scale was shown to produce Her2 antibody containing M9 glycan by Kristina Han. The complementarity-determining regions of the Her2 antibody may mimic protein folding intermediates that bind to UGGT.

3.3.2 UGGT glucosylation assay

To assay the enzymatic activity of purified UGGT, a glucosylation assay was performed. Frozen UGGT stocks stored at -80 °C were used in this assay. Commercial bovine pancreatic RiboB that contained a small portion of the M9 glycan was used in the assay as limited recombinant RiboB expression was observed when expressed with kifunensine. The 50-µL reaction mixture contained: 10mM HEPES pH 7, 10 mM CaCl2, 0.2 µCi UDP-Glc* (approximately 300 nM), 7µM UGGT and acceptor substrate. The substrate concentration of RiboB was approximately 2 mM and the substrate concentration of Her2 was approximately 10µM. 4-vinylpyridine alkylated RiboB had been previous reported as a good substrate for UGGT (77) but it was almost insoluble. The reaction was incubated at 37 °C for 3 days. A shorter incubation time (i.e. 1 d) was tested but the activity of UGGT towards substrates were not significant enough compared with controls. The mixture was loaded onto a Sep-Pak C18 cartridge and washed with 50 column volumes of water to remove free UDP-Glc*. The acceptor substrate (and attached Glc*) was eluted with 70% isopropanol and the [3H] was quantified with a scintillation counter. Each reaction was performed in triplicate.

At high concentration, UGGT activity towards commercial RiboB was observed and the result was validated by calcium dependence tests (Figure 17B). The Her2 experiments showed that UGGT had a higher activity towards Her2-kif than Her2-S. This result is consistent with the preference of UGGT for the M9 glycan.

1 2 3 4

UDP-Glc* + + + +

UGGT + +

A RiboB + +

1 2 3

UDP-Glc* + + +

UGGT + + +

RiboB + + +

Ca2+ + B EDTA +

1 2 3 4 5

UDP-Glc* + + + + +

UGGT + +

Her2-S + +

C Her2-kif + +

Figure 17. UGGT glucosylation assay.

A shows UGGT activity towards RiboB. B tests the calcium dependence of UGGT glucosylation. C compares UGGT activities towards Her2-S and Her2-kif.

Chapter 4 Discussion 4.1 Expression, purification and crystallization of UGGT

4.1.1 Mammalian expression of UGGT

In this study, we have established stable expression of UGGT using a PiggyBac transposon- based mammalian expression system developed in our lab (122). This system is designed for large-scale stable protein production under doxycycline induction. Transient transfection is used for short term expression and we use transient expression to test the expression of new constructs. Classical integration methods that stably insert the transgene of interest into the genome are susceptible to position effects which lead to transcriptional variation from one cell to the next. The PiggyBac transposon recognizes random TTAA sites and inserts multiple copies of the transgene at different sites in the genome, which minimizes position effects (127).

We used a mammalian expression system because it contains the enzymes and chaperones required to assist in the folding of our glycoprotein targets. UGGT is a large protein (approximately 175 kDa) with one third of the protein predicted to be unstructured (84). UGGT has one reported N-glycosylation site (78) that requires proper glycosylation and several cysteine residues that require proper formation of disulfide bonds.

4.1.2 Proteolysis of UGGT

In this study, I found that proteolysis of UGGT occurred during storage as shown in Figure 8A and I established an effective procedure to prevent the proteolysis of UGGT. The cOmplete™ protease inhibitor cocktail (Roche) was shown to be most effective against proteolysis among the several conditions tested (Figure 8B). Addition of the protease inhibitor cocktail during purification and storage of UGGT reduced the rate of proteolysis. In addition, freezing in liquid nitrogen was shown to preserve the UGGT enzymatic activity upon storage at -80 ºC over a period of one year. Therefore, we should include protease inhibitor cocktail along with UGGT to minimize degradation and freeze UGGT at -80 ºC for long term storage.

The proteolysis of UGGT was also reported when recombinant rat UGGT was purified by Parodi and his colleagues (77). In their report, discreet bands at 175, 150, 135, 92, 80 and 37 kDa

UGGT were observed on SDS-PAGE (77). Proteolysis was also found to be a problem in a later report studying UGGT from Rattus norvegicus, Schizosaccharomyces pombe and Drosophila melanogaster (79). In this study, Parodi and his colleague showed that UGGT contained at least two domains with a linkage extremely sensitive to endoproteolysis (79). The distinct proteolytic pattern is explained by the prediction that the five domains in UGGT are connected by unstructured linker regions that account for one third of the protein length (84). I observed a similar proteolytic pattern upon storage at 4 ºC over weeks.

4.1.3 Crystallization of UGGT

Crystallization of UGGT has been attempted in several groups and proven to be difficult (128). The large protein size with one third of it predicted to be flexible linker regions may contribute to the difficulty in crystallizing UGGT. The third Trx-like domain from the thermophilic fungus Chaetomium thermophilum, that survives up to 60 ºC, has been determined by X-ray crystallography (84).

We have tried to crystallize UGGT on its own as well as in complexes with Fabs but failed to obtain crystals. Crystallization of UGGT on its own was set up at 2 mg/ml using the NeXtal Classics suite (Qiagen). Crystal trials of UGGT in equal molar mixture with Fab F1, E4, E5 and F7 were set up using NeXtal ProComplex suite (Qiagen). No crystal formation was detected in any of the trials. Either clear drops or amorphous precipitates were observed.

Several factors could contribute to the failure to crystallize. The flexible linker regions in UGGT will generate conformational heterogeneity that prevents crystallization. The low solubility of UGGT sets limits for the concentration of UGGT in crystal trials. Although Fab binding creates additional opportunities for crystallization, the binding of Fabs to a flexible linear epitope may not promote crystal lattice formation (129). In contrast, Fabs that bind to conformational epitopes are expected to have less fluctuation between the protein and the bound Fab. We attempted to use a dot blot to check if our Fabs bind to conformational epitopes but failed as our Fabs have weak binding and the signals were washed off.

4.2 Generation of Fabs

Antibody fragments have been shown to be an effective tool in protein crystallization (130). The binding of Fabs to the protein of interest provides additional polar surface for crystallization and

47 the Fab complex may be more soluble than the free protein. In addition, the binding of Fabs to the protein of interest may reduce conformational flexibility of the target protein.

There are two approaches to generating Fabs against a target antigen: the phage display technique and the hybridoma technique. We chose the phage display technique as it provides a cheap and efficient way of generating Fab. The properties of phage display Fabs largely depend on the design. For instance, the complementarity determining region of our Fabs are rich in hydrophobic residues such as Leu, Phe and Trp (120), which may result in high affinity binding to hydrophobic epitopes on the protein.

4.2.1 Bacterial expression of Fabs

Following approaches reported in the literature, our first attempts at producing the phage display Fabs were based on bacterial expression (119). One advantage of a bacterial expression system is that it allows rapid expression and analysis of protein as E. coli cells grow at a very fast rate in comparison to mammalian cells. However, the expression of Fabs in the reducing environment of the cytoplasm is problematic as the disulfide bonds of Fabs are not properly formed. This problem is solved by using leader sequences to direct the secretion of the Fabs to the periplasmic space of bacteria (131). The periplasmic space is an oxidizing environment with chaperone-like molecules (124) and disulfide isomerases (125) that help the folding of Fabs. It is known that Fabs are susceptible to periplasmic proteases and a specially engineered E. coli 34B8 strain with deletions in the periplasmic proteases ompT and degP has been used to express Fabs for this reason (119). As the bacterial expression of Fabs resulted in low yields (Table 3) and had potential problems with folding and proteolysis, we switched to mammalian expression. The mammalian expression system is the natural expression system for human Fabs, which is expected to improve the quality and quantity of the expressed Fabs.

4.2.2 Mammalian expression of Fabs/antibodies

We have explored generating Fabs by mammalian expression of Fabs and by papain digestion of mammalian expressed antibodies. The former provides straightforward production of Fabs without further modification required. The latter requires additional papain digestion and separation steps to generate the Fabs.

I generated Fabs from antibodies by papain digest and optimized the papain digest conditions as shown in Figure 13. For our specific antibodies, papain digestion at neutral pH, at room temperature for 5 hours and at a papain-to-antibody mass ratio of 1:20 was optimal. The pH optimum that I determined is consistent with the reported optimum pH range (pH 5-7) of papain (132).

The separation of Fab from Fc is usually achieved with Protein A affinity chromatography as Fc binds to Protein A. Our phage-display Fabs have a special framework that also binds to Protein A (123), which increases the difficulties of separation. Therefore, we used direct mammalian expression of Fabs as the way to generate Fabs for our work.

By comparing the yields of bacterially expressed Fabs, mammalian expressed antibodies and mammalian expressed Fabs (Table 3), it is clear that mammalian expressed Fabs have the highest yields. It is not surprising that mammalian expression levels are much higher than bacterial expression levels as the mammalian expression system is the natural system for the folding of human Fabs/antibodies.

4.2.3 Fab UGGT affinity assay

In the Biacore experiment, most of the Fabs did not reach saturation in the binding curves within their solubility limits, except for Fab E5 (Figure 15). The Kd value of 14 µM calculated based on a simple one site binding model for Fab E5 is expected to be close to the actual Kd value as Fab E5 reaches saturation in the binding curve. The binding curve of Fab F3 almost reaches saturation (Figure 15) so that the calculated Kd value of 2 µM may approximate the actual Kd value of Fab F3. The calculated Kd values for Fab E4 (2 µM), F7 (32 µM), F11 (63 µM) and F1 (106 µM) may be less close to their actual Kd values as the binding curves of these Fabs are far from reaching saturation (Figure 15). To perform a good binding study, we need to prepare Fab concentrations starting from 10x below the Kd value to 10x above the Kd value so that the binding curves are complete and can reach saturation. These Fabs do not have high enough affinities to UGGT so that we cannot get to high enough concentrations to do a complete binding study.

We may need to screen more phage display Fabs in order to obtain high affinity Fabs. It is possible that we simply did not screen enough phage display Fabs to obtain high affinity Fabs.

Another reason for the low affinities of the Fabs is that these Fabs recognize flexible linear epitopes on UGGT. Iwata et al. proposed that antibodies raised against denatured proteins may recognize flexible linear epitopes (129). Fabs that recognize linear epitopes tend to have lower affinities (133). As one third of UGGT is predicted to be unstructured, a large portion of the phage display Fabs against UGGT may bind to flexible linear epitopes and have low affinities.

We chose to use the Biacore instrument for measuring the affinity of Fabs for UGGT. The Biacore system provides a continuous real-time measurement of binding based on surface plasmon resonance. It has been widely used to measure the binding affinity between antigen and antibodies produced from phage display (134) or hybridoma (135). By comparison with isothermal titration calorimetry, the Biacore system was reported to require 100-1000 fold less protein (136).

4.3 Design of RiboB constructs

RiboB, a small (124 amino acids) monoglycosylated protein, has been used to derive substrates for UGGT since UGGT was first purified (137). Initial work utilized urea denatured RiboB to mimic the protein in a late folding conformation, characteristic of a UGGT substrate (137). Trombetta and Helenius proposed using RiboB S-protein as a substrate for UGGT (100). RiboB S-protein (residues 21-124) is generated by subtilisin cleavage and removal of the N-terminal S- peptide (residues 1-20). As the RiboB S-protein contains all four intrachain disulfide bonds, it retains a native conformation to a large extent (138). It was found that RiboB S-protein was well behaved, soluble and monomeric (100).

We want to use RiboB as an acceptor substrate to build on this work. Only a small fraction of the commercially available bovine pancreatic RiboB molecules contain the M9 glycan that is required for UGGT enzymatic activity. We decided to express RiboB rather than just use commercially available material so that we could control the glycoform of RiboB and facilitate production of the protein-modified forms of RiboB with the correct M9 glycoform. One design of the RiboB construct is to use an enterokinase cleavage site to replace the subtilisin cleavage site. This design has been proposed by Raines and his colleagues as subtilisin is a non-specific protease (99). Enterokinase, on the other hand, is a specific enzyme that recognizes a DDDDK sequence and cleaves at the C-terminal of the DDDDK sequence (139). I have demonstrated that our RiboB with the DDDDK sequence can be expressed in mammalian cells and cleaved by

50 enterokinase as shown in Figure 16. Another design of the RiboB construct is to include a C- terminal transglutaminase recognition sequence. Transglutaminase catalyzes the formation of an isopeptide bond between lysine and glutamine residues (140). We want to use transglutaminase to attach fluorescent probes to the RiboB so that the UGGT substrate interaction can be measured by frontal affinity chromatography. In addition, I also made the RiboB construct combining the DDDDK sequence and the transglutaminase recognition sequence that allows both enterokinase cleavage and transglutaminase derivatization. Both constructs were expressed and purified and the enterokinase cleavage was performed on the purified RiboB with the enterokinase site (Figure 16). Expression of both constructs in the presence of kifunensine to give the M9 N-glycan resulted in very limited protein production, which prevented the use of the constructs in the enzyme assay.

4.4 Preparing M9 glycan containing protein

One of the major hurdles in studying the UGGT substrate interaction is preparing M9 glycan containing substrates. M9 glycan containing protein is transiently present in vivo as the terminal mannose residues are hydrolyzed by ER alpha-mannosidase I and other mannosidases as shown in Figure 1B. Two approaches have been reported so far to generate M9 glycan containing protein. Thomas and his colleagues (77) have engineered a special yeast strain, DT111 with triple deletions in MNS1 (ER α-mannosidase), MNN1 (Golgi α-1,3-mannosyltransferase) and OCH1 (α-1,6-mannosyltransferase) to produce M9 glycan containing protein. The DT111 strain that we received from the Thomas group showed impaired growth likely due to the triple deletions and was difficult to transform.

We attempted to use kifunensine, a potent alpha-mannosidase I inhibitor (115), as a way of generating M9glycan containing UGGT substrates. Kifunensine has been used to generate the Man9 glycoform for many applications, such as crystallization of glycoproteins (117) and the production of therapeutic antibodies (141). Almost homogenous Man9 glycan was reported with 2 µg/ml kifunensine in the tissue culture medium (141).

We encountered a very significant reduction in the protein yields obtained from large scale expression of stably transfected 293S GnTI- cells grown in the presence of kifunensine. In the experiment, we included kifunensine at the reported 2 µg/ml (approximately 10µM) concentration in 30 ml FreeStyle 293 expression media (Invitrogen) over a week and scaled up to

1 liter while maintaining kifunensine concentration for several weeks. After scaling up to 1 liter, the cell culture with kifunensine showed aberrantly low cell density by comparison with the non- treated cell culture. We attempted to prolong the productive interval of media collection but no significant improvement was observed. Very limited protein was obtained under kifunensine treatment (Figure 16D).

Incubation with kifunensine abolishes the removal of α-1,2-mannoses from N-glycans on misfolded glycoproteins (115) and therefore interferes with the recognition by the ER-associated protein degradation system (142). Previous work using a mammalian expression system to express secreted glycoproteins showed that HEK293T cells with kifunensine only survive for 4 days (143). In another report, it was shown that kifunensine at 1 µg/ml concentration reduced cell growth and could not be used while expanding the culture (144), which is consistent with our experience. Finally, it has been shown that when proteins are expressed using stable cell lines, the cells need to grow to full confluency before replacing the media with kifunensine containing media (145).

4.5 UGGT glucosylation assay

As mentioned previously, UGGT is highly sensitive to proteases and proteolytic degradation of UGGT cannot be completely inhibited. An enzyme assay is required to monitor the status of UGGT and is also a basis for future characterization of UGGT substrate interaction.

In this study, we showed that UGGT had activity towards commercial RiboB and a Her2 antibody containing M9 glycans. With the assumption that commercial RiboB was in its native form during the experiment, we demonstrated that native glycoproteins may also be glycosylated by UGGT. We will compare the kcat and KM values of UGGT towards native RiboB and toward RiboB S-protein once we improve the expression of RiboB in the presence of kifunensine. If the kcat values are the same, this will suggest that misfolded protein does not enhance the catalytic activity of UGGT and does not explain its preference for misfolded glycoprotein substrates.

Perhaps more likely is that the KM for misfolded substrates will be lower than that for native folded substrates. This would support the suggestion that the N-terminal domain of UGGT serves to recognize misfolded glycoproteins and increases the local substrate concentration for the glucosyltransferase domain to act on.

Our results also support that fact that UGGT prefers M9 glycan containing proteins. By comparing the activity of UGGT towards the Man5 glycan containing Her2 antibody (Her2-S) and the M9 glycan containing Her2 antibody (Her2-kif), it is clearly seen that UGGT has a higher activity towards Her2 with M9 glycan than Her2-S (Figure 17C). This result is consistent with the literature and supports that idea that Her2-kif, when it contains the M9 glycan, serves as a substrate for UGGT (59).

Chapter 5 Future Directions 5.1 Fragmentation of UGGT

It was observed that UGGT is extremely sensitive to proteases and that it needs to be protected with a protease inhibitor cocktail (Figure 8). Proteolysis may reduce protein yields during expression and purification, inactivate the enzyme in the assay, and generate heterogeneous species detrimental to crystallization. Despite these problems, proteolysis provides some insights into the structure of UGGT that can be utilized in future studies.

The knowledge of UGGT domain structure was mostly acquired based on studying proteolytic fragments (79) and from bioinformatic prediction (84). We can use sequence alignments to predict the domains of human UGGT1 based on the domains of Chaetomium thermophilum UGGT (84). Once we have defined the domains in human UGGT1, we can clone and express a single domain or combination of domains for structural and functional characterization. Crystallization of a single domain of UGGT may be more fruitful as a single domain has less flexibility than the full-length UGGT. Limited proteolysis may also be used to remove any remaining loops in the single domain to improve crystallizability. In addition, we can also monitor binding of Fabs to each domain using surface plasmon resonance and try to crystallize a single domain or multiple domains with Fabs.

5.2 Fabs as a tool for characterization

As part of the effort to characterize UGGT, we want to use phage display Fabs as a tool for crystallization. No hits were observed in the6 crystallization trials8 of UGGT with Fabs F1, E4, E5 and F7. Affinity studies using surface plasmon resonance showed only Fab E5 reached saturation in the binding curve with a reliable Kd value of 1 µM while the rest Fabs could not reached saturation within their solubility limits.

It has been suggested that antibodies raised against denatured proteins recognize flexible linear epitopes and seldom facilitate crystal formation (129). In this report, Iwata et al. developed protocols for generating conformational antibodies against membrane proteins using liposome immunization, liposome enzyme linked immunosorbent assay (ELISA), denatured dot blot and

54 binding affinity selection by surface plasmon resonance (129). As one third of UGGT is predicted to be unstructured, a significant portion of the phage display Fabs against UGGT may recognize flexible linear epitopes. For future studies, we may need to obtain more Fabs with higher affinities that recognize conformational epitopes. We may also use in vitro affinity maturation that utilizes several rounds of mutation and selection to generate Fabs with increased affinities. The selection procedures mentioned in the report (129) may also be adopted to select conformational Fabs with high affinities against UGGT. In addition, we can express and use UGGT domains instead of full-length UGGT for phage display screening so that Fabs against flexible linker regions are not obtained.

Fabs can be used not only as a tool for crystallization but also as probes for functional studies. We can include each Fab in the enzyme assay and monitor the effect on the UGGT activity. Fabs that inhibit UGGT activity may be used in in vivo studies to block UGGT activity and examine the cellular effects. Crystal structures of inhibitory Fabs with UGGT, or UGGT domains, may provide insight into the mechanism of UGGT glycosylation.

5.3 Preparation of Man9 containing substrates

We have encountered a reduction in the yields of proteins expressed with kifunensine and it was reported that kifunensine reduced cell growth and could not be used when expanding the culture (144) as we also observed. For future expression with kifunensine, we will grow cells to full confluency on a liter scale before adding kifunensine. The kinetics of protein production after kifunensine addition will be monitored by western blot using antibodies against the Protein A tag. By plotting the protein yields against the time after kifunensine addition and comparing this with non-treated experiments, we will determine the optimal time point to harvest the protein after kifunensine addition.

5.4 RiboB substrates

In this study, we have made several RiboB constructs that can be used for studying the UGGT substrate interaction. The transglutaminase recognition sequence in the C-terminus of the RiboB constructs allows for the addition of fluorescent dyes to RiboB using transglutaminase. The RiboB UGGT interaction can be monitored by frontal affinity chromatography. The RiboB S- protein generated by enterokinase cleavage of RBEK can be used for a binding assay or an

55 enzymatic activity assay when expressed with kifunensine. Crystallization of the RiboB S- protein with UGGT will not only provide the 3-dimensional structure of UGGT but also reveal motifs/residues in UGGT responsible for binding to its acceptor substrates.

My results showed that UGGT can transfer glucose to native RiboB at a high RiboB concentration (i.e. 2 mM) (Figure 17). It will be interesting to determine the affinity between native RiboB and UGGT using surface plasmon resonance or frontal affinity chromatography. If binding is not too weak we may be able to set up crystallization trials with the goal of obtaining a UGGT acceptor substrate complex using native RiboB at high concentration (i.e. 10-fold over the Kd value).

We will also try to determine the kcat and KM values of UGGT towards native and misfolded

RiboB. As discussed above, if the kcat values are the same, native RiboB will be used to study catalytic mechanism (e.g. metal ion dependence and catalytic residues by mutagenesis).

5.5 UGGT Sep15 interaction

As mentioned in the introduction, Sep15 forms a tight complex with UGGT with a Kd value of 20 nM (86). The binding of Sep15 to UGGT may improve the solubility of UGGT as well as provide additional opportunities for crystallization. We will express Sep15 as a tool to assist in the characterization and crystallization of UGGT. To prepare the complex of UGGT and Sep15, we will add Sep15 in excess of UGGT and isolate the complex through gel filtration chromatography. The crystal structure of UGGT with Sep15 is of particular interest as Sep15 is proposed to serve as a structural extension of UGGT (84).

Sep15 is reported to enhance UGGT activity when assayed with fluorophore-conjugated N- glycans (87). We will examine the effect of Sep15 on UGGT activity using glycoprotein substrates to further investigate the role of Sep15 in the UGGT substrate interaction.

Conclusions

UGGT is one of the key enzymes in ER quality control. The X-ray crystal structure of the Trx3 domain of UGGT has been solved (84), but the 3-dimensional structure of full-length UGGT is still unknown. UGGT is known to recognize M9 glycan-containing glycoproteins in a molten globule state but the molecular interactions implicated in the recognition are not fully understood.

In this study, I attempted to solve the X-ray crystal structure of full-length UGGT using Fabs as a tool. Although UGGT failed to crystallize, I gained experience in expression and purification of UGGT and Fabs. The binding studies using surface plasmon resonance indicated that all of the Fabs showed weak binding to UGGT and that we may need to screen more Fabs to obtain Fabs with high affinity to UGGT. To ensure that the purified UGGT was active, a glucosyltransferase assay was performed. We identified UGGT activity towards native commercial RiboB at a high concentration.

It will be informative to compare the kcat and KM values of UGGT towards native and misfolded

RiboB. If the kcat for misfolded protein is higher than the kcat for folded protein, this will suggest that misfolded protein activates UGGT. If the kcat values stay the same while the KM for misfolded substrates is lower than the KM for native substrates this will suggest that the N- terminal domain of UGGT serves to increase the local substrate concentration for the glucosyltransferase domain. This would explain the preference of UGGT for misfolded substrates.

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