Biochemical and biophysical investigations of N-linked glycosylation pathways in archaea

Biochemical and Biophysical Investigations of N-linked Glycosylation Pathways in Archaea

by

Michelle M. Chang B.S. Chemical Biology

University of California, Berkeley, 2008

MASSACHUSETTS INSTITUTE OF TECHNOLOLGY

MAY 192015

LIBRARIES

in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

at the

Massachusetts Institute of Technology

December 2014 [Febrtary _2c1 2014 Massachusetts Institute of Technology

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Signature of Author: Certified by:

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Accepted by:

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Robert W. Field Haslam and Dewey Professor of Chemistry Chairman, Committee on Graduate Students

This doctoral thesis has been examined by a committee of the Department of Chemistry as follows:

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Alice Y. Ting Ellen Swallow Richards Associate Professor of Chemistry Thesis Chair

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Barbara Imperiali Class of 1922 Professor of Chemistry and Biology Thesis Supervisor

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Alexander M. Klibanov Novartis Professor of Chemistry and Bioengineering

Biochemical and Biophysical Investigations of N-linked Glycosylation Pathways in Archaea

by

Michelle M. Chang

Submitted to the Department of Chemistry on December 11, 2014 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

Abstract

Asparagine-linked glycosylation is an abundant and complex protein modification conserved among all three domains of life. Much is known about N-glycan assembly in eukaryotes and selected bacteria, in which the oligosaccharyltransferase (OTase) carries out the

en bloc transfer of glycans from polyprenyl-PP-linked donors onto asparagine side chains of

acceptor proteins.

The first aim of this thesis is to elucidate the biochemical details of archaeal N-linked glycosylation, specifically through in vitro analysis of the polyprenyl-P-dependent pathway of the methanogenic archaeon Methanococcus voltae. The archaeal OTase, known as AglB, utilizes a-linked dolichyl-P-trisaccharide substrate as the glycosyl donor for transfer to the acceptor protein. This dolichyl-P-glycan is generated by an initial retaining glycosyltransferase (AglK) and elaborated by additional glycosyltransferases (AglC and AgIA) to afford Dol-P-GlcNAc-Glc-2,3-diNAcA-ManNAc(6Thr)A. Despite the homology to other bacterial or eukaryotic OTases that exploit polyprenyl-PP-linked substrates, the M. voltae AglB efficiently transfers disaccharide to model peptides from the Dol-P-GlcNAc-Glc-2,3-diNAcA monophosphate. While this archaeal pathway affords the same asparagine-linked

P-glycosyl

A second focus of this thesis involves biophysical studies to probe structural features and conformational dynamics of AglB. An intramolecular LRET experimental system was developed to report on substrate binding and the resulting structural transformations in AgIB. There is a strong need for detailed studies on the mechanistic and functional significance of archaeal adaptations of N-linked glycosylation, especially exploring differences between AglB and other OTases that allow AglB to utilize these unique polyprenyl-P-linked substrates.

Lastly, a cell-free expression system was established for the efficient synthesis of Alg5, a yeast dolichyl-phosphate glucosyltransferase that shares high sequence similarity to AglK, the first glycosyltransferase in the M. voltae pathway. Dol-P-Glc was generated and examined to unambiguously characterize the stereochemistry of the product of Alg5.

Thesis Supervisor: Barbara Imperiali

Acknowledgements

I would like to start by thanking my advisor, Barbara Imperiali, for giving me the incredible opportunity to be a part of her lab and for her guidance, patience, and support throughout my graduate studies. Thank you for assembling a wonderful group of people who are always willing to collaborate and help one another become better scientists. Thank you also to my thesis committee members, Professors Alice Ting and Alex Klibanov, for their help and advice through the years.

I have had the pleasure of working with a fantastic group of labmates over the past six years. I am thankful for everyone who welcomed Mike and me into the lab in our first year. Even though I didn't overlap very long with them, Galen, Elvedin, Mark, and Nelson all helped make the lab a wonderful place in my first year. Angelyn, Meredith, Wendy, and Brenda were the four fourth-year students who helped me in countless ways as a new graduate student. I am especially thankful to Angelyn for taking the time to mentor me and teach me about the world of membrane protein preparations, OTase assays, HPLC maintenance, and so much more. I am also grateful to Meredith for her generous and sage advice and for teaching me about nanodiscs and the joy of volleyball. I am thankful for Wendy's help with my fluorescent peptide experiments. I worked on PglB with Marcie, and I am so thankful that I had her to discuss science with and for all of her help with LBTs. I appreciate being able to walk through grad school with Mike as a classmate and labmate, and I love that he hosted an annual Thanksgiving meal for our classmates. I am happy to have shared most of my grad school duration with Vinita, who has been a great friend and someone to commiserate with in purifying our sugars and lipid-linked oligosaccharides. I am thankful to Stephanie for helping me with yeast expression and for deciphering my horrible drawings. I am delighted that Sonya joined our lab and enjoyed our time working on cell-free translation. Austin always spoke with great enthusiasm for science and never failed to ask about my day, and I am sorry that his time in our lives was cut short.

I also need to acknowledge the many postdocs throughout my time, starting with Matthieu, Jay, and Cliff who were always generous with their advice. I am particularly thankful to Matthieu for teaching me about peptide synthesis. I would also like to thank postdocs Andrew, Elke, Laura, Marthe, Julie, Joris, Debasis, Silvano, and Cristina who have been brilliant scientists and great resources throughout the years. I am glad to see Monika continuing the LBT-PglB project, and I am excited to see how it compares to AglB. I am thankful for Garrett's help with Dol-P synthesis and NMR and for being my hockey and early lunch buddy. Joris has been my late lunch partner. James, Philipp, and Carsten have each been my podmate at some point, and I appreciate all of their vastly different personalities and value our various conversations about science and life.

I would like to thank members of the Stubbe lab for allowing me to use their French press, as well as members of the Drennan lab for helping me with crystallography. I would also like to thank Traci, Daniel, Chiara, Seymour, and Kaspar for their time working in our lab. I have also enjoyed the time Will, Thais, Pi, Hui, and Natalie have spent in our lab as undergrads. And I would be remiss if I did not thank Debby for her work in the BIF and Elizabeth for all her work to keep the lab running.

Lastly, I would never have made it this far if it weren't for the love and support of my family and friends. Thanks to my mom and dad who are always proud of me and concerned for my academic and personal well-being without ever pressuring me with their expectations. Thanks to my amazing sister, Liana, who is always there for me whenever I need to talk. Thanks

to the MIT Graduate Christian Fellowship, Hope Fellowship Church, and Tang small group communities for fortifying my faith and for all the adventures we've had together. And thank you to my current and former roommates (Stephanie, Heather, Wei-Shan, Nan, Ange) who have made Tang a special place filled with laughter, shared experiences of failures and triumphs, rousing conversations about God and relationships, and jigsaw puzzles.

Table of Contents

Abstract ... 3

Acknow ledgem ents ... 4

Table of Conttents ... 6 List of Figures ... 9 List of Tables... 12 List of A bbreviations... 13 Chapter 1: Introduction...15 Introduction ... 16

Eukaryotic N -linked glycosylation... 17

Bacterial N -linked glycosylation... 19

A rchaeal N -linked glycosylation... 20

Thesis objectives ... 28

References ... 29

Chapter 2: Identification and characterization of archaeal glycosyltransferases...33

Introduction ... 34

Results and D iscussion... 41

AglH is not the first enzyme in the M. voltae N-linked pathway... 41

A glK is a specific D ol-P-GIcN Ac synthase ... 42

D ol-P-GIcN A c exhibits an a-glycosidic linkage ... 47

A giK m utagenesis and inhibition studies... 50

Com parison of A glK with other glycosyltransferases ... 51

Observation of Dol-P-glycan in M . maripaludis cells ... 53

A gIC is a U D P-Glc-2,3-diN A cA glycosyltransferase... 56

Identification of D ol-P-glycans in other archaeal species... 60

Conclusions ... 62

A cknow ledgem ents ... 63

Experim ental M ethods ... 64

Cloning of A glH , A glK and AgiC ... 64

Protein expression and purification... 64

Synthesis of (C55-60) (S)-dolichols... 65

Synthesis of (S)-dolichyl-phosphate ... 66

Synthesis of (C55-60) Dolichyl-PP-GlcNAc and Dolichyl-PP-GaINAc ... 67

Synthesis of U D P-[3H ]G lc-2,3-diN AcA ... 67

A glK and A glC glycosyltransferase assays ... 67

A glK reaction product identification... 69

A glK D X D m utagenesis... 69

N M R and M S characterization of Dol-P-glycans ... 70

M ethanococcus maripaludis LLO preparation ... 70

Pyrococcusfuriosus LLO preparation ... 71

References ... 72

Chapter 3: Characterization of an archaeal oligosaccharyl transferase ... 76

Introduction ... 77

Results and discussion...82

Characterization of oligosaccharyl transferase activity ... 82

AgIB and its lipid-linked glycosyl donor substrate... 87

Investigation of the role of AgIB His-597... 89

AgLB crystallographic studies ... 92

Conclusions ... 97

Acknowledgem ents ... 98

Experim ental m ethods... 99

AglB expression and purification... 99

Oligosaccharyl transferase assay... 100

Purification of glycopeptides ... 101

AglB H597 m utagenesis... 101

Purification of AglB for crystallography ... 101

AglB peptide substrates... 102

AgIB crystallography ... 103

References ... 104

Chapter 4: Efforts towards establishing an experimental system for investigating AgiB conform ational dynam ics by LRET ... 108

Introduction ... 109

Results and discussion... 115

Generation of LBT-AgIB construct...115

Generation of LBT-AgIB cysteine mutants ... 117

LRET with fluorophore-labeled LBT-AgIB ... 121

Lum inescence studies with LBT-AgIB and peptide substrate ... 124

N ew peptide library for LRET experim ents... 127

Luminescence studies with LBT-AgIB and short acceptor peptides ... 131

Conclusions ... 132

A cknowledgem ents...133

Experim ental m ethods... 133

LBT-AglB construct cloning, expression, and purification ... 133

LBT-AglB and LBT-Ub lum inescence experim ents ... 135

LBT-AgIB Cys m utagenesis ... 135

LBT-AgB fluorophore labeling...136

AglB peptide substrate synthesis... 137

Km determ ination of AglB peptide substrates ... 138

References ... 139

Chapter 5: Characterization of Alg5 activity and product stereochemistry ... 141

Results and discussion... 145

A lg5 expression in E. coli ... 145

Alg5 expression in S. cerevisiae ... 150

Cell-free translation of Alg5 ... 151

Conclusions ... 156

Acknowledgem ents ... 157

Experim ental m ethods... 157

A lg5 expression in pGEX-4T3, pET-47, and pE-SUM O vectors... 157

Alg5 expression in pBAD vector ... 158

A lg5 expression from pET(Alg5) plasm id... 160

Alg5 expression in S. cerevisiae ... 160

Alg5 activity assay ... 162

Cell-free expression of Alg5 ... 162

Dol-P-Glc synthesis and analysis... 164

List of Figures Chapter 1

Figure 1-1: Schematic overview of the five different types of protein glycosylation. ... 17

Figure 1-2: N-linked glycosylation pathway in S. cerevisiae. ... 18

Figure 1-3: N-linked glycosylation pathway in C. jejuni...20

Figure 1-4: N-glycan structures from Halobacterium salinarum, Thermoplasma acidophilum and Pyrococcusfuriosus... 22

Figure 1-5: N-linked pathway in H. volcanii in high and low salinity. ... 23

Figure 1-6: N-linked glycosylation pathway in M. voltae. ... 25

Figure 1-7: N-linked glycosylation pathway in M. maripaludis...26

Figure 1-8: N-glycosylation pathway in S. acidocaldarius. ... 27

Chapter 2 Figure 2-1: Representative structures of the two main GT folds...35

Figure 2-2: Catalytic m echanism s in GTs... 37

Figure 2-3: N-linked glycosylation in Methanococcus voltae. ... 39

Figure 2-4: General assay for PGT and GT activity.. ... 41

Figure 2-5: Topology prediction for M. voltae proteins AgIK and AglC ... 43

Figure 2-6: SDS-PAGE and Western blot analysis of purified AglK and AglC proteins ... 43

Figure 2-7: A glK activity assay. ... 45

Figure 2-8: Normal phase HPLC purification of Dol-P-GlcNAc. ... 45

Figure 2-9: ESI-M S of purified Dol-P-GlcNAc... 46

Figure 2-10: Capillary electrophoresis of AglK reaction products... 46

Figure 2-11: Alignment of AglK from M. voltae with S. cerevisiae Alg5 ... 47

Figure 2-12: 1H-NMR spectrum of Dol-P-GlcNAc... 48

Figure 2-13: Activity assay for AgIK DI05A and D107A mutants...50

Figure 2-14: AgiK inhibition observed with UDP-(5F)-GlcNAc...51

Figure 2-15: Alignment of AglK from M. voltae with AgIJ from H. volcanii or MMP 1170 from M m arip alud is. ... 53

Figure 2-16: N-linked glycan structures from M voltae and M. maripaludis ... 54

Figure 2-17: Mass spectrometry analysis of M maripaludis total lipid extraction ... 55

Figure 2-18: Biosynthetic pathway of UDP-ManNAc(3NAc)A ... 57

Figure 2-19: AglC donor and acceptor substrate specificity...58

Figure 2-20: Normal phase HPLC purification of Dol-P-Glc-2,3-diNAcA ... 58

Figure 2-21: ESI-MS of Dol-P-GIcNAc-Glc-2,3-diNAcA ... 59

Figure 2-22: Reverse phase LC/MS analysis of P. furiosus whole cell extractions confirms the presence of Dol-P-heptasaccharide and other Dol-P-glycan intermediates...61

Figure 2-23: M voltae N-linked glycosylation pathway enzymes ... 63

Chapter 3 Figure 3-1: N-linked glycosylation pathway in Saccharomyces cerevisiae. ... 77

Figure 3-3: N-linked glycosylation pathway in Campylobacterjejuni...80

Figure 3-4: Sequence alignment of 16 archaeal Stt3 homologs, depicting the region surrounding the critical W W D X GX m otif... 82

Figure 3-5: Comparison of the archaeal OTases expression by Western blot...82

Figure 3-6: Topology prediction for M voltae AgiB... 83

Figure 3-7: Purified AglB analyzed by SDS-PAGE and Western blots ... 83

Figure 3-8: Standard assay for OTase activity... 84

Figure 3-9: AglB activity assay using the peptide Ac(YKYNESSYKpNF)NH 2 as the acceptor substrate and Dol-P-GlcNAc or Dol-P-disaccharide as the donor substrates ... 85

Figure 3-10: Dependence of AglB activity on divalent metal cations ... 86

Figure 3-11: Reverse-phase HPLC purification of peptide and glycopeptide product...86

Figure 3-12: ESI-MS of the glycopeptide produced by AgIB ... 87

Figure 3-13: Sequence alignment of the WWDXGX motif of representative OTases...90

Figure 3-14: Activity of AglB His-597 mutants with Dol-P-GIcNAc-[3H]Glc-2,3-diNAcA and Ac(YKYNESSYKpNF)NH 2 as the donor and acceptor substrates. ... 91

Figure 3-15: AglB WT, H597A, H597F, H597N, H597Y expression in membrane fractions .. 91

Figure 3-16: AglB wild type and AgIB H597Y mutant activity with Dol-P-GlcNAc-Glc-2,3-diNAcA and Und-PP-Bac-GaINAc... 92

Figure 3-17: Gel filtration chromatography of AgIB with Triton X-100 and LMNG...94

Figure 3-18: Structure of LM NG amphiphile. ... 95

Figure 3-19: AglB acceptor peptide library screen ... 96

Figure 3-20: Crystallization of AgIB in the presence of peptide ... 97

Figure 3-21: M voltae AglB catalyzes the oligosaccharyl transfer from a Dol-P-glycan onto the Asn residue within the NXS/T sequon of acceptor proteins. ... 98

Chapter 4 Figure 4-1: N-linked glycosylation across the three domains of life ... 110

Figure 4-2: Jablonski diagram of a ligand-lanthanide complex ... 111

Figure 4-3: Lanthanide binding tag... 112

Figure 4-4: Intram olecular LRET of AglB ... 114

Figure 4-5: LBT-AglB purification and luminescence of LBT-AglB upon Tb3+ titration ... 116

Figure 4-6: Membrane topology prediction plot for AgIB, illustration of Cys to Ser mutants, and structure of BODIPY-TM R m aleim ide... 118

Figure 4-7: LBT-AglB-C35S/C590S purification and OTase assay... 119

Figure 4-8: LBT-AglB-C35S/C590S/P356C and LBT-AgIB-C35S/C590S/T367C purification and labeling with BODIPY-TM R fluorophore. ... 120

Figure 4-9: Emission spectrum of LBT-AglB-T367C-BODIPY-TMR with or without gating 121 Figure 4-10: Time-gated emission spectra of BODIPY-TMR labeled P356C upon Tb3+ titration. and luminescence decay measured at 543 nm ... 123

Figure 4-11: Time-gated emission spectra of BODIPY-TMR labeled T367C upon Tb3+ titration and lum inescence decay measured at 543 nm ... 124

Figure 4-12: Emission spectra and luminescence decay of LBT-AglB-T367C-BODIPY-TMR and Tb3+ ion exhibit quenching upon addition of Ac(YKYNFTSYKRR)NH 2 peptide...125

Figure 4-13: Loss of luminescence signal with addition of peptides Ac(YFNFTGRR)NH2 and Ac(YKYNFTSYKRR)NH 2to LBT-Ub... 127

Figure 4-14: AgiB acceptor peptide library screen with peptides having N-terminal acetylation and or free N -term inal am ines... 130 Figure 4-15: Emission spectra of LBT-Ub with Tb3 _{and addition of peptides Ac(TFNFTS)NH}

2

and Bz(TFNETS)NH2.... ... . . . ... 132

Chapter 5

Figure 5-1: Dolichol pathway of N-linked glycosylation in Saccharomyces cerevisiae...143 Figure 5-2: Alignment of AgiK from M. voltae with S. cerevisiae Alg5 ... 144 Figure 5-3: Topology prediction for S. cerevisiae Alg5. ... 145 Figure 5-4: GST-Alg5 and His6-Alg5 expression detected by SDS-PAGE and Western blot.

Activity assay of GST-Alg5, His6-Alg5 constructs ... 146

Figure 5-5: Expression of His6-SUMO-Alg5 in C41 (DE3), C43 (DE3), BL21 (DE3) RIL,

Rosetta2 (D E3), and Lem o2l (DE3) cell lines ... 146 Figure 5-6: Alg5-Hisio expression levels detected at various times after induction with variable

concentrations of L-arabinose ... 147 Figure 5-7: Activity of cell lysates Alg5-Hisio expressed from pBAD vector in BL21 (DE3) RIL cells at various times after induction with 0.2% L-arabinose. ... 148 Figure 5-8: TRX-His6-Alg5 expression is detected by Western blot analysis...148

Figure 5-9: Alg5 expression as CEF from pET(Alg5) under different conditions. Activity assay of A lg5 C E F s... 149 Figure 5-10: HPLC fractions of the organic extraction of the Alg5 reaction products... 150 Figure 5-11: Alg5 yeast expression in yeast strains INVSc1, W303a, or W303c...151 Figure 5-12: Glucosyltransferase activity assay with membrane fractions of Alg5 expression in

IN V Sc1, W 303a, and W 303c ... 151

Figure 5-13: Western blot showing cell-free expression of Alg5-NT and Alg5-CT ... 153 Figure 5-14: Western blot of Alg5-NT and AglC expression with additives, or Alg5-NT and

A lg5-CT solubilization after expression. ... 154 Figure 5-15: Glucosyltransferase activity assay with Alg5-NT and Alg5-CT ... 155 Figure 5-16: 1H-NMR and 3_{'P decoupled} 1_{H-NMR spectrum of Dol-P-Glc... 156}

List of Tables Chapter 1

Table 1-1: Features of archaeal N-linked glycosylation in select archaeal species. ... 28 Chapter 2

Table 2-1: 1H- and 13_{C-NMR assignments of Dol-P-GlcNAc structure ... 49}

Chapter 4

Table 4-1: Amino acid preference at each position of XO-X1-X2-X3-X4-X5 peptide. ... 131 Table 4-2: Apparent Km values of best AglB peptide substrates. ... 131

List of Abbreviations

Standard 3-letter and 1-letter codes are used for the 20 natural amino acids. Standard 1-letter codes are used for the 4 common DNA bases.

AcCoA ATP B-OG BODIPY CAM CE CEF CHAPSO DDM DIPEA DMF DMSO Dol-P Dol-PP DMPC DTT EDTA ER ESI-MS Fmoc Fos-12 FRET GDP-Man GST GT IPTG HEPES Km Kd LB LBT LDAO LLO LMNG LRET NDP Ni-NTA NMR NP-HPLC OTase acetyl-coenzyme A adenosine 5'-triphosphate n-octyl

P-D-glucoside

capillary electrophoresis cell envelope fraction

3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate n-dodecyl--D-maltoside diisopropylethylamine dimethylformamide dimethylsulfoxide dolichyl-phosphate dolichyl-diphosphate 1,2-dimyristoyl-sn-glycero-3-phosphocholine dithiothreitol

extinction coefficient for molar absorptivity ethylenediaminetetraacetic acid

endoplasmic reticulum

electrospray ionization-mass spectrometry 9-fluorenylmethoxycarbonyl

n-dodecyl phsophocholine

fluorescence resonance energy transfer guanosine diphosphate mannose glutathione S-transferase

glycosyltransferase

isopropyl f-D-1-thiogalactopyranoside

4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid Michaelis constant

dissociation constant Luria-Bertani broth

lanthanide binding tag lauryl dimethylamine oxide lipid-linked oligosaccharide lauryl maltose neopentyl glycol

luminescence resonance energy transfer nucleotide diphosphate

nickel nitrilotriacetic acid nuclear magnetic resonance

normal phase high performance liquid chromatography oligosaccharyltransferase

PAL PDB PEG-PS PGT pNF POPC Pren-P Pren-PP PSUP PyBOP Ro RP-HPLC SDS-PAGE T-TFA TIS TLC TMHMM Triton X-100 TRX TUPS UDP UDP-Bac UDP-Glc UDP-GlcNAc UDP-Gal UDP-GalNAc UDP-Glc-2,3-diNAcA UMP Und-P Und-PP Xaa 5-(4'-aminomethyl-3',5'-dimethoxyphenoxy)valeric acid Protein Data Bank

polyethylene glycol-grafted polystyrene phosphoglycosyltransferase

4-nitrophenylalanine

1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine polyprenyl-phosphate

polyprenyl-diphosphate pure solvent upper phase

benzotriazol- 1 -yl-oxytripyrrolidinophosphonium hexafluorophosphate

FOrster distance

reverse phase high performance liquid chromatography sodium dodecyl sulfate polyacrylamide gel electrophoresis lifetime

trifluoroacetic acid triisopropylsilane

thin layer chromatography

Tied Mixture Hidden Markov Model for transmembrane prediction t-octylphenoxypolyethoxyethanol

thioredoxin

theoretical upper phase with salt uridine 5'-diphosphate UDP-2,4-diacetamido-2,4,5-trideoxy-ax-D-glucose UDP-glucosamine UDP-N-acetyl-D-glucosamine UDP-galactosamine UDP-N-acetyl- D-galactosamine UDP-2,3-diacetamido-2,3-dideoxy-D-glucuronic acid uridine 5'-monophosphate undecaprenyl-phosphate undecaprenyl-diphosphate

Introduction

Post-translational modifications of proteins help to expand the proteome far beyond what is directly encoded by the genome.' Glycosylation is one of the predominant and most complex protein modifications found in nature, occurring in all three domains of life - Eukarya, Bacteria, and Archaea.2 _{The enormous diversity of sugars, sugar linkages, sugar-protein linkages, and} glycan size leads to a seemingly endless number of possible protein-linked glycan structures. Glycosylation can profoundly impact protein function, playing important roles in biological processes such as protein folding and stability, intracellular targeting, cellular signaling and adhesion, and the immune response.3

Five major types of protein glycosylation have been observed in nature and are classified based on the manner in which the glycan is linked to the modified protein (Figure 1-1). N-linked glycosylation involves the en bloc transfer of an oligosaccharide from a lipid carrier onto the side chain amide nitrogen of asparagine residues within a conserved sequon of acceptor proteins.4-6 O-linked glycosylation involves the transfer of monosaccharide units onto the side chain hydroxyl oxygen atom of either serine or threonine residues, and to a lesser extent, other residues such as tyrosine, hydroxylysine and hydroxyproline. Glycosylphosphatidylinositol (GPI) anchoring involves the transfer of a GPI moiety to the C-terminus of a target protein, tethering it to the cell membrane.' Phosphoglycosylation involves the addition of sugar I -phosphates to the hydroxyl oxygen atom of serine or threonine residues.9 C-mannosylation involves the transfer of mannose to the indole C2' carbon atom of tryptophan residues resulting in a C-C linkage.10'11 N-linked, 0-N-linked, and GPI-anchored glycosylation have been well-studied biochemically, while

0-glycosylation P-glycosylation C-glycosylation GPM-anchor

zon W

Figure 1-1: Schematic overview of the five different types of protein glycosylation. Taken from Jarrell, et al.'

Eukaryotic N-linked glycosylation

N-linked glycosylation is an essential part of the protein maturation process in eukaryotes and is estimated to occur in more than half of all eukaryotic proteins.'2 Genetic and biochemical characterization of this process has provided the best understanding of this pathway in

Saccharomyces cerevisiae (Figure 1-2A), but N-glycosylation is remarkably conserved in all

eukaryotes, from yeast to man.'3 _{The first phase of N-glycosylation occurs on the cytoplasmic} face of the endoplasmic reticulum (ER) membrane. The assembly of the lipid-linked oligosaccharide (LLO) is initiated by a phosphoglycosyltransferase, followed by a series of membrane-bound glycosyltransferases in the asparagine-linked glycosylation (Alg) family that catalyze the transfer of each monosaccharide unit from nucleotide sugar donors onto a dolichyl-diphosphate (Dol-PP) carrier. Dolichol is a long chain, a-saturated linear polyisoprene that typically varies in size between 14 and 21 units depending on the cell type and species.'4 _After the completion of the branched heptasaccharide, the Dol-PP-GlcNAc2Man5 intermediate is

flipped across the membrane to face the ER lumen in an ATP-independent manner. Yeast genetic Man-4cwwif o Galrncto. (Gd * kn(I * N'A*My~uCOGsamN(G4A SGuWsmri (GICN) *Muano (an Upid asaw S DO Gooh ftycopm-.i Torg~t gtyoop-ew

analysis previously implicated Rftl as the purported flippase, but recent studies show that Rftl-null cells do not stall at the flippase step, and Rftl may instead act as a chaprone.15"1 6 The flipped LLO is further elaborated by the transfer of mannose and glucose residues from Dol-P-mannose and Dol-P-donors, respectively. The fully assembled branched tetradecassachride, Glc3Man9GlcNAc2, is transferred by the oligosaccharyltransferase (OT) onto asparagine residues

within the N-X-S/T sequon, where X can be any amino acid except proline (Figure 1-2B).

A

I

j

I A,

IrA

I

A1g98 a Ag6 a A1IgQ CLa A1g12

T 4 . 1. -4a

i

L

0-

Ci

IIcLc

B

A1 A

-

Figure 1-2: (A) N-linked glycosylation pathway in S. cerevisiae. (B) Structure of the eukaryotic N-linked glycan, Glc3Man9GlcNAc2.

1--GDP-11 A191

Newly formed glycoproteins are processed by glycosidases and glycosyltransferases in the ER and Golgi apparatus before being secreted or distributed to various cellular locations. This downstream modification results in the vast diversity of N-linked glycans observed in eukaryotes." Although the Glc3Man9GlcNAc2 tetradecasaccharide core is widely conserved in

eukaryotes, several species of protists have been found to assemble only truncated forms of the glycan.18 N-glycans play an important role in the quality control of protein folding in the ER, serve as identity and localization tags to direct proteins to the proper cellular destination, help regulate the mobility of cell membrane proteins through interactions with lattice-forming lectins, impart structural rigidity, and may provide protection against proteolysis.19

Bacterial N-linked glycosylation

The first bacterial N-linked glycosylation system was discovered in Campylobacter

jejuni, a gram-negative human gut mucosal pathogen.2 0_'2 1 _{Glycan assembly occurs on the} cytoplasmic face of the periplasmic membrane by the protein glycosylation (Pgl) enzymes (Figure 1-3A). This pathway resembles the first half of the dolichol pathway in S. cerevisiae, but with several differences. A phosphoglycosyltransferase initiates the LLO assembly and then a series of glycosyltransferases add monosaccharide units from nucleotide sugar donors onto an undecaprenyl-diphosphate (Und-PP) carrier rather than onto Dol-PP. Undecaprenol is a long chain, unsaturated polyisoprene comprising 11 isoprene units.2 2 Instead of the GlcNAc residue found in eukaryotic N-glycans, the first sugar in the C. jejuni glycan is N,N'-diacetylbacillosamine (Bac). After the completion of the heptasaccharide, the Und-PP-BacGalNAc5Glc intermediate is flipped across the membrane to the periplasmic face by an

(PglB) onto asparagine residues within the extended sequon, D/E-X1-N-X2-S/T, in which an

acidic residue occupies the -2 position (Figure 1-3B).

A

PgIF PgIE

UDP-U - UDP-8 UDP-O Cytoplasm PgIC P91A

Perplasm

B

I

I

_]

o

Figure 1-3: (A) N-linked glycosylation pathway in C. jejuni. (B) Structure of the N-linked glycan, GlcGalNAc5Bac.

Over one hundred glycoproteins have been identified in C. jejuni and are associated with a wide range of cellular functions.2 4 _{Mutations of the pgl locus resulted in impaired host cell}

adhesion and colonization, establishing a link between N-glycosylation and pathogenicity.20,25,26 Other important roles for N-linked glycosylation may include modulation of protein-protein interactions, enhancement of protein stability, and protection of C. jejuni from osmolytic stress.27 28

Archaeal N-linked glycosylation

In 1977, recognition of major differences in the structure and genetics of prokaryotes led to the classification of archaea and bacteria into two distinct domains.29Archaea share common

features with both bacteria and eukaryotes while also possessing several unique characteristics. Like bacteria, archaea are single-celled organisms, have circular chromosomes and no nucleus or membrane-bound organelles, but they also possess several genes and metabolic pathways that are more closely related to those of eukaryotes, especially the enzymes involved in transcription and translation. Archaea contain ether-linked phospholipids in their cell membranes instead of the ester-linked phospholipids found in bacteria and eukaryotes. In addition, archaea are able to

30 utilize a wide range of energy sources and frequently inhabit extreme environments.

N-linked glycosylation is predicted to occur in nearly all of the archaeal species with sequenced genomes.3 1 _{While many archaeal N-glycans have been identified in S-layer,}

archaellin, and other cell-surface glycoproteins, not many N-glycan structures have been fully characterized and even fewer biosynthetic pathways understood.. The structures that have been characterized over the years demonstrate the incredible variety of archaeal N-glycans (Figure 1-4).3-3 There is a high diversity of archaeal N-linked glycan structures in terms of size, the degree of branching, the identity of the linking sugar, the presence of unique sugars, and further modification of sugar moieties by methylation, sulfation, and even addition of pendant amino acids. Such diversity is far beyond that found in bacteria and eukaryotes. However, the chemical structure of the glycan and the nature of the glycan linkages to protein have often remained undetermined and the pathways for N-glycan assembly unclear. The recent availability of complete genome sequences for nearly 200 archaeal species and the development of appropriate molecular tools for genetic manipulation have facilitated the detailed delineation of several of these pathways.31

,36 _{Since the first discovery of archaeal N-linked glycoproteins in}

limited to the euryarchaeal Haloferax volcanii, Methanococcus voltae, and Methanococcus

maripaludis, and the crenarchaeal Sulfolobus acidocaldarius.

A B HO 0 0>(. 0 0 H H1CO R'H $O3 RAO R- RO H R30 30 R30 330 RO ~ .~ R=H, SOO A2N -o IN0 1 H 0 AcN HO

/10-13

HOOH HIH 6ic OH 040

TOHOH OH OilH~

high-mannose-type linked glycan of Thermoplasma acidophilum. (D) Structure of the N-linked heptasaccharide in Pyrococcusfuriosus.

There are two pathways of N-glycosylation in the extreme halophile Haloferax volcan ii. Under normal high salt conditions, the process begins with the sequential addition of the first four sugar subunits of the pentasaccharide from nucleotide-activated donors to a Dol-P carrier at the cytoplasmic leaflet of the cytoplasmic membrane (Figure 1 -5A).38-44 _{In contrast to the}

eukaryotic (C70-Cl o) Dol-PP lipid carriers, (C5 5-C6o) Dol-P serves this role in H. volcanii. The

Dol-P-tetrasaccharide intermediate and a separate Dol-P-mannose are flipped across the membrane, and the tetrasaccharide is transferred by the oligosaccharyltransferase (AgiB) to the asparagine residue within the N-X-S/T sequon of the target S-layer glycoprotein or archaellins. The final mannose subunit is then delivered from its flipped Dol-P carrier onto the glycoprotein-linked tetrasaccharide, resulting in the final glycoprotein N-glycoprotein-linked to the pentasaccharide. At higher salinities, both Dol-P charged with the first four pentasaccharide sugars and Dol-P charged with final mannose sugar have been observed by mass spectrometry analysis.

A NDP-U

AgIM NDPU NDP-U NDP-U AgIF

AgIJ q AgIG AgIl

Cytoplasm a. - a.- C. ON C Exteror Dolichol OH - 7-8 2 NDP-U AgIE -+ I SAM AgIP -- + o-Agli2 Ag Il AIj~iAg l2 AgI5 P AgI8 Ag l3

Ag6 qtAplasmss Ag7Ag19 i Agli4 !soi

Cytoplasm a. - a.- e L . -. C.. - a - a. Exteror Ag ? AI HH, 115 0 , NDP-u AgID

!

N Hexuronic acid (HexA) * Mannose (Man)

O Rhamnose (Rha)

I

Figure 1-5: (A) linked pathway in H. volcanii cells grown in 3.4 M NaCl. (B) A second N-linked pathway in H. volcanii cells grown in 1.75 M NaCl. The exact structures of the N-N-linked glycans on S-layer glycoproteins or archaellins have not yet been determined.

When H. volcanii cells are grown at lower salinity, S-layer glycoproteins display a distinct tetrasaccharide, comprising a sulfated hexose, two unmodified hexoses, and a rhamnose. Dol-P charged with this tetrasaccharide has also been observed by mass spectrometry.43 The current working model in this low-salt environment implicates an entirely different set of enzymes for glycan assembly (Figure 1-5B). A currently unidentified oligosaccharyltransferase, not AglB, is believed to be responsible for the transfer of this glycan to a different asparagine residue on the S-layer glycoprotein. This second pathway appears to be an adaptive response and is recruited when the first pathway is compromised.45

Chapters 2 and 3 of this thesis detail the biochemical characterization of the N-linked glycosylation pathway of Methanococcus voltae, a marine methanogenic mesophile. This pathway is initiated at the cytoplasmic face of the cell membrane by a series of glycosyltransferases that add monosaccharide units from nucleotide-activated sugar donors onto

a Dol-P carrier (Figure 1-6A).46 49 The Dol-P-trisaccharide is then flipped across the membrane

to the exterior surface of the cell, where AglB transfers the trisaccharide onto the asparagine residue within the N-X-S/T sequon of target proteins. In our studies, AgIB efficiently transfers a disaccharide from a Dol-P carrier to acceptor peptides, but the glycan that is natively N-linked to archaellins and S-layer proteins is a trisaccharide, ManNAc(6Thr)A-Glc-2,3-diNAcA-GIcNAc (Figure 1-6B).

A UDP -U UDP-U UDP--U _GlcNAc

Cytoplasm cL A1K AgIC Ag1A Glc-2,3-diNAA

[LC

[Dolichyl-phosphate

Figure 1-6: (A) N-linked glycosylation pathway in M voltae (B) Structure of the N-linked

archaellar glycan, ManNAc(6Thr)A-Glc-2,3-diNAcA-GlcNAc.

The N-linked glycosylation pathway in another methanogen, M maripaludis, shares similarities with that of the closely related M voltae (Figure 1-7A).50 _{Chapter 2 of this thesis}

posits that the first glycosyltransfer step of the M maripaludis pathway is initiated by MMP 1170, an enzyme that has yet to be characterized but exhibits extremely high homology to

AglK, the first glycosyltransferase of the M voltae pathway.49 _{Three other glycosyltransferases}

add monosaccharide units from nucleotide-activated sugar donors onto a P carrier. The Dol-P-tetrasaccharide is then flipped from the cytoplasmic face of the cell membrane to the exterior, where AglB transfers the tetrasaccharide onto the asparagine residue within the N-X-S/T sequon of acceptor proteins. Strong evidence for a Dol-P carrier comes from our mass spectrometry analysis of M maripaludis cells, in which we observed the presence of a Dol-P-trisaccharide intermediate bearing the first three sugars of the N-linked glycan identified in M maripaludis archaellar and pilin proteins, Sug-ManNAc(3NAm6Thr)A-Glc-2,3-diNAcA-GaNAc, where the terminal Sug is 2-acetamido-2,4-dideoxy-5-0-methylhexosulo-1,5-pyranose (Figure 1-7B).

A _{U GaINAc} NDP-U NDP-U NDP-U NDP- M Glc-2,3-diNAcA

MMP1170?

!

Cytplsm .. -- c. - . 0- -- + O. - + O. c 2NAc(4deoxy)hexulose Dolichyl-phosphate (Dol-P) Exterior AgIB Dolichol OH B HO 0 OH NHAc 0 --<NHc ~CiO 0 OH OH

OCH3 AcHN AcHN

Figure 1-7: (A) N-linked glycosylation pathway in M maripaludis (B) Structure of the N-linked

archaellar glycan, 2NAc(4deoxy)hexulose-ManNAc(3NAm6Thr)A-Glc-2,3-diNAcA-GaNAc.

Crenarchaeal N-linked glycosylation has been best studied in the thermophilic Sulfolobus

acidocaldarius, but the pathway enzymes are only partially identified (Figure 1-8A).5

2-54 The actions of a phosphoglycosyltransferase and subsequent glycosyltransferases are thought to build up a tribranched glycan on a Dol-PP carrier, which is flipped from the cytoplasmic to the exterior surface of the cell membrane, before transfer of the glycan onto the asparagine residue of target proteins. S-layer, archaellin, and cytochrome b5 5 8/5 6 6 proteins are N-glycosylated with

heterogeneous family of glycans, with the largest comprising GlcNAc2GlcMan2 and an unusual sulfated sugar called sulfoquinovose (Figure 1-8B). The dolichols observed in S. acidocaldarius are unusually short and display a higher degree of saturation of internal isoprene units than eukaryotic or other archaeal dolichols. While genetic complementation data suggest that Dol-PP is the lipid carrier, only Dol-P and Dol-P charged with different hexoses have ever been detected

by mass spectrometric analysis of lipid extracts.5 3 _{No Dol-P or Dol-PP bearing complex glycans}

the M voltae pathway illustrates that genetic complementation studies alone are insufficient in characterizing enzyme activity.47'49 This ambiguity suggests that the S. acidocaldarius pathway could in fact be more like the euryarchaeal pathways, with Dol-P being the lipid carrier instead of Dol-PP. Interestingly, S. acidocaldarius is the only archaeal organism studied thus far in which N-linked glycosylation appears to be essential.54

A UDP-* Gic

Dol-P-U Agl3 Dol-P-U DoI-P-U U G2cNAc

or ,, I or or EM an

NDP-1 qNDP- NDP-U UDP-U NDP-U NDP-U _{0 Sulfoquinovose (QuiS)}

Cytoplasm 0 A CL (L a. Dolichyl-phosphate (Dol-P)

Exterior Dolichol AgIB

OH B HO OH HO H OH H010 0 _OH HHO O OH OH 0O 'jOH HO H

Figure 1-8: (A) Current understanding of the N-glycosylation pathway in S. acidocaldarius. (B) Structure of the tribranched hexasaccharide N-glycan containing the 6-sulfoquinovose.

Progress has been made in recent years to identify highly unusual archaeal N-glycans and the enzymes involved in their biosynthesis and transfer. Much of this progress has come through deletion of genes potentially involved in a pathway and analysis of the downstream effects on protein glycosylation through mass spectrometry. Unfortunately, there is a dearth of biochemical validation of proposed pathways and in vitro enzymatic assays are difficult when the identities of the substrates are often unknown. Even when the glycan identity is known, questions remain about the lipid-linked carrier (Dol-P vs. Dol-PP) and the mechanism of transfer to acceptor

proteins. Archaeal oligosaccharyltransferases (designated as AglBs) are easily identifiable in archaeal genomes, and many archaeal species encode multiple AgiBs. The physiological significance of this observation remains unclear, as multiple AgiBs could reflect differences in substrate or target preference, possibly as a function of local growth conditions. A few AglB crystal structures have been published, but there are still major gaps in our knowledge of the AgiB mechanisms (Table 1-1). All known oligosaccharyltransferases contain a WWDXGX2

motif, and sequence variability in this motif may reflect different activities of the various versions of the protein in the same species.55 It would be highly noteworthy if a link could be established between the type of AgiB and the lipid-linked oligosaccharide substrates it uses.

Table 1-1: Features of archaeal N-linked glycosylation in select archaeal species, highlighting the WWDX1GX2 motif of AglB. Verified oligosaccharyltransferases are marked with (*).

Species AgIB WWDX1GX2 Observed LLO Crystal structure

H. salinarium OE2548F WWDYGH Dol-P-HexHexA"'

Dol-PP-tetrasaccharide56

H. volcanii HVO 1530 WWDYGH Dol-P-tetrasaccharide43 _

M. voltae Mvol_1038* WWDNGH

M. maripaludis MMP1424* WWDNGH Dol-P-trisaccharide4 9

S. acidocaldarius Sacil274 WWDYGY Dol-P-Hex"

P. furiosus PF0156* WWDYGY Dol-P-heptasaccharide PFO 156 C-term"7 PF0411 WWDWGH (Chapter 2)

P. horikoshii PH0242 WWDYGY PH0242 C-term8

PH1271 WWDWGH

A. fulgidus AF0380* WWDYGH AF0380 C-term, 5 ful5

AF0329 WWDYGN AF0329 C-term58

AF0040 WWDYGN AF0040 C-term58

Thesis objectives

The overall goal of this thesis is to gain a deeper understanding of the process of N-linked glycosylation in archaea. Chapter 2 details the identification and characterization of the activities of AglK and AgIC, the first two glycosyltransferases in the M voltae N-glycosylation pathway.

Chapter 3 discusses the biochemical characterization of AgIB activity using purified substrates, which represents the first biochemical demonstration of archaeal oligosaccharyltransferase activity. Chapter 4 describes our progress towards developing a new system to investigate the

conformational dynamics of AglB upon substrate binding using a lanthanide-based luminescence resonance energy transfer technique. Finally, Chapter 5 describes our efforts to express Alg5, a dolichyl-phosphate glucosyltransferase, and the stereochemical analysis of the product.

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48. Chaban, B., Logan, S. M., Kelly, J. F. & Jarrell, K. F. AgIC and AgiK Are Involved in Biosynthesis and Attachment of Diacetylated Glucuronic Acid to the N-Glycan in Methanococcus voltae. J. Bacteriol. 191, 187-195 (2009).

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52. Peyfoon, E. et al. The S-Layer Glycoprotein of the Crenarchaeote Sulfolobus acidocaldarius

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53. Guan, Z., Meyer, B. H., Albers, S.-V. & Eichler, J. The thermoacidophilic archaeon

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54. Meyer, B. H. & Albers, S.-V. AgIB, catalyzing the oligosaccharyl transferase step of the

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59. Matsumoto, S. et al. Crystal structures of an archaeal oligosaccharyltransferase provide insights into the catalytic cycle of N-linked protein glycosylation. Proc. NatL. Acad Sci. 110, 17868-17873 (2013).

Chapter 2: Identification and characterization of archaeal glycosyltransferases

A portion of the work described in this chapter has been published in the following:

Larkin, A., Chang, M.M., Whitworth, G.E., Imperiali, B. Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis. Nat. Chem. Biol. 2013, 9, 367-373.

Introduction

Nature exhibits an extensive and complex variety of glycan structures in part due to the enzymatic action of glycosyltransferases (GTs), which catalyze glycan assembly through the transfer of individual monosaccharide units. Due to the diversity of monosaccharide units, multiplicity of glycosidic linkages, branching of oligosaccharides, and processing by glycosyltransferases and glycosyihydrolases, the structure of glycans on mature glycoproteins can be highly heterogeneous.' GTs most commonly use sugar nucleotide derivatives (e.g. UDP-Glc, GDP-Man, CMP-NeuAc) as glycosyl donors, but they can also use lipid-phosphate sugars (e.g. Dol-P-Man) and unsubstituted phosphate sugars as activated donors. Acceptor substrates can be other sugars, proteins, lipids, nucleic acids, antibiotics, or other small molecules.2

Currently, the Carbohydrate-Active EnZYmes (CAZy) database includes over 148,000 unique GTs that have been identified and classified into 96 families based on sequence similarities.3 These family classifications are continually updated as new sequences are added to the CAZy database and new structures are deposited into the Protein Data Bank (PDB). The first X-ray crystal structure of a GT was reported for the bacteriophage T4-glucosyltransferase in 1994.4 There are now X-ray crystal structures available for over 100 GTs in at least 38 GT families.! A comparison of these GT structures shows that while these enzymes have low sequence homology, they exhibit a surprisingly high structural homology, with two major structural folds, called GT-A and GT-B (Figure 2-1).2 Both GT folds are largely composed of ca/p/a sandwiches. GT-A folds contain a single Rossmann fold, in which the active site is located between two

s-sheets,