Structure-Function Relationships in Human P-Hexosaminidase

YONGMIN HOU

A Thesis submitted in conformity with the requirements for the Degree of Doctor of Philosophy Department of Laboratory Medicine and Pathobiology University of Toronto

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Yongmin Hou, Ph-D., 1999 Department of Laboratory Medicine and Pathobiology University of Toronto

Abstract Human bhexosarninidase (Hex) isozymes are dimeric composed of a and/or P subunits, i.e. Hex A (aJ3),Hex B (PP) and Hex S (aa).Only Hex A has a unique in vivo function, i.e., of GM~.Thus, in the either a or $ subunit of Hex A can result in ganglioside storage and two major forms of gangliosidosis, i.e. Tay-Sachs or Sandhoff diseases, respectively.

The goal of this thesis is to study structurefunction relationship in human Hex. The first project was to characterize a novel missense (fPro504Ser) associated with the chronic form of using both the patients' and transfected cells. Biochemical studies of mutant Hex A demonstrated that this substitution dkctly affects the ability of Hex A to hydrolyze its natural substrate and also intracellular transport, producing chronic Sandhoff disease. The second project was to examine the validity of the molecular modeling of human Hex based on the X-ray structure of bacterial chitobiase. To achieve this, I first developed a new method for the generation of a C-terminal H.s6-tagged form of pro-Hex B in transfected cells. Re-examination of $kg21 1Lys using this highly purified form of mutant Hex B indicated that the substitution resulted in an with a more than 10-fold increase in its apparent Krn and over a 1000-fold decrease in its Vmax. Thus &kg21 1 plays a critical role in substrate binding and in orientating the substrate in the .

Next I used the same approach to analyze the candidate conserved active acidic residues in human Hex B, The f3Glu355Gln substitution reduced Vmax >8000-fold with a slight lowering of the Km, consistent with Glu355 being the catalytic acid group in human Hex. The mp354Asn mutant also had an almost normal Km but a 6000-fold Vmax decrease, also conforming with Asp354 functioning as a "base" to aid in the proposed substrate-assisted catalysis. PAsp241, along with mp240 and PAsp290, is involved in substrate binding; whereas $Asp196 is important in the initial folding or dimer formation of the &subunits rather than any involvement in its active site. Taken together, I concluded that the chitobiase model is correct in light of the active site residues in human Hex characterized thus far. The studies of these active site residues suggest that human Hex and probably all the Family 20 enzymes have similar structures and a conserved substrate-assisted catalysis mechanism. Acknowledgments

I would like to thank my supervisor, Dr. Don Mahuran, for his advice and support throughout my graduate work Without his encouragement I would not have completed my Ph.D. thesis. His relaxed attitude along with his excellent computer knowledge made my research work full of fun and interesting. I wish to achowledge Dr. John Callahan and Dr. Brian Robinson for their guidance and for serving as members of my advisory committee. I would also like to thank other members of my thesis committee members, Dr. Andrew Bognar, Dr. Janet Forstner and Dr. Clifford Lingwood for their time and advice.

Special thanks to Amy Leung for her great technical assistance. I would also like to thank other collaborators, Dr. Roderick Tse, Dr. Beth McInnes, Dr. Aleksander Hinek, and Dr. Steve Withers (UBC). I enjoyed the discussions with Dr. Sunqu Zhang, Rick Bagshaw, Benny Biao Cha, Scott Bukovac. I appreciate the assistance of Mana Chow for organizing my thesis meetings.

Most of all I would like to thank my wife and my son for their love, continued support, and encouragement during the time.

iii Thesis Abstract ...... i ... Acknowledgments ...... II.I ... List of Tables ...... VI.U List of Figures...... ix Publications...... xii . . ... Abbrevra~ons...... m wter_1 General Introduction

Introduction ...... *...... 1 . 1 Structure and Function of PHexosaminidase ...... 1-2 Hex isozymes and activator ...... 1-2 Biosynthesis. processing and intracellular transport ...... 1-6 to Golgi...... 1-6 Lysosomal incorporation...... 1. 12 Hex substrates and protein purification ...... *.....1 . 12 Artifkid Artifkid substrate.....*...... 1. 13 Natural substrate ...... 1. 13 Hex purification ...... 1-19 Enzymatic function ...... 1. 19 Hydrolysis of GM~ganglioside requires the interaction of three proteins ...... 1. 19 Genetics and Molecular Defects in Gangliosidoses ...... 1-20 Structure of HEXA and HEXB genes...... 1-20 The gangliosidoses...... 1-23 Clinical phenotypes of GM~Gangliosidoses ...... 1-24 Critical threshold model ...... 1-25 Animal models for the gangliosidoses ...... 1-26 Mutations causing -2 gangliosidosis ...... 1-27 Partial gene deletions ...... 1-28 Mutations producing early stop codons ...... 1-28 Mutations affecting mRNA processing ...... 1-29 Missense mutations ...... 1-31 The B 1-variant of Tay-Sachs disease...... 1-33 Molecular Modeling of the Active Sites in Human Hex ...... 1-36 The active sites of chitobiase ...... 1-36 Molecular modeling of human Hex ...... 1-37 Modeling of Streptomyces Hex ...... 1-45 Catalytic mechanism ...... 1-45 Active site studies of human Hex ...... 1-46 Thesis Objectives ...... 1-49 References ...... 1-50

-ter 2 A Pro504Ser Substitution in the P-Subunit of Hexosaminidase A Inhibits amsubunit Hydrolysis of GM~Ganglioside. Resulting in Chronic Sandhoff Disease

Introduction...... 2-1 Materials and Methods ...... 2-5 Preparation of genomic DNA...... 2-5 RNA isolation and reverse transcription ...... 2-5 DNA amplification and direct sequencing...... 2-5 Generation of mutant constructs ...... 2-6 Cell culture and DNA transfection ...... 2-7 . . Hex actnaty assay...... 2-7 Western blotting ...... 2-7 Separation of the Hex isozymes...... 2-7 Kinetic analysis ...... 2-8 hydrolysis assay ...... 2-8 Thermal stability study ...... 2-8 Intracellular localization of P proteins quantified using indirect immunofluorescence ...... 2-9 Results ...... 2-10 Detection of a novel missense mutation ...... 2-10 Patients' Hex activity and protein levels ...... 2-10 Effect of the mutation on stability and intracellular transport ...... 2-16 Hydrolysis of artificial and natural substrates by a$(Pro504Ser) Hex A ...... 2-27 Discussion ...... 2-27 References ...... 2-33

mter 3 Identification of PArg211 as a Residue Critically Involved in Substrate Binding and Orientation Using a C-terminal Epitope-tagged Form of Pro-Hex B Introduction ...... 3-1 Materials and Methods ...... 3-6 DNA construction...... 3-6 Cell culture...... 3-7 Transfec tion ...... 3-7 Enzyme assays ...... 3-8 Western blot analysis ...... 3-8 Ni-NTA chromatography ...... 3-8 Determination of kinetic parameters ...... 3-9 Hex affinity chromatography ...... 3-9 Results ...... 3-10 Expression and purification of C-terminal His6 Hex B in CHO cek ...... 3-10 Biochemical properties of Pro-Hex B and Pro-Hex %His6 ...... 3-14 Examination of the role of PArg211 in the active site of Hex B ...... 3-20 Discussion ...... 3-22 References ...... 3-25

Cha~ter4 Analysis of Candidate Active Acidic Residues in Hex B Introduction ...... -4-1 Materials and Methods ...... 4.5 Protein homology alignment and modeling ...... 4.5 Site-directed mutagenesis and vector construction ...... 4-5 Transfection of mutant constructs ...... 4-9 Assays of enzyme and protein levels ...... 4-10 -cation of Hiss-tagged mutant Hex B protein ...... 4.10 Kinetic studies...... -4-10 CNAG affinity binding assay ...... 4.11 Results ...... 4-11 Expression and puification of mutant proteins ...... 4. 11 The effect of mutations on their enzyme specific activity ...... 4. 14 Kinetic analysis of putative active site residues ...... 4. 19 Confiation of each residue's role in either substrate binding or catalysis.. 4-19 Discussion...... -4-22 References...... -4-26

Summary and Future Prospects

summary...... 5-1 Future Dzrectlons...... -5-4 Analysis of candidate active aromatic midues based on the chitobiase model...... 5-5 Location of the active sites with respect to the subunit-subunit interface.. ... -5-5 Identification of residues involved in binding a-specific negatively charged substrate...... 5-9 Studies of protein-protein interaction...... -5-9 Large scale production of Hex B for crystallization ...... -5- 1 1 References ...... -5-12

Appendix ...... a Hou Y, Tse R,and Mahuran DJ (1996) Biochemistry, 353963-3969 (Abstract)...... a Hou Y, Vavougios G,Hinek A, Wu KK,Hechtrnan P, and Mahuran DJ (1996) American J. of Human Genetics ,59:52-58 (Abstract)...... b Tse R, Vavougios G,Hou Y and Mahuran DJ. (1996a) Biochemistry a 3963-3969 (Abstract)...... c Tse R, WuYJ, Vavougios G, Hou Y, Hinek A and Mahuran DJ. (1996b) Biochemistry 2 10894-10903 (Abstract) ...... d

vii List of Tables

Chapter 1 Table 1.1. Active, acidic residues in the chitobiase and their aligned residues in Streptomyces plicatus hex & human hex, a-and P-subunits.

Chapter 2

Table 2.1 .Unitsa of Hex A present in fibroblasts measured after isozyme separation by Chromatofocusing.

Table 2.2. Biochemical characterization of the effwts of the PPro504Ser substitution.

Chapter 3 Table 3.1. Assay of Hex binding to Ni-NTA column from the medium and lysate of mock and pDNA-kHis6-transfected CHO cells using MUG substrate. Table 3.2. Analysis of kinetic parameters and affhty binding to CNAG for mature Hex B, pro-Hex B, pro-Hex B-His6 and mutant pro-Hex B *-His6 (R21 IK).

Table 3.3. Km a values for preparations of imidazole elution from the media of cells expressing pro-Hex B-His6 and pro-Hex B*-His6 (R211K)utilizing MUG substrate.

Chapter 4 Table 4.1. Active acidic residues in the chitobiase and their aligned residues in Streptomyces plicatus hex & human hex, a and P subunits.

Table 4.2. Oligonucleotides Used to Mutate PDNA. Table 4.3. Determination of kinetic parameters and affinity binding to CNAG for Hex B, Hi%-tagged pro-Hex B and pro-Hex B* mutant.

Chapter 5 No Table

viii List of Figures

Chapter 1 Figure 1.1. The HEX gene system. Figure 1.2. Biosynthesis and intracellular transport of Hex A. Figure 1.3.Structure of the artificial substrates used to assay Hex A and 8 activity; 4- Methylumklliferyl-GlcNAc (4-MUG) (Panel A-used to assay Hex A, B or S); 4- Methylumbelliferyl-GIcNAc-6-sulfate (4-MUGS) (Panel B-used to assay isozymes containing an a-subunit, i.e. Hex A and S).

Figure 1.4. Structure of -2 ganglioside. Figure 1.5. Model for the lysosomal catabolism of GM~ganglioside.

Figure 1.6: Sequence alignment for the catalytic domain III of chitobiase with the a- and P- subunits of human Hex as well as Sp-Hex. Figure 1.7. Molecular modeling of human Hex from the atomic structure of chitobiase. Figure 1.8. The proposed substrate-assisted catalytic mechanism-Figure 2.1. Autoradiography of nucleotide sequencing gels: Direct sequencing of PCR products from A) genomic DNA (sense strand); B) cDNA (antisense strand).

Chapter 2 Figure 2.2. Direct restriction digest assay (see diagrams at the top of each panel) for the presence of the C1510T transition in PCR fragments from A) genomic DNA, and B) cDNA.

Figure 2.3. Western blot analyses using an anti-human Hex A antibody of the a and polypeptides in the total cell lysates from; co-transfected CHO cells (with wild type acDNA and mutant &DNA, P*); normal fibroblasts (Normal), and fibroblasts from Sandhoff patients presenting with chronic (Chr.* one of the subjects of this report, line 2400) subacute (Subac., line GM 2094) and acute (Acute, line GM 294) forms of GM~gangliosidosis (the genotype of each patient is given at the bottom of the figure below the corresponding sample lane).

Figure 2.4. Western blot analyses using an anti-human Hex A antibody of the a and P polypeptides in the total cell lysates (the amount of protein loaded is given directly above the sample lane) from fibroblasts of one of our chronic patients (line 2400) and a normal individual. Cells were grown in the presence, +, or absence, -, of leupeptin which inhibits lysosomal degradation of proteins. Figure 2.5. Indirect immunofluorescencemicroscopy using an anti-human Hex B antibody of A) untransfected CHO ceb, or CHO cells co-transfected with, B) cDNAs encoding wild type prepro a and P polypeptides, C) cDNAs encoding wild type a and pAsp208Asn polypeptides or D) cDNAs encoding wild type a and $ ProS04Ser polypeptides.

Figure 2.6. Graphical representation of the percentage of various pproteins; wild type (Wild), or containing either an Asp208Asn (D208N)or a Pro504Ser (P504S) substitution (X-axis); in either the ER or endosomd in permanently transfected CHO cells. Figure 2.7. Natural substrate, GM~gangliosidd activator complex, assay of DMseparated Hex A (Fig. 2.3) fi-om untransfected CHO cells (endogenous CHO cell Hex A), solid squares; or CHO cells co-transfected with either cDNAs encoding wild type prepro a and P polypeptides, solid circles, or wild type a and PPro504Ser prepro-polypeptides, solid diamonds.

Chapter 3 Figure 3.1. Western blot analysis using an anti-human Hex B IgG in lysates from mock CHO cells (CHO), CHO cells transfected with pEFNEO-P (P), pcDNA-P-His6 @His) or pcDNA-His-P* encoding a R211K substitution @*-His). Figure 3.2. Coomassie blue staining (A) and Western blot (B) of purified proteins from the serum free medium of untransfected CHO cells (CHO), CHO cells transfected with pcDNA-His-P @-His) or mutant pcDNA-His-P* encoding a R211K substitution @*-His), or cells transfected with pEFNEO-P (P). Figure 3.3. Kinetic analysis of the mature Hex B, pro-Hex B, pro-Hex %His6 and mutant pro-Hex B*-Hi%containing R211K using the neutral MUG (mM) substrate.

Chapter 4 Figure 4.1. Western blot analysis using an anti-human Hex B Ig G in lysates from nontransfected CHO cells (CHO), CHO cells transfected with pcDNA-P- His6 (WTj or mutant pcDNA-P*-His6 encoding one of the following substitutions: D196N, D241N, D354N, E355Q and E491Q. Figure 4.2. Coomassie blue staining (A) and Western blot (B) of purified proteins from the serum free medium of the CHO cells (CHO), CHO cells transfected with pcDNA- pHis6 (WT) or mutant pcDNA-P*-Hisciconstructs.

Figure 4.3. Kinetic analysis of wild type pro-Hex B-His6 (WT) and mutant pro-Hex B*-His6 using the neutral MUG (mM)substrate. Chapter 5

Figure 5.1. Proposed models for formation of the active Hex A dimer from the inactive a and $ monomers in the ER. "+" indicates the positive site unique to a that allows Hex A (a$)and Hex S (aa)to bind negatively charged substrates. Publications and Manuscripts

Hou Y, McInnes B, Karpati G, Hinek A, and Mahuran DJ (1998). A Pro504Ser Substitution in the &Subunit of &Hexosaminidase A Inhibits a-Subunit Hydrolysis of GM~Ganglioside, Resulting in Chronic Sandhoff Disease. J. Biol. Chern, 273 (33): 21386-21392 (Chapter 2)

Hou Y, Mahuran DJ (1999). Identification of Ng211 as a midue critically involved in substrate binding and orientation using a C-terminal. epitope-tagged form of Pro-Hex B. Biochemistry, to be submitted (Chapter 3) Hou Y, Mahuran DJ. (1999). Analyses of candidate active site midues in human hexosaminidase B . J. Biol. Chem., to be submitted (Chapter 4)

Chapter 1 (Most in M.Sc. thesis): Hou Y, Tse R, and Mahuran DJ (1996) The direct determination of the substrate speciticity of the a- active site in heterodimeric f3-hexosaminidase A. Biochemistry, 353963-3969 Hou Y, Vavougios G, Hinek A, Wu KK, Hechtman P, Kaplan F, and Mahuran DJ (1996). The aV192L substitution mutation in the a subunit of Hex A is not associated with the B1-variant of Tay- Sachs disease. American J. of Human Genetics ,5952-58

Collaborative Work: Tse R, Vavougios G, Hou Y, Mahuran DJ (1996). Identification of an active acidic residue in the catalytic site of P-hexosaminidase. Biochemistry 35 (23) : 7599-7607 Tse R, Wu YJ, Vavougios G, Hou Y, Hinek A and Mahuran DJ (1996). IdentEcation of functional domains within the a-and p subunits of P-hexosaminidase A through the expression of a-P fusion proteins. Biochemistry 35 (33) : 10894-10903

xii Abbreviations

4-MU 4-MUG 4-MUGS 4-methylumbelliferyl-~N-acetylglu~0samine-6-~uIfate Activator activator protein CHO Chinese hamster ovary cell line CRM cross reacting material ER endoplasmic reticulum FCS fetal calf serum GalNAc N-acetylgalactosamine GlcNAc N-acetylglucosamine GM2 GM~ganglioside, GalNAcP(1-4)- meuAca(2-3)-] -GalP(I -4)-Glc-cerarnide GM3 GM~ganglioside, WeuAca(2-3)-J-GalP(1-4)-Glc- GA~ GA~ganglioside, GalNAcp(1-4)-Galp(1-4)-Glc-ceramide Hex Hex A Hex B @hexosarninidase B HPLC high performance liquid chromatography kb kilobase pair kDa kilodaltons Man mannose MF'R mannose 6-phosphate receptor Mr apparent molecular weight NeuNAc N-acetylneuraminic acid nm/hr nanomoles per hour RER rough endoplasmic reticulum SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis EF elongation factor promoter TGN trans Golgi network wt wild type

xiii Chapter 1

Introduction INTRODUCTION The existence of an N-acetyl-PD-glucosaminidase (NAG) in mammalian tissue was described over 50 years ago (Watanabe 1936). It was later found that unlike bacterial NAGS, the mammalian enzyme has an acidic pH optimum, and is locaked predominately in . It cleaves the terminal non-reducing, p- 1,Clinked, glycosidic bonds of both amino sugars, N-acetyl- D-glucosamine (GlcNAc) and N-acetyl-D-galactosamine (GalNAc). Thus, the appropriate name of the enzyme is bhexosarninidase or simply Hex (EC 3.2.1.52), and its primary natural substrates are oligosaccharides, , , glycopmteins and glycosaminoglycans. The increased interest in Hex came from its association with Tay-Sachs disease (TSD). This disease was first described late in the last century (Sachs 1887; Tay 188 I), and thus is one of the oldest known lysosomal storage diseases. The clinical phenotype associated with TSD results from the intralysosomal accumulation of a2ganglioside (GM~),primarily in neurond ceh where gan~osidesare most abundant (reviewed in (Gravel et al. 1995)). It was later found that two other related diseases, i.e. Sandhoff disease and AB- variant, also present similar clinical phenotypes. Therefore these three diseases are termed "forms of GM~gangliosidosis". It is now known that in vivo only Hex A (ap) can hydrolyze GM~but it also requires the presence of the GM~activator (Activator) protein. Thus, defects in either the a- or j3-subunit, or the activator protein result in TSD,Sandhoff disease or the AB-variant, respectively. The current era of investigation was stimulated by the development of a Hex specific affi'itnity ligand (2-acetamido-N-E-aminocaproyl-2-deoxy-e)(Geiger et al.

1974), which resulted in much greater yields and purity of the isolated isozymes. This made it possible to define the protein structure more pmisely and to analyze the amino acid sequence of the polypeptide chains. Such structural data lead to the isolation of cDNA and genomic clones encoding both of Hex A's subunits, as well as those encoding the Activator protein (reviewed in (Gravel et al. 1995)). The elucidation of naturally occurring, disease associated mutations in each of the three companding genes has made genotype-phenotype (biochemical and clinical) correlation possible. Some of these correlations have led to a better understanding of the cell biology of Hex and given clues to the location and mechanism of its active sites. Others have posed more general questions that remain to be answered dealing with mRNA processing, transport and stabiity, and the "quality control" mechanism(s) involved in the retention and degradation of "abnormal" and/or massembled proteins in the ER. The recently reported crystal structure of bacterial chitobiase allows one to perform homology-based modeling of the human Hex- Comparisons of biochemical data from mutations and active site studies in human Hex with those predictions from chitobiase have shown both some agreements and disagreements. Additionally, since the chitobiase functions only as a NAG, has a neutral pH optimum, and is a monomer, the accuracy and validity of the bacterial model needs to be examined.

STRUCTURE AND FUNCTION OF j3-HEXOSAMINIDASE

In 1968, Robinson and Stirling showed that human spleen hexosaminidase activity could be separated into two PI-forms, an acidic (PI-4.8), heat-labile form, Hex A, and a basic (PI-6.9), heat-stable form, Hex B (Robinson and Stirling 1968). Immunological and biochemical analyses later indicated that both isoforms were composed of a and P subunit dimers and were thus two isozymes. These subunits were designated a and P, respectively. Whereas Hex A is composed of an acidic a and a basic j3 subunit, Hex B consists of two p subunits (Beutler et d. 1976; Srivastava and Beutler 1973). The two-subunit theory was confirmed in human-mouse somatic cell hybrids containing partial complements of human chromosomes. It was shown that expression of human Hex B required only the presence of , whereas Hex A required chromosomes 5 and 15 simultaneously (Gilbert et al. 1975; Lalley et al. 1974). This confirmed that the a and p subunits were encoded by distinct genes, now named HEXA (encoding a subunit), and HEXB (encoding P subunit) and A and B forms were isozymes (Fig. 1.1). Early investigations into the structure of Hex. however, were hampered by the need for many protein purification steps which may have resulted in the partial degradation of the enzyme. Figure 1.1. The HEX gene system. Three polypeptides, each encoded by a different gene, m needed for the degradation of ganglioside GM~:the a and subunits of the Hex A isozyme and the activator protein, which binds the ganglioside and "presents" it to the enzyme. Gene HEXA HEX8 GM2A Cht 5q13 Chr 5q32-33 Chr 15q23-24

Polypeptide El Activator

om a$ BB Hex S Hex A Hex B

Substrates Physiologic 1 1 1 G lycop roteins Glycosaminoglycans? Ol~gosaccharides ~gcosaminoglycans Glycolipids

complex - formation

Synthetic 6-sulfated Neutral (MUG) Neutral (MUGS) 6-sulfated (MUGS) (MUG) With the development of a specific affinity ligand for Hex by Geiger et a1 (Geiger et al. 1974), it becarne possible to purify enough enzyme for dkct investigation of the polypeptide composition of the a and subunits. Mahuran and colleagues purified Hex from human placenta and found that the a subunit of Hex A contained a single polypeptide (54 kDa), while the fl subunit in Hex A or Hex B consisted of at least two smaller polypeptides (-25 kDa) (Mahuran and Lowden 1980). They later demonstrated that the two P chains were non-identid, but because they appeared to be produced from a single locus they were called pa (acidic PI) and Pb (basic PI), 20-30 kDa (Mahuran et d. 1982). Thus the structure of Hex A and Hex B were a(p$b) and 2(paPb) (Fig. 1.2). An important finding by Hasilik and Neufeld using pulsechase techniques was that both the a and P subunits are synthesized as large precursor propolypeptides (63 kDa), which were proteolytically processed to their mature fonns in the lysosome or late endosome, a, 56 kDa; P, 28, 26, 24 kDa & other smaller fragments (Hasilik and Neufeld 1980) (Fig. 1.2). The 26, 24 kDa and "smaller fragments" where later shown to be caused by carbohydrate heterogeneity in the Pb chain (Sonderfeld-Fresko and Proia 1988). Cleavage of the large precursor P chain approximately in half explained how two nonidentical polypeptides (pa & Pb) could be generated from a single gene product. The presence of additional small peptides of Mr 7-12 kDa in the a and fl subunits has also been demonstrated (Hubbes et al. 1989). These peptides are composed of the original N-terminus of the pro a- and P-polypeptides, and are retained by disulfide bonds in the mature a and p subunits (Fig. 1.2) (Quon et al. 1989; Sonderfeld-Fresko and Proia 1989). Therefore the mature a subunit is composed of one minor and one major chain (apam), whereas the mature p subunit contains one minor and two major chains (ppPapb). Thus, the complete structure of Hex A is (%am)(babPa)and that of Hex B is 2(&bbPa) (see Fig. 1.2). For simplicity, the Hex isozymes are referred to by their subunit structures, Hex A (up) and Hex B (PP). A third unstable isozyme,

Hex S, was discovered to account for the low level of residual activity in the tissues of patients with Sandhoff disease (Sandhoff 1969; Sandhoff et al. 1971). Hex S is a homodimer of a subunits. The activator was identified in 1977 (Hechtman 1977; Hechtman and LeBlanc 1977) and purified in 1979 (Conzelmann and Sandhoff 1979). It is a small, Mr ~22,000,acidic (PI-4.8) heat stable monomer. Like the subunits of Hex it is a which is &rived from the N- terminal proteolytic cleavage in the lysosome of a larger precursor, M*24,000 (Burg et al. 1985). It is not related to the other activator proteins, i.e. SAP A-D, which are derived from a single multi-activator precursor encoded by a gene on (reviewed in (Fiirst and Sandhoff 1992)). Instead, the GM~activator is encoded by a separate gene on chromosome 5,

GM2A (Heng et al. 1993; Xie et al. 1992a). The protein specifically promotes the hydrolysis of glycolipids, particularly ganglioside, by Hex A (Meier et al. 199 1). A deficiency of the Gm activator is responsible for the group of GM~gangliosidosis patients, AB-variant, who have neither Hex A nor Hex B deficiency (Conzelmann and Sandhoff 1978) (reviewed in (Fiirst and Sandhoff 1992)).

Endaplasmic reticulum to Golgi The Signal peptide. Like secretory, plasma membrane and other lysosomal proteins, the Hex isozymes are which are synthesized and processed through a complex biosynthetic pathway via the rough endoplasmic reticulum (ER) and Golgi apparatus (reviewed in (Gravel et al. 1995)). Two molecular forms of a and f3 chains are associated within these compartments, the prepropolypeptides and the propolypeptides (or precursors). Both subunits of Hex are synthesized on ribosomes bound to the rough ER as prepropolypeptides. Targeting to the ER is achieved through hydrophobic N-terminal signal peptides (the a and P pre-sections), which are cleaved co- translationally. The signal peptides for the prepro- a and p chains were determined to be 22 or 42 amino acids in length, respectively (Neote et d. 1990a; Stirling et al. 1988). With the removal of Figure 1.2. Biosynthesis and intracellular transport of Hex A. Posttranslational modifications occurring in subcellular compartments arre shown. The first frame represents the full- length sequence of the prepro-a (529 residues) and prepro-P (556 residues) chains. It includes the signal peptides, depicted as shaded boxes. The oligosaccharides are composed of mannose, M; N-acetylglucosamine, Gn; Glucose, GI. The second frame shows pro-Hex A after monomer folding, phosphorylation of mannose residues, and subunit association occurring in the late ER/salvage compartment and cis-Golgi. A "?" is used to show that the composition of the indicated oligosaccharide is not known. The circled P represents the phosphorylation of the indicated oligosaccharides (lightly shaded boxes). The third frame represents the structure of the mature subunits in lysosomal Hex A. The polypeptides are linked by disulfide bonds, indicated "S-S". Molecular weight values are calculated from the deduced primary structures and include the Mr contributed by the oligosaccharides. - 500 Amino Acids *

The "quali control system" . - Pre Pro-&Chain Golgi network

+ 1 rGn? rGn? Golgi

trans I Golgi network LGn2Man6-7@ I I these small peptides, it is predicted that the pro-a chain has 507 amino acid residues, Mr = 58,328 and the pro+ contains 514 residues, MI= 58,749. In order for protein synthesized in the ER to exit the cis Golgi three events must occur (Edgington 1992; Newman and Ferro 1990): 1) The polypeptides are glycosylated at selected Asn- X-SedThr (where X can be any amino acid except for Asp or Pro generally found in pturns) (Kornfeld and Kornfeld 1985; Kornfeld 1986). 2) The propolypetides must fold to their near native conformation (Pelham 1989). 3) If required for functionality, multimen must be formed (Hurtley and Helenius 1989). If either of the latter two events do not occur, the unfolded andlor unassembled polypeptides are retained and eventually degraded in the ER (Edgington 1992; Hurtley and Helenius 1989; Proia et al. 1984). Glycosylation of the nascent polypeptides. The glycosylation of polypeptides is the first step in targeting the pro-isozymes to the lysosome. The pro- a or fi subunits undergo cotranslational glycosylation at three (Asn 115, 157 and 295), or four selected sites (Asn 84, 142, 190, and 327) (Fig. 1.2). This glycosylation step involves dimt transfer of a mannose-rich moiety, Glc3Man9NAc2, from a -linked intermediate to the nascent polypeptide. The first step in the initial processing of the high-mannose structures (i.e. the removal of the terminal triglucosides) also occurs in the ER. The resultant high mannose structures are subsequently susceptible to phosphorylation in the cis Golgi network. Protein folding. Like secretory and plasma proteins, lysosomal proteins are synthesized in the oxidizing environment of the ER. Thus, information on the transport of these two relatively more abundant types of proteins can be extrapolated to lysosomal proteins. Intracellular trafficking studies of secretory proteins have shown that misfolded polypeptides, e.g. due to mutation, are retained in the ER and eventually degraded (reviewed in (Ladish 1988)). In addition, even some properly folded subunits of multimeric proteins are retained in the ER. The retention of misfolded and unassembled proteins is achieved through interaction with specific ER resident proteins known as chaperones (Ferreira et al. 1994; Melnick et al. 1994). These resident proteins are believed to be recycled between the ER and the cis Golgi network. There are four major chaperones of the ER: PDI (protein disulfide ); BiP, a member of the Hsp 70 family; calreticulin, a lumenal protein; and calnexin, a transmembrane protein (Bergeron et al. 1994). These molecular chaperones promote protein translocation, folding, and oligomerization, and thus form a "quality control system" in the ER. The difference in rates of secretion between various proteins is directly related to the speed at which the newly synthesized protein can exit the EWcis Golgi network, i. e. folded andor forming multirners (Pelham 1989). One retention mechanism for soluble chaperones and any protein they may be competed with is mediated by a specific retention signal on the chaperone. This signal consists of a C-terminal KDEL (Lys-Asp-Glu-Leu) sequence or other related tetrapepticies. It is suggested that KDEL receptors, found in the cis Golgi network, bind such resident proteins and recycle them back to the ER (Hurtley and Helenius 1989). Although the degradative mechanism of misfolded or unassembled proteins in the ER is poorly understood, there is some evidence that a few of these proteins are degraded through the ubiquitin-proteasome pathway. For example, it has demonstrated that the plasma membrane protein CFI'R carrying APhe508 mutation is polyubiquitinated in the cytosol. The polyubiquitinated mutant protein then serves as the substrate of 16s protease. These data suggest that some unidentified retrograde transport pathway exists between the lumen of the ER and the cytosol. Assembly of the active Hex dimer. Proper folding is a prerequisite for both carbohydrate phosphorylation and subunit association. Pulse-chase studies in human fibroblasts indicated that both a and P precursors become phosphorylated before they associate to form ap dimers. Interestingly, the pro-a chain remained in a monomer state for at least five hours, i.e. twice as long as the pro-P chain. It is probable that a large pool of properly folded pro-u monomers is generated and retained in the ER andfor cis Golgi network so that mass action will force the pro-a monomers to associate with newIy synthesized pro-P chains to form heterodimeric Hex A, rather than the more stable homodimeric Hex B (reviewed in (Mahuran 1991)). This model would explain why only small amounts of Hex S (aa)are found in Sandhoff fibroblasts, despite the presence of normal amounts of a-mRNA (Korneluk et al. 1986); whereas large amounts of Hex B (PP)accumulate in Tay-Sachs fibroblasts (Sandhoff 1969). Phosphorylation of mannose residues in carbohydrates. For many glycoproteins destined for the lysosome, the critical modification is phosphorylation (Hasilik et al. 1980). The generation of the mannose-6-phosphate tag is initiated in the cis Golgi network, and is accomplished by the sequential action of two enzymes. First, UDP-G1cNAc:lysosomal enzyme N- acetylglucosarnine- 1-phosphotransferase, transfers GlcNAc- 1-phosphate from UDP-GlcNAc to selected mannose residues on the al,6-linked branch of a high mannose oligosaccharide. The initial phosphorylation may occur in the cis Golgi network, which is believed to bridge the ER and cis Golgi. Then, in the cis Golgi compartment, a second phosphorylation can also occur at a mannose residue in the a1 3-linked branch. Both phosphorylation steps require a properly folded protein domain for recognition by the enzyme and give rise to phosphodiester intermediates. The second enzyme involved in the generating the man-6-P signal is N-acetylglucosamine- 1- phosphodiester-a-N-acetylglucosaminidase,which is located in the cis GoIgi compartment and removes the terminal GlcNAc residue. Golgi sorting. The newly synthesized, properly folded and dirnerized pro-Hex isozymes enter the cis Golgi through the cis Golgi network. Transport from the cis Golgi to the medial, trans, and trans Golgi network compartments is via bulk-flow. In these compartments Hex and other proteins may undergo a variety of posttranslational modifications to their oligosaccharide moieties resulting in the formation of complex type oligos. However, the great majority of these processing steps is blocked if the oligosaccharide contains a M-6-Pgroup. The phosphomannosyl residues serve as the essential component for the high affmity binding of lysosomal proteins to one of two MPR (mannose-6-phosphate receptor), i.e. the MPR-CI (cation independent) and the MPR-CD (cation dependent) (Dahms et al. 1989). The lysosomal protein- MPR complex then exits the trans Golgi network via a coated vesicle and is delivered to a late endosome. Dissociation of the receptor-ligand complex occurs by acidification of this organelle. The receptors are then recycled back to the trans Golgi network andlor plasma membrane to ferry additional ligands. However, some of the pro-Hex molecules do not bind to the MPRs, presumably because of underphosphorylation and/or saturation of the receptor pool. This results in the enzyme following the cell's default secretory pathway, i.e. appearing in cell culture medium or in body fluids (Fig. 1-2).

Lysosod incopration In the lysosome, a number of pmteolytic as well as glycosidic processing events occur to the pro-Hex isozymes (Fig. 1.2) (reviewed in (Mahuran 1991)). The glycosidic processing of Hex isozymes in the lysosome involves the rapid removal of the mannose 6-phosphate tag dong with most of the exposed carbohydrate residues, reducing the Mr and pI heterogeneity due to the oligosaccharides (OfDowd et al. 1988). The proteolytic modifications result in the Ioss of an excess of basic amino acids, and give rise to the mature subunit structure of Hex A and Hex B. Thus the mature forms are slightly more acidic than their precursor counterparts. The 65 kDa a- precursor is processed to generate the 7kDa unglycosylated a,,chain and the glycosylated 56 kDa a, polypeptide (Fig. 1.2). Similarly, the 63 kDa P-precursor is cleaved into three mature polypeptides, the glycosylated pp (1 1-14kDa), pa (28-26 kDa), and Pb (24-22 kDa) chains. The two (apam) and three (babPa)polypeptides chains are held together in their respective mature subunits by disulfide bonds (Fig. 1.2). An important implication of these Hex modifications is that the mature subunits are easily distinguishable from their respective pro-fonns after reduction and separation by SDS-PAGE, and make the molecular weight change an excellent lysosome incorporation marker.

Lysosomal Hex cleaves the glycosidic bond of terminal, non-reducing, $-linked GalNAc or GlcNAc residues from numerous &xcial and natural substrates. Whereas the a and P subunits in any of these dimeric forms are able to hydrolyze many of the same neutral natural substrates, e. g. N-linked oligosaccharides, and artificial, e. g . 4-Methylum belliferyl-GlcNAc (4-MUG), substrates, only those containing an a subunit can hydrolyze negatively charged natural, e.g. GlcNAc-6-sulfate terminal MPS (mucopolysaccharide) and Gmganglioside, and &cia& e.g. 4- MUGS (4-MUG-6-sulfate), substrates. However, in vivo only the heterodimer can hydrolyze GM~and requires the Activator as a substrate-specific co-factor. Because of the complexity and low Vmax of these natural substrate assays, in vitro Hex activity is assayed using artificial substrates. These artificial substrates are commercially available and are hydrolyzed at a rate -1000-fold faster than the natural substrate; however, they axe not as isozyme and co-factor specific as the naniral substrates.

A rticial substrates The most common artificial substrate used to assay Hex is the non-fluorescent PGlcNAc derivative of the fiuorophor compound 4-MU (4-methylumbelliferone), i.e. 4-MUG.Since 4- MUG is an uncharged substrate (Fig. 1.3), it is recognized by all the Hex isozymes. An a-subunit specific (Hex A and S) negatively charged substrate, 4-MUGS, has recently been developed (Bayleran et al. 1984). MUGS contains the 6-sulfate derivative of N-acetylglucosamine (Fig. 1.3). The level of enzyme activity is measured by the fluorescence of the MU produced after incubation at pH 4.3. Optimal conditions for Hex assays have been studied extensively. The pH optimum is usually between 4.1 and 4.5. Acetate buffers inhibit Hex; thus the assays are normally performed in citrate-phosphate buffers (reviewed in (Mahuran et al. 1985)). Generally for purified or partially purified enzyme 0.1-0.3% human albumin, pretested for residual Hex activity, is included in the assay to prevent binding of the enzyme on vessel walls.

Natural substrates While Hex A can hydrolyze natural substrates such as glycolipids, gangliosides, glycoproteins and glycosaminoglycans, ganglioside GM~is the compound stored in all three GM~ gangliosidoses (Tay-Sachs, Sandhoff and the AB-variant form) and thus is the most useful natural substrate. -2 is a consisting of a hydrophilic end, a tetrasaccharide unit, GalNAc~(l-4)-(NANAa(2-3)-Ga~(1-4)-Glc,attached to a hydrophobic lipid moiety, ceramide (Fig. 1.4). In vivo in the presence of the -2-activator protein Hex A cleaves the terminal B- GalNAc residue from GM~to generate GM~.Radiolabeled GM~is used to increase the sensitivity Figure 1.3. Struchm of the artificial substrates used to assay Hex A and B activity; 4- Methylumbelliferyl-GlcNAc (&MUG) (panel A-used to assay Hex A, B or S); 4-

Methylmbelliferyll-GIcNAc-6-sulfate (4-MUGS) (Panel B-used to assay isozymes containing an a-subunit, i.e. Hex A and S).

Figure 1.4. Structure of GM~ganglioside. GmAc Gal Glc ceramide of in vitro enzyme assay. Generally, Hex A activity is determined by the radioactive counting of the 3~GalNAcresidue released from labeled Gmafter the separation of the product (neutral) from the substrate (negative) by ion-exchange chromatography. Both Hex A and Hex S are capable of degrading GM~in vitro when activator protein is

replaced by a bile salt detergent, e.g., Na Taurocholate. However, in contrast to assays utilizing the activator, which are linear for 25 to 40 hours, detergent-based assays lose linearity after about 1 hour (Enberger et al. 1980). In the latter case the loss of linearity is caused by a loss of enzyme activity since enzyme is slowly denatured in the presence of the detergent. Therefore, activator- based assays not only offer increased specificity, but also produce a linear rate of hydrolysis over a longer period of time. In addition to glycolipids such as ganglioside GM~,oligosaccharides also serve as natural substrates for Hex (reviewed in (Mahuran et al. 1985)). GIycosarninoglycans are oligosaccharides composed of disaccharide units with varying degrees of sulfation. One such compound is keratan sulfate, which contains pl-3-linked GlcNAc-6-S04. Hex A can directly hydrolyze the GlcNAc-6-

SO4 motif from keratan sulfate; however, there is also a specific sulfa- present which can produce a neutral tenninal PGlcNAc residue, which is a substrate for Hex B as well as Hex A

(Kresse et al. 1981; Ludolph et al. 1981). Bayleran et a1 (Bayleran et al. 1984) used this observation to develop the a-specific artificial substrate, MUGS. Other oligosaccharides that can be hydrolyzed by Hex include asparagine-linked oligosaccharides on glycoproteins (containing terminal P-GlcNAc residues), and - and threonine-linked oligosaccharides (containing both PGlcNAc and P-GalNAc residues) (Schachter 198 1). Glycoproteins are degraded primarily in the liver. Unlike the lower abundance MPS substrate, defects in Hex A and B activity in Sandhoff disease result in storage and excessive excretion of some partially degraded oligosaccharide. It is believed that in Sandhoff disease the low level of Hex S is sufficient to hydrolyze the MPS substrate. Hex punjCication Initial purification of Hex from human placenta or liver includes the separation of Hex and other glycoproteins from the total soluble protein fraction by Concanavalin-A-Sephmse affinity chromatography. A highly specific ligand, 2-acetamido-N-&-aminocapoyl-2-deoxy-PD- glucopyranosylamine, coupled to Sepharose 4B or Sephacryl S-200 is then used as a specific Hex affinity column (Geiger et al. 1974; Mahuran and Lawden 1980). The final separation of Hex isozymes can be achieved by any pHcation technique that separates proteins based on their pf, i.e., charge differences. Anionic exchange, DEAE chromatography is the method predominantly used for preparative isozyme separation (Mahuran and Lowden 1980). Whereas Hex B does not bind to the DEAE column at pH 6.0, Hex A and any minor Hex S can be separated with a NaCl gradient (Hou et al, 1996). Other methods used to separate the Hex isozymes include isoelectrof~using(IEF) (Sandhoff 1969) and chromatofocusing (O'Dowd et al- 1986). IEF has shown that Hex B, A and S have pls of 6.9,4.8 and 3.5, respectively.

Hydrolysis of GM~gangliosidk requires the interaction of three proteins It appears that only the Hex A isozyme has a unique in vivo function, i.e. to catabolize the GM~ganglioside. Because Hex A is a heterodimer, and GM~hydrolysis also requires a substrate- specific , the Activator, this reaction involves three proteins. A model of GM~hydrolysis by Hex A was proposed by Sandhoff ((Sandhoff et al. 1989), Fig 1S). In this model, the water- soluble Hex A alone cannot degrade membrane-associated GM~ganglioside, owing to steric hindrance by the membrane surface. That task is accomplished by the enzyme acting on a gangliosiddActivator complex. The Activator protein acts to extract or lift the ganglioside from the membrane surface and to present it to the water-soluble enzyme for hydrolysis. The Activator and Gw ganglioside form a 1:l complex strong enough to survive native PAGE (Conzelmann et al.

1982). Thus, the complex is the true in vivo substrate for Hex A. After hydrolysis. the cleavage product, -3, is reinserted into the membrane and the Activator is available for another round of catalysis (Fig. 1S). The fact that only heterodimeric Hex A, not homodimeric Hex B or S, can hydrolyze suggests that elements of both the a and P subunits in Hex A are necessary for correct Activator:- binding. Whereas the a subunit provides the catalytic site for GM~hydrolysis (Hou et al. 1996a), the f.3 subunit is somehow necessary for the correct binding or orientation of the activator/ganglioside complex. To directly demonstrate that the f3 active site does not participate in GM~hydrolysis, I did two different experiments. First, I generated a novel form of Hex A with an inactive p* (R211K) subunit and demonstrated that the up*-Hex A had the same level of GM~ hydrolysis as did the WT Hex A (Hou et al. 1996a). Second, when I tested another HEXB gene mutation encoding PProSO4Ser, I found that ap*(P504S)-Hex A from either transfected cells or patients' fibroblasts had 3-fold less activity towards the activatorlG~ganglioside complex as compared to its MUGS activity, suggesting P*(P504S) may be involved in the binding of the complex (see Chapter 2, Hou et al, 1998).

GENETICS AND MOLECULAR DEFECTS IN GM~GANGLIOSIDOSES

Structure of HEXA and HEXB Penes In the mid-1980s' the structural characterization of Hex A and Hex B isozymes were given a major boost with the isolation of cDNA clones encoding the a and B subunits (Komeluk et al. 1986; Myerowitz et al. 1985; Myerowitz and Proia 1984; O'Dowd et al. 1985). Subsequently, the structures of the HEXA and HEXB genes were determined. The HEXA gene is 35 kb long and contains 14 exons (Proia and Soravia 1987). The promoter region is GC-rich with possible TATA and CAAT box motifs. HEXA maps to chromosome l5q23-qU (Nakai et al. 199 1). The HEXB gene is about 45 kb long and also contains 14 exons (Neote et a.. 1988; Proia 1988). Its promoter area has been confirmed experimentally. It contains several GC-box-like sequences, as well as others matching consensus sequences of various promoter elements including steroid Figure 1.5. Model for the lysosomal catabolism of GM~ganglioside. Hex A does not directly interact with the membrane-bound ganglioside. Instead, the ganglioside is extracted from the membrane by the Activator protein. The xesulting water-soluble activator-lipid complex is the substrate for the Hex A enzymatic reaction. After the reaction, the lipid product, gangliosik %3 is re-inserted into the membrane and the activator protein is available for another round of catalysis. GaNAco 04~eu4c 4 GMGangliodde [ Phospholipids responsive elements (Neote et al. 1988). HEXB gene locates on chromosome 5q13 (Bikker et al. 1988). A cornpatison of the deduced primary sequences from the cDNAs revealed an overall -60% identity. As well, the structures of the HEXA (a subunit) and HEXB (P subunit) genes show a striking degree of homology in both the number and the placement of exohtronjunctions. These data suggested that HEXA and HEXB arose from a common ancestor (Korneluk et al. 1986; Proia 1988). An important implication of the primary structural homology between the a and f3 subunits and the common evolutionary origin of the two genes, is that functional domains and "active" residues will be conserved within the aligned sequences. One such functional residue, a-Arg 178, which aligns with J3-Arg211, provides experimental support for this hypothesis. Both of these residues have been shown experimentally to be necessary for the catalytic activity in their respective subunit (Brown and Mahuran 1991).

Degradation of Gmrequires the interaction of the a and P subunits of Hex A in the ER as

well as Hex A and the activator protein in the lysosome. Defects in any one of these proteins result in excessive intralysosomal accumulation of ganglioside GM~and one of the three forms of GM~

gangliosidosis: Tay-Sachs disease, Sandhoff disease and AB variant (reviewed in (Gravel et al. 1995)). Tay-Sachs disease is due to deficiency of a subunit caused by mutations in the HEX4 gene (Bartholomew and Rattazzi 1974). Thus, these patients are deficient in Hex A and Hex S (Okada and O'Brien 1969). Biochemically, this disorder is characteri~dby normal or elevated leveIs of Hex B and has therefore also been referred to as the "variant B fom of GM~gangliosidosis (Sandhoff et al. 1971). A few patients with a novel form of Tay-Sachs disease have been found to synthesize an a subunit that associates almost normally with the P subunit but is devoid of catalytic activity. The resulting dirner, a$,behaves like nonnal Hex A in some respects such as isoelectric point and catalytic activity toward several synthetic substrates, but it is inactive towards the a- specific sulfated artificial substrate (e.g. MUGS) and the physiological substrate %(Kytzia et d. 1983; Li et al. 1981). The unique biochemical characteristic of these patients makes them a subtype of TSD or variant B, i.e. the "B 1variant". Sandhoff disease mults from HEXB gene mutations and affects the P subunit, the common subunit in both Hex A and Hex B (Sandhoff et al. 1971). Thus, these patients are deficient in both isozyme activities. This variant has been designated the "variant 0" form, indicating zero Hex activity. There is however a small amount of residual Hex activity (el% of normal) associated with this variant produced from the labile a homodimer, Hex S (Beutler et al. 1975). Since a CRM (cross-reacting material) is very low even though a mRNA levels are unaffected in Sandhoff disease, it is possible that the vast majority of a chain protein is unable to form dimers and exit the ER (reviewed in (Mahuran 1991)). Al3 variant is caused by deficiency of the GM~activator protein due to the mutations in the GM2A gene (Conzelmann and Sandhoff 1978; Schroder et al. 1993; Xie et al. 1992b). In this variant, the Hex A and Hex B isoenzymes are not affected, which suggested the classification "variant AB".More recently, cDNA clones encoding the GM~activator have been isolated (Klima et al. 1991; Xie et al. 1991). The known structure of the GM2A gene, which remains incomplete at the 5' end, suggests that it is a small gene of at least 16 kb, The gene contains at least four exons and has been mapped to chromosome 5q31.3-33.1 (Heng et al. 1993).

Clinical phenotypes of GM~gmgEiosidoses The clinical phenotypes of GM~gangliosidoses show great variability. On the basis of their age at onset of clinical symptoms, patients are generally classified into acute (classical infantile type), subacute (late infantile and juvenile types) and chronic forms (adult and chronic types) (reviewed in (Mahuran 1991)). Generally, the earlier the onset of symptoms the more severe the resulting disease. AU three forms of GM~gangliosidoses have a similar course within a given phenotype (although only the infantile form of AB-variant has been described). The most common type of the GM~gangliosidoses is the acute form, which is also the clinicaUy and biochemically least variable. While affected infants generally appear completely normal at birth, they usually begin to show mild motor weakness at 3 to 5 months of age. More profound neurological symptoms are evident later in the first year of life and thereafter motor and mental deterioration develop rapidly. Death usually occurs by the age of 4 years. The subacute form generally presents as ataxia and progressive psychomotor retardation between 2 and 10 years of age. A vegetative state with decerebrate rigidity develops by age 10 to 15, followed by death within a few years, usually due to infection. The chronic form is the most clinically variable, even between members of the same family (Argov and Navon 1984). However, in virtually all cases, the= is evidence of widespread central nervous system involvement. Symptoms include spinocerebellar and lower motor neuron dysfunction and psychosis in 33% of patients. Some individuals are clinically asymptomatic but have very low Hex A levels by standard assay procedures (Conzelmann et al. 1983). These variant forms are sometimes referred to as 'pseudo- deficiencies'. Thus individuals with very similar low levels of Hex A activity can be asymptomatic or present with dramatically different clinical phenotypes.

Whereas mutations that cause the complete loss of Hex A activity, e.g. partial HEXA or

HE=XB gene deletions (see below), give rise to the acute form of GM2 gangliosidosis, it is believed that mutations leaving even very small levels of residual Hex A activity give rise to diseases of later onset and milder course. The large variations in subacute and chronic phenotypes are believed to be associated with residual Hex A activity in patient samples >O but 40% (Sandhoff et al. 1989). Thus, when mutations do not totally preclude the production of active Hex A, other unrelated gene products can modulate its residual activity and/or the rate of substrate synthesis, even by small amounts. This may result in patients with .the same genotype presenting with different clinical phenotypes. It is also very difEcult to characterize the mutant Hex A isozymes in patient cells because of these very low residual enzyme activities (Gravel et al. 1995). The lack of clinical symptoms in the 'pseudo-deficiencies' may arise from their higher residual Hex A activity, i.e. 210% of normal.

Critical threshold model The critical threshold model was proposed on the basis of the correlation between the residual activity of a lysosomal enzyme, e.g. Hex A, and the turnover rate of its substrates, e.g. ganglioside. This model also proposes some simplified conditions, i.e. a substrate to be degraded is enclosed within the lysosome and there is no feedback regulation or influence of the substrate concentration on other biochemical pathways. Utilizing an assay system employing the Activator protein and radiolabeled GM~ganglioside as a substrate, a correlation between residual Hex A activity and the severity of the resulting disease has been detexmined (Conzelmann et al. 1983). Activities found for acute, subacute, and chronic patients were 0.1 %, 0.596, and 2-4% of normal controls, respectively. Two clinically healthy probands with low Hex A activity were found to possess activities of 11 and 20% (Dreyfus et al. 1977). Thus, this data suggests a "critical threshold", i.e. the minimum amount of Hex A activity required to keep the rate of GMZ hydrolyzed greater than or equal to the rate of ganglioside transport and incorporation into the lysosome. For Tay-Sachs and Sandhoff disease this is between 5 and 10% of normal Hex A activity (Conzelmann and Sandhoff 1984).

Animal models for the GM~gangliosidoses Mouse models have been produced for both Tay-Sachs and Sandhoff disease. While the knockout Sandhoff mouse carries a clinical phenotype closer to that observed in human patients, the Tay-Sachs mouse does not develop a clinical phenotype even though it has biochemical and histological evidence of GM~ganglioside storage (Phaneuf et al. 1996; Sango et al. 1995). The suggested explanation for this was that mice have a sialidase, with a substrate specificity diffenmt from that of its human counterpart. The mouse sialidase may be able to hydrolyze the sialic acid from GM~ganglioside to produce GA~,which is an in vivo substrate (although a poor one) of Hex B. Interestingly, both the Tay-Sachs and the Sandhoff mutant mice a fertile in the early part of their lives, and the two mutant mice have been bred to produce offsprings with a total lack of Hex activities. The double mutant mice display a severe clinical phenotype of gangliosidosis and mucopolysaccharidosis, i.e. accumulation of glycosarninoglycan (Sango et al. 1996). These data indicated that glycosaminoglycans, like gangliosides, are critical substrates for Hex. In the case of Sandhoff disease, the low residual level of Hex S must be sufficient to hydrolyze these substrates in mice and humans, a hypothesis initially suggested by Kresse et aL (Kresse et al. 1981; Ludolph et al. 1981). In addition, despite the death of these mutant mice, when bone marrow fiom nonnal mice was transplanted to Sandhoff mice, the mutant mice have double their life expectancy with a slower neurological progression of the disease (Norflus et al. 1996). This study supports the idea that bone marrow transplants will not be an effective therapy for lysosomal storage diseases with neurological involvement because of the inability of the secreted enzyme to cross the blood-brain barrier. The AB-Variant mouse produced a clinical phenotype intermediate to that of the other two. Storage of GM~was only slightly increased over the Tay-Sachs mouse. However, in this case some GA~storage was also evident, but still at a level -10-fold less than that of the Sandhoff mouse (Liu et al. 1997). The lower level of GA~storage suggests that this ganglioside can be

hydrolyzed in the absence of the activator, but that its presence is likely necessary for an optimal rate of degradation. Although earlier work had suggested the possibility that mouse Hex B could hydrolyze GM~in the presence of activator (Burg et al. 1983), a more recent study with the fully

purified Hex isozymes from mice and recombinant mouse activator, demonstrated that it can not (Yuziuk et al. 1998). This study went on to show that in contrast to its human analog, the mouse activator can effectively stimulate the hydrolysis of GA~by mouse Hex A and as well, but to

much lesser extent by mouse Hex B. Thus these mice models have demonstrable species-specific differences in the activator, Hex A, Hex B, and lysosomal sialidase, and indicate that making extrapolations from them to the human condition is risky.

Various mutations have been characterized in the HEM and HEXB genes (primarily in the HEXA gene) from patients with gangliosidoses. These genotypic changes can be placed into a number of broad categories. Pdgene&letions Three partial gene deletions have been described, one in the HEXA gene (Myerowitz and Hogikyan 1986; Myerowitz and Hogikyan 1987), and two in the HEXC) gene (Neote et al. 1990b; Zhang et al. 1995). The HEX4 deletion is 7.6 kb long, containing exon 1 and 7 kb of flanking DNA. This mutation occurs frequently in the French Canadian population (De Braekeleer et al. 1992). In the case of HEXB gene deletion mutations, the most common one thus far identified is a 16-kb deletion spanning the promoter, exon 1 through 5, and part of intron 5 (Neote et al. 1990b). This mutation makes up a surprising 25% of all Sandhoff alleles. Another HEXB mutation is a 50 kb deletion from 25 kb upstream of the promoter to intron 6 (Zhang et aI. 1995). The 7.6 kb HEXA or the 16 kb HEXB gene deletions have been well analyzed. Patients homozygous for either of these deletions have been described and present with the classical acute form of GM~gangliosidosis; indicating that this clinical phenotype results from the total lack of all

Hex A activity. This conclusion also explains why the acute form shows the least clinical variation since there is no possibility of any variation in residual Hex A activity.

Mutations proriucing early stop coclons There are numerous cases of GM~gangliosidosis caused by premature stop codons. Remature stop codons can be generated directly through a nonsense point mutation, or indirectly through a fhne shift, small deletions or insertions. The effect of early stop codons rn usually expected to produce a truncated protein that may have some residual activity depending on how close to the C terminus the new Stop occurs. However, this assumption has proven to be incorrect for HEX mutations because of the following two characteristics. First, a unique 'quality control system' in the ER allows chaperones to recognize abnormal proteins (including the mutant proteins resulting from early stop codons), retain and ultimately degrade them. Second, like many other mRNAs transcribed from mutant genes, HEX rnRNA containing a premature stop codon has a greatly reduced stability, often making it undetectable by Northern blotting. An example of frame shift, producing a Stop (at codon 431), is a 4 bp insertion, which occurs quite frequently in the Ashkenazi Jewish population (81% of mutant alleles in a population with -1:30 carrier frequency) and in the Cajun population (70% of mutant alleles) (McDowell et al. 1992)). No steady-state -A was detected in these patients' cells even though nuclear run-off experiments confirm that the mutant HEXA gene is transcribed at a normal rate (Paw and Neufeld 1988). To date there have been six nonsense mutations reported. None of them produced detectable levels of mutant mRNA as measured by Northern blot. One exception to the above observation is a single bp deletion mutation in exon 13 of the HEX4 gene, AC15 10. This mutation results in normal steady state levels of amRNA encoding 4 new amino acids before generating a STOP at codon 507, i.e. the mutant mRNA encodes a loss of 22 residues from the C-terminus of the proa chain. However, despite the normal mRNA levels, only low levels of the mutant proa chain can be detected (Lau and Neufeld 1989). These data suggest that the closer the STOP codon is to the 3' end of the coding region the more stable may be the mutant rnRNA.

Mutations affecting mRNA processing This type of mutation is at or near an introdexon splice junction and thus affects mRNA processing. The general consensus sequences of the 5' and 3' splice sites are well established (Krawczak et al. 1992). Four of these nucleotides are invariant, which include the first and the last two nucleotides of an intron (intervening sequence, IVS), i. e. TVS + lg and +2u of the 5' splice site, as well as, IVS -2a and -1g of the 3' splice site (Padgett et al. 1986). Among the thirteen mutations of this type identified in the HEM and HEXB genes, seven of them that affect mRNA splicing involved one of these 4 nucleotides. Patients hornozygous for this type of mutation have no detectable mRNA and present with the acute phenotype (reviewed in (Gravel et al. 1995)). For mutations beyond these 4 nucleotides, the consensus sequences have only a limited usefulness in predicting biochemical and clinical phenotypes. The majority of the other splicing mutations allow some normal mRNA to be produced, resulting in detectable residual Hex A activity and milder clinical phenotypes. Of interest are two HEXB gene mutations outside of the above 4 nucleotides, which affect mRNA splicing. In both cases some properly spliced mRNA is produced with detectable Hex A activity ranging from 5% for two patients with subacute Sandhoff disease, to 10% for asymptomatic individuals. Thus they serve as good examples to demonstrate the validity of the critical threshold model. The first HEXB gene mutation, associated with subacute Sandhoff patients, was identifled as a 'g-26a' substitution at the 3' end of intron 12. This substitution created a new 3' splice site, leading to a mRNA encoding an extra 8 amino acids between exons 12 and 13 (Nakano and Suzuki 1989). These patients' cells produce normal steady-state levels of

PmRNA, of which 95% encodes the additional 8 residues. The remaining -5% is properly spliced

PmRNA and results in the 5% residual Hex A activity, i.e. no Hex B is formed from the residual normal p subunits (Dlott et al. 1990). The second HEXB mutation is associated with patients previously designated as "Hexosaminidase Paris" due to their Hex A+/Hex B- isozyrne pattern @reyfus et al. 1977). These individuals are clinically asymptomatic, producing approximately 10% of normal Hex A activity. The mutation causing this biochemical phenotype is a duplication of 16 bp of intron 13 and the fust 2 bp of exon 14 producing an inframe insertion of 6 amino acid codons. Like the previous mutation, normal steady-state levels of mRNA can be detected, but 90% of the mRNA encodes the additional six residues. Again the remaining 10% encodes normal

P-subunits which preferentially form into Hex A heterodirners rather than the more stable Hex B homodimer, resulting in the characteristic Hex A+/Hex B- biochemical phenotype (Dlott et al. 1990; McInnes et al. 1992). Both of above mutations cause an in-frame insertion of several codons in 90-95% of the mRNA transcripts encoding elongated pro-P chains. However in both cases only small amounts of the elongated pro-P chains can be detected immunologically in patient cells. These mutant pro-P chains do not contain phosphorylated mannose residues and are recognized by antiserum specific for the unfolded form of the P-chain. Thus, the elongated pro-P-chains are detained in the ER and degraded (Dlott et al. 1990), i.e. a fate similar to that of most missense mutations (see below). Missense mutations To date the= have been at least 60 point mutations encoding amino acid substitutions described in the HEX4 and HEXB genes. All missense mutations at all but two codons (B1 mutations, see below) in either HEX gene result in normal levels of mutant mRNA coupled with a dramatic reduction in both mature andlor a protein (CRM),and Hex B andor Hex A activity in patient cells. As previously discussed, many data indicate that mutant forms of either subunit caused by missense mutations, as well as the wild type a chain in cells deficient in P, may undergo rapid degradation at an earlier stage in their biosynthesis and/or intracelllar transport. These deficiencies are caused by the ER which has a tight "quality control system". Most missense mutations likely alter the folding, decrease subunit affinities, or at least change the surface properties of the affected subunit sufficiently so that it is recognized by one or more chaperones as being "abnormal" and/or monomeric proteins. Thus, the major deleterious effect caused by most HEX missense mutations may be at the level of intracellular transport rather than structure- function. This hypothesis is supported by the biochemical data from three HEXA missense mutations, i.e. aGly269Ser, aGly250Asp and aGlu482Lys, associated with the chronic, subacute and acute forms, respectively. When these different mutant acDNAs were individually overexpressed in COS-1 cells along with the wild type @DNA, surprisingly high levels of Hex A activity were detected as compared to activity levels in patient cells (Brown and Mahuran 1993). This is due to the fact that COS cells over express protein encoded by inserts in vectors containing the large T antigen. This results in transfected cells expressing 200- to 400-fold more of the protein than the corresponding endogenous protein in nontransfected COS cells (Brown and Mahuran 1993). The effect of over-expression would be to greatly increase the concentration of Hex pro- polypeptides (mutant or wild type) in the ER. This increased concentrations could either help drive protein folding and/or herformation through a form of mass action (Alber 1989; Dill and Shortle 1991; Pakula and Sauer 1989), and/or more simply by saturating the chaperone(s) that recognk the mutant protein as "abnormal". A similar effect was also observed in COS cells transfected with

wild type acDNA alone. Unlike the situation in Sandhoff patient cells, reasonably high levels of

Hex S in the transfected COS ells were detectable. However when the above three mutant

mDNAs were transfected without the wild type P, little or no activity was detected (Brown and

Mahuran 1993). Thus, the over expression of the proa chain alone can enhance the fonnation and

intracellular transport of wild type Hex S, but it is not strong enough to overcome the added

destabilizing effect of an a missense mutation.

Biochemically, a rnissense mutation that affects the packing of the hydrophobic core of a protein causes the greatest disruption to its overall folding pattern whereas those affecting residues located on the surface of a protein lead to the least disruption (Alber 1989; Goldenberg et al. 1989). In general, a missense mutation resulting in a mutant protein, which retains some abiliw to fold, produces a less severe clinical phenotype. Expression of such mutations in COS cells often result in reasonably high levels of Hex that may also be heat labile. Examples of mutations affecting heat

stability include aGly269Ser, aTyrl80His, PArgSOSGln, all of which are associated with the

chronic form of gangliosidosis (reviewed in (Mahuran 1997)).

Two missense mutations in exon 7, aArg247Trp and aArg249Trp, present with a

pseudodeficiency of Hex A. They are responsible for most of the false positives obtained by

enzymatic-based Tay-Sachs screening procedures. In these tests carrier status is defined as Hex A activity -40% of the total Hex activity (Cao et al. 1993; Triggs-Raine et al. 1992). These

substitutions were shown to significantly reduce the a-subunit protein levels by affecting its

stability in vivo, rather than by affecting the processing of the mutant a-subunit (Cao et al. 1997).

The small amount of Hex A is probably fully functional and can hydrolyze GM~(Cao et al. 1997) at a level above the "critical threshold" (Conzelmann and Sandhoff 1984) in order to prevent the storage of ganglioside. The BI-variant of Tay-Sack disease An interesting, yet rare mutational effect results in the B1-variant form of Tay-Sachs disease. The "B 1 vatiant fond' of Tay-Sachs disease represents missense mutations that produce a unique biochemical phenotype. Mfected individuals produce nonnal levels of Hex A and Hex B activities when assayed using 4MUG and the heat denaturation assay. Therefo~,these patients were initially thought to have GM~activator defects (the AB variant form of GM~gangliosidosis). However, when assayed with LGMUGS or GM~ganglioside in presence of the activator protein, Hex A levels are barely detectable. The explanation for this unique biochemical phenotype is that

the mutation primarily affects the a-active site without affecting subunit folding or association and

thus transport to the lysosome. The result is a Hex A with only an active P subunit. Thus, the study of these types of mutations can lead to a localization of the residuedarea in the a sequence that produce its functionality, i.e., its active site domain.

The most common B 1 variant mutation was an aArg178His substitution, located in exon 5 of HEXA gene (Ohno and Suzuki 1988) (reviewed in (Suzuki and Vanier 1991)). Sequence comparison indicated that aArg178 is invariant among 15 species as diverse as scotobacteria (Tse

1996). Patients homozygous for this substitution are associated with a subacute phenotype (Dos Santos et al. 1991). Two other mutations at the same Arg 178 codon have been identified, i. e. Argl78Cys and Argl78Leu flanaka et al. 1990; Triggs-Raine et al. 1991). In both cases the patients show the more severe, acute phenotype. This increased severity is consistent with the observation that these patients samples have much less Hex A activity (as assayed with MUG) and aCRM, presumably because the less conservative amino acid substitutions also affect protein folding.

Linking a missense mutations associated with the B 1 phenotype to specif~cfunctions is complicated by the presence of the normal functional P subunit in the Hex A heterodimer. For instance, it was not known from the initial data from B1 patients samples whether the affected a

subunit had undergone a partial change in substrate specificity (i.e. become Plike) and could no

longer bind negatively charged substrates, but could still hydrolyze neutral substrates; or whether it had lost its catalytic ability entirely. Furthermore, subtle changes to the stability, capacity for

intracellular transport, or processing of the a subunit could well be masked by the presence of the

normal p subunit. On the other hand, expression of a mutant a cDNA alone in hopes of producing

mutant Hex S for analyses would be problematic due to mutant a subunits' over-sensitivity to any

amino acid substitution; as demonstrated by its failure to form aGly269Ser Hex S (associated with

the mild adult and chronic forms of Tay-Sachs disease) (Brown and Mahuran 1993; Navon and Proia 1989). To overcome this problem, Mahuran and colleagues developed an alternative method to analyz the classic aArgl78His, the B1 mutation that involved mutating the codon for the aligned Arg2ll residue in the P subunit. This approach took advantage of the common evolutionary origin of the a- and P- subunits which indicates a conservation in structure-function relationship. By expression of the mutant PArg211His construct in monkey COS cells they were able to examine the effect of this mutation on P subunits which are normally capable of forming stable, active homodimers, Hex B. It was found that the mutant P homadimers were formed and incorporated into the lysosome in a manner nearly identical to that of normal Hex B, but caused a near total loss of normal Hex B activity (Brown et al. 1989). Consistent with the initial report of this substitution and a computer-prediction on its effects on the secondary structure of the a subunit (reviewed in (Suzuki 1991)), Brown and Mahuran also noted small additional changes in the stability and rate of maturation of the mutant Hex B protein. These additional effects were totally eliminated by a more consewative Lys substitution (f3Arg211Lys). Through kinetic studies

and the use of Arg-specific modifying agents, they were able to conclude that the pArg2ll and, by

extrapolation aArg178, are active site residues, probably part of the catalytic sites (normal Km, but

only 0.2% of normal Vmax for MUG) (Brown and Mahuran 1991).

The second mutation associated with B1 variant, olAsp258His, is within exon 7 of the

HEXA gene. It was identified in two patients with acute Tay-Sachs disease (Fernandes et al. 1992). Both patients were found to retain -16% of their Hex A activity with MUG, but less than 1% with MUGS (F3ayleran et al. 1987). Using the similar approach to aArgl78His, we analyzed a conservative substitution at the analogous P-residue, PAsp290Asn. Kinetic analysis demonstrated that the mutant PAsp290Asn homodimer had 2.5-fold higher Km and the reduced apparent Vmax (3% of normal) as compared to the wild type Hex B. We also found that this decreased Vmax primarily resulted from a reduction in mature P-CRM (Tse 1996). Thus, the PAspZgOAsn, and by extension the aAsp258His (His being a much less conservative substitution) appears to affect both substrate binding and intracellular transport. A mutation at a third residue, uVall92Leu, was also reported to link with the B1 phenotype. This mutation was located in exon 6 of the HEXA gene. However, aVal192 corresponds to PAla224 and therefore is not conserved in the P subunit. The absence of homology suggested that it is not a catalytic-site residue, but could be part of the for a-subunit- unique, negatively charged substrates. To test the effects of this mutation on its substrate binding or catalysis I developed a new strategy for analyzing a-chain mutations. This method still avoided the production of the unstable Hex S isozyme while eliminating the problem of a functional P subunit in Hex A. I cotransfected the wt or mutant acDNA with a P*cDNA encoding the $kg21 lLys substitution. This allowed me to generate a novel form of ap*-Hex A with inactive J3Arg2l lLys subunit. Thus, any enzymatic activity from the a$*-Hex A must come from the a subunit I demonstrated that when this procedure is used with a normal acDNA, a Hex A (ap*) with nearly identical kinetic properties to the wild-type Hex A is produced. These properties include the ability to hydrolyze GM~ganglioside in the presence of human activator protein (Hou e$ al. 1996a). Using this approach, I was surprised to find that the aVal192LRu substitution produced a pro-a chain that did not form a dirners and was not transported to the lysosome. I verified these results by reexamining the enzymatic activity and Hex protein levels in the fibroblasts from the original patient. Thus, aVall92LRu did not result in a B1-like biochemical phenotype, but was typical of a folding mutation (Hou et d. 1996b).

MOLECULAR MODELING OF THE ACTIVE SITES IN HUMAN HEX

e actwe. sites. of bac&rial chit&* Both Hex and chitobiase belong to the glycosyl "Family 20" enzymes. In this family only the structure of chitobiase from Serratia rnarcescens has been determined at atomic level so far. All members of a given hydrolase family are thought to be evolutionarily related and to have similar three-dimensional structures. Thus, homology modeling of human Hex becomes possible based on the chitobiase structure. The accuracy of such modeling is variable and dependent on the degree of primary structure similarity between the protein with the known structure and the protein being modeled. Sequence alignment between human Hex and chitobiase shows that there is only a 26% identity in its active site region, and this is achieved only with many large gaps inserted into the human sequence at predicted loop structures (Fig. 1.6 and 1.7). Outside this area there is little similarity. In addition, chitobiase is a monomer and functions at neutral pH. Therefore it is necessary to evaluate the validity of the modeling of human Hex. Data on mutations and the active site show both areas of agreement and disagreement with the predictions from the bacterial model. Unlike human Hex, chitobiase is a monomeric protein with a predicted Mr. 98,500. As well there are no cofactors, metals or other ligands in the native enzyme. The enzyme contains four domains and its active site is located at the C-terminal end of the cen~al(m)8-barrel (Fig. 1.6, 1.7 and Table 1.1). Among the potential 16 active site residues, the carboxylate amino acid, Glu 540, acts as the catalytic acid in chbiase. It binds to the glycosidic linkage at the distance of 2.9 A. Interestingly, Glu 540 is invariant among family 20 enzymes and there is no second carboxyl group at an appropriate distance to serve as the classic nucleophile. It is noticed that Asp539 H- bonds to N2A of the acetamido-group in the non-reducing sugar NAGA to assist it to bend towards the anomeric CIA. Another critical active site residue is Arg 349, which sits at the base of the binding pocket. It tightly anchors and orientates the NAGA residue through hydrogen bonds with its the 0IBA and OH4A groups. These data suggest that Arg 349 directly participates in substrate binding. Arg349 aligns with PArg2 11 and aArg178.

Comparison of the molecular modeling data from bacterial chitobiase with those from our evaluation of the B 1-variants suggests that the model must be at least partially correct. Two B 1- variant residues, aArg178/PArg211 and aAsp258/PAsp290, seem to fit the model. The aAtg178/$Arg211 correspond to chitobiase c-Arg349. c-Arg349 is an active site residue, directly involved in substrate binding rather than catalysis as we predicted for PArg211. These data are not inconsistent with the results from our lab if the very low level of irnmuno-precipitated Hex activity detected in the mutant Hex B (Z$ArgZllLys) was from the endogenous COS cell background.

Due to the much higher Km of the mutant human Hex B being expressed and low solubility of the MUG substrate, it may have been impossible to increase the substrate concentration sufficiently to detect activity from the mutant human enzyme particularly if Vmax was also affected by the mutation. The aAsp2581PAsp290 aligns with chitobiase c-Asp448, which is only indirectly involved in substrate binding. These observations are also consistent with our data from PAsp290/Asn mutant human Hex B. We have shown that PAsp29OIAsn has 2.5 fold higher Km than that of wild type Hex B with only a small decrease in Vmax. Finally, aVal lW/PAlaZlS corresponds to chitobiase c-Glu 363. In agreement with our data, the model places c-Glu 363 far from the active site on the N-terminal end of the (m)8barrel. Hence the predictions made from the model conform reasonably well with the biochemical characterization of the mutations associated with the B 1- Figm 1.6: Sequence alignment for the catalytic domain III of chitobiase with the a- and B- subunits of human Hex (Tews et al. 1996b) as well as Sp-Hex (Mark et al. 1998). The alignment was performed with CLUSTAL W program based on the secondary structure of human Hex or Sp-Hex, which are predicted by PHD program (Tews et al. 1996b; Mark et al. 1998). The chitobiase domain III is a ($a)8-barrel where the active site is

located. Residues identified in chitobiase (denoted by %-') and implicated in human Hex

as being involved in substrate biding or catalysis are underlined, see table I. A previously identifed area of homology (without introducing gaps) (Somerville and ColweU 1993; Tse et al. 1996) is interlined with dots. Identical residues in all three

sequences are indicated by I*'. DSPRFSH RGILIDTSN QFRRFRH. EGkb-bRTSM * **** ** ** * DAPRFPY RGL FLQVm DTPRYAW RSAMLDVSN

------SL SHVYTPNDVR MVIEYARLRG IRVLPEFDTP

------me- PV THIYTAQDVK EVIEYARLRG IRVLIAEFQTP ** *** * ** *** HDLSETTCLL PQYGQGPDVY GGFFSRQDYI DIIKYAQARQ IEVIPEIDMP v------GGGP GGYYTKAEYK EIVRYAASRH LEVVPEIQMP

PINPTLNTTY SFLTTFFKEI SEVF-----P DQFIHLGGDE VEF------PVNPSLNNTY EFMSTFFLEV SSVF-----P DFYLHLGGDE VDF------** * * * * **** YLNPCLDSSQ RFVDKVIGEI AQMHKEAGQP IKTWHFGGm AKNIRLGAGY SLCVDKDVTY DFVDDVTGEL AALT-----P GRYLHIGGDE AHS------

VLDIIATINK G-SIVWQEVF DDKVKLA--- -PGTIVEVWK -DSA- -YPEEL LLDIVSSYGK G-YVVWQEVF DNKVKIQ--- -PDTIIQVWR EDIPVNYMKEL * * * * * VSKLVKAHGI DRMQAYQDGL KDAESSKAFA TSRVGVNFW TLYWG-GFDSV RVQPIVAKYG KTVVGWHQLA GAEPVEG--- -ALVQYWGLD RTGDA-EKAEV p-435 SRVTASGFPV ILSAP--WYL DLISY------GQ DWRKYYKVEP a-405 ELVTKAGFRA LLSAP--WYL NRISY------GP DWKDFYVVEP * * * * * CHB651 NDWANKGYEV WSNPD- DFPYEVNPDE RGYWGTRFS DERKVFSFAP Sp375 AEAARNGTGL ILSPADRTXL DMKYTKI)-TP LGLSNAGYV- EVQRSYDWDP

P-502 LWPRASAVGE RLWSS P-516 a-473 LWPRAGAVAE RLWSN a-487 *+* *** Jr f CBH751 IFPRALSVAE RSWHR CHB-765 Sp456 IFPRLPGVAN WAGPR SpHex-470 Figure 1.7. Molwular modeling of human Hex from the atomic structure of chitohiase (Tews et

al. 1996a). The cartoon here is adapted from Mark et a1 (Mark et al. 1998). For ease of comparison the corresponding residue numbers we based an the primary structure of human Hex B. 1 Chi tobiose Table 1.1. Active acidic residues in the chitobiase and their aligned residues in Streptomyces plicatus hex & - Sp-Hex SpHex Experimental Data CHB function news et al. 1996) 3xperimental data from Hex (Brown and (Mark et al. 1998) blahuran 1991; Tse el al. 1996) I.

No function postulated 3D196N: normal Km,Vmax4.2Q Re-examination in Chapter 4 I.

Holds R349 in place by polar 3D208N: Only monomeric precursor formed interaction, H-bonds to term. amino and imino groups -. 162R R162His:Km(O.l4mM, Docks heGlcNAc by H-Binding PR211K: Normal M-protein MUG) increased by 40-Fold, at OH3 & OH4,sits at the base of Vmax reduced 5-fold, altered the binding pocket Re-examination in Chapter 3

pH profile -,

191D Holds R3.19 in place by polar D240N: Normal M-protein, Km increased 10- interaction, H-bonds 10 H452 fold. Vmax= 1 1Q I

Ilolds R349 in place by polar Study in Chapter 4 interaction, Coordinates water mol. with E380 - Holds R349 in place; H-bonds to Not consenvcdin human Hex amino and irnino groups, coordinates water mol. with D379I- 246D D246N: Km increased 1.2- H-bonds to D539, indireclly H- D290N: Lower M-protein; Km increased 2.5 fold, vmax decreased Zfold bonds substrate through a H20 fold, Vmau=71%

Acce~tsH-Bonds from 0~1 H262 H294

H-bonds to acetamido-N2 to help- D322 D354 Study in Chapter 4 distorl it towards C1

variant. In several other regions be chitobiax model also tits with biochemical dati. The model suggests a general scheme: the morc severe mutations are located deeper in the protein core; the less sevexv and benign mutations are placed on the protein surfiace.

odeline of Strentom~cesHex Recently, comparative moIecular modeling of the Streptornyces plicatus (Sp-Hex) has been also performed (Mark et al. 1998). The modelcd structure of Sp-Hex is similar to that of chitobiase. It also forms an eight-strinded dP-barrcl containing the active site pocket on its C- terminal end, and thus the active sites of the Sp-Hex udthe chitobiue are predicted to be highly conserved (Fig 1.6, T~hlc1.1). For example, the catalytic acid Glu540 in chitobiwe aligns with Sp-Hex Glu3 14. In Sp-Hcx the pmlonated carboxyl group of Glu3 14 is within hydrogen bonding distance (2.5 A) of the oxygen in thc glycosidic linkage of the substmte. Thus. Glu3 14 is perfectly situated to donate ics proton to lhc glycosidic oxygen, thereby aiding in the hydrolysis of the substrate. Chitohiaw Arg.349 aligns with Sp-Hex Arg 162. The 6-guanido group of Arg 162 appears to directly hind thc suhamtc by lbrrning two H-honds, each at a distance of 2.6 A, to OH- 3 and OH-4 of the NAGA sugar of the chitohiose. In addition, three TITIresidues (Trp344, 442 and 408) in Sp-Hex are part of active site pockct as they pack against the hydrophobic hexose rings of the chitohio.~structure. Furtheimor~=the active site model of Sp-Hex appears to lack an appropriate rcsiduc that could act as thc nuclcaphilic ha.* in drtssical acidbase catalysis. Mark et a1

(Mark et a1. 1998) wcnt on to examine Glu3 14, Arg 162 and Asp246 by mutational analysis and expression in E-coli. The lattcr two residues align with codons involved with thc human £31 of Tay-Sachs disease aArg 178 and aAsp258. Their kinetic data are consistent with tho.se predicted from the Sp-Hcx modcl (sce also Chapter 4).

The mechanism of cleavagc of glycoside bonds by Hex remains unclear. It has been proposed that glycosyl hydr~lascsemploy a pneral acid-catdysis mechanism involving two acidic residues. One acid, protonnted. e.g.C-Glu540 (J'd~le 1.1) acts as an acidha% catalyst and the other, unprotanakd, as a nucleophile responsible for stahiliation of the substrate's transient carbonium ion by forming a covalent cnzyme-suhstmk intermediate. It has heen shown that most glycosyl , e.g I ysozyme (Malcolm et al. 1WW), P-galac tosidme (McCmter et al. I 997) and a-glucosidase (Hermans et al. 1991), use this classical mechanism for catalysis. Recently a second mechanism, substrate-assisted catalysis, was proposed for Hex (Fig. 1.8). At least two lines of evidence support this mechanism. First, it was found that a potentid nitrogen-containing imversible inhibitor for Hcx liom bovine kidney and jack bean did not form the expected covalent bond with the enzyme (making it an imeversible inhibitor), but instead was a competitive inhibitor and "pseudosuhstratc" for Hex. Bused on these data, the authors conclude that a carbonium ion intermediate was not part of its catalytic ~ncchanism(kgler and Bollhagen 1992). They proposed that a residue in Hex functioning as a base, hydrogen-bonds with the proton from the acetimido group of the bound suhstl';ltc producing an intcmd substrittc oxazoline (C-1 joined to the ncetamido-oxygen, Fig. 1.8) structure as tlw reactive ink~mediaw(Knapp et al. 1996; kgler and Bollhagen 1992). Secondly, the data from the crystal structure of chitohiase-substrate complex also indicates that this is the mechanism by which chitobia.~cleaves its suhstrites and identified the unprotonatcd c-Asp539 as thc "hnsc" (Tews ct al. 1996a) (Tahle 1.1 and Fig. 1.8).

Active site studies of human Hex

The area we and others have previously identified as king the most conserved without the introduction of gaps, in 15 aligned Family 20 sequences, p189-254 (Fig. 1.6) (Somerville and Calwell 1993; Tse et A. 1996). is not predicted by the chitchiast: model to contain the a or P catalytic acid or base residue. The rcsidues that align with Lhe protonated acid residue in chitobiase are aGlu323 and PGlu355. Those that align to the basic residue (unprotonatcd acidic) are aAsp322 and PAsp354. Interestingly, PGIu355 was ~Isc) laheled with a substr'dte malog containing a photo-dl'inity label in the aglycone position and initially postulated as being involved in substrate binding (Liessem et al. 1905). This position is not normally considered important for either Hex substrate binding or catalysis and thc lab1 itself reacts non-spwikically with any nearby sidechain. NevertheIcss, Fernandes et a). (Femandes et al. 1997) have implicated the aligned Figure 1.8. The proposed substrate-assisted catalytic mechanism (Legler and Bollhagen 1992; van Scheltinga et ill. 1995). (A) The protonated (catalytic) acidic residue donates a proton to the P(1.4)-glycosidic oxygen while another residue (B-), functioning as a bast: (kept unprotonated by the micro-environment of the active site), =moves a proton from the acetamido group of the hound suhstrik producing an internal oxazolinium (axial position of the C-1 atom covalently joined to the carbonyl oxygen of the N-acetyl group) intermediate (B). (C) Thc intermediate remains stable long enough for the aglycon (HO-

R) to dit'fusc away which is then folIowcd by an incoming water molccule to perform a second nuclcophilic substitution at the carbohydrate C-1 (P position). (D) The acidic

residue and the basic group return to their initial state. =?Acidic residue aGlu323 and Pennybacker et al. the PGlu.355 (Pennybacker et al. 1997) as catalytic residues in expression studies. Previous to these reports, Tse et a1 in our lab identified another candidate acid residue in the p 189-254 area, PAsp 196 (Fig. 1.6, Table 1.1) which when mutated to N produced a normal Km and level of mature PCRM, hut had a Vm of 0.2% (Tse et al. 1996). This residue was not analyzed by the other groups and we did not analyze PGlu355. Furthermore, each group used different expression systems which a11 shared the common problem of some contamination by endogenous Hex. Therefore I analyzed PGlu355 and PAsp196 along with other acidic candidate active-site residues using a novel expression system (see Chapter 4).

THESIS OBJECTIVES

The objective ol' this thesis is to idcntil'y structure-function relationships in human Hex. To this end, I biochemically charactc~i~xda novcl mutation in patients associated with chronic Sandhol'f disease (Chapter 2). I examined the cl'li=ct of this mutation on intracellular trmsport, heat stability, kinetics of arti!icial suhstriites, and most importantly, the hydrolysis of natural substrate GM~ganglioside. To tcst the validity of ~nolecularmodeling of human Hex from the X-ray structure of chitohiase, I developed a new system for the generation of an epitope-tagged form of

Hex B and thus specilically purily it away from endogenous Hex from the host cells (Chapter 3). Re-examination of bAsg211 using this new method sevculed it has a role different from our previous finding hut consistent with the chitobiue model (Chapter 3). Next I analyzed the candidate acidic active sitc residues in Hex B prcdictcd from the chitohiase structure using the same approach (Chapks 4). These studies vdidatcd thc molecular modeling of human Hex from the bacterial enzyme and indirectly continn thc suhstr~tcmechanism of action for human Hex. REFERENCES

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Bartholomew WR, Rat- MC (1974) Immunochemical charactt=rization of human P-D-N- act=@ hexosaminidase from normal individuals and patients with Tdy-Sachs disease. 1 Antigenic difference ktwem hexosaminidw A and hexosaminidase 8. Int. Arch. Allergy Appl. Immunol. 46: 5 19-524 Bayleran J, Hwhtman P, Kolodny E, Kahack M (1987) Ttly-Sachs disease with hexosminidase A: Characterization of the defective enzyme in two patients. Am. J. Hum. Genct. 41 : 532-548 Bayleran J, Hech tman P, Silray W (1 984) Synthesis of 4-methylum helliferyl-beta-D-N- acetylglucosamine-6-s~1lf5teand its use in clusil~crrtion of GM~gangliosidosis genotypes. Clin. Chim. Acta. 143: 73-89

Bergwon JJM, Brenner MB, Thomas DY, Williams DB ( 1994) Calnexin: a mem brine-bound chaperone of the cndoplasmic reticulum. TIBS 19: 124-128 Beutler E, Kuhl W, Comings D (1975) Hcxosaminidase isozyme in type 0 GM2 gangliosidosis (Sandhoff-Ja~zkcwitzdisease). Am. J. Hum. Genet. 27: 628-638

Beutlcr E, Yoshida A, Kuhl W, Lec JE (1976) The subunits of human hexo~aminid~eA. Biochem. J. 159: 54 1-543

Bikker H, Meyer MF, Merk AC, deVi.jlder JJ, Bolhuis PA (1988) XmnI RFLP at 5q13 detected by a 049 Xmn I fragment of human hexosaminidase (HEXB). Nucleic Acids Res. 16: 8 198-8 198

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A ProSO4Ser Substitution in the P-Subunit of P-Hexosamindase A Inhibits a-Subunit Hydrolysis of GM~ Ganglioside, Resulting in Chronic Sandhoff Disease INTRODUCTION The hydrolysis of ganglioside (GM~)requires the proper synthesis, intracellular transport and protein-protein interactions of three different gene products. Two of these, encoded by the evolutionarily related HEXA (15q23-q24 (Nakai et al. 199 1)) and HEX7 (5q 13

(Bikker et al. 1988)) genes, a~ the a and B subunits of heterodimeric fbhexosaminidase A (Hex

A), respectively. The third gene product is a small heat stable protein, the &2 activator protein (activator), encoded by the GM2A gene (Sq31.3-33.l(Heng et al. 1993)). Mutations in any one

of these genes can result in the storage of -2 and one of the family of human diseases known as the GM~Gangliosidoses. HEX4 mutations are associated with Tay-Sachs disease, HEXB with Sandhoff disease and GM2A with the AB-variant form [reviewed in (Gravel et al. 1995)J. The Gmgangliosidoses show extreme variability in clinical expression. Typically, the earlier the age of onset of clinical symptoms the more severe the disease. A nomenclature based on the different clinical phenotypes and recognizing the dominance of the encephdopathy (rather than only the age of onset) has been suggested (Gravel et al. 1995); acute (the classical infantile form), subacute (late infantile and juvenile forms), and chronic (adult and chronic forms). The most common, acute form is a severe neurological disorder that usually results in death within 4 years. Mutations associated with the classical phenotypes prevent the formation of any functional Hex A. It is now generally believed that the broad range of less severe phenotypes result from small variations in the levels of residual Hex A activity, on the order of 0-5% (Leinekugel et al. 1992). Healthy individuals with -10% residual Hex A activity have been described (reviewed in (Gravel et al. 1995)). While patients with the acute forms of GM~gangliosidosis are deficient in Hex A activity, their total Hex activity, as measured by neutral artificial substrates, is significant. Tay- Sachs patients often have near normal levels of activity due to the presence of the homodimeric

Hex B isozyme (PP), and Sandhoff patients have 1-5% of normal levels from homodimeric Hex S (otos) (reviewed in (Gravel et al. 1995)). Since any dimeric combination of a and/or subunits

produces an active isozyme, each subunit must contain a potential active site. The characteristics of the two sites have been examined in the two homodirners (Kytzia and Sandhoff 1985) and in a

novel form of Hex A with an inactive P-subunit due to a Arg211Lys substitution (Hou et al.

1996a). These data indicate that the presence of the subunit affects the Km and V- of the a

active site towards neutral substrates (Hou et al. 1996a). Whereas both subunits of Hex A are

equally capable of hydrolyzing neutral FGlcNAc or P-GaWAc containing substrates, e.g. 4-

methylumbelliferyl-f3-N-acetylglucosamine(MUG), only isozymes containing an a subunit can

efficiently hydrolyze negatively charged P-GlcNAc-6-sulfate containing substrates, e.g.

methylumbelliferyl-$-N-acetylglucosamine-6-sulfate (MUGS), i e. the MUG/ MUGS hydrolysis

ratio for Hex B is -300, for Hex A-4, and for Hex S -1 (Hou et al. 1996a). The specificity of the Hex isozyrnes for G1132 ganglioside indicates that it should also be considered as a negatively charged substrate, presumably due to the sialic acid residue attached to the penultimate Gal

residue. However, whereas Hex S as well as Hex A, but not Hex B can hydrolyze GM~in the

presence of detergents, only Hex A is functional in vivo with the Gmactivator/ -2 ganglioside complex (reviewed in (Gravel et al. 1995)). Thus some component(s) of the P subunit are necessary for the hydrolysis of GM~in viva The exact role of the activator remains controversial (Li et al. 1995; Sandhoff et al. 1995; Wu et al. 1994). However it is generally agreed that it binds both the lipid and oligosaccharide portions of GM~extracting or at least lifting the ganglioside out of the membrane, then the complex interacts with Hex A for hydrolysis (Meier et al. 1991). Hex B can hydrolyze the neutral, asialo derivative of GM~,GA~, in the presence of detergent, but has little activity in the presence of activator. Furthermore it has been reported that the activator, even in the absence of Gw,can slightly inhibit the hydrolysis of MUGS by both Hex A and Hex S (reviewed in (Fiirst

and Sandhoff 1992; Sandhoff et al. 1989)). These data indicate that at least a portion of the

binding site for the complex is also located in the a-subunit, The required elements of the j3-

subunit may function by increasing the afFinity of Hex A for the complex and /or correctly orientate the complex, allowing the efficient hydrolysis of the terminal sugar from the

ganglioside. Furthermore, these P-elements may act directly, by interacting with the complex, or

indirectly by affecting the conformation of the a subunit. Other functions associated with the P-

subunit include; greatly increasing the stability of the resulting dimer, and facilitating the

transport of the a-subunit out of the ER (reviewed in (Gravel et al. 1995; Mahuran 1991)).

To date, all missense mutations except those at two codons, in either HEX gene result in

normal levels of mutant mRNA but paradoxically with a dramatic reduction in both mature P

and/or a protein and Hex B and/or Hex A activity in patient cells. This is believed to be the

result of a strict "quality control system" in the endoplasmic reticulum (ER) that prevents the transport and increases the degradation rate of misfolded proteins or unassembled subunits

(unlike B subunits, a subunits have an apparently low affinity for each other) [reviewed in

(Gravel et al. 1995; Mahuran 1991)J. In several cases it has been demonstrated that subunits with missense mutations, even those associated with the most severe clinical phenotype, are not totally incapable of forming a partially functional Hex A, but may be prevented from doing so by their retention and degradation in the ER (Brown and Mahuran 1993; De Gasperi et al. 1996). Thus, the major detrimental effect caused by most HEX missense mutations is at the level of intracellular transport rather than structural changes that specifically affect some aspect of enzyme-function. The exceptions to this conclusion are the missense mutations at aArg178

(Ohno and Suzuki 1988; Tanaka et al. 1990) and aAsp258 (Fernandes et al. 1992) which produce the B 1 biochemical phenotype. Patients with the most common Arg l78His substitution were originally thought to have an activator defect because they express both normal levels of

Hex A and B activities, as assayed with neutral (common) substrates, e.g. MUG. However, unlike the normal Hex A found in the true AB-variants, Kytzia et al. found that B1 variant-Hex

A was inactive toward an a-specific GlcNAc-6-sulfate containing substrate (as well as Gm ganglioside even in the presence of added activator protein), and they suggested the presence of a mutation at or near the active site of the a subunit (Kytzia et d. 1983). This hypothesis has been demonstrated to be correct for substitutions at either residue based on mutational and expmssion studies of the aligned bresidues, i. e. P-analogs, PArg2 11 (Brown and Mahuran 1991; Brown et al. 1989) and PAsp290 (Tse et aI. 1996a), and on molecular modeling of human Hex using the structure of bacterial chitobiase pews et al. 1996). We described ten years ago two sisters of French Canadian ancestry with a chronic Sandhoff phenotype (Rubin et al. 1988). We have also previously reported that these patients are heterozygous for the common 16kb 5' HEXB deletion allele which does not transcribe PmRNA

(Neote et al. 1990). In this report we characterize the second mutant allele in these patients, a missense mutation in exon 13 of the HEXB gene that results in a ProSO4Ser substitution. This mutation produces a novel biochemical phenotype that impacts directly on the ability of Hex A to hydrolyze Gm.This is the first report of a mutation in the f3 subunit that affects the ability of

Hex A to hydrolyze its natural, but not its artificial substrates; and localizes essential elements of the p chain for natural substrate hydrolysis to its C-terminus. Cultured fibroblast. were lysed by directly adding 1.0 ml of DNAZOL Reagent (GIBCO

BRL) to the 10 cm2 culture dish. The lysate was then transferred into an Eppendorf tube and insoluble cell debris was removed by brief centrifugation. The genomic DNA in the supernatant was precipitated with ethanol and dissolved in 10rnM Tris-HC1 buffer containing 1mM EDTA pH 7.4 (McInnes et al. 1992).

Total RNA was isolated by using TRIzoI Reagent (GIBCO MU),as described by Hou et al. (Hou et al. 1996b). Two pg total RNA were used to synthesize the single strand cDNA according to the SUPERSCRIPTTM I1 procedure (GIBCO BRL). Briefly, RNA was first denatured at 70°C for 10 min and then incubated at 420 C for 50 min with 200 units of SUPERSCRIPT I1 and 0.2 pg random primers in 20 pl of 50 mM Tris-HC1 (pH 8.3), 75 mM KCI, 3 mM MgC12, 20 mM DTI' and 0.5 mM each of four dNTP's. Two pl of this mixture were directly used for PCR reaction to designed synthesize and amplify double stranded cDNA.

DNA arn~lificationand direct seauencing Amplification of exon and introdexon junctions from genomic DNA, and cDNA fragments was performed by PCR as previoudy described (Mclnnes et at. 1992). The reactions were carried out in 100 pl volume containing 0.1-0.5 pg genomic DNA or 2 p1 cDNA (by reverse transcription), 10 mM Tris-HCI (pH 8.3), 50 mM KCI, 1.5 mM MgCI2, 0.01% gelatin,

0.2 mM each of four dNTP's, 0.5 pg of each primer and 2.5 U AmpliTaqTM Taq polymerase.

Amplification was achieved by incubation in a DNA Thermal Cycler (Perkin Elmer Cetus) for

30 cycles, each consisting of 30 s denaturation at 940C, 30 s annealing at 55-600C, 1-3 min extension at 720C. The region around exon 13, found to be heterozygous for the Pro504Ser mutation in genomic DNA and homozygous in cDNA was amplified by PCR using oligonucleotides 129 (exon 10, sense; GGTTTTGGATATTAmGCAACCATAAA) and 14A (3' UTR, antisense; TCAATCAATAAAAATATT%). The resulting PCR products from genomic DNA and cDNA were 799 bp and 716 bp, respectively. PCR products were purified by utilizing a Geneclean Kit (Bio 101 Corp.) and direct sequencing was performed with

[a35S]dATP using a modification of the SequenaseTM protocol (U.S. Biochemical Corp.), as

described by McInnes et al. (McInnes et al. 1992).

The wild type constructs, pREP4-a and pEFNEO-P have been reported (Hou et d.

1996a). The mammalian expression vectors pREP4 (InVitrogen) and pEFlVEO (kindly supplied to us by Dr. Anson) (Anson et al. 1992), have hygromycin B and neomycin (G418) resistance markers, respectively. To introduce the mutant cDNA into the pCD vector, a 636 bp product

generated by RT-PCR from patient fibroblast RNA (as described above) containing PPr0504Ser

was digested with P'MI at a site 5' to the mutation and Ban I at a site 3' to the mutation. The

middle fragment of 387 bp was purified and subcloned into pCDP43 (Brown and Mahuran 1991)

treated with PfoMUpartial Banl. To generate the mutant pEFNEO-PProS04Ser, a 2.0kb

fragment, partially digested by Bad1from pCDPProSWSer, was isolated and subcloned into

the BamHF site of the pEl3WO-P vector. The mutation was verified by DNA sequencing. A construct encoding an Asp208Asn substitution in the @DNA insert of pEFNEO has previously been reported (Tse et al. 1996a). In permanently transfected CHO cells this construct produces only soluble, monomeric, precursor P subunits (Tse et al. 1996a). We now used this transfected clonal CHO cell line as a control for the ER-retention of mutant P protein. CHO cells were grown in MEM with 10% FCS and antibiotics at 370C in 5% C@. Transfections were performed using Lipofection from GIBCO-BRL, as previously described (Hou et al. 1996a). Transfected cells were also grown in serum-free media containing lOmM NH4CI which diverts proteins targeted to the lysosome to the secretory pathway. After 1 and 2 days the Hex activity from medium was measured using MUG.

Cells were lysed in a buffer of 10 mM Tris-HC1 pH 7.5 and 5% glycerol through five sets of freeze-thaw cycles. Protein from cell lysate was quantitated by the Lowry method (Lowry et

al. 1951). Hex activity from cell lysates was determined using a a-chain specific substrate

MUGS and the common substrate MUG (Brown and Mahuran 1993).

The protein (amounts loaded are indicated in each figure) from cell lysates or DEAE fractions were resolved by SDS-PAGE using a Bio-Rad mini-gel system (Laemmli 1970).

Proteins were transferred to nitrocellulose overnight at 40C. The filter was blocked in 5% skim

milk and then incubated overnight with primary antibody, rabbit anti-human Hex A (Hou et al. 1996a; Hou et al. 1996b). Nitrocellulose was washed 4 times with 1% skim milk and incubated with a secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG for 1 hr. The filter was developed using the Amersharn ECL system and exposed to Hypedlm.

on of the Hex ISOW Proteins (3 mg) from lysates of patient or normal fibroblasts, or control or transfected CHO cells were applied to a 1.0 ml column of DEAE CL-6B (Pharmacia). The unbound Hex B fraction was collected with 10 mM sodium phosphate pH 6.0. Hex A was eluted by applying 0.15 M NaCl in 10 mM sodium phosphate pH 6.0 (Hou et al. 1996a). Three ml fractions were collected and assayed for Hex activity. The Km value was determined by varying the concentration of the substrates from 0.125 to 4.0 mM for MUG, and from 0.05 to 2.5 mM for MUGS. As well, 10 experimental points were used for each Kin determination. The normal and mutant Hex A from transfected CHO or patient cells were purified away from the other Hex isozymes by DEAE ion-exchange chromatography (see above). Kinetic constants wexe calculated using a computerized nonlinear least squares curve fitting program for the Macintosh, KaleidaGraphTM 3.0 (Hou et al. 1996a).

3H-~2ganglioside (20 nmoles), labeled in the C6-position of its N-acetylgalactosamine moiety (Novak et al. 1979), was incubated in the presence of 2.0 pg recombinant activator protein from bacteria (Klima et al. 1993; Smiljanic-Georgijev et al. 1997) at 370C for 18 hr in 10 mM citrate buffer (pH 4.1), 0.5% human serum albumin, and 10 mM GlcNAc (carrier), with 0, 50, 100, and 200 Units of Hex A, nmol of MUGS hydrolyzed /hr, from; normal or patient fibroblasts or, produced from human cDNAs (normal a with normal or mutant P) in transfected

CHO cells (final volume of 100 pl). The hydrolyzed product from GM~,i.e. {3H)GalNAc, was separated from the unreacted GM~substrate by passage through a positively charged ion exchange mini-column of 0.6 ml AG3 X4 (acetate form) resin. The unbound fraction containing

(3H)GalNAc was determined by liquid scintillation counting, as described previously (Hou et al. 1996a).

shady The wild-type and mutant Hex A or Hex B isozymes, which had been separated by DEAE chromatography, were added to 700 pl preheated citrate phosphate buffer (pH 4.1) with

0.3% human serum albumin. The heat denaturation was performed at 450C and aliquots (100 pl) were removed at intervals of 0, 15,30,45,60,75,90 min for Hex A, and 0,30, 60,90, 120, 150, 180 min for Hex B, placed on ice, and assayed for enzyme activity. The wild-type and mutant Hex A from transfected CHO cells were also tested for their residual MUGS activity after incubation at 370 C for 18 hr, under conditions that mimicked the natural substrate assay, above. . . locCr in ' . n n

Non-transfected, or CHO cells transfected with constructs encoding a) the wild-type

PcDNA (Iysosomal-localization control), b) the ProSO4Ser substitution, or c) a Asp208Asn substitution (ER-localization control) were grown at 370C in 5% CO2 on glass slide covers in a

10 cm2 culture dish. After 24 hr of incubation, the cells were fixed and gently permeabilized with

100% cold methanol at -200 C for 30 min. The fixed cells were then washed in PBS, blocked with 1% BSA, and incubated with the primary polyclonal anti-Hex B antibody (Redonnet- Vernhet et al. 1996), diluted 1:200 for 1 hour. The secondary antibody, a green fluorescein- labeled goat-anti-rabbit IgG F(ab)2 ), diluted 1: 100, was then added for 1 hour, either alone or in combination with 1: 10,000 dilution of propidium iodide, which in addition to nuclear DNA also stains the cytoplasmic RNA and marks the position of the ER with the red fluorescence. The cells were then washed 3X with PBS and mounted with elvanol. In control cultures the pre-immune rabbit IgG substituted for the primary antibody. The slides.were analyzed and the proportion of P protein present in the ER or endosome/ lysosome determined using a fluorescent microscope (Olympus Vanox-AH-3, magnification 800 X) and two narrow band filters to detect the green and red fluorescence separately. An additional broad-spectrum filter was also used for the simultaneous detection of the fluorescein-tagged green Hex B and the nucleic acids labeled with red propidium iodide fluorescence. In this setting the overlapping of the red and green labels in the cytoplasm is marked by yellow fluorescence and indicates the colocalization of Hex B and ER (Hinek et al. 1996). Multiple images of the same cell obtained with all the above mentioned filters were captured with the CCD camera (Optronix), stored in Macintosh 9500 computer and quantitatively analyzed using the Image Pro Plus program (Media Cybernetics. MD) according to manufacturers instructions. In each of the three experimental groups (wild type P, PPro504Ser, and PAsp208Asn), images of 50 cells were mdy7zd, and results were statistically evaluated to

give quantitative measurements of the percentage of each J3-protein that resides in the ER and, or

Iysosome.

etection of a novel missense mutation Direct sequencing of the exons and exodintron junctions of the HEXB gene revealed that the patients were heterozygous for an C15 10->T transition in exon 13 (+2bp from intron 12) at the codon t'or Pro504, which results in its conversion to a Ser codon (Fig. 2.1A). We have previously reported that the patients were also heterozygous for the common 16kb 5' partial HEXB deletion allele, A16kb (Neote et al. 1990). To conklrm that this missense mutation was not part of the deletion allele we also xyuenced the P-cDNA. In this case the patients appear to he homozygous for thc missense mutation (Fig. 2.1B). Since a HaeIII site was predicted to be lost in the presencc of the C->T trilnsition, rhc direct sequencing results from both genomic DNA (Fig. 2.2A) and cDNA (Fig. 2.2B) wcrc confirmed by HaeITI-digestions of a strategic PCR- fragment from both patients and 11orm;ll individuals. In addition to the genomic PCR-fragments from the 5 normal individuals shown in Fig. 2.2A, samples from at least 45 other normal individuals were analy7xd and found not to contain this mutation (data not shown). Thus the C- >T transition in the Pro504 codon is not present in either thel6kb deletion or any of the 100 normal HEXB alleles we analymd.

Patients' Hex activitv and nrotein levcls Interestingly, the residual Hcx A activity present in the patients' fibroblasts, -20% is only about half that found in cells from an ohligate carrier of Sandhoff disease (acute form), 5-10 fold higher than the average Ievels of 5 cell lincs from subacute patients (Table 2.1). and even slightly Figure 2.1. Autoradiography of nucleotide sequencing gels: Direct sequencing of PCR products from A) genomic DNA (sense strand); B) cDNA (antisense strand). The mutation is indicated by "*". T. T Val so9 ; Genomic A i Ah508 c , 2399 normal f \ 1 ScrlOl G cxon A \GATC GATC

inmm inm 12 12

;/ cDNA normal B Pro 500 GATC Pro 500

Arg 501 Ag 501

Lcu 502 Leu 502

Trp 503 Trp 503

Pro so4 Ser 504

Arg 505 Arg 505

Ah506 Ala 506

Ser 507 Scr SO7

Ala SO8 Ala 508 Figure 2.2. Direct restriction digest assay (see diagrams at the top of each panel) for the presence of the C151OT transition in PCR liagments from A) genomic DNA, and B) cDNA. M-l (line 2399) and M-2 (lint: 2400) are samples from our two patients' fibroblasts, N and N-1 to N-5 are samples from the fibroblasts of 6 different normal individuals, Std. are Mr standards with the indicated number of base pairs (left). Haelll A (DNA) Normal E3bp 550 bp

~r nV 703 bp Std. Uncut Cut

Normal 339 bp Haelll-

Std. Uncut Cut Std. N M-1 M-2 Table 2.1: Unitsa of Hex A present in fibroblasts measured after isozyme separation by Chromatot'ocusing (Mclnnes et al. 1992; O'Dowd et al. 1986).

MUG MUGS~ Cell Line Hex B Hex A Hex A %Norma1 %Normal %Norma1

a. Unils are in nmol MU llrl mg-l of the indicated substrate hydrolyzed by Hex A or Hex B

h. These data whilc determined during the O'Dowd et al. 1986 study, have not heen previously reported

c. From (O'Dowd ct al. 1986)

d. The Chromatofocusing profile of lysatc from one of these cell lines has previously been reported (McInnes et al. 1992)

e. The range of numbers presented is the range of activities measured in the two affected sisters' cell lines. f. This MUGIMUGS ratio, 1.5: 1 (Hou ct al. 1996a) plus the slightly lower pH of elution

(O'Dowd et al. 1986) suggc=ststhis activity is fi-om a small amount ol' pro Hex S, mature Hex S is well separated from Hex A hy this procedure (McInnes et al. 1992; O'Dowd et al. 1986) (the proachain loses basic amino acids during maturation (Mahuran 1990)). higher than those reported for asymptomatic individuals with low Hex A activity (10-15%) (Dlott et al. 1990; Kytzia et al. 1984; Leinekugcl et al. 1992). We also investigated the levels of a and f3 CRM in the patient's cells and comparccl thcm to levels found in cell lines from a

normal individual, a subacute patient (2.5% residual Hex A activity), and an acute patient (0% residual Hex A activity) (Fig. 2.3). The apparent levels of mature P CRM in these simples were

consistent with the decreased Hex A and B activities rcported in Table 2.1, indicating that the specilk activity of the mutant Hex isozymes for artilkcia1 substrates had not changed. However, it was also apparent that thcre was a grcat incrcasc in the ratio of pn=cursor/ mature forms of the a and/ or p polypcptidw. suggesting that tlx PPro504Ser mutation results in the retention of a significant amount of newly synthesized prop chains in the ER (d'Azzo et al. 1984; Hasilik and

Neukld 1980) and Iikcly a more rapid turn over rite (Ashkenas and Byers 1997). To confirm that the mutant mature polypeptides wcre not hcing degraded in the lysosome, normal and patient cells wcre grown in media containing Icupcptin, which has been shown to inhibit the turn over of mutant P-chains in thc lysosomc (Brown and Mahurnn 1991; Brown et aI. 1989). No dramatic incrwse in cithcr Hcx activity or rnalurc p CRM was ohserved (Fig. 2.4). Effect of the mutation on stahilitv and intracellular transnort

To fully characterize the biochemical eflkcts 01' the Pro504Ser mutation, CHO cells were permanently co-transfected with two cDNAs encoding the normal a and the mutant P polypeptides. A high-producing clonc was isolated and grown (Fig. 2.3). The Hex isozymes from the lysatc of these cells werc separated hy ion-exchange chromatography (Fig. 2.3). Since several mutations linked to the chronic form 01' GM~gangliosidosis have been shown to produce a less heat stable isozyme, as well as an increased retention of the mutant subunit in the ER

(Bolhuis et al. 1993; Brown and Mahumn 199.3; Dc Gaspcri et al. 1996; De Gasperi et a). 1995), Figure 2.3. Western blot analyses using an anti-human Hex A antibody of the a and P polypeptides in the total cell lysates (the mount of protein loaded is given directly below the sample lane) from; co-trtlnskcted CHO cells (with wild type acDNA and mutant PcDNA, P*); normal fibroblasts (Normal), and fibroblasts from Sandhoff patients

presenting with chronic (Chr.* one of the subjects of this report, line 2400) subacute (Subac., Iine GM 2094) and acute (Acute, line GM 294) forms of GM~gangliosidosis (the

genatypc of each patient is given at thc hottom of the figure below the corresponding sample lane). Analyses of the polypeptides present after isozyme separation (Hex B and

Hex A; Hex S was not eluted liom thc column) by ion-exchange chromatography (DEAE-

Separation) 01' co-transkctcd CHO cell lysutcs are also shown. Transfected @pt) CHO Cell Extr. Fibroblast Extracts Total DEAE-Separated Normal Sandhoff Patients Hex B HexA Chr.' Subac. Acute

pg Protein: 5 5 50 50 50 N 16kb l6kb 16kb 16kb (~504s) (IVS- 12) Figure 2.4. Westcrn blot analyses using an anti-human Hex A antihody of the a and P polypeptides in the total cell lysates (the amount of protein loaded is given directly above the sample lane) from fibroblasts of one of our chronic patients (line 2400) and a nonnal individual. Cells were grown in the presence, +, or absence, -, of leupeptin which inhibits lysosomal degradation of proteins. Total Hex specitlc activities, using the MUG or MUGS artificial substrates, are given at the bottom of the figure. Chronic Patient Normal

Leupeptin - + + - (pmol MU) (hr)-l (mg total protein)-1 MUG 2.5 2.3 19 16 MUGS 0.75 0.78 2.8 2.2 the T1/2s of both hex isozymes carrying the mutant suhunit was determined at 45 "C (Table

2.2). Consistent with these previous observations, the Pro504Ser substitution decreases the heat stability of both the A and B isozymes (TahIe 2.2).

The ability of the PPro504Ser substitution to inhibit ER to Golgi transport was directly

confirmed by immuno-f.luorescence microscopy (Fig. 2.5). CHO cells permanently transfected

with either the wild-type (Fig. 2.5B) or mutant PcDNAs encoding the Pro504Ser (Fig. 2.5D) or

Asp208Asn (Fig. 2.5C) substitutions were examined. The latter suhstitution results in only

monomeric, precursor p chains in trnnsfwtcd cdls (Tsc ct al. 1996a), and thus serves as a control

for ER-retention (Iiasilik and Ncui'cld 1980; Proia et al. 1984). Quantitative measurements of

the green versus yellow (ovwlappinp 01' thc red and grecn labels in the cytoplasm, i.e. ER)

fluorescence in 50 cells from each gmup indicated that virtually all of the Asp208Asn P-protein

(97+ 2%) is present in the ER of transfected cclls. compared with 60rl: 10% of the pprns@%r

protein and 152 5% of the wild-type P chain (Fig. 2.6). Furthermore, CHO cells co-transfected

with the wild type a and P and those transl'cctcd with a and PPro504Ser were grown in iOmM

NH4CI and the MUG activity measured in the tnedia. Wild-type transfected cells secreted I. 1

~104nmoVhrImL on day I, and 2.4 ~1(@Units on day 2. Cell with the mutant PcDNA secreted

0.18 ~104Units 011 day I, and 0.28 X 104 Units on day 2. Thus diverting the mutant Hex from the lysosomal incol-porition to thc sccrctory pathway did not result in a large increase in activity, confirming that thc loss ot' activity and PCRM occurs at an early point in protein transport, i.e. the ER. Table 2.2. Biochemical characterization of the e~fcctsof the PPro504Ser substitution

Isozyme KmiL Krn" 101 X GM~45°C Tin Remaining (MUG, mM) (MUGS, mM) IMUGSa (rnin)h MUGS after 18hr 37Wb CHO(-)C ND~ ND oo1 1 ND ND

Hex A (ap) 0.80A).OS 0.26A).(12 1.3H.01 2W )A 10 81+4%

Hex A* (ap*)" 0.74d1.03 0.29+0.02 0.48~0.02 60+5 66-c3%~ Hex B (PP) 0.7 1&.05 ND ND UDf ND

Hex B (P*P*) ND ND ND 12( 125 ND

Fibroblast Hex A*6 ND ND ( 1.44ko.03 ND ND

a. Standard csrar of thc cxperimcntal points as calculated lSromthe best lit curve base on the Michadis-Mentcn equation (R vaIucs were all 20.098), or line (Fig. 2.7) b. Mean and standard deviation of values from 3 independent experiments c. Unttansfected CHO cells; endogenous CHO cell Hex A does not hind the GM~/human

activator complex, i.e. this is a species-spccific reaction (Wou et al. l996a). d. Not determined e. Hex A was isolated by ion-exchangc chromatography from CHO cells co-transfected with cDNAs encoding wild-type a and Pro504Scr substituted P, P*, subunits. f. Undctecttlhle loss in cnzymc activity over the cntirc incubation period

g. Mutant Hex A isolated l-rom thc patient's fibroblasts by ion-exchange chromatography Fi yre 2.5. Indirect immunotluorcmmce microscopy using an anti-human Hex B antibody of A) untransfected CHO cells; or CHO cells co-transfected with, B) cDNAs encoding wild type prepro a and P polypeptides, C) cDNAs encoding wild typa a and PAsp208Asn polypeptides or D) cDNAs encoding wild typa a and PProSO4Ser polypeptides.

Figure 2.6. Graphical rcpresen~ationoC the percentage of various P-proteins; wild type (Wild), or containing either an Asp208Asn (D208N)or a Pro504Ser (P504S)substitution (X-axis); in either the ER or endosome/ lysosome in permanently tmnsfected CHO cells. Bars

represent the sttindard deviation of each value and were obtained from the analyses of 50 cells per experimental group. .._'..'. . ER I Endosorne & Lysosome I

Construct Hvdrolvsis of artiticial and natural substrates hv ap(Pro504Ser) Hex A

Due to the relatively high levels of residual, mutant Hex A activity, i.r. apPro504Ser

(ap*,Hex A*), we found in both our patients' cells and in co-transfected CHO cells, it remained

difficult to explain why the patients should present with any disease phenotype. One possibility

would he that the P mutation had some direct effect of the function on the Hex A* isozyme. To

address this question we first examincd the kinetic hchovior of Hcx A* with the common and a-

specific substrates, MUG and MUGS rcspectivcly. Kinetic analysis confirmed that Hex A* has the same apparent Kms as the wild typc isozyrne liw thcse artificial substrates (Table 2.2). We next tested the ability of the Hcx A* to hydrolyze its natural substrate, the GM~activator: GM~ ganglioside cornplcx. Using samp1cs 01' Hcx A and Hex A* that contained the same number of MUGS units, we fi~undthat the mutant isozyme is 3-fhki less active towards the nriturd substrite than is the wild type Hex A (Fig. 2.7, Tahlc 2.2). Furthermore, we confirmed that the residual Hex A in the paticnt's L'i hcohlasts also had a decreased activity towards GM~as compared to MUGS (Table 2.2, GM~/MUGSratio). Finally wc tested the stabiIity of the wild type and mutant Hex A over thc 18 hr, 37°C i~~cuhationpcriod used in the GM~hydrolysis assay. Both forms of Hex A lost some activity towards MUGS over this time period; however, at the end of 18 hr. the rcsidual activity 01' thc mutant li~rrnwas only 15% Icss than the wild type (Tahle 2.2).

DISCUSSION We have previously dernonstratcd that two French Canadian patients with chronic Sandhoff disease arc l~etcrc.)zygousfor the common A 16kh HEXB allele (McInnes et al. 1992;

Neote et al. 1990). Sinw this allclc products no PmRNA, the uncharacterized, second allele must be responsible for the 15-25% rcsidual Hex A activity (using MUG) we reported to he present in the patients' librohlasts, and thcir mild cl~ronicphenotype. Western blotting with Figure 2.7. Natural substrate, GM~gunglioside/ activator complex, =say of DEAE separated

Hex A (Fig. 2.3) from untransfccted CHO cells (endogenous CHO cell Hex A), solid squares; or CHO cells co-transfected with either cDNAs encoding wild type prepro a and P polypeptides, solid circles, or wild type a and PPro504Ser prepro-polypeptides, solid

diamonds. For each form of Hex A, three samples containing 50, 100 and 200 units of

MUGS were assayed and the best 13 straight line drawn. The slopes of each line k its calculated standard error is given in Table 2.2. Note that for this assay the human activator

is "species-specitk", i.e. endogenous CXO cell Hex A is virtually non-functional.

anti-p antiserum has also indicated a similar reduction in the amount of mature P protein

(McInnes et al. 1992). In this report we identify the second allele as a C1510T transition encoding a Pro504Ser substitution. We demonstrate that this substitution is not found in either the 16 kh deletion allele or in 100 normal HEXB alleles we examined, indicating it is the major

cause of the patients' biochemical and clinical phenotypes. This conclusion was strengthened by

studies of the PPro504Ser mutant Hex A and Hcx B isozymes produced in CHO cells

permanently tlnnsltctcd with thc wild typc a and/or mutant P cDNAs. The mutant P subunit

produced isozymes with decrcascd heat stability (Table 2.2) and increased retention in the ER, 60&10% as compared to 15+5% for the wild type (Fig. 2.5, 2.6). The latter data indicate that the mutation reduces thc P-containing Hex isozyme content in lysosome by 30-60%. These data are also consistent with thc cft'ccts ol' other mutations producing the chronic phenotype i.e. aGly269Ser (Brown and Mahuran 1993; Navon and Proia 1089) aTyrl8OHis (De Guperi et al.

1996), PArg505GIn (Bolhuis et ul. 1993), and PAlil543Thr (De Gasperi et al. 1995).

Dlott et al. characterized thc HEXB mutation associated with clinically asymptomatic individuals whose hiochcrnical phcnotypc 01' low levds of rcsidual Hex A and undetectable levels of Hex B, i.'. Hex A+/ Hcx B-, had p~.eviousIy heen designated as "Hexosaminidase Paris" (Dreyt'us ct al. 1977) . In the sarnc rcpcwt they chtlracterizcd another mutation associated with subacute Sandhofl' disease which was also characterized by the Hex A+/ Hex B- biochemistry. Both of these patients wew also hetcrozygous for the A16 kb allele and thus, their biochemical phenotype was duc to thcir second allele (Neo:e et al. 1990). In both cases the second allele produced a partial splicing dclbct in the HEXB gene encoding an elongated P polypeptide, i.e. a duplication of -16->+2 hp of IVS-13->oxon 14 (asymptomatic ) and g-2% IVS-12 (subacute) (Diott et al. 1990). It was also shown that the residual activity present in these patients' samples was from a small amount of properly spliced PmRNA encoding the wild

type protein. Activity measurements indicatcd that the asymptomatic individuals had twice as much residual Hex A activity as the subacute patients, 10%~and 5% respectively (Dlott et al. 1 990). In this and other reports (McInnes et al. 1992; Neote et d. 1990; O'Dowd et al. 1986) we

have included the cell line from the above suhacute patient (g-2% IVS-12; A16kh) in our analyses. In our hands the residual Hex A activity in this line is 2-3% of normal, using artificial substrites (Table 1) (McInnes et al. 1992; O'Dowd et al. 1986). This would suggest that Hex A levels of 4-64) of normal should prevcnt GM~storage and disease. This estimate is close to that set as the "critical threshold" by SandholT and collcagucs (Kytzia et a]. 1984; Leinekugel et al. 1992). Given this critical threshold and our previous Hex A activity data, it has heen difficult to explain why our two paticnts present with chronic GM~gangliosidosis. Two possibilities were considered. First, that the P mutation is somchow aSL'ecting the a active site, lowering its activity towards MUGS and GM~,r.g. a new typc of B1-variant. Second, the mutant P subunit is affecting the ability of Hex A to hind the GM~activator/ GM~ganglioside complex.

We now reprwt the rc-examination ol' sesidual Hex A* activities using the a-specitic

MUGS substrate (Tahlc: 2.1) and t,hc evaluation of both a and P CRM levels in patients' cell using an anti-Hex A antiserum (Fig. 2.3 and 2.4). These analyses confirmed our previous data; pa~.ticularlythe lcvcls of' MUGS activity I'mrn Hex A* is 9-23% of normal, and the P-CRM present in the cell line l'rom thc afoscmcntinncd suhacutc patient is much less than half that present in cells from our patient (Fig. 2.3). Thus, this suhstilution docs not appear to specifkcally affect the a active site, e.8. through some induced conl'ormational change. However, to fully eliminate this possibility we determined the Km of Hex A* for both the MUG and MUGS suhstrites. These were found to be normal (Table 2.2). Finally we assessed the ability of the Hex A* produced in transfected CHO cells and semi-purified from one of our patient's tibroblasts to hydrolyze its natural substrate, the GM~ activator/ GM~ganglioside complex (Fig. 2.7, Table 2.2). These data demonstrate that the

PProSO4Ser mutation reduces the ability of the Hex A* to hydrolyze ganglioside in the presence of human activator by 3-fold (Table 2.2). If this three fold reduction in the specific activity of Hex A* towards GMZganglioside, hut not MUG or MUGS, is factored in to our residual Hex A activity measurement (Table 2.1), the patients' Hex A* activity is reduced to 3-9% of normal. This is very close to the critical threshold values we discussed above and is consistent with the chronic phenotype observed in the patients. Recently we (Tse et al. 1996h) and others (Pennybacker et al. 1996) have reported the characterization of a-P fi~sionproteins. Although some of our conclusions differed, both studies concluded that the C-terminus of the P-polypeptide is important for the correct binding of the activator/ ganglioside complex. The charclcterization of this novel, naturally occurring mutation strengthens these conclusions and identifies the region surrounding Pro504 as the area in the C- terminus most likely to he responsible for this function. REFERENCES

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Ashkenas J, Byers PH (1997) Gene Regulation '97. The final stage of gene expression: Chaperones and the regulation of protein fate. Am. J. Hum. Genet. 6 1: 267-272 Bikker H, Meyer MF, Merk AC, devijlder JJ, Bolhuis PA (1988) XmnI liFLP at 5q13 detected by a 049 Xmn I fragment of human hexosaminidase (HEXB).Nucleic Acids Res. 16: 8198-8198 Bolhuis PA, Ponne NJ, Bikker H, Baas F, Vianney de Jong JMB (1993) Molecular basis of an aduIt form of Sandhoff disease: Substitution of glutamine for arginine at position 505 of the beta-chain of beta-hexosaminidax results in a labile enzyme. Biochim. Biophys. Acta 1182: 142-146

Brown CA, Mahuran DJ (1991) Active arginine residues in P-hexosaminidase: Identification through studies 01' the B l variant ol'Tay-Sachs disease. J. Biol. Chem. 266: 15855-15862

Brown CA, Mahurnn DJ (1993) P-hexosaminidase isozymes from cells co-transfected with a and p cDNA constructs: Analysis of a subunit missense mutation associated with the adult form of Tay-Sachs disease. Am. J. Hum. Genet. 53: 497-508

Brown CA, Neote K, Leung A. Gravel RA, Mahuran DJ (1989) Introduction of the a subunit mutation associated with the B 1 variant of Tay-Sachs disease into the P subunit produces a P-hexosaminidase B without catalytic activity. J. Biol. Chem. 264: 21 705-2 17 10

dlAzzo A, Proia RL. Kolodny EH, Kaback MM, Neukld EF (1984) Faulty association of a-and P-subunits in some forms of j3-hexosaminidase A deficiency. J. Biol. Chem. 259: 11070- 11074 De Gasperi R, Gama Sosa MA, Battistini S, Yeretsian J, Raghavan S, Zelnik N, Leshinsky E, Kolodny EH (1996) Lateonset Gm2 gangIiosidosis: Ashkenazi Jewish family with an exon 5 mutation (Tyrl XO-->His) in the Hex A alpha-chain gene. Neurology 47: 547-552 De Gasperi R, Gama Soszl MA, Grcbner EE, Mansfield D, Battistini S, Sartorato EL, Raghavan SS, Davis JG, Kolodny EH (1895) Substitution of alanine543 with a threonine residue at the carboxy terminal end of the bcta-chain is associated with themolnbile hexosaminidase B in a Jewish family of Oriental ancestry. Biochem. Mol. Med. 56: 31-6 Dlott B, DiAzzo A, Quon DVK, Neufeld EF (1990) Two mutations produce intron insertion in mRNA and elongated P-subunit of human P-hexosaminidase. 3. Biol. Chem. 265: 17921- 17927 Dreyfus JC, Poenaru L, Vibert M, Ravise N, Boue J (1977) Characterizntion of a variant of beta- hexosaminidase: "Hexosaminidase Paris". Am. J. Hum. Genet. 29: 287-293 Fernandes M, Kaplan F, Natowicz M, Prence E, Kolody E, Kaback M, Hechtrnan P (1992) A new Tay-Sachs disease BI allele in exon 7 in two compound heterozygotes each with a second novel mutation. Hum. Mol. Genet. 1: 759-761 Fiirst W, Sandhoff K (1992) Activator proteins and topology of lysosomal sphingolipid catabolism. Biochim. Biophys. Acta 1126: 1- 16 Gravel RA, Clarke JTR, Kahack MM, Mahuran D, Sandhoff K, Suzuki K (1995) The GM2 gangliosidoses. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The Metabolic and Molecular Bases of Inherited Disease, 7 edn, vol 2. McGraw-Hill, New York, pp 2839- 2879 Hasilik A, Neufeld EF (1980) Biosynthesis of lysosomal enzymes in fihrohlasts: synthesis as precursors of higher molecular weight. J. Bioi. Chem. 255: 4937-4945 Heng HHQ, Xie B, Shi X-M,Tsui L-C,Mahuran DJ (1993) Refined mapping of the GM~ activator protein (GM2A) locus to 5q 3 1.3-33.1, distal to the spinal muscular atrophy locus. Genomics 18: 429-43 1 Hinek A, Molossi S, Rabinovitch M (1996) Functional interplay between interIeukin-1 receptor and elastin binding protein regulates Cibronectin production in coronary smooth muscle cells. Exp. Cell Res. 225: 122- 131 Hou Y, Tse R, Mahuran DJ (1996a) The Direct Dctermination of the Substrate Specikity of the a-Active site in Heterodime~icP-Hexosuminidase A. Biochemistry 35: 3963-3969 Hou Y, Vavougios G, Hinek A, Wu KK, Hcchtman P, Kaplan F, Mahuran DJ (1996h) The ~allg*~euMutation in the u Subunit of P-Hexosaminidase A is not Associated with the B1-Variant Form of Tay-Sachs Disease. Am. J. Hum. Genet. 59: 52-58 Klima H, Klein A, Van Echten G, Schwarzmann G, Suzuki K, Sandhoff K (1993) Over- expression of a functionally active human GM2-activator protein in Escherichia coli. Biochern. J. 292: 571-576

Kytzia H-J, Sandhoff K (1985) Evidence for two different active sites on human p- hexosaminidase A. J. Biol. Chem. 260: 7568-7572 Kytzin HJ, Hinrichs U, Maire I, Suzuki K, Sandhoft' K (1983) Variant of GMzgmgliosidosis with hexosnminidase A having a severely changed suhstrate specificity. EMBO J. 2: 1201- l2O5 Kytzia HJ, Hinrichs U, Sandhoff K (1984) Diagnosis of infantile and juvenile forms of GM~ gangliosidosis variant 0 Residual activities toward natural and different synthetic substrates. Hum. Genet. 67: 4 14-4 1 8 Laemmli UK (1970) Cleavage of structusal proteins during the assembly of the head of Bacteriophage T4. Nature 227: 680-685 Leinekugel P, Michel S, Conzelmann E, Sandhol'f' K (1992) Quantitative correlation between the residual activity of beta-liexosaminidase A and and the severity of the resulting lysosomal storage disease. Hum.Genet. 88: 5 13-523 Li SC, Wu YY, Sugiyama E, Taki T, Kasama T, Cilsellato R, Sonnino S, Li YT (1995) Specific recognition of n-acetylneuraminic acid in the GM2 epitope by human GM2 activator protein. J. Biol. Chem. 270: 24246-2425 1 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275

Mahuran DJ (1990) Characterization of human placental P-hexosaminidase 12: Proteolytic processing intermediates of hcxosaminidaw A. J. Biol. Chem. 265: 6794-6799 Mahuran DJ (1991) The Biochemistry of HEX4 and HEXB Gene Mutations Causing GM2 Gangliosidosis. Biochim. Biophys. Acta 1096: 87-94 McInnes B, Potier M, Wakamatsu N, Melsncon SB, Klavins MH, Tsuji S, Mahuran DJ (1992) An unusual splicing mutation in the HEXB gene is associated with dramatically different phenotypes in patients from different racial backgrounds. J. Clin. Invest. 90: 306-314 Meier EM, Schwarzmann G, Furst W, Sandhol'f K (1991) The human GM2 activator protein. A substrate specific cohctor of r)-hcxosaminidase A. J. Biol. Chem. 266: 1879- 1887

Nakai H, Byers MG, Nowak NJ, Shows TB (1 99 1) Assignment of beta-hexosaminidase A alpha- subunit to human chromosomal region 15~23->q24.Cytogenet. Cell Genet. 56: 164-164 Navon R, Proia RL (1989) The Mutations in Ashkenazi Jews with Adult GM2 Gangliosidosis, the AduIt Form of Tay-Sachs Disease. Science 243: 147 1- 1474 Neote K, McInnes B, Mahuran DJ, Gravel RA (1990) Structure and distribution of an Alu-type deletion mutation in Sandhoff'clisease. J. Clin. Invest. 86: 1524-1531 Novak A, Lowden JA, Gravel YL, Wolk LS (1979) Preparation of radiolabelled GM2 and GA2 gmgliosides. J. Lipid Res, 20: 678-680 O'Dowd BF, Klavins MH, Willard HF, GrsveI R, Lowden JA, Mahuran DJ (1986) Molecular heterogeneity in the in Fin tile and juvenile forms of Sandhoff disease (0-variant GM2 Gangliosidosis). J. Biol. Chem. 26 1: 1 2680- 12685 Ohno K, Suzuki K (1988) Mutation in GM~-GangliosidosisB 1 variant. J. Neurochern. 50: 3 16- 318 Pennybacker M, Liessem B, Moczall H, Til'lt CJ, Sandhoff K, Proia RL (1996) Identification of domains in human beta-hexosamiiiidase that determine substrate specificity. J. Biol. Chem. 271: 17377-17382

Proia RL, dlAzzo A, Neulkld F (1984) Association of a- and P-subunits during the biosynthesis of P-hexosarninidase in cultured f'ibmhlasts. J. Bid. Chem. 259: 3350-3354 Redonnet-Vernhet I, Mahuran D, Salvayre It, Dubas F, Levade T (1996) Significance of two point mutations present in each HEXB allele of patients with adult GM2 gangliosidosis (Sandhoff disease): Homozygosity of 11do7->~alsubstitution is not associated with a clinical or biochemical phenotype. Biochim. Biophys. Acta. 1317: 127- 133 Rubin M, Karpati G, Wolfe LS, Carpenter S, Klavins MH, Mahuran DJ (1988) Adult onset neuronopathy in the juvenile typi of hexosaminidase A &d B deficiency. J. ~euro.Sci. 87: 103-1 19 Sandhoff K, Conzelmann E, Neufeld EF, Kahack MM, Suzuki K (1989) The GM2 gangliosidoses. In: Scriver CV, Beaudet AL, Sly WS, Valle D (eds) The Metabolic Basis of Inherited Disease, 6 edn, vol 2. McGraw-Hill, New York, pp 1807- 1839 Sandhoff K, Harzer K, Fiirst W (1995) Sphingolipid activator proteins. In: Scriver CR, Beaudet AL, Sly WS, Vallc: D (eds) The Metabolic Basis of Inherited Disease, 7 edn, vol 2. McGraw-Hill, New York, pp 2427-244 1 Smiljanic-Georgijev N, Rigat B, Xie B, Wang W, Mahuran DJ (1997) Characterization of the affinity of the GM2 activator protein for glycalipids by a fluorescene dequenching assay. Biochim. Biophys. Acta 1339: 192-202 Tnnaka A, Ohno K, SandhofC K, Mairc;: I, Kolodny EH, Brown A, Suzuki K (1990) GM~- gangliosidosis B 1 variant: Analysis oT P-hexosaminidast: a gene abnormalities in seven patients. Am. J. Hum. Genet. 46: 329-339 Tews I, Perrakis A, Oppenheim A, Dauter 2,Wilson KS, Vorgias CE (1996) Baterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay-Sachs disease. Nature Struc. Bid. 3: 638-648 Tse R, Vavougios G, Hou Y, Mahuriln DJ (1996a) Identification of an active acidic residue in the catalytic site of P-hexosaminidasc. Biochemistry 35: 7599-7607 Tse R, Wu YJ, Vavougios G, Hou Y, Hinek A, Mahuran DJ (199th) Identitkation of Functional Domains within the or arid p Subunits of P-Hexosaminidase A Through the Expression of a-p Fusion Proteins. Biochemistry 35: 10894- 10903 Wu YY, Lockyer JM, Sugiyama E, Pavlova NV, Li Y-T,Li S-C (1994) Expression and specificity of human GM2 activator protein. J. Bid. Chem. 269: 16276-16283 Chapter 3

Identification of PArg211 as a Residue Critically Involved in Substrate Binding and Orientation Using a C-terminal Epitope-tagged Form of Pro-Hex B 4 ' INTRODUCTION

The human lysosomal hexosaminidase (Hex) isozymes are dimeric enzymes composed of a [encoded by the HEXA gene 15q23q243 (Nakai et al. 1991) and/or $ [encoded by HEXB gene

5q13 (Bikker et al. 1988)l subunits. The combination of these subunits generates three active isozymes: Hex A (up),Hex B(PP), and Hex S (m).Defects in the a (due to mutations in the

HEXA gene) and B (due to mutations in the HE&B gene) subunits result in the two major forms of -2 gangliosidoses, i.e. Tay-Sachs disease and Sandhoff disease, respectively. These inheritable neurodegenerative diseases are characterized by lysosomal accumulation of Gm mainly in neuronal tissues where gangliosides are most actively synthesized (reviewed in (Gravel et al. 1995)).

Since the primary structures of the a and P subunits share 60% sequence identity, both are evolutionarily and therefore structuralIy related (Korneluk et al. 1986). Furthermore it is believed that all the members of a given hydrolase family are evolutionarily related and have similar three-dimensional stnrctures, Thus molecular modeling of human Hex has been done based on the 3-D structure of bacterial chitobiase, as both are part of the glycosyl hydrolase "Family 20". However, the accuracy of such modeling is variable and dependent on the degree of primary sequence similarity between the protein with the known structure and the protein being modeled. Sequence alignment shows that there is only a 26% identity of amino acid sequence between human Hex and the bacterial enzyme in their active site region. Furthermore, this degree of identity is based on an alignment that is only achieved by generating many large gaps in the human sequences, although these gaps are predicted to be at loop structures (Tews et al. 1996). Outside of this region there is little sequence similarity. In addition chitobiase is a monomer and functions at neutral pH. Thus, whereas the modeling of bacterial chitobiase may be helpful for characterizing the active site of human Hex, the validity of the model and the accuracy of the alignment on which it is based must be determined experimentally. Recently, comparative molecular modeling of Streptomyces plicatus (Sp-Hex), a member of Family 20 enzymes, has also been made from the chitobiase structure (Mark et al. 1998). The overall three-dimensional structure that was generated for Sp-Hex is similar to the above model of human Hex (Tews et al. 1996). However, the Sp-Hex shares only 25% and 30% sequence identity to human Hex and chitobiase, respectively. Interestingly, characterization of two residues in Sp-Hex that align with those associated with human B1 variant of Tay-Sachs disease by site mutagenesis and expression, produced data consistent with those predicted from the chitobiase model (see below). Analysis of missense mutations, associated with GM~gangliosidoses in hopes of identifying active site residues in human Hex, has not been as informative as initially predicted since most of these point mutations result in the retention of the mutant protein in the endoplasmic reticulum (ER), due to the ER's tight "quality control system" (reviewed in

(Mahuran 1991; Mahuran 1997)). Only two missense mutations at a-Arg178 (Kytzia et al. 1983;

Ohno and Suzuki 1988) and a-Asp258 (Fernandes et al. 1992) have produced some clues to the active site residues in Hex. These mutations are associated with the B1 variant form of Tay- Sachs disease. B1 patient samples have a unique biochemical phenotype. They express reasonable levels of Hex A activity, i.e. above what is thought to be the critical level of activity needed to prevent Gmstorage, when assayed with neutral substrate, e-g. MUG. However, unlike the normal Hex A, the B1 variant-Hex A is inactive toward the a-specific substrates, e.g.

MUGS. This unusual phenotype leads to the hypothesis that these mutations affect residues at or near the active site of the a-subunit (see below). However, analysis of a subunit in the context of heterodimeric Hex A is not straightforward, because of the presence of a normal, active P subunit. For example, it was not known whether the affected a subunit had undergone a partial change in substrate specificity, i.e. become $-like and could still hydrolyze neutral substrates, or if it had entirely lost its catalytic ability. Furthermore we have demonstrated that the normal J3 subunit in heterodieric Hex A retains approximately 1.33% of a's activity toward the so-called a-specific MUGS substrate (Hou et al. 1996a). The normal Hex B in Tay-Sachs cell lines was also found to generate 1-6% normal level of MUGS activity (Fernandes et al. 1997). In addition, expression of a mutant acDNA alone in hopes of producing mutant Hex 3 for analyses is also problematic due to mutant a dimers' over-sensitivity, i.e. lack of dimers formation, to any amino acid substitution even when over-expressed in COS cells (Brown and Mahuran 1993). To overcome these problems, we developed three methods for analysis of these a-mutations. The most recent one I developed as part of this thesis takes advantage of the first method developed by Brown et a1 (Brown et al. 1989). For a-residues that are not conserved in P, I transfected the mutant a with a cDNA encoding an inactive but otherwise normal PArg211Lys subunit (see below) to effectiveIy eliminate the interference of activity in Hex A (Hou et al. 1996a). For residues that are homologous in both the a and P subunits, Brown et a1 developed a method that analyzes the biochemical consequences of the a mutation by a conservative substitution in the aligned codons in the P-subunit, i.e. PArg2 11Lys for /aArg 178 (Brown and Mahuran 199I), and later Tse et a1 applied this method to the other codon associated with the B1 variant,

PAsp290Asn for a Asp258 (Tse et al. 1996). Kinetic analysis produced data that indicated the

PArg211Lys Hex B has a normal Km and 0.2% Vmax, suggesting PArg2 11/01 178 takes part in substrate catalysis (Brown and Mahuran 1991). The apparent Km for PAsp29OAsn was increased to 2.5-fold with a Vmax of 7 1% of wild-type Hex B, indicating the involvement of PAsp290 in substrate binding (Tse et al. 1996). The above observations are generally in agreement with the molecular modeling data for human Hex as well as Sp-Hex that are predicted from the structure

of bacterial chitobiase (Mark et al. 1998; Tews et al. 1996). The PAsp290/a258 corresponds to chitobiase Asp448 that is only indirectly involved in substrate binding (Tews et al. 1996). In Sp- Hex, Asp 246 aligns with these residues. When a cDNA encoding an Asp246Asn substituted protein was expressed in E. coli, the purified mutant protein was shown to have 3-fold higher

Km relative to the wild-type enzyme and Vmax decreased by 2-fold. Human fJArg21 Ya178 aligns with Arg379 in chitobiase and kg162 in Sp-Hex. From the chitobiase model it is known that Arg379 sits at the base of a binding pocket for NAGA and plays the most critical role of any residue in substrate binding and orientation. Mutational analysis of Argl62His in Sp-Hex revealed that its Km had increased by 40-fold and its Vmax was reduced by 5-fold. Thus the Arg residues in chitobiase and Sp-Hex are "active residues", directly involved in substrate binding, rather than catalysis as we suggested for w11in human Hex. Because of the low Vmax and the apparently normal Km of Hex B* (2PArg211Lys), activity could have arisen from a very low level of contaminating endogenous COS or CHO cell Hex, i.e. a "signal to noise" problem (see below). The above problem of the "signal (from translated human cDNA) to noise (endogenous Hex)" ratio is shared by all the other laboratories studying human Hex through mutation and cellular expression (Fernandes et al. 1997; Pennybacker et al. 1997). Pennybacker et a1 reported signal to noise ratio of 30:l for expression of wild-type Hex B in insect cell (Pennybacker et al.

1997). In another study, Fernandes attempted to increase the signdnoise ratio by analyzing a mutations in a human Tay-Sachs disease cell line. Since this method relies on the recruitment of an endogenous p subunit by the a produced from transfected cDNA, its signal to noise ratio is dictated by the level of p activity in the a*p dimer towards MUGS. As mentioned above, the normal p subunit presented in Hex A produces 1.33% of the normal Hex A MUGS activity, i.e.

45:1 signahoise ratio. We previously reported a 50-100 fold of signahoise ratio for the Hex B

activity from cells transfected with the wild-type BcDNA (transient or permanently transfected

COS or CHO cells) using a "human Hex-specific" immunoprecipitation assay (Brown et al. 1989). For purpose of analyzing naturally occurring mutations associated with Gm gangliosidosis the above signal to noise ratio from cells expressing human Hex cDNA is more than sufficient (Akalin et al. 1992; Brown and Mahuran 1993; Hou et al. 1996b; Trop et al. 1992). However site-directed mutagenesis studies of other glycosyl hydrolases indicated that specific activity, Kcat, is lowered over 6000-fold if the catalytic acid is neutralized, e.g. (Malcolm et al. 1989) and CenA from Cellulomonasfimi (MacLeod et al. 1994). This extremely lowered Kcat is characteristic of the loss of this group. To date, none of the above methods for studying the human Hex active site has reached this ratio of signalhoise. Thus with only our present methods an unequivocal identification of the catalytic residue in Hex would require a signal to noise increase of 60-fold. As well, when a residue in Hex involved in substrate binding is mutated and its mutant activity is decreased to near the background level of the non-transfected cells, it is also impossible to distinguish the Km value of the human expressed mutant enzyme because of contamination by the endogenous enzyme. In order to resolve the above problems, a new system is needed to more fully remove the endogenous Hex contamination from the samples of mutant human Hex. In the present study, we report a method for the generation of a C-terminal His6-tagged Pro-Hex B in transfected CHO cells. We demonstrate that this novel form of Hex B is secreted and not retained in the ER as other tagged constructs were (data not shown), and is easily purified away from the endogenous Hex contamination. We further demonstrate that the kinetic properties of both pro-Hex B and pro-Hex B-Hi% are the same as the mature form. As an initial attempt to test the validity of the bacterial chitobiase model with this highly purified form of Hex

B, we re-evaluated the conservative f3Arg211Lys substitution. In addition we analyzed the ability of the mutant to bind the CNAG affinity ligand. From these new data, we are able to definitely

assign a role to residue BArg211 in substrate binding and orientation, which adds weight to the

validity of the bacterial chitobiase model as extrapolated to human Hex. MATERIALS AND METHODS

A Cons- Cloning procedures were as described by Sambrook et al. (Sambrook et al. 1989). In order to construct DNA encoding pro $-Fxa-His6-COOH, a 1.8 kb fragment, containing pchain cDNA and Factor Xa-His6 sequences, was amplified using the sense primer (5'- AGTAAGCTTGCGGCCGCAGAAGTCGGGTCCCGAGGCT-3') and the antisense primer (5'CGGGTCTAGAGCGGCCGC'ITCAATGATGATGATGATGATGTCTACCCTCGATCAT GTI'CTCATGGTTACA-3'). The reactions were performed in a 100 pl mix containing 10 ng plasmid DNA, 20 mM Tris-HC1 (pH 8.8), 10 mM KCl, 10 mM (m4)2S04,2 mM MgS04,O. 1% Triton X-100,0.2 mM each of dNTPs, 0.4 plkl of each primer and 1 unit Vent DNA polymerase (BioLabs Inc., Beverly, MA). The cycling steps used were as follows: 1 cycle of heat denaturation at 95 OC for 5 min, 28 cycles of each consisting of denaturation at 95 OC for 30 sec, annealing at 56 OC for 1 min, and extension at 73 OC for 1 min, 1 cycle of 73 OC for 7 min, in a

Perkin Elmer-Cetus thermal cycler 2400. The PCR product was digested with Xba 1, purified with Geneclean kit (Bio 101 Inc., Vista, CA), and subcloned into expression vector pcDNA3.1HisA-1, which contains neomycin gene as a selective marker. pcDNA3.1HisA-1 is modified from pcDNA3.1HisA (Invitrogen Inc., Carishad, CA), in which the Hind III-Xba I polylinker sequence was replaced with Hind 111-Xba I-Not I-Xba I linker to facilitate the subcloning procedure. The resulting fusion expression plasmid is designated as pcDNA-P-His6.

For construction of pcDNA-P*-His6 encoding a Arg211Lys substitution, a 1.5 kb Bst X I fragment was excised from pHexB43 (kg211Lys) (Brown and Mahuran 1991) and used to replace the corresponding DNA fragment in pcDNA-P-His6. The mutation and orientation of the insert were verified by DNA sequencing using T7 DNA polymerase sequencing kit (Phannacia) before transfeztion of the DNA into CHO cells.

l2fuhmz CHO cells were maintained in a-MEMsupplemented with 10% fetal bovine serum, 100 pglml streptomycin, and 100 pglml penicillin, at 37 OC in 5% Ca. Transfected cells were grown in the same media containing 400 pg/ml G418. For large-scale purification of Hex, cells were plated and grown in the 850 cm2 roller bottle (Becton Dickinson Labware, Lincoln Park,

NJ) at 37 OC in 5% C@ incubator, When cells reached confluence, they were then washed twice with PBS and replaced with serum free media (GIBCO-BRL). The media were collected for subsequent protein purification (see below).

Transfections were performed using Superfect Reagent (Qiagen Inc., Valencia, CA) by essentially following the manufacturer's instructions. CHO cells were grown overnight until they were about 40% confluent. Ten pg of DNA were mixed with 40 pg of Superfectm reagent (Qiagen) in 800 pl serum-free MEM. The mixture was incubated for 10 minutes at room temperature to form DNA-Superfect complex. The complex was then added drop-wise to the culture dishes and incubated for 2-hr at 37 OC. Next, the cells were washed and refed with a-

MEM plus 10% FCS for 2 days. Following this, the cells were trypsinized and replated at a 1:8 dilution in a-MEMwith 10% FCS and 400 pglml neomycin. Two weeks later, drug-resistant colonies were picked and grown in 24-well plates. The medium and lysate from the surviving cells were assayed for Hex activity and those producing the highest levels were selected for further growth and analyses. For cells transfected with pcDNA-P-Hiss* encoding a R2 11K substitution, G418 resistant colonies were screened by Western blot analysis using human anti- Hex B antibody. Elmmawu Cells were harvested and Iysed in a buffer of 10 mM Tris-HCI (pH 7.5) and 5% glycerol through five sets of fhze-thaw cycles. For protein purification purpose, cells were directly lysed in a native binding buffer (10 mM sodium phosphate pH 7.8, 0.5 M NaCl) for Ni-NTA chromatography or 20 mM citrate-phosphate buffer (pH 4.5) containing 0.5 M NaCl for CNAG affinity chromatography (see below). Protein concentration was determined by the Lowry method (Lowry et al. 1951). Human Hex activity from cell lysates and media was measured using a MUG substrate based on a MU fluorescent assay (Brown et al. 1989).

estern Equal amounts of total protein from each sample of cell lysate or purified protein were resolved by SDS-PAGEusing the Laemmli gel-buffer system (12.5% gel) and a Bio-Rad mini- gel system (Laemmli 1970). Proteins were transferred to nitrocellulose and the filter was blocked with 5% skim milk, as described previously (Hou et al. 1998; Hou et al. 1996b). The primary antibody was a rabbit anti-human Hex A. A horseradish peroxidase-conjugated goat anti-rabbit XgG (1: 10,000 dilution, Irnmux) was used as the secondary antibody. The nitrocellulose was developed using the Amersham ECL system and exposed to Hyperfilm.

I-NTA chrm The Pro-Bond beads (Invitrogen Inc., Carlsbad, CA) were prepared by washing twice with sterile water and three times with the native binding buffer. One ml of gel was packed in small column and equilibrated in the same buffer. Cell lysates or media from control CHO or transfected cells were supplemented with NaCl to a final concentration of 0.5 M to reduce the non-specific binding before they were directly loaded on to the Pro-Bond column. Nonspecifically bound proteins were removed by washing the column twice with 3 ml native binding buffer and three times with 3 ml native wash buffer (10 mM sodium phosphate pH 6.0, 0.5 M NaCl). H.s6-conta.ining proteins were eluted with increased concentration of imidazole (30 mM, 80 mM and 500 mM) in the native wash buffer. The purity of the proteins was examined through SDS-PAGEfollowed by Coomassie Blue staining. *m *m . . . . The apparent Km and Vm,, values were determined for the MUG substrate using concentrations ranging from 0.1 to 4 mM (Hou et al. 1998). These constants were calculated using a computerized nonlinear least square curve fitting program for the Macintosh, KaleidaGraphTM 3.0. Thus the individual substrate concentrations and their corresponding initial velocity measurements were directly fitted to the Mic haelis-Menten equation, Vi=Vmax

*[S]/(Km+[S]), making possible the calculation of an accurate standard error (Tommasini et at.

1985a). Kcat values were calculated based on the enzyme's calculated specific activity at Vmax (MU released/hr.mol of each purified enzyme).

ex The affinity ligand CNAG was obtained from Dr. Withers's lab at the University of British Columbia. The ligand was coupled to SephacryX S-200 according to Mahuran (Mahuran and Lowden 1980). For purification of pro- and mature Hex B from Tay-Sachs fibroblasts, the media or lysate samples were dialysed in 10 mM sodium phosphate buffer (pH 6.0) containing 0.2 M NaCl before loaded on a CNAG column. After washing to remove nonspecific proteins with three gel volumes of 10 mM sodium phosphate buffer, 0.5 M NaCl, the Hex protein was eluted with 10 mM Tris-HC1 (pH 8.5). For substrate binding assay, the affinity ligand beads were pretreated with 0.1% RNase A overnight to block unspecific sites. After washing the beads three times to remove the RNase A, 10 pg of purified Hex proteins were loaded on a 0.3 ml minicolumn. The column was then washed twice with 2 ml of 10 mM sodium phosphate buffer pH 6.0, 0.2 M NaCl (wash buffer). Finally the specifically bound Hex protein was eluted with the same wash buffer containing 150 pM S-lactone (a competitive inhibitor). The Hex binding affmity was assessed by the Hex protein recovery, which was calculated on the basis of the total eluted protein versus the total protein loaded on the column as determined by Lowry method (Lowry et al. 1951).

RESULTS

Bxnression and nuritkcation of C-terminal His&Hex B in CHO cells

A fusion construct containing p cDNA and C-terminal His(, tag (prop-Fxa-Hisc,-COOH)

was generated by PCR. A fiictor Xa site was inserted between pro-P and Hisl; sequence to

facilitate removal of the tag. This DNA fragment was verified by sequencing and then subcloned

into mammalian expression vector pcDNA3.l HisA to produce pcDNA-p-His6. The DNA

construct was tnlnstkcted into CHO cells to establish a permanent cell line from which individual clones were selected hued on the Hex activity found in their media (Hou et al. 1996b). To examine the expression and intracellular transport of the His-tagged Hex B protein, Western hlot analysis was performed using equal amounts of lysate proteins from cells

transfccted with pcDNA-P-Hisc and cells transltcted with pEFNE0-P (Tse et al. 1996). which contains the p cDNA alone and thus serves as a control. Both of these cells produced similar levels of immunorenctive hands corresponding to mature P polypeptides (-30 kDa) (Fig. 3.1, lmes p and &His), indicating that the His(, fusion enzyme can he incorporated into the lysosome.

We next attempted to purily the HisC,-tilgged proteins from cell Iysates by Ni-NTA chromatography under native conditions. After washing the column several times with a low concentration of imidazole to remove unspecific and endogenous Hex contaminating proteins, the elution was carried out with 500 mM imidazole. Unexpectedly, the level of Hex activity eluted from cell lysatc was very low, i.e. only 22 of Hex activity could he recovered from NP2 column (Table 3.1). This result indicated that the His6-tag is cleaved when Hex reached the lysosome. Since high level of Hex activity is also secreted from the trmsfected cells, we Figure 3.1. Western blot analysis using an anti-human Hex B IgG in lysates from mock CHO

cells (CHO). CHO cells transfected with pEFNEO-P (j3J pcDNA-P-His6 @-His) or

pcDNA-His-P* encoding a R211K substitution (P+-His). Samples of each lysate contain

equal amounts of total protein (20 pg). The positions corresponding to the prof3 (65 kDa)

and mature p chains are indicated on the right. Protein standards are shown on the left. CHO P P-His P*-His KDa Table 3.1. Assay of Hex binding to Ni-NTA column from the medium and lysate of mock and pcDNA-P-His6-~nsfectedCHO cells using MUG substrate

Enzyme Medium Lysate Totala ~ree~Boundc Totala ~ree~Boundc

CHO 9.2 8.4 0.0008 1.03 0.9 0.0001 pcDNA-P-His6 54 10.9 38.3 3.75 3-21 0.08

Note: The medium and lysatc were rotiitcd with Ni-NTA heads overnight at 4'~.The heads were then packed into a minicolumn for wash and elution (see material and methods). Total Hex activity (endogenous and expressed human) in the medium or lysate from the same batch of cells. Hex activity remaining in the supernatiint, i.e. unbound proteins containing endogenous Hex

and some human expressed mature Hex B in lysate in which His6-tag was cleaved.

Hex activity (expressed human Hex B with n C-terminal Hisc,-tag)eluted from Ni-NTA heads after wash with low concentration of imidazole. attempted to purify the pro-Hex B-Hisc, from the expression medium. Seventy-one percent of

pro-Hex B-His6 activity was recovered after Ni column chromatography (Table 3.1). SDS-PAGE

followed by Coomassie blue staining detected a single band corresponding to pro-P (65 kDa)

polypeptide (Fig. 3.2A). Western blot using anti-human Hex 8 antibody further confirmed the

identity of the 65 kDa poIypeptide as the P subunit of Hex (Fig. 3.2B). In contrast, Iess than

0.01% of endogenous protein in nontransfected CHO cells nonspecifically hound to the Ni column from either medium or lysate. Thus, Ni-NTA chromatography resulLs in the isolation of the expressed human Hisr,-tagged pro-Hex B that is over 99.99% pure and therefore free of contamination by the CHO enzymes.

Biochemical nronerlies of Pro-Hex B and Pro-Hex B-Hisc,- I next determined if the pro-Hex B-Hisr, isozyme has similar biochemical properties to the pro- and mature forms of Hex B. Pro- and mature forms were purified from the medium and lysate of CHO cells permanently transfected with pEFNEO-P (Hou et al. 1996a) or from Tay-

Sachs fibroblasts hy CNAG affinity chromatography, a wcll established and classic method for Hex purification (Mahuran and Lowden 1980). Determination of the specitk activity using these purified enzymes revealcd that pro-Hex B-Hisr, had a specific activity similar to that of either pro-Hex B or mature Hex B lib~rn(Table 3.2). Next the kinetic constants for the MUG substrate were determined (Fig. 3.3). The apparent Km and Vmax values for both precursor forms were identical to those determined with the mature form of Hex B isolated from ttansfected cells

(Tahla 3.2) or human placenta (Tommnsini at al. 1985h), i.e. Km=-0.7 mM and ~max=-107 nmol/hr/mg Hex protein, respectively. Taken together, these data demonstrate that; (I) C- terminal His6-tag in pro-Hex B does not have an effect on it,s kinetic parameters; (2) both precursor forms (pro-Hex B and His-pro-Hex B) have the same biochemical characteristics as the mature isozyme. Figure 3.2. Coomassie blue staining (A) and Western blot (B) of purified proteins from the serum free medium of untrilnsfected CHO cells (CHO), CHO cells transfected with

pcDNA-His-P @-His) or mutant pcDNA-His-P* encoding a R211 K substitution (P*-His),

or cells transfected with pEFNEO-P (P). The purification of Hex for the former three (lanes

CHO, P-His and P:l:-His ) was carried out using Ni-NTA column under native conditions

and pro-Hex B (lam P) was purified by CNAG afflnity chromatography. The identities of

these purii'ied proteins were veritkd by using an anti-Hex B IgG (B). The Mr standard and

pro-P location are indicated. CHO P P-His P*-His STD -148

- 60

- 42

- 30

KDa

CHO P P-His P*-~is

17 KDa Figure 3.3. Kinetic analysis of the mature Hex B, pro-Hex B,pro-Hex &Hisr, and mutant pro-

Hex B*-Hisfi containing R211 K using the neutral MUG (mM)substrate. The experimental

data paints are shown and were directly fitted to the Michaelis-Menten equation. The equation with its calculated best-fit constants (Km and apparent Vmax) were then used to generate the lines joining the data points on the graph. The R values for all of the experiments were >0.995. Thc actual Km and Vmax values are given in Table 3.2. Kinetic analysis of wt and mut. Hex B (R211 K)

F 20 -

Hex B + Pro-Hex6

0 0.5 1 1.5 2 2.5 3 3.5 4 MUG (mM)

Examination of the role o f PArrz211 in the active site sf Hex B

Previous studies indicated that Arg 21 1 is an active site residue in Hex B and that it is probably involved in catalysis (Brown and Mahuran 1991); whereas it was predicted from chitobiase model that this residue is directly involved in substrate binding. To determine the

exact role of PArg2 1 1, pcDNA-P*-Hise encoding a Arg2 1 1Lys substitution was transfected into

CHO cells. Western blot verified the trimsfected cells can produce the mature, lysosomal form of

mutant Hex B (Fig. 3.1, lane P*-His), most of which is not retained on the His6-tag (data not

shown). Therefore, expression media was utilized for puritkation of the mutant Hex B as well as wild type pro-Hcx B-HisG. The elution was carried out with increasing imidazole concentration. Kinetic parameters for the MUG substrate were determined (Table 3.3).

Interestingly, the Km value for the pool of mutant enzyme that eluted at 30 mM imidazole was 0.65 mM, which was the same value as the endogenous CHO Hex. The pool eluting at 80 mM imidazole had an apparent Km of 1.5 rnM, and the pool eluting at 500 mM had an apparent Km >8 mM (no further Hex being eluted at higher concentration). These results are consistent with the idea that the 80 and 30 mM iiactions of enzyme contained increasing amount of endogenous as compared to mutant human Hex B. The 10-fold increase of Km in mutant Hex B as compared to wt enzyme indicated that Arg 21 1 is indeed involved in substrate hinding. To verify that the mutant Hex B encoding an R211K substitution affects substrate hinding as well as Vrnax, a CNAG affinity minicolumn was prepared. The ligand beads were

blocked with RNase proteins hefore the purified Hex was loaded. After washing of Km-altered mutant proteins, the elution was performed with the high af'finity competitive inhibitor, 8- lactone(Mahuran and Lowden 1980). If a mutant alters its suhstrate binding sites, it would also be expected to have a lower affinity for the ligand. As shown in table 2, over 70% of wt Hex from either Hex B or pro-Hcx B-Hisl; binds to CNAG. In contrast, only 7.4% of mutant protein binds to the column, consistent with its higher Km for MUG substrate. Table 3.3. Km"values for preparations of imidazole elution from the media of cells expressing pro-Hex B-His6 and pro-Hex B*-His6 (R2 1 1 K) utilizing MUG substrate

lmidazoleb Pro-Hex B-His6 Pro-Hex B*-His6

0 mMC 0.65k0.08 0.65k0.10 30 mM 0.70k0.10 0.68M.07 80 mM 0.68k0.09 1 .52+0. 12 5W mM 0.66k0.07 >8"

a. Km values art: cxpresscd in millimolar and thc standard crror described as A, calculated from the hcst lit curve based on the Michaelis-Mcnten equation (R values were all 0.998). b. Imidazale in a wash buffer (pH 6.0) (see methods). c. Sample was ohtained in wash preparation without imidazole, mast of which contains endogenous enzyme. d. No exact Km value was obtained due to low solubility of MUG substrate. DISCUSSION In vitro mutagenesis and mammalian cell expression is now a routine approach to link mutations associated with GM~gangliosidosis to their biochemical functions and characteristics.

However, all the laboratories expressing human Hex retain a small amount of the endogenous

Hex from these host cells. This rr=sult.s in I100 of signal/noise ratio. A >60-fold increase in this signavnoise ratio is needed to reach the level that has been required to identify the catalytic acid group in other glycosyl hydralases. Ta circumvent the interference of endogenous Hex we engineered a His6 DNA sequence into either the 5'- or 3'-end of HEXB gene and transfected each of them into CHO cells. Whelcas N-terminal His6-tag affected the ability of the Hex protein to exit the ER (data not shown), the Hex B containing the C-terminal Hisfiepitope did reach lysosomes (Fig. 3.1). However, the C-terminal His(, tag itself is apparently removed in the lysosome along with other prepropcptides during the formation of the mature Hex B form. Fortunately, large amounts of lysosomal protcin encoding human Hex are secreted into media of transfected cells. Therel'ore we were able to purify the human pro-Hex B-Hisc, from the expression medium by Ni-NTA chromatography under native conditions. This purified pro-Hex B-His(, was found to be 99.99%, Sree from the contamination of endogenous CHO Hex. The MUG specific activities for the purilled pro-Hex B, pro-Hex B-Hisc, and the mature Hex B forms were found to be nearly identical, i.e. 10.5, 13.8 and 10.1 mmoUhrImg, respectively (Table 3.2). Furthermore, kinetic analysis with MUG substrate revealed that their kinetic constants (Table

3.2) for the pro-Hex B (Krn=0.70&0.07, Kcat=4.9), pro-Hex B-His(, (Kmd1.66f 0.07, Kcat=5.9) were also similar to those determined for mature Hex B (Km=0.71 40.05, Kcat=3.5) (Tommasini et al. 1985h). Thus, the hiochemicnl properties 01' the precursor forms (pro-Hex B, pro-Hex B- Hisc;) are not dif'lkrent from the mature Hex B form. Consequently this novel Hex construct could he used for muritional analyses, in particular, studies of the isoenzyme's active site, which requires the absolute ~.c=mavulof endogenous Hex. Our previous data revealed that PArg211 was an active site residue likely part of catalysis

in Hex B (Brown and Mahuran 1991). However, the recent molecular modeling from bacterial chitobiase suggests that the aligned residue in chitobiase, Arg349, is directly involved in

substrate binding, interacting with OH3 and OH4 of a B-GlcNAc substrate, docking the substrate

in its proper orientation in the active site (Tews et al. 1996). It is possible that apparent normal

Km from Hex B* (2PArg211Lys) was misinterpreted due to a very low level of endogenous

COS or CHO cell Hex co-immunoprecipitated with the mutant human Hex B. This conclusion was confirmed by the kinetic analysis of Hex fractions eluted with a lower imidazole concentration. Under these conditions the apparent Krn for the Arg211Lys mutated protein was normal (Table 3.3). Kinetic analysis of the highly purified 500 mM elution fraction resulted in an apparent Km for MUG of this mutant increased by at least 10-fold as compared to wild-type Hex B with a Vmax reduced to only 0.07% of normal. These data indicated that Arg211 has a critical role in substrate binding and suggested that it is required to properly orientate the substrate in the active site. The very low apparent Vmax further suggested that without an Arg at this position substrate molecules can be bound incorrectly and act as inhibitors. To further verify the above observation, a substrate affinity column was used. We found that only 7.4% mutant Hex B could be bound and eluted from the column with a strong competitive inhibitor, &lactone. This result indicated that the substrate binding site on the mutant protein was altered so that the mutant Hex can no longer efficiently bind the Hex affinity ligand. These data are also consistent with those from molecular modeling followed by mutational analysis of Arg162 in Sp-Hex (Mark et al. 1998). Thus, the experimentally determined role for the Arg residue in both human and Streptornyces Hex was correctly predicted by the chitobiase model. The data contained in this report have also important implications for future experiments aimed at studying other active residues in Hex based on the chitobiase model. The acidic residues will be the first targets as we previously indicated that acidic residues were involved in active sites in Hex B (Tse et al. 19%). Furthermore it remains to k determined whether human Hex uses a general acid catalysis or a substrate assisted catalysis mechanism. The latter was proposed based on the chitobias structure and also appeared to be supported by recent inhibitor studies of mammalian Hex. Controversy dso exists over the identity of the catalytic acid group hecause all previous methods for mutational analysis did not obtain an unequivocal signaVnoise ratio and no one group examined all candidate residues. Therefore characterization of these acidic amino acids, i.e. Asp241, Asp354 and Glu 355 of human Hex B, using the approach presented in this report is required. If the full characterization of these active site amino acids validates the chitohiase rnodcl then it would he apparent that human Hcx also shares this substrite-assisted mechanism Ihr hydrolnscs. REFERENCES Akalin N, Shi H-P,Vavougios G, Hechtman P, Lo W, Scriver CR, Mahuran D, Kaplan F (1992) Novel Tay-Sachs disease mutations from China. Hum. Mut. 1: 40-46 Bikker H, Meyer MF, Merk AC, devijlder JJ, Bolhuis PA (1988) XmnI RFLP at 5q13 detected by a 049 Xmn I fragment of human hexosaminidase (HEXB). Nucleic Acids Res. 16: 8 198-8198

Brown CA, Mahuran DJ (1991) Active arginine residues in P-hexosaminidase: Identification through studies of the B1 variant of Tay-Sachs disease. J. Biol. Chem. 266: 15855-15862

Brown CA, Mahuran DJ (1993) P-hexosaminidase isozymes from cells co-transfected with a and p cDNA constructs: Analysis of a subunit missense mutation associated with the adult form of Tuy-Sachs disease. Am. J. Hum. Genet. 53: 497-508

Brown CA, Nwtt: K, Leung A, Gravel RA, Mahuran DJ (1989) Introduction of the a subunit mutation associated with the B1 variant of Tay-Sachs disease into the P suhunit produces a P-hexosaminidase B without catalytic activity. J. Bid. Chem. 264: 21705-217 I0 Fernandes M, Kaplan F. Natowicz M, Prence E, Kolody E, Kahack M, Hechtman P (1992) A new Tny-Sachs disease B1 allele in exon 7 in two compound heterozygotcs each with a second novel mutation. Hum. Mol. Genet. 1: 759-761 Fernandes MJG, Yew S, hclerc D, Henrissat B, Vorgiils CE, Gravel RA, Hechtman P, Kaplan F (1997) Identil'ication of candidate active site residues in lysosomrrl beta-hexosaminidase A. J. Biol. Chern. 272: 814-820 Gravel RA, Clarke JTR, Kahack MM, Mahuran D, Sandhol'f K, Suzuki K (1995) The GM2 gangliosidoscs. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The Metabolic and Molecular Bases 01' Inhcritcd Disease, 7 edn, vol 2. McGrtlw-Hill, New Yosk, pp 2839- 2879

Hou Y, McInnes B, Hinek A, Karpati G, Mahulaan D (1998) A ~ro5%er substitution in the P- suhunit of P-hexosaminidase A inhibits a-subunit hydrolysis of GM2ganglioside, resulting in chronic SandhoSf disease. 3. Biol. Chem. 273: 21386-21392

Hou Y, Tse R, Mahuran DJ (1996a) The Direct Determination oC the Suhstrrtte Specificity of the a-Active site in Heterodime~icP-Hexosaminidase A. Biochemistry 35: 3963-3969

Hou Y, Vavougios G, Hinek A, Wu KK, Hechtman P, Kaplan F, Mahuran DJ (1996h) The ~a11'12LeuMutation in the cx Subunit of P-Hexosaminidase A is not Associated with the B I-Variant Form of Fay-Sachs Disease. Am. J. Hum. Genet. 59: 52-58 Korneluk RG, Mahuran DJ, Neote K, Klavins MH, OfDowd BF, Tropilk M, Willard HF, Anderson M-3, Lowden JA, Gravel RA (1986) Isolation of cDNA clones coding for the alpha subunit of human P-hexosaminidase: Extensive homology betwem the a and P subunits and studies an Tay-Sachs disease. J. Biol. Chem. 261: 8407-8413 Kytzia HJ, Hinrichs U, Maire I, Suzuki K, Sandhoff K (1983) Variant of GM2-gangliosidosis with hexosaminidase A having a severely changed substrate specit'icity. EMBO J. 2: 1201- 12O5 LaemmIi UK (1970) Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 227: 680-685 Lowry OH, Rosehrough NJ, F;irr AL, Randall RJ (1951) Protein measurement with the FoIin phenol reagent. J. Biol. Chem. 193: 265-275 MacLeod AM, Lindhorst T, Withers SG, Warren RA, J, (1994) The acid/base catalyst in exoglucanase/ from Ccblrdonons .fim.i is 127: Evidence from detailed kinetic studies of mutants. Biochcmistry 33: 637 1-6376 Mahuran DJ (1991) The Biochemistry of HEXA and HEXB Gene Mutations Causing GM2 Gangliosidosis. Biochim. Biophys. Acta 1096: 87-94

Mahuran DJ (1997) GM 2 gangliosidosis and Structure-Function Relatioships in P- Hexosaminidase. In: Swallow D, Edwards Y (eds) Protein Disfunction in Human Genetic Disease, 1 edn. Bios Scientitic, Oxford UK., pp 99-1 17 Mahuran DJ, Lowden JA (1980) The subunit and polypeptide structure of hexosaminidase from human placenta. Can. J. Biochem. 58: 287-294 Malcolm BA, Rosenherg S, Cowy MJ, Allen JS, de Baetselier A, Kirsch JF (1989) Site-Directed Mutagenesis of the Catalytic Residues Asp-52 and GIu-35 of Chicken Egg-White Lysozyme. Proc. Natl. Acad. Sci. 86: 133- 137 Mark BL, Wasney GA, Ssllo TJS, Khan AR, Cao ZM, Rohhins PW, James MNG, Triggs-Raine BL (1998) Structural and functional characterization of Strt=ptomyces plicatus beta-N- acetylhexosaminidase by comparative molecular modeling and site-directed mutagenesis. J. Bid. Chem, 273: 196 18- lC)A24

Nakai H, Byers MG, Nowak NJ, Shows TB (1991) Assignment of heta-hexosaminidase A alpha- subunit to human chromosomal region 15q23->q24. Cytogenet. Cell Genet. 56: 164- 164 Ohno K, Suzuki K (1988) Mutation in GM~-GnngliosidosisB1 variant. J. Neurochem. 50: 316- 318 Pennybacker M, Schuette CG, Liessem B, Hepbildikler ST, Kopetka JA, Ellis MR, Myerowitz R, Sandhoff K, Proia RL (I 997) Evidence f'or the involvement of Glu-355 in the catalytic action of huinsn beta-hexosaminidase B. J Biol Chem 272: 8002-8(H)6 Samhrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, second edn. Cold Spring Harhor Laboratory Press, Cold Spring Harbor, N.Y. Tews I, Perrakis A, Oppenheim A, Dauter 2,Wilson KS, Vorgias CE (1996) Baterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay-Sachs disease. Nature Struc. Biol. 3: 638-648 Tommasini R, Endrenyi L, Taylor PA, Mahuran DJ, Lowden JA (1985a) A statistical comparison of parameter estimation for Michaelis-Menten kinetics of human placental hexosaminidase. Can. J. Biochem. Cdl Biol. 63: 225-230 Tommasini R, Mahuran DJ, Lowden JA (198%) Discrimination of the isozymes of human placental hexo~aminid~eby kinetic parameter estimation. Can. J. Biochem. Cell. Biol. 63: 2 19-224 Trop I, Kaplan F, Brown C, Mahuran D, Hechtman P (1992) A G1~~~~-->Aspsubstitution in the a subunit of Hexosaminidase A causes juvenile-onset Tay-Sachs disease in a Lebanese- Canadian hmily. Hum. Mut. 1: 35-39 Tse R, Vavougios G, Hou Y, Mahuran DJ (1996) Idcntil'ication of an active acidic residue in the catalytic site of P-hcxosnminidase. Biochemistry 35: 7599-7607 Chapter 4

Analysis of Candidate Active Acidic Residues in Hex B INTRODUCTION Mammalian Hex is a lysosomal enzyme which cleaves terminal non-reducing p1-4 linked glycosidic bonds of the amino sugars, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine, and thus in vivo it is xesponsible for the catabolic degradation of the ganglioside GM~.Defects in

human Hex cause lysosomal accumulation of -2 mainly in neuronal tissues (where their synthesis is greatest), resulting in inheritable neurodegenerative diseases, known as GM~

gangliosidoses (reviewed in (Gravel et al. 1995)). However, to date the mechanism of cleavage of glycoside bonds by Hex is still poorly understood. It is thought that all glycosyl hydrolases employ a general acid-catalysis mechanism involving two acidic residues. One acid, protonated, acts as an acid catalyst and the other, unprotonated, as a nucleophile. The latter is responsible for the stabilization of the substrate's transient carboniurn ion though the formation of an enzyme- substrate covalent intermediate. Recently a second mechanism, substrate-assisted catalysis, was proposed for Hex. Four lines of evidence support this mechanism. First, it was found that the compound (*)-6-acetamido- l,2-anhydro-6-deoxy-myo-inositol,a tight-binding inhibitor for Hex from bovine kidney and jack bean, did not form a covalent bond with the enzyme (Legler and Bollhagen 1992). Based on these data, the authors conclude that a carbonium ion intermediate was not part of its catalytic mechanism. They proposed that a residue in Hex functioning as a base (nucleophile), hydrogen-bonds with the proton from the acetamido group of the bound substrate allowing the production of an internal oxazohe structure by the substrate itself which is the reactive intermediate (Legler and Bollhagen 1992). Secondly, the same type of internal oxazolinium intermediate was demonstrated for the Family 18, plant chitinadysozyme, hevarnine, by determining its X-ray structure with a bound inhibitor (van Scheltinga et al. 1995). Thirdly, the data from the crystal structure of a chitobiase-substrate complex from the same Family 20 as human Hex, also displayed the same mechanism. In chitobiase a protonated acidic residue, i.e. c-Glu540 (Table 4.1), functions as the catalytic acid while a residue functioning as a base, i.e. unprotonated c-Asp539, forms hydrogen bonds with the polar acetamido group of the sugar substrate to assist the catalytic reaction. Finally, recent mutational studies also suggest that conserved active site structurr: exists in Streptomyces plicatus Hex (Sp-Hex), also a "Family 20" enzyme. All the members of a given hydrolase family are believed to be evolutionarily related and thus have similar three dimensional structures. This assumption allowed molecular modeling of human Hex and recently the Sp-Hex based on the chitobiase structure. The overall 3-D structure that was generated for Sp-Hex (Mark et al. 1998) was similar to the previously reported model for human Hex (Tews et al. 1996). However, the related monomeric bacterial chitobiase has only 26% sequence identity to its human counterpart which occurs only within their active site region. Outside of this region there is IittIe sequence similarity. As well, the Sp-Hex is a monomer and has only 25% sequence identity to human Hex (Mark, 1998). Thus, the modeling data of the human Hex active site need to be validated experimentally. Previous hopes of identifying structure-function relationships in human Hex through analysis of mutations associated with GM~gangliosidoses have not been realized because most naturally occurring mutations first affect protein-transport out of the ER. As well, many of these mutations are not consewative substitutions and therefore likely cause some additional structural changes in the protein. For example, the B1-substitution aArgl78His was predicted (by computer analysis) to cause a change in the secondary structure of the a polypeptide (Ohno and Suzuki 1988). When Brown et a1 (Brown et al. 1989) analyzed the mutation in an aligned codon in the f! subunit, pArg2llHis, they found that it caused a small change in the rate of processing of the mutant protein. Two other B 1 mutations at the same codon, otArgl78Cys (Tanaka et al. 1990) and aArgl78Leu (Triggs-Raine et al. 1991), were shown to have lower levels of mutant a chain which are still able to foxm heterodirners and exit the ER. When a more conservative PArg211Lys substitution was introduced in the aligned P subunit a nearly normal level of P-CRM was observed (Brown and Mahuran 1991). Therefore the data concerning mutations at aArg 178 or the aligned fig211 correlate with the degxee to which the substitutions are conservative, i.e., CRM for the chain with Arg=Lys>His>>CyS>Au (Taylor 1986). Recently in vitro conservative mutations were made on the evolutionarily conserved residues in aligned Hex-related sequences in hopes of identifying the active site. Whereas most of these mutants did not interfere with Hex transport and produce mature Hex proteins, their functions as determined kinetically did not completely conform to the data predicted from chitobiase modeling. For example, mutational studies showed that fMspl96Asn substitution produced a Hex B (nonnal levels of matme j3CRM) with a normal Km and a Vmax of only 0.2% whereas the aligned residue in chitobiase is not in the active site. PhotoaEiity labeling has been used by Liessem et a1 (Liessem et al. 1995) in an attempt to identify active site residues in Hex. However, because of the placement and chemical properties of the labe1 this approach would not reliably or accurately identify active residues in Hex. The label was placed at the aglycone region of the inhibitor, an area where Hex shows little or no specificity (Liessem et al. 1995). As well, the photoaffinity labed is non-specific and would not necessarily bind to an "active residue". Based on the results of their study PGlu355 was identified as a residue likely involved in substrate binding (Liessem et al. 1995). Nevertheless from more recent data obtained through mutational analysis and baculovirus/insect cell expression by the same group, it was concluded that QGlu355 is involved in catalysis (Pennybacker et al. 1997). The latter

conclusion was based on the observation that the mutant protein has a normal Km (-0.60 mM) but a reduced Vmax value, i.e. near the background level of the untransfected cell. However, the Km for the MUG substrate of Hex from insect cells, the host for the expression of above mutant cDNA, is also -0.60 mM whereas the human enzyme is 0.7-0.9 mM. Thus, it remains unknown whether this normal Km resulted from a small amount of endogenous insect cell Hex or was &y from the mutant human Hex protein. The authors themselves could not resolve this question. In another study, Fernandes reported aGlu323 (which aligns with PGlu355) was a catalytic site residue based on the expression of aGlu323Gln cDNA in a human fetal Tay-Sachs disease neuroglial (TSD-NG)cell line (Fernandes et al. 1997). Since this method relies on the recruitment of an endogenous P subunit by the transfected a,its kinetic analysis of the a mutant is interfered with by the level of endogenous $ activity towards "a-specific" MUGS. It has been shown that normal p subunit present in this line contained approximately 1.33% residual activity toward MUGS substrate. Hex B has a MUGMUGS ratio of -300/1, Hex A of -441 and Hex S of 41 (Hou et al. 1996a). AU of the above analyses of the active site of human Hex using in vitro mutagenesis and cellular expression encountered the problem of endogenous Hex contamination (Cao et al. 1997; Femandes et al. 1997; Pennybacker et al. 1997) (Chapter 3). Thus, no absolute conclusion has been reached as to the validity of the model of human Hex based on the structure of chitobiase. In order to resolve this issue, when examining candidate active site residues an expmsion and purification system must result in a very high signal (from translated human cDNA) to noise (endogenous Hex) ratio. GeneraIfy a 6000-fold signallnoise ratio has been required to unequivocally identify the catalytic acid group of other glycosyl hydrolases, e.g. lysozyme

(Malcolm et al. 1989) and CenA from Cellulomonasfimi (MacLeod et al. 1994). We have mxntly reported a method that solves the above signallnoise problem by generating a novel epitope-tagged form of Hex B that can be purified away from all endogenous Hex (Chapter 3). We introduced a sequence that encodes a factor X site and Hiss sequence at the 3' end of fkDNA, which was then permanently transfected it into CHO cells. We found that the Hiss-tagged pro-Hex B is secreted and not retained in the ER as other tagged constructs were (data not shown); however the tag was lost once the protein entered the lysosome. Thus we demonstrated that pro-Hex B-Hiss can be purified directly from the medium of the transfected cells using Ni-NTA chromatography under native conditions with a purity of over 99.99%. We further demonstrated that this purified pro-Hex B-Hiss has the identical biochemical properties as the mature form. Using this highly purified fonn of Hex B we initially test the validity of the molecular modeling of human Hex from chitobiase structure by reexamining the effect of the PArg211Lys substitution. In a combination of analyses of the mutant's binding capability, i.e. Km for MUG and its ability to bind the affinity ligand CNAG, we were able to assign PArgZll to a role in substrate binding and orientation rather than catalysis as we previously predicated. In this report, we extend our investigation of the validity of the chitobiase model by examining other candidate active site residues in Hex B. We analyzed the acidic residues which are conserved in a and B subunits of human Hex as well as bacterial chitobiase, including Asp 241. Asp354, Glu491, Asp196 and Glu355. The former three amino acids have not been studied before and the proposed catalytic acid Glu355 and Asp 196 has not been studied in context with other candidate acidic residues, which would act as controls. We introduced conservative mutations into all these residues and expressed them with the C-teminal His-tag. Kinetic analysis of these purified mutant proteins from the b-ansfected CHO cells followed by CNAG affinity binding assays demonstrates that the chitobiase model is valid.

MATERIALS AND METHODS

Sequence alignment of the catalytic domain of chitobiase, Sp-Hex and human Hex a and subunits was made based on a molecular modeling system that uses secondary structure predictions to calculate gaps (Tews et al. 1996)(Mark, 1998). The alignment covers only the active site area of the enzymes since there is little similarity in the sequences outside of this region. Candidate acidic residues involved in the active site of human Hex are shown in Table 4.1. ite-dmcted. . d vector construcnon

The mutations were made in plasmid pHexB43 (Brown et al. 1989). FDNA encoding an

Asp l96Asn substitution was constructed in our previous report (Tse et al. 1996). However, its correct sequence was reconfirmed. For all other mutations (Table 4.2), mutagenesis was performed by a two-step PCR procedure (Hou et al. 1996b), which involved the utilization of three different oligos. Briefly, in step 1, amplification was achieved by an oligo containing the appropriate nucleotide change (Table 4.2) and a 3' end oligo. This PCR product (termed

“intermediate") containing the point mutation of interest then served as a "mega-primer" in conjunction with the 5' end oligo to yield the fmal product of the second round PCR reaction. The 5' and 3' end oligos each carry proper restriction sites to facilitate the ligation of the final PCR Table 4.1. Active acidic residues in the chitobiase and their aligned residues in Streptomyces plicatus hex & human hex, a and p subunits

Sp-Hex Sp-Hex Experimental Data CWB function (Tews et al. 1996) Experimental data from Hex (Brown and park et al. 1998) Mahuran 1991; Tse et al. 1996)

No function postulated BDl96N: normal Km, Vmax=0.2%

Re-examination in this studv

Holds R349 in place by polar PD208N: On1 y monomeric precursor formed interaction, H-bonds to term. amino and imino groups

Holds R349 in place by polar D240N: Normal M-protein, Krn increased 10- interaction, W-bonds to H452 fold, Vmax=ll%

Holds R349 in place by poIar This study interaction, Coordinates water mol. with E380

Molds R349 in place; H-bonds to Not conserved in human Hex amino and imino groups, coordinates water mol. with D379 ------

D246N: Km increased 1.2- H-bonds to D539, indirectly H- D2WN Lower M-protein; Km increased 2.5 fold, vmax decreased 2-fold bonds substrate through a H20 fold, Vmax=7 1% mol.

H-bonds to acetamido-N2 to help This study distort it towards C1 314E E314Q: Km decreased 7-fold, PE355: Labeled by a photo-affinity probe Vmax decreased 296-fold (Liessem et al. 1995); aE323 and PE355: altered pH profile Implicated as an active residue by expression (Fernandes et al. 1997; Pennybacker et al. 1997);

Re-examination in this study

E739 H-bonds to incoming H20 E462 This study molecule, H-bonding 05B Table 4.2. Oligonucleotides Used to Mutate @DNAa number oligonucleotides change substitution induced

1 5'-TCCACCA'ITA'IT~CTCTCCAAGGGT'IT-3' GAT+MC Asp t96Asn 2 5'-GGCACATAGTIYjAT&CAG'KTI'T-3' GAC+AAC Aspm Asn 3 5'-mGGGAGGmGAAGTGGAA?T-3' GAT+MT AspmAsn 4 5'-TITGGGAGGAGAT-GTGGAATT-3' GAAjCAA GlumGln 5 5'-A-A-TATGTGGATGCAAC-3' GAA-XAA Glu491Gln 6 5'-AGATAGTGTATGCCCAGGGGTAT-3' 3' primerb none 7 5'-GGTITCTACAAGTGGCATCAT-3' 5' primerb none 8 5'-CCTCCAATCITGTCCATAGCTA-3' 3' primer none

9 . 5'-CCAAATGATGTCCGTATGGTGA'IT-3' 5' primer none 10 5'-TGGTTTGTCCAAA-ATCAATGTA-3 3' primer none 11 5'-GGmATATTA'ITGCAACCATAA-3' 5' primer none a. The nucleotide changes are underlined, and the changes consequently made in the deduced amino acid sequence of the bsubunit are also shown. b. Oligo #6 and 7 serve as the 5' and 3' end primers to create the Asp241Asn substitution by the two-step PCR procedure whereas oligo #10 and 11 are utilized for generation of the

Glu491Gln substitution (see text). The 5' and 3' end primers used for generation of either the Asp354Asn or Glu355Gln mutation are oligo #8 and 9. AU these primers contain proper restriction enzyme sites for facilitation of subcloning the PCR fragment back into the pHEXB43 (see details in methods), product containing the derived mutation into pHexB43. To remove any contamination of the residual oligos used in step 1 of the reaction, the "intermediate" product (generated from step 1) was purified using Geneclean kit (Bio 101 Inc., Vista, CA) before it was used for the step 2 amplification. Amplifications were achieved using Vent polymerase (BioLabs Inc., Beverly, MA) to increase the fidelity. The reactions were carried out in 100 pl. Each reaction mix contained 50 ng DNA template, 20 mM Tris-HC1 (pH 8.8). 10 mM KCI, 10 mM (NH4)2S04, 2 mM MgS04, 0.1% Triton X-100, 0.2 mM each of dNTPs, 100 ng of each oligo and 2 units Vent DNA polymerase (BioLabs Inc.). The cycling conditions were described in chapter 3. Generation of the Asp241Asn substitution included the following procedure: (a) 1" round of amplification of the 260 bp DNA fragment using oligo #2 and #6 (Table 4.2); (b) 2ndround of arnplifcation of the 669 bp fiagment using the 260 bp intermediate product and oliga #7 flable 4.2); (c) Digestion of the 669 bp DNA with EcoR1 and subcloning it into pHEX843 to =place the corresponding wild-type segment. Similarly, a 582 bp EcoR1-Bsu36 I DNA fragment containing a Asp354Asn or Glu355Gln mutation was generated by two-step PCR using oligo #3, 8 and 9 or oligo #4, 8, and 9, respectively. Subsequently, either of the above fragments was ligated into pHEXB43 that was treated by Bsu36 I and partial EkoR1 digestion. To mteGlu491Gln substitution, oligo #ST 10 and 11 were utilized to amplify a 715 bp BamHl DNA fragment. AU the mutant cDNA inserts in pHexB*43 were fully sequenced and verified by T7 sequencing kit (Pharmacia). Next, the 1.5 kb Bst X I inserts, each containing a substitution described above, were subcloned into pcDNA-p- )IiS6 (Chapter 3) to replace its corresponding segment. The mutation and orientation of the insert in the resulting pcDNA-P*-Hi% were re-verified by DNA sequencing before transfection of the DNA into CHO cells. Transfection of m@mt construc&

CHO cells were maintained and propagated in a-MEM supplemented with 10% FCS and antibiotics at 37 OC in 5% C02. Transfections were canied out using Superkt Reagent (Qiagen, Valencia, CA), as described in Chapter 3. Neomycin (400 &ml) was present in all the stable transfectants. Since no significantly increased level of Hex activity was detected in the media of some cells transfected with mutant constructs, Western blot analysis was used to assess their level of cellular expression. Assavs of enzvme and nrotein levels

Harvested cells were lysed in a buffer of 10 mM Tris-HC1 (pH 7.5) and 5% glycerol through five sets of freeze-thaw cycles. Protein concentrations were measured by the Lowry method (Lowry et aI. 1951). Human Hex activities from the cells' lysates and media were determined using the MUG substrate (Brown et al. 1989). Western blotting was done essentially as described previously (Hou et al, 1998) (see also chapter 3). The primary and secondary antibodies used werc a rabbit anti-human Hex A (made in our lab) and a horsersldish peroxidase- conjugated goat anti-rabhit IgG (Immux), respectively. The nitrocellulose membrane containing the proteins WLS devclopcd and exposed to Hyprl'ilm using the ECL system (Amersharn). Purification of His6-twed- mutant Hex B nrotein

The preparution of small columns 01' Pro-Bond gel (Invitrogen, Carlsbad, CA) was as described in chapter 3. For reduction of nonspecitlc binding NCI was added to the medium before it was passed through the Ni-NTA column. Nonspecitically hound proteins were eliminated by first washing the column with 10 ml of native wash buffer (10 mMsodium phosphate pH 6.0. 0.5 M NaCl), and then with 15 ml each of native huflkr containing 30 mM or 80 mM imidazole- Specifically bound pmteins were eluted with the same huf'fcr containing 50() mM imidazole. The purity of each mutant was dete~minedby SDS-PAGE analysis followed by Coomassie Blue staining. Kinetic studies

The concentrations of MUG substrate were varied from 0.1 to 4 mM for the determination of the Km and Vm;, values. Because of the limited solubility of MUG, 4 mM represents the near maximum concentration of suhstrate achicvahle. The kinetic constants were calculated using a computerized nonlinear least squares curve tltting program for the Macintosh, KdeidaGraphm 3.0 (Hou, 1996). The purified enzyme allowed us to directly calculate the Kcat vdue based on the specific activity (MUGhydrolyzed mmol-1) of each purified form of Hex B and a Mr of 130,000. CNAG affinitv bindinrr assu

The affinity ligand CNAG was synthesized by Dr. Withers's lab at the University of British Columbia, and it was coupled to Sephacryl S-20() according to Mahum (Mahuran and Lowden 1980). The ligand beads we= pretreated with 0.1% RNse A overnight to block nonspecific sites. These belids were washed with 20 mM citrate buffer (pH 4.3) for removal of residual RNase A hefore 0.3 ml heads was packed in a minicolumn. Fifteen pg of purified Hex proteins was loaded on the colt~mn.After washing twice with 2 ml of 10 mM sodium phosphate buffer pH 6.0,0.2 M NaCl (wash hul-fer), the specitkally bound Hex protein was eluted with 150 pM &lactone (a competitive inhibitor) in the same wash buffer. The Hex binding rrfiinity was calculated on the basis of the total eluted protein versus the total protein loaded on the column as determined by Lowry method (Lowry et al. 1951).

RESULTS

Exnression and ~urif'icationof mutant proteins

To determine the role of candidate active and conserved acidic residues of Hex B (Table 4.1) in substrate hinding or catalysis, a conservative substitution was made at these residues (Table 4.2). These suhstitutions included an Aspl96Asn, Asp241Asn, Asp354Asn, Glu355Gln or Glu491Gln. In addition, a sequence encoding a Hisr, tag and factor Xa was also added into the 3'- end of each mutant cDNA for ticilitation of purification. Thew mutant constructs were then permanently transfccted into CHO cells and highly expressing, G418-resistant clones were picked. Western blot analysis (Fig.4.1) of their cell lysates indicated that all the mutant constructs but one Figure 4.1. Western blot analysis using an anti-human Hex B Ig G in lysates from nontransfsted

CHO celis (CHO), CHO cells transfected with pcDNA-P-His6 (WT) or mutant pcDNA-P*-

His6 encoding one of the following substitutions: D196N, D241N1 D354N1 E355Q and

E491Q. Equal amounts of total proteins (20 pg) from each lysate were loaded. The positions

corresponding to the prop (65KDa) and mature p chains are indicated on the right. Protein

standards are shown on the left.

(PAspl96Asn) produced mature (lysosomal) P-CRM at a level similar to that of the wild-type P construct. Only precursor kchain was detected intrac(=llularly in cells expressing the cDNA construct encoding PAsplgBAsn, indicating that this mutant protein does not exit the ER. To further confirm the Hi& tag in the PAsp196Asn has no effect on the mutant protein processing, a cDNA containing PAspl96Asn alone was trmsfected into CHO cells. Western blot revealed the

' identical result, i.e. detecting only precursor not mature P-chain from the cell lysates (data not shown). Thus, Asp196Asn affects the protein transport, and consequently we focused our subsequent experiments on the other mutants. We have previously demonstrated that the C-terminal His, tag is only present in the precursor form not in the lysosomal rnntutc Hex protcin. Large quantities of this pro-Hex B-His, enzyme is .secreted into medium of transfectcd cells and therefore can be purified by Ni-column

under native conditions. The exclusive removal of the CHO endogenous Hex requires a wash with

80 rnM imidamle. A similar procedure was utilirxd here to purify the mutant enzymes from the media of transfected cells expressing each substitution. After electrophoresis on SDS-gels and staining with Coomassie blue. a single hand with Mr 65 kDa (corresponding to the size of pro-P

polypeptide) was dctected horn the wild type and cach purified mutant protein (Fig. 4.2A). This 65 kDa polypeptide was proven to be the P subunit by virtue of its reactivity with human Hex B antibody (Fig. 4.2B). No comparable peptide was found in the medium from the untransfected CHO cells (Fig. 4.2B). The effect of mutations on their enzvme switic activitv

The purified protcin enabled us to directly calculate the specific activity of wild type and each mutant enzyme. The mutation bat made the largest change in the specific activity of a mutant Hex B was the GIu355Gln substitution, lowering it 820()-fold as compared to the wild-type value (Table 4.3, column 1). The Asp354Asn substitution produced a Hex B with the second lowest specific rctivi ty, 6500-fold lower. W herreas the Glu49 1Gln suhstitu tion produced a similar level of Figu~4.2. Coomassie blue staining (A) and Western blot (B) of purified proteins from the serum

free medium of the CHO cells (CHO), CHO cells transfected with pcDNA-P-His6 (WT) or

mutant pcDNA-P*-Hiss constructs. The mutant constructs encoded a D24 1 N, D354N,

E355Q or E491Q substitution. The Hex proteins are purified by Ni-NTA chromatography under native conditions and their identities m confirmed by immunohlotting using an anti-

Hex B Ig G (B). Equal amounts of puritied protein from each sample were loaded for Fig. A

(4 pg) and Fig. B (0.1 pg). The Mr standard and pro-P location are indicated. CHO WT D241N D354N E355Q E491Q STD

B CHO WT D241N D354N E3SQ E491Q Table 4.3. Determination of kinetic parameters and affinity binding to CNAG for Hex B, His6-tagged pro-Hex B and pro-Hex B* mutant Enzyme SAa Ihb vrnax" Kcat" ~cat/Km~ % of CNAG

Placental Hex B 10.lM.5 0.7 11t0.05 29.8H.92 3.9a. 14 5.5ko.4 75k5.1 WT 13.8M.8 0.66a.07 46k2 5.98a.3 9.0~1.4 71k4.2 ~211~' 0.007~.0004 >8 ~0.025 <0.0032 4.0004 7.4kl.l Il240Ng 0.78 8&2 3.7d.4 0.4d.02 0.054.015 NDh

D2W 3.7 1.9M.2 18.94.86 2Sd.1 1.324.2 ND D241N 0.48a.03 1.87s0.11 1.4M.04 0.18a.005 0.096&.008 6.921.2 D354N 0.0021~.0001 0.68d.04 0.00724.0001 0.00093~.00001 0.0015~0.0001 68d.2 E355Q 0.0016~.0001 0.59s0. 10 0.0056~.OOOl 0.00072~.00001 0.001 1+0.0001 7 6i4.1 E491Q 9.8a.8 0.69d.08 38k1.6 4.941t0.2 7.2k1.2 70k4.6 a. Specific activity (mmoyhrlmg) was measured at 1.6 mM MUG substrate. S.D. value from three independent experiments. b. Hex activity was determined using MUG substrate and the Km values are given in mM with the standard error reported as *, caculated by the KaleidaGraph 3.0 model fitting Macro (directly fitting the initial velocity versus substrate concentration data to the Michaelis-Menten equation). The Vmax values are given as mol of MU/hr/mg Hex B protein. c. Kcat is expressed as maximum mol of MU released/hr.mol purified Hex B protein (Mr. 130,000)~10~,hilxlO'. d. KcatKm is given as hf'.~'lxl0'~. e. Percent of Hex biding to CNAG was caculated on protein recovery, i.e. protein eluted with &lactone (a competitive inhibitor) in the total protein loaded on CNAG minicolumn. f. Data from Chapter 3. g. Data from Tse et a1 (Tse et al. 1996), The SA and Vmax values were recaculated to mmol/hr/mg for comparison. h. ND: not determined. activity to the wild-type enzyme, the Asp241Asn reduced the spilic activity to 30-fold lower

(Table 4.3, column 1). . . Krnebc analvsis of putative active site residua

We next determined the kinetic parameters for the purified normal and each of the mutant enzymes using the MUG substrate. Consistent with previous data, the pro-Hex B-His,, purified from the CHO cells transfected with wild-type j3-Hisfi cDNA, displayed the identical Km and Kcat to those determined for purified placental Hex B (Table 4.3). A Glu491Gln substitution served as our control as the aligned residue in chitobiase hydrogen bonds to 05B group of its substrate through incoming H,O (Hcx substrates have no OSB group). As expected, it had the same kinetic constanB as the wild-type cnzyme. Thc Glu355Gln suhstitution produced a normal Km value; however, the Kcat value was dccreixscd hy over 80()0-fold (Fig. 4.3, Table 4.3). Thus, the PGlu355 is directly i~ivolvcdin catalysis, not in suhstratc binding. Similarly, the Asp354Asn suhstitution generated a normal Km hut largely reduced the level of Kcat value (over 6oOMold) as compared wih the wild-type enzyme (Fig. 4.3, Fable 4.3). Hence PAsp354 also appears to he involved in catalysis prohahi y through interacting with the acetardo-group of the substrate as suggested by the chitobiax model (discussed below). On the other hand, analysis of kinetic parameters of the Asp241Asn mutant revealed 3-fold increase in Km (Fig. 4.3, Table 4.3), a value similar to that which was Sound for Asp29OAsn (Tse et al. 1996). The Kcnt value determined for the Asp241Asn suhstitution was also reduced. Thus, PAsp241. is likely involved in substrate binding.

We have rcccntly shown that CNAG binding can he used ;IS an assay to differentiate the effect of a mutation on suhstratc binding versus catalysis (Chapter 3). To confim the kinetically detined roles of the above residues, fit'ken pg of purilied normd or mutant protein was loaded on a CNAG affinity minicolumn. The control Glu491Gln mutant exhibited the same level of binding affinity (70%) as either the wild-type enzyme (7 1%) or placental Hex B (75%). The normal Km Figure 4.3. Kinetic analysis of wild type pro-Hex B-His6 (WT)and mutant pro-Hex B*-His,

using the neutral MUG (mM)substrate. The mutant Hex B contains a D241N, D354N, E355Q or E491 Q substitution and only the former three Hex B mutants are shown here. The Hex proteins are puritied from the media of the trmsfected ells using Ni-NTA column. The experimental data points are shown and were directly fitted to the Michaelis-Menten equation.

The quation with its calculakd best-fit constants (Kin and apparent Vmax) were then used to

generate the lines joining the dillil points on the graph. The R values for all of the experiments were >0.995. The actual Km and Vmax values are given in Table 4.3. Kinetic analysis of Wt and Mutant Hex 8

0 0.5 1 1.5 2 2.5 3 3.5 4 MUG (mM) mutants, Asp354Asn and Glu355Gln, also bound the Hex-specific ligand in a manner similar to that of the wild type, i.e. 68% md 76%. respectively (Table 4.3, column 5). By comparison, the replacement of Asp241 with Asn produced a Hex B with a much lower binding capacity, 6.9%, relative to the wild type enzyme, consistent with the observed higher Km value. DISCUSSION

We previously showed that evolutionarily conserved acidic residues arc= involved in the active sites in Hex (Tse et ill. 1996). Recently moll=cula.r modeling of human Hex from the structure of chitobiilse predicts that there art= eight conserved active residues (Table 4.1). Among these, Asp208, Asp240 and Asp290 in Hex B have hwn charickrized (Tse et al. 1996).

Substitution of PAsp208 to Asn was found to only produce monomeric precursor. The

Asp24OAsn mutant was shown to have I 1-fold increase in Km with its Vmax decreased by -10- fold- The PAsp29OAsn substitution had a 2.5-fold higher Km than the wild type enzyme with a slightly reduced apparent Vmax. Thus, whereas Asp208 is critical for initial prokin folding andlor dimer formation, and both Asp240 and Asp290 are involved in substrate binding. These conclusions ai-t: consistent with the molecular modeling of human Hex based on chitobiase structure. As we11, our previous conclusion as to the role of PAsp290 has gained further support from a recent comparative moleculur modeling of Sp-Hex from the chitohiue structure and follow- up mutagcnesis experiments (Table 4.1) (Mark, 1998). Human PAsp290 aligns with Asp246 in

Sp-Hex. When Asp246 was converted to Asn and the enzyme purified from transformed E. coli, its Km was 2-fold higher than the wt enzyme and its Vmax was also decreased by 2-fold. In the prcscnt study we examined the role of the other live candidatc active acidic amino acids using our wcently developed procedure that results in a very high signal to noise ratio (Chapter 3). Wc hund that the control Glu49 I Gln mutant protein behaves in the same manner as the wild-type enzyme in all aspects investig~ted.Since contradictory roles have ken postulated for Glu355 (Liessem et al. 1995; Pennyhacker et al. 1997), re-examination of this residue was necessary. A 28000-fold duction in the specific activity for the Glu3SSGln substituted Hex B was detected. This is the largest reduction in Hex specific activity from a point mutation ever documented and is consistent with Glu355 king the catalytic acid group in human Hex B. This

hypothesis was further strengthened by the kinetic data. Substitution of Glu355 to GIn led it a Hex B with a normal Km for MUG but with a Kcat value only 0.0 12% of nomd (Table 4.3). Despite the large decrease of its Kcat and Vmax, the mutant protein bound the Hex affinity ligand as well as the wild-type enzyme, contiming its role in catalysis hut not in substrate hinding. These data are also consistent with the recent mutational analysis of the aligned residue Glu314 in Sp-Hex. Conversion of Glu3 14 to Gln in Sp-Hex has ken shown to reduce its Vrnax by 296-fold with 7- fold decrease in it! Km. The Km dcclcase caused by this substitution was suggested to result from the mutant enzyme becoming saturclted with the suhstrate mare readily than the wild type enzyme (Mark et al. 1998).

Human PAsp354 aligns with Asp539 in chitobiase. The latter is postulated to form hydrogen bonds with the acetamido group of the non-rcducing sugar ring NAG-A in the diNAG (A+B) substrate to assist the cahlytic reaction. Our data also support the critical involvement of

PAsp354 in the catalysis of Hex B. Mutagencsis of Asp354 into Asn resulted in over a 6500-fold reduction in Kcat hut with an unaltered Km value. Tts unaltered substrate-binding ability was further veritied by its apparently normal binding affinity for the Hex-speciik ligand.

The aligned rcsidue to human Hex thr Asp379 in hacteiid chitohiue is PAsp241.

Chitobiase Asp379 appears to he indirectly involved in suhstrilte hinding as it holds the critical Arg 349 in place by polar interaction. Arg349 sits at the base of the binding pocket and therefore plays the most important role of any i*esidue in substrate hinding. This residue aligns with PArg211, which we have shown plays just as critical a rdc in the substrate-binding site in human Hex B. As well Arg211 orients the suhstrate in its correct position for hydrolysis. This role is likely the cause of the large decrease in Vmllx and Kuclt we ohserved with the PArg2 1 1Lys mutant (Chapter 3). Kinetic analysis of the Asp241Asn suhstitution was shown to also increase Km, but only by a factor of 3 with a reduced 33-fold Kcat reduction. A Km-change mutant would also be expected to bind less efticiently to the Hex aflinity gel, which is what was observed within Hex B containing this mutation (Table 4.3).

The aligned residue of human PAsp196 is Asp334 in chitobiase, which is not suggested as part of the active sites. Our data herein indicated that PAsp196 is like Asp208, very important in the initid folding andor dimer formation of the prop subunit. These events are prerequisites for the exit of the enzyme hmthe ER (Proia et al. 1984). Western blot detected only the prop form of the subunit containing the Asp196Asn substitution. We have concluded that some unexplained experimental error was Ihc cause (IS our previous report of the Aspl96Asn suhstitution which produced nolmal lev& of matulc P chain in ~clnsfcctedcells (Tse et al. 1996). To ensure that no additional mutation cxcu~~cdin our mutant insert, we re-sequenced the entire mutant PcDNA. No error outside ol' the Aspl96Asn substitution was found. Thus, our experimental data from

PAsp196 are consistent with those predicted from the modeling studies (Tahle 4.1).

Our mutational examination of the candidate active residues in human Hex thus far is concordant with the data pmiictcd fi-otn the chitobiase moIecular model. Therefore the active site structure modelcd Ihr human Hcx and its associated suhstr~teassisted mechanism for catalysis appears to be valid. Our data separate the active groups into two general classes: (1) the catalytic resid~~esPGlu35S and PAsp354; PGlu355 serving as catnlytic isid and proton donor whereas the unprc~tonatedform of PAsp354 functioning as u base to help the catalysis likely through interacting with the N-acetyl group in the suhstrate. (2) the residue that directly interacts with the substrate,

PArg211 (chapter 3), and residues that are indirectly involved in substrate hinding, including

Asp240, Asp241 and Asp290 in Hex B. Signilicantly, the conservation of these active site residues strongly indicates that human Hex and probably all the Family 20 enzymes have similar structure and catalytic mechanisms. The latter, substrate-assisted catalysis, deviates from what was thought to he universal glycosidic mechanism modeled from other hydrolases such as lysozyme. Brown CA, Mahuran DJ (199 1) Active arginine residues in P-hexosaminidase: Identification through studies of the B1 variant of Tay-Sachs disease. J. Biol. Chem. 266: 15855-15862

Brown CA, Neok K, hung A, Gravel RA, Mahum DJ (1989) Introduction of the a subunit mutation associated with the B 1 variant of Tily-Sachs disease into the P subunit produces a P-hexosaminidase B without catalytic activity. J. Biol. Chem. 264: 2 1705-2 1710 Cao ZM, Petroulakis E, Salo T, Triggs-Raine B (1997) Benign HEXA mutations, C739T(R247W) and C745T(R249W), cause beta-hexosarninidase A pseudodeficiency by reducing the alpha-subunit protein levels. J. Biol. Chem. 272: 14975- 14882 Fernandes MJG,Yew S, kclerc D, Henrissat B, Vorgias CE, Gravel RA, Hechtman P, Kaplan F (1997) Identification of cmdidate active site residues in lysosomal bet&-hexosaminidase A. J. Bid. Chem. 272: 8 14-820 Gravel RA, Clarke JTR, Kahack MM, Mahuran D, Sandhoff' K, Suzuki K (1995) The GM2 gangliosidoses. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The Metabolic and Molaular Bases of Inherited Disease, 7 edn, vol 2. McGraw-Hill, New York, pp 2839- 2879

Hou Y, Tse R, Mahurin DJ (1996a) The Direct Dete~minationof the Substrate Speciticity of the a- Active sitc in Hetcrodimeric P-Hexosaminidase A. Biochemistry 35: 3963-3969

Hou Y, Vavougios G, Hinck A, Wu KK, Hwhtman P, Kaplirn F, Mahuran DJ (1996b) The ~all')2LcuMutation in the or Subunit of P-Hexosaminidast: A is not Associated with the B 1- Variant Form of Tay-Sachs Disease. Am. J. Hum. Genet. 59: 52-58 Legler G, Bollhagen R (1 992) (&)-6-Acetamido- l,2-anhydro-6-deoxy-myo-inositol: A tight- binding inhibitor and pseudosubstrate for N-acetyl-beta-glucosnminidase~~Carbohydr. Res. 233: 113-123 Liessern B, Glornhitza GJ, Knoll F, khmann J, Kellermann J, Lottyxich F, Sandhoff K (1995) Photoaffinity laheling of human lysosornal beta-hexosaminidase B - Identification of Glu- 355 at the suhstratc binding sitc. J. Bid. Chem. 270: 23693-23699 Lowry OH, Rosehrough NJ, Fan AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275 Machod AM, Lindhorst T, Withers SG, Warren RA, J, (1994) The acidbase catdyst in exoglucmase/xylana.u: from CuNulomrms fimi is glutamic acid 127: Evidence from detailed kinetic studies of mutants. Biochemistry 33: 6371-6376 Mahurm DJ, Lowden JA (1980) The subunit and polypeptide structure of hexosaminidase from human placenta. Can. J. Biochem. 58: 287-204 Mrtlcolm BA, Rosenhwg S, Corey MJ, Allen JS, de Bac=tselier A, Kirsch JF (1989) Site-Directed Mutagenesis of the Catalytic Residues Asp-52 and Glu-35 ol' Chicken EggWhite Lysozyme. Proc. Natl. Acad. Sci. 86: 133-137 Mark BL, Wasney GA, Salo TJS, Khan AR, Cm ZM,Robbins PW, James MNG, Triggs-Raine BL (1 998) Structural and functional chwxkrization of Streptomyces plicatus beta-N- acetylhexosaminidw by comparative molwular modeling and sitedirected mutagenesis. J. Biol. Chem. 273: 1961 8-19624 Ohno K, Suzuki K (1988) Mutation in GM~-GangliosidosisB1 variant. J. Neurochem. 50: 316- 318 Pennyhacker M, Schuette CG, Liessern B, Hephildikler ST, Kopetka JA, Ellis MR, Myerowitz R, Sandhoff K, Proia RL (1997) Evidence for the involvement of Glu-355 in the catalytic action of human beta-hexosaminidase B. J Biol Chem 272: 8002-8006

Proia RL, d'Azzo A, Neuf'ld F (1984) Association of a- and P-subunits during the hiosynthesis of P-hexosaminidase in cultured fibroblasts. J. Biol. Chem. 259: 3350-3354

Tinaka A, Ohno K, Sandhoff K, Main: I, Kolodny EH, Brown A, Suzuki K (1990) GM~- gangliosidosis B 1 variant: Analysis of P-hexosaminidast: a gene abnormalities in seven patients. Am. J. Hum. Gcnet. 46: 329-339 Taylor WR (1986) The classitlcation of amino acid conscrvation. J. Theor. Bid. 1 19: 205-218

Tews I, Pelrakis A. Oppnheim A, Dauter 2, Wilson KS, Vorgias CE (1996) Baterial chitohiwe structure provides insight into catalytic mechanism and the hasis of Tay-Swhs disease. Nrtture Struc. Bid. 3: 638-648 Triggs-Raine BL, Ake~manBR, Clarke JRT, Grwd RA (199 1) Sequence of DNA tlanking the exons of the HEXA gene, and identitication of mutations in Tay-Sachs disease. Am. J. Hum. Genet. 49: 1041- 1054 Tse R, Vavougios G, Hou Y, Mahuran DJ (1996) Identikation of an active acidic residue in the catalytic site of P-hcxosaminidase. Biochemistry 35: 7599-7607 van Scheltinga ACT, A~mansS, Kalk KH, Isogrti A, Henrissat B, Di.jkstra BW (1995) S~rewhemistryof chitin hydrolysis hy plant chitinuse/lyso7,yml=ysymand x-ray structure of a complex 2nd allosamidin: Evidence for substrate assisted catalysis. Biochemistry 34: 156 19- 15623 Chapter 5

Summary and Future Prospects The objective of this thesis was to study structure-function relationships in human Hex by

identifying residues directly involved in the hydrolysis of GM~ganglioside (h)and to

experimentally test the validity of a molecular model of human Hex based on the X-ray structure of bacterial cchitohiase. Chapter 2 described the identification of a missense mutation associated with chronic Sandhoff disease and the biochemical characterization of this mutation's role in both the patients' and trimsfwted cells. My data indicated that the PPro504Ser mutation results in the increased retention of' 45% of newly synthcsizcd pro-P chains in the ER. However the amount retaincd was not sul'llcien~to produce a clinical phenotype based on the critical threshold calculations made by Sandhoff et ill (Conzdmann et al. 1983). Kinetic analysis demonstrated that the mutation does not afl'ect Hex A's Km for neutral or charged artificial substrates.

However, I found that the mutant p subunit rcduccd thc specific activity of Hex A for its natural substrate, the GM~gangliosidc1Activator complcx, as compared to its specific activity for artificial substrates, by 3-fi>ld. These data suggest that PPro504 is part of Hex A's Activator binding site. Thus I concluded that the chronic phenotype of the patients result hm a combination of this 3-fold I-eduction in GM2-hydrolysis activity and the reduction in mature lysosornal protein due to dccrcascd intrrtccllultlr trrinsport.

During my gr~duatework, several rcpor~swcrc published that studied the active sites of human Hex mainly hascd on the analysis of citlicr natural occurring mutations (Brown et al.

1989) or in vitro mutations 01' evolutionarily conserved residues identified by sequence alignments (Femandes et al. 1997; Pennyhacker et a1. 1997; Tse et al. 1996a). All these studies by mutational analysis and subsequent ccllular expression had <10(): 1 signal to noise ratio due to endogenous Hex contamination from host cells. Later the three dimensional structure of bacterial chitobiase was puhlished and allowed for the molwular modeling of the active site of human

Hex. Controversy existed particularly over the identity of the catalytic acidic residue, hut

because of the low signal/noisc= ratio existing in above studies no absolute conclusion could be

reached. Generally a 6000-fold signaWnoise ratio has been required to unequivocally identify the

catalytic acid group of other glycosyl hydrola.ws, e.g. lysozyme (Malcolm et al. 1989). Thus, in

order to resolve the controversy, a ncw expression and purification system was needed to more

fully remove the endogenous Hex contamination from samples of mutant human Hex.

In Chapter 3, I dcvelopcd a mcthod for thc generation of a Hisc,-tagged form of Hex B that was not retaincd in thc ER of trmsf'cctcd CHO cells. This form of Hex could then be purified via a ~i+'-columnto eliminate cndogcnous Hex protein, and allow a more precise re- examination oS the PArg2 1 1 Lys and othcr substitutions in Hex B. Whcreas the C-terminal His6- tagged protein reached the lysosome, the tag was not rctained in the mature lysosomal Hex El form. Thus, all my work using the His6-thrm 01' Hex had to he done using the precursor form of the enzyme (pro-Hex B). Since large amounts of pro-Hex B are normally secreted into medium of transfected cells, I was able to usc the expression media as the source of mutant enzymes which were then purified by Ni+'-NTA chromatography under native conditions. The purified human Hex B is >99.99%1, L'rw l'rom thc contamination of endogenous CHO Hex. I further demonstrated that this pro-Hex B-His, had the same biochemical characteristics as the mature form of Hex B. In an initial study to tcst the validity of the human Hex model from chitohiase using this highly puril'icd form of Hcx B, I re-invcstigated the PArg211Lys mutation. Kinetic analysis indicated that the npparcnt Km of Hcx B containing the PArg211Lys substitution was increased more than 10-Sold, whcreas its Kcat was reduced by 1800-fold. Consistent with this finding, this mutation greatly lowcred the isozyme's binding nftinity for an immobilized Hex- specific ligand. Thus, I concluded that $Arglll has a critical role in substrate binding and in

orientating the substrate in the active site. This conclusion adds weight to the validity of the

bacterial chitobiase model as extrapolated to human Hex.

Our data with respect to the function of pArg2ll is also supported by recent comparative

modeling and subsequent mutational analysis of the aligned Arg162 in Sp-Hex. Conversion of

Arg162 to His resulted in a large increase (40-fold) of its Km and a decrease (5-fold) of its

Vmax. Thus the molecular modeling data, based on chitobiase structure, for the Arg residue in

both human and Streptomyces Hex are valid.

In Chapter 4, I extended the examination of the validity of the chitobiase model by

investigation of other candidate active site residues in Hex B. I analyzed the acidic residues

which are conserved in a and P subunits of human Hex as well as in the aligned active-site

model of bacterial chitobiase. These included Asp 241, Asp354, Glu491, Asp196 and GIu355

(Table 4.1). The former three amino acids had not been studied before and the proposed catalytic

acid residue Glu355 and Asp196, had not been analyzed together or in context with other

candidate active acidic residues, which would act as controls.

The approach used here is similar to that in Chapter 3. Conservative mutations were

made at each of the above residues, followed by the addition of nucleotides encoding the C-

terminal His, tag to each mutant cDNA. Kinetic analysis demonstrated that the PGlu355Gln

substitution had a slightly decreased Km but a Vmax >8000-fold Iess than the wt isozyme. This

is the largest Vrnax reduction in human Hex from a point mutation ever reported and is

consistent with Glu355 being the catalytic acid group in human Hex. This conclusion is further strengthened by the normal binding ability of Glu355Gln to the Hex affinity ligand. A similar observation was also found in the PAsp354Asn Hex B, which displayed a normal Km but slightly higher reduction in Vmax, 6000-fold. This result is in agreement with the idea that

Asp354 functions as a "base" in the substrate-assisted mechanism proposed for chitobiase and

Hex. On the other hand, kinetic studies of the puritied PAsp241Asn Hex B indicated that the isozyme had a 3-fold increase in its Km and a 33-fold rcduction in its Kcat. The increased Km of this mutation was also verilkd by it.s lower level of binding to the Hex-specific ligand relative to the wild-type enzyme. In combination with our previous data, Asp240, Asp241 and Asp290 are critical for efficient substrate binding. However, in comparison with Arg2 1 1 (Chapter 3) these three residues play lcss important roles in thc suhstratc hinding. In addition, this data corrected a misconception with respect to the role of PAsp196. We fi~idthat PAspl96 is important for initial folding and/or dimerization, an clYcct similar to that seen with PAsp208Asn Hex B (Tse et al.

1996n). rather than any involvement in catalysis. Taken together, I concluded that the chitobiase model is vaIid in light (If' thc active sitc rcsiducs in human Hex identil'ied experimentally thus Far.

The characterization al' these activc rcsiducs suggests that the human Hex and prohahly all the

Family 20 enzymes have similar structures and a conserved catalytic mechanism. Thus human

Hex likely hydrolyzes its subs tmtc through a suhstrate-assisted mechanism, rather than what previously was hclicved to he the "universal" acid-catalysis mechanism based on lysozyme.

FUTUREDIRECTIONS Whcrcas the structure ol' monomeric chiiohiasc: provides a starting point to define the structure-function sclationships within thc activc site of human Hex, it can not answer the questions regarding the domain involvcd in dimerimtion of the subunits in human Hex and area of protein-protein interaction hctween Hex A and the ActivatorIGryr2 complex. The future experiments described below may answer some 01- these questions. Clearly the determination of the X-ray structure of human Hex would dlow better interpretation of the biochemical data and

ultimately allow us to elucidate most of the structure-function relationship in human Hex. . . active aromatic res ducss hueds on the ch- model While some acidic residues are located in the active site pocket of chitohiase, several

aromatic amino acids are also predicted to he part of the binding pocket (Tews et al. 1996). In

chitobiase, Trp 737 stacks against GlcNAc ring whereas Trp 616 and 639 stabilize the essentially

planar N-acetyl group in the GlcNAc residue of the substrate. As well, Tyr 669 forms hydrogen

bonds with acctamido-07 of the NAGA-NAGB substrate. Recent comparative modeling of Sp-

Hex from the chitohiase structure suggests similar functions for the aligned conserved aromatic

residues (Mark et al. 1998). The aligncd aromatic amino acids in human Hex B are Trp 405,424,

489 and Tyr 450 (Tahlc 1.1). Thc investigation ol' the potential role of these residues in substrate

binding can he accomplished using the same approach as described in Chapter 3 and 4. To minimize the structuri~lchange each of above Trp residues will be substituted with a Tyr and/or

IIe while Tyr 450 will he converted to TIP and/or Ile. This can he followed by the introduction of the C-terminal His, sequcnce, kinctic analysis of each purified mutant enzyme, and the confirmational binding assay using thc Hex ull'inity ligand (CNAG). Generally, if a residue is involvcd in the suhstsatr: binding, the substituted protein would be expected to have an increased

Km value for MUG and consequently a lowcr binding affinity to the Hex-specific lipand.

Location of the active sites with rcsncct to the subunit-subunit interface

Although it has long hcen known that the dimerimtion of monomeric inactive pro-a and pro-P subunits is essential for the activi~y01' Hex A. the mechanism of this dimer formation remains to he defined. There are two models that explain the need for dirner formation in order to

"activate" each subunit's active site. In the first model, each active site is totally contained within its associated subunit. Therefore dimerization causes a conformational change that indirectly

activates each site (Fig. 5.1A). In the second model the active sites are directly activated by

dimerimtion, because they are 1oc;lted at the subunit-subunit interface. Thus a small part of the

a-active site is donated by the P-subunit, and vice versa (Fig. 5.1B). Two pieces of evidence that

we have generated support the latter model. 1) In monomeric CHB, c-Asp344 is involved in

substrate binding; however, when we mutated its aligned residue in P, i.r. Asp208Asn, we

produced a P suhunit that was unable to form dimers (Table 1.1) (Tse et al. 199th). 2) Through kinetic studies of Hex A* with an inactivc p* suhunit, I demonstrated that the a active site has different parameters for MUG dcpcnding on thc idcntity 01' its paired subunit, i.e. Hex A*, a@* or Hex S, aa (Hou t=t al. 1996a). The mutant p-constructs that could be made for the above aromatic residues along with those I have made (described in Chapter 3 and 4) could be used to differentiate between the two models. 11' thc second model is true, some of the mutations we generate in the p suhunit bascd on thc mono~nericCHB active site, may have an effect on the active site of the paired suhunit. Thus, we could co-cxprcss the wild-type acDNA with each mutant p-construct that all'ccts Hcx B activity such as PAsp241, into CHO cells. Ion-exchange chromatography can be pc~formedto scparatc Hex A from other isozymes. Next, the aa*-His,

(Hex A*) can be puril'ied away liom the endogenous CHO Hex A through the ~i"-column and then analyzed kinetically using a-spccific MUGS. If Hcx A* gives n large reduction of MUGS activity, then the residue in the p subunit could be playing a role in the a active site and could indeed be located at the a and P suhunit intcrli~ce. Figure 5.1. Proposed modds Tor formation of the active Hex A Aimer from the inactive a

and p monomers in the ER. "+" indicates the positive site unique to or that allows Hex

A (ap)and Hex S (act)to bind negatively charged substrates.

. . IderltrfiCatmn- .. ot ~sldues. involved in bind in^ a

It has heen shown that only the a-active site can hydrolyze eftkiently negatively charged substrates such as MUGS and GM2 (Baylerlm et al. 1984; Hou et al. 1996a). We have p~viously identified a middle section of a that is associated with its ability to hydrolyze MUGS by expressing oc/P fusion proteins (Tse et al. 1996h). We will couple this knowledge with the structure of chitobitlse containing its hound substrate. Although chitobiase does not hydrolyze

GlcNAc-6-sulfate containing substrates, its molecular three-dimensional model containing a bound substrate can give clucs to the arca in thc a that interact with the C6-sulhte group. By downloading these atomic coordinates from PDB,we are able to view and manipulate this model using the RasMac v2.6 program. This allows us to identify two residues in chitohiue that are located near the C6 position of its NAGA-substrate. The aligned residues in the asuhunit,

Am424 and Arg425, are not conserved in P subunit. We will make mutations, i.e. aAsn424Asp and aArg425Lys and express the mutant acDNA either alone to produce Hex S, or co- transkcting it with catalytically inactive p* (PGlu355Gln) to produce Hex A*, respectively. If the a-mutant has lost its ability to hyddyze MUGS hut not MUG, then these amino acids are responsible for a-specific substrate hindi ng.

Studies of protcin-protein intcrsction

These studics ;1rc hasod on thc surprising degree of species-specificity that has been demonstrated for both thc formation of the Hcx dimcrs and for the interaction of Activator with

Hex A. Recently we addressed the qi~estionof possible formation of interspecies heterodimers, r.g. CHO-P or CHO-a with human-P; hecause such heterodimers would be expected to immunoprecipitate in our previous immunospecil'ic human Hex assay and confound our mutational analyses. A careful examination of our published diitil demonstrates that there is na

detectable heterodimer formation (Hou et al. 1996a). Most importantly, when a P*cDNA

construct (P*Arg2 1 1Lys) is exprc=ssed,it produces virtually inactive, dimeric, lysosomal Hex B *

(P*P*), i.e. the immunoprecipitnted protein does not produce activities >2-fold that of the negative control cells for either MUG or MUGS (Hou et al. 1996a). Thus, the human P* subunits do not recruit a signitlcant amount of active CHO cell-a and/or -P subunits into dimer formation.

On the other hand, if a normal human acDNA is co-transfected with P*cDNA, active Hex A*

(ap*)is produced. Furthermore we have Ihund that CHO-Hcx A has little or no activity toward

3~-~~2in the presence ol' human activator (Hou st al. 1996a). Others haw also ohserved this with mouse-Hex A and human activator (Yuziuk et al. 1998).

To localize the arca(s) in p involved in thc formation of Hex B in the ER, we will make human/ mouse fusion PC-proteins by virtue of mutation techniques utilizing either common restriction sites or a PCR method that allows fusions to he made at any point in the aligned sequences (Hou et al. 1W6h; Tse et al. 1996b). Thesc chimeras, PC, will he co-tmnsfected into

CHO cdls with P*-Hisfi cDNA (sec ahovc). Thc Hise-tagged protcin will be isolated on a Ni+2 column and assayed for Hcx B activity. Sincc 2(P*-Hisri) Hex B* is inactive, the presence of even low levels of MUG activity will indicate ~hcformalion of PC-P*-Hisl, dimers. The same procedure can he uscd with the acDNA; howcver it would he expected that the areas of interaction would he in thc samc aligned positions within the two subunits.

In all the above st~ldics,we would expect that the functional residues in the areas we identify would not he conserved in tlic mouse proteins. Such residues will he tested by in vim mutagenesis and expression. . . reduction of Hex B for -trllll/,d~l~n. .. ..

For determination of detailed crystal structure of Hex B, large amounts of protein are

required to explore methods of crystallizntion. Hex B was chosen because of its stability. Given

p's 60% sequence identity with a. the structure of Hex A can be determined through molecular

modeling. Using Hcx B proteins that we prepared from human placenta, Church et a1 (Church et ill. 1992) have crystallized it and obtained low definition X-ray structure. The major problem encountered was the variation in crystal-formation between preparations, likely the result of heterogeneity in N-linked sugars and polypeptide structures due to glycosidic and prokolytic processing in the lysasome (Fig. 1.3). To producc lnrge quantities of the homogeneous precursor form of Hcx B, the pcDNA-P-Hisc, translbctcd CHO cclls I made can he cultured in a biowactor.

The His,-tagged pro-Hex B protcins can hc puril'icd from the mcdia through a ~i+'-columnand used for crystrtllization. Such a procedure has been successfully used for a glycosylated lysosomal suifatasc and resulad in a high-resolution structure. A1 ternatively, haculoviruslinsrct expression system could he employcd to incrcasc the yield of pro-Hex B. Whereas Proia's group recently showed that pro-Hcx B can bc I'unctionally cxprcssed in insect cells (Pennybacker et al.

1997). they would hcc the same problem of heterogeneity (in N-linked sugars and polypeptide structures) as well as the contamination of inscct Hex (described in Chapter 3 and 4). The modification will he made by construction ol' DNA encoding pro-P -Fxa-His, in baculovirus trmslcr vcctor, e-g. pBlucBac (Invitmgcn). Again culturing the insect cells in a Bio-Reactor after intkction with the rccomhinant virus would rcsult in production of lnrge amounts of secreted pro-

Hex protein l'mrn the media, which can then hc used for crystallization. Once ohtained, these structures will allow us to confirm and expand our biochemical data, producing an accurate model of Hex catalytic mechanism. REFERENCES

Bayleran J, Hechtman P, Saray W (1984) Synthesis of 4-methylumbelliferyl-heta-D-N- acetylglucosamine-6-sulfate and its use in classification of GM~gangliosidosis genotypes. Clin. Chim. Acta. 143: 73-89

Brown CA, Neote K, bung A, Gravel RA, Mahurtln DJ (1989) Introduction of the a subunit mut~tionassociated with the B 1 variant of Tay-Sachs disease into the subunit produces a P-hexosaminidase B without catilytic activity. J. Biol. Chem. 264: 2 1705- 21710 Church WB, Swenson L, Jamcs MNG, Mahuran D (1992) Crystallization of human P- hexosaminidase B. J. Mol. Biol. 227: 577-580

Conzelmtlnn E, Kytzia H-J, Navon R, Sandhol'l' K (1983) Ganglioside GM2N-acetyl-beta-D- galactosaminidase activity in culturcd fibroblasts of late-infantile and adult GM2 ganpliosidosis ptlticnts and of healthy prohands with low hexosaminidase level, Am. J. Hum. Genct. 35: 900-9 1 3 Fernandes MJG, Yew S, Lcclcrc: D, Hcnrissat B, Vorgias CE, Gravel RA, Hechtman P, Kaplan F (1997) Identil'ication of candidate active site rcsidues in lysosomal heta- hexosaminidase A. J. Biol. Cliem. 272: 8 14-820

Hou Y, Tse R, Mahuran DJ (1996a) Thc Direct Dcte~minationof the Substrate Specificity of the a-Active sitc in Hcterodimcric P-Hcxosaminidasl= A. Biochemistry 35: 3963-3969

Hou Y, Vavougios G, Hinck A, Wu KK, Hcchtman P, Kaplan F, Mahuran DJ (1996b) The ~nl192LeuM~~tation in the a Subunit of P-Hcxosaminidase A is not Associated with the B 1-Vtlrian t Form of' Tiy-Stlchs Diseasc. Am. J. Hum. Genet. 59: 52-58

Malcolm BA, Roscnherg S, Ccmy MJ, AIIcn 35, dc Baetselier A, Kirsch JF (1989) Site- Directed Mutagenesis ol' the Catalytic Residues Asp-52 and Gh-35 of Chicken Egg- White Lysozyme. Proc. Natl. Acad. Sci. 86: 133- 137

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Tews I, Perrakis A, Oppenhcim A, Dauter 2, Wilson KS, Vorgias CE (1996) Baterial chitohiase structure provides insight into catalytic mechanism and the basis of Tay- Sachs discuse. Nature Struc. Biol. 3: 638-648 Tse R, Vavougios G, Hou Y, Mahurin DJ (1996a) Identilkcation of an active acidic residue in the catalytic site of ~hexosuminidase.Biochemistry 35: 7599-7607

Tse R, Wu YJ, Vavougios G,Hou Y, Hinek A, Mahuran DJ (1996h) Identification of Functional Domains within the a and P Subunits of P-Hexosaminidase A Through the Expression of a-P Fusion Proteins. Biochemistry 35: 10894-10903

Yuziuk JA, Bertoni C, Beccan T, Orlacchio A, WuY-Y, Li S-C, Li Y-T (1998) Specificity of mouse GM2 activator protein and P-N-acetyl-hexosminidase A and B: Similarities and differences with their human counterparts in the catabolism of GM2. J. Biol. Chem. 273: 66-77 The Direct Determination of the Substrate Specificity of the a-Active Site in Heterodirneric P-Hexosaminidase A

ABSTRACT The P-hexosaminidase isozymes are produced through the combination of a and P subunits to form any one of three active dimers (monomeric subunits are not functional). Heterodimeric hexosaminidase A (ap) is the onIy isozyme that can hydrolyse GM~ganglioside in vivo, requiring the presence of the GM~activator protein. Hexosarninidase S (aa)exists, hut is not considered a physiological isozyme. Although the hexosaminidase B (PP) is present in normal human tissues, it has no known unique function in vivo. However, an unique function for the P-active site present in both hexosaminidase A and B has been indicated in a previous study of the various substrate specificities of the homodimeric forms of hexosaminidase (S and B). It was concluded that the a-active site is only able to efficiently hydrolyse negatively charged substrates, and the :.-active site is only able to hydrolyse neutral substrates. When this model of non-overlapping a- and P- substrates is extrapolated to heterodimeric hexosarninidase A, it has a major effect on the interpretation of recent results relating to the mode of action of the (342 activator protein. In this report I established a new system to directly examine these substrate specificities using a novel form of hexosaminidase A containing an inactive P subunit, produced in permanently tunsfcaed CHQ cells. I demonstrate that; whereas the P- active site has the same substrate spccil'icities in cither its A-heterodimeric or B-homodirneric forms, the a-active site in the A-heterodimcr has dil'lkrent kinetic parameters than the a-active site in the S-homodimer. I conclude that the a and P suhunits in hexosaminidilse A participate equally in the hydrolysis of neutral substrates. The aV192L Substitution Mutation in the a Subunit of Hex A is not Associated with the B 1-variant of Tay-Sachs Disease

ABSTRACT Substitution mutations adversely affecting the a suhunit of P-hexosaminidase A (ap)result in Tay-Sachs disease. The majority affect the initial folding of the proa chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a "B1 mutation" that specilkally affects an "active" residue necessary for substrate binding and/or catalysis. In this case hexosaminidase A is present in the lysosome, but it lacks all a-specific activity. Kinetic analysis of suspected B 1-causing mutations is complex because hexosaminidase A is heterodimeric and both subunits possess similar active sites. In this report I examine a previously idcntificd I31 mutation, aVall92Leu. CHO cells were permanently co-transfwted with an acDNA construct encoding the substitution and a mutant PcDNA (PArg21 lLys), encoding a P suhunil that is inactive, hut normal in all other respects. Surprisingly I found that the Val l92Leu substitution produced a proa chain that did not form a-P dimers and was not transported to the Iysosolme. Finally I re-examined the hexosaminidase isoxymes in the fibroblasts li-om the original patient. These data were also not consistent with the biochemical phenotype of the B 1 variant of Tay-Sachs disease previously reported to he present. Thus, I conclude that the Vall92Leu substitution does not specifically affect the a-active site. Identification of Functional Domains within the a and P Subunits of P-Hexosaminidase A Through the Expression of a-p Fusion Proteins

ABSTRACT There are three human P-hexosaminidiue isozymcs which are composed of all possible dimeric combinations of an a andor a p subunit, i.e. A (ap), B (PP), and S (aa).The amino acid sequences of the two subunits are 60% identical. The homology between the two chains varies with the middle> the carboxy terminal>> the amino terminal portions. Although dimerization is required for activity, each subunit contains its own active site and differs in its substrate specil'icity and thermal stability. The presence of the P subunit in hexosaminidase A influences the substrate specificity ol' the a,cJ.g. in vivo onIy the A-heterodimer can hydrolyze GM~ganglioside. In this report we localize functional regions in the two subunits by ceIlular expression of a/P fusion proteins joined at adjacently aligned residues. First, a chimeric dP chitin was made by replacing the least well conserved amino-terminal section of the P-chain with the corresponding a-section. The biochemical charncteristics of this protein were nearly identical to hexosaminidase B. Therefore, the most dissimilar regions in the subunits are not responsible for their dissimilar biochemical propcrtics. A second fusion protein was made that, so that it included the more homologous rniddlc section ol'the a chain. This protein expressed the substrate specificity unique to isozymcs cont~iningan a-subunit (A and S). We conclude that the region responsible hrthe ability of the a-subunit to bind negatively charged substrates is located within residues a 132-283. Interestingly, the remaining carhoxy-terminal section from the P-chain, p316-556, was sut'l'icicnt to allow this chimeri to hydrolyse GM2-ganglioside with 10% the specific activity of hcterodimeric hexosaminidase A. Thus the carhoxy terminal section of each subunit is Iikely involved in subunit-suhunit interactions. Identification of an Active Acidic Residue in the Catalytic Site of P-Hexosaminidase

ABSTRACT Human P-hexosaminidnse A and B are dimeric lysosomal glycosidases composed of evolutionarily related a and/ or P subunits. Both isozymes hydrolyse terminal P-linked GalNAc or GlcNAc residues from numerous artificial and natural substrates; however in vivo GM~ganglioside is a substrite for only the heterodirneric A isozyme. Thus, mutations in either gene encoding its aor p subunits can result in GM~ganglioside storage and Tay-Sachs or Sandhoff disease, respcctivcly. In gcnerill, all glycosyl hydrola,ses ilre believed to have one or more acidic residues in their catalytic site. We demonstrate that incubation of hexosnminidase with a chemical modifier specific for carhoxyl side chains produces a time dependent Ioss of activity, and that this ct'fcct can he blocked by the inclusion of a strong competitive inhibitor in the reaction mix. We hypothesized that the catalytic acid residue(s) should be located in a region of overall homology and be invariant within the aligned deduced primary sequences of the human a and p suhunits, as well as hcxosaminidases from other species, including bacteria. Such a region is encoded by exons 5-6 of the HEXA and HEXB genes. This region includes PArg211 (invariant in 15 sequences), which we have previously shown to be an active residue. The region also contains two invariant and one conserved acidic residues. A fourth acidic residue, PAsp290, in exon 7 was also investigated in this study because its aligned residue in the a-subunit, aAsp258, has heen associated with the B 1 variant of Tay-Sachs disease. Conservative suhs~itutionswere made at each candidate residue by in vim) mutagenesis of a PcDNA, followed by cellular cxpression. 01' these, only the PAsp 196Asn substitution decreased the kcat (350-910-fold) without any noticeable effect on the Km. Mutagenesis of either PAsp240 or PAsp290 to Asn decreased kcat by 10- or 1.4-fold, but also raised the Km of the enzyme 11- or 3- fold, respectively. The above results strongly suggest that PAspl96 is a catalytic acid residue in P-hexosaminidase.