• Carbohydrates• Glycosyltransferases and

glycosidases• Protein glycosylation• Glycobiology of bacteria• Glycan binding and the

role of glycans• Research at Caltech

‘Glycobiology’ coined by Raymond Dwek (Oxford) in 1988

BMB 170 Lecture 18Carbohydrates, Nov. 28th

GT-B Fold

(1bgt)

GT-A fold

(1foa)

GT-C fold• 12 Families• Large integral membrane

protein having between 8 and 13 transmembrane helices

• Most utilize lipid phosphate-activated donor sugar substrates

• No apparent common fold besides transmembrane component

• First structure of a piece came from archael STT3 soluble domain

Igura et al (2008) EMBO J 27:234 (2zai)

PglB structure from C. lari• First membrane

protein glycosyltransferase

Locher group: Lizak et al (2011) Nature 474:350 (3rce)

Peptidoglycan glycosyltransferase• Important potential as

drug target• Structure of the soluble

domain of PBP2 from S. aureus

• Not a Rossman fold, more orphan classes?

Transpeptidase

Glycosyltransferase

Lovering et al (2007) Science 315:1402 (2olv)GT domain from A. aeolicus; Yuan et al (2007) PNAS 104:5348 (2oqo)

Cellulose synthase

• Cellulose is the worlds most abundant polymer

• Is both a GT and a pore• Forms a complex rosette pattern

that facilitates the higher order cellulose structure

• Is connected to the cytoskeletal machinery

• Cellulases are one of the most important biotechnology enzymes

Bacterial cellulose synthase

Morgan, Strumillo & Zimmer (2013) Nature 493:181 (4HG6)

• Generating nucleotide substrates

• Building oligosaccharides

• Generate glycoconjugates that are biologically accessible– Modify biological

function– Labeling

Bertozzi & Kiessling (2001) Science 291:2357

Exploiting glycosyltransferases

Labeling glycans• Bertozzi lab (UC

Berkeley)• Using glycoconjugates

and chemistry to metabolically label sugars with reactive groups

• Can label specific sugars spatially and temporally

Laughlin et al (2008) Science 320:664

Glycosidases• Also called

glycosylhydrolases (GH)• 113 Families (~80

characterized structurally)

• Over 2400 entries in the PDB (400 unique)

• Different from GT in that they have many folds

• Established the catalytic mechanism (inverse of GT)

Davies & Henrissat (1995) Structure 3:853Henrissat et al (2008) Curr Opin Struct Biol 18:527

Tamiflu• Oseltamivir -

neuraminidase inhibitor of Roche

• Viral neuraminidase– Glycosidases– Found on the surface of

flu viruses– Cleaves sialic acid to

release virus from cell• Mimics transition state

Bossart-Whitaker et al (1993) JMB 232:1069 (1nnb)

ABO Blood groups• Distinction discovered in 1900• Represents presentation of

antigens A & B• Difference characterized by

terminal sugar on oligosaccharide on erythrocytes

• ABO blood groups are distinguisheded by differences at the amino acid level of a single enzyme GT expression

• Goal: find a glycosidase that can efficiently cleave both terminal sugars in blood

• Solution(?): E. meningosepticum α-N-acetylgalactosaminidase

α1,3-N-acetylgalactosaminyltransferase

α1,3-galactosyltransferase

Liu et al (2007) Nature Biotech 25:454

Protein glycosylation• N-linked glycosylation

– Attached at asparagine– Occurs in the ER

• O-linked glycosylation– Attached at serine or threonine– In secretion

• Occurs later in golgi• Role in extracellular matrix and mucosal secretions

– Also plays role in signaling

N-linked Protein Glycosylation• The majority of secreted eukaryotic proteins are

glycosylated• Improper glycosylation is linked to many diseases• Glycosylation plays many roles

– Protein stability– Molecular trafficking– Receptor interaction sites– Signal transduction– Cell adhesion– Endocytosis

• Important problem for pharmaceutical industry

Glycosylation of Proteins

• Monitorsproperfolding• Stabilizesproteinfold• Protectshydrophobicsurfaces

• Aidsinproteinfolding• Localizesprotein

Hemagglu<nin(2viu)–DonWiley

Sugarshideprotein

• HarrisonetaldeterminedthestructureofGP120ofSIVinana<vestate(fullyglycosylated)

• Sugarsdominatethesurface

• 40kDprotein,~20kDofsugars

Chen..Harrison(2005)Nature433:834(2bf1)

N-link glycan affects conformation• Linked sugars lead to

new conformational constraints

• Different sugars can result in different structures

O’connor & Imperiali (1998) Chem & Biol 5:427

Simple model for N-linked glycosylation

N-linked glycosylation pathway

Asparagine-Linked Glycosylation proteins

First Alg protein structure – Alg13• Forms a complex with

Alg14• Similar to the C-

terminal Rossman-like fold in GT-B family

• Alg14 (also partial GT-B fold) could complete catalytic binding pocket

Wang et al (2008) Structure 16:965 (2jzc)

Congenital Disorders of Glycosylation

Many of the ALG proteins have mutations linked to disease.

Diseases linked to both N- and O- linked glycosylation.

Jaeken & Matthjis (2007) Annu Rev Genomics Hum Genet 8:261

CDG phenotypes

Haltwanger & Lowe (2004) Annu Rev Biochem 73:491

Gain of glycosylation diseases

Reviewed in Voigt et al (2007) Curr Opin Gen Dev 17:245

OST Complex

Problem 8 Subunits!

STT3 contains the enzymatic function and is the functional subunit in eukaryotes

STT3 Structure

Locher group: Lizak et al (2011) Nature 474:350 (3rce)

WWDYG Motif

Locher group: Lizak et al (2011) Nature 474:350 (3rce)

The OST in other speciesLeishmania major STT3 can substitute for the whole yeast OST.

Nasab et al (2008) Mol Biol Cell 19:3758

Kelleher et al (2006) Glycobiology 16:47R

Different roles for different complexes

Yan & Lennarz (2005) Glycobiology 15:1407

Ost4p• First structure of a

component of the OST

• Solved by NMR• 1rkl

Zubkov et al (2004) PNAS 101:3821

Ost3p/6p• Show site specific

dependence for glycosylation

• Lumenal domain has thioredoxin fold

• Cysteines play a role (i.e. oxidoreductase activity)– Conformational change of

loop based on disulfide bond formation

– Reduce substrates?– Pausing intermediate?

Schulz, Stirnimann, Grimshaw..Aebi PNAS (2009) 106:11061(3ga4)

EM model of the yeast OST

Li et al (2008) Structure 16:432

• 12Å map of a tagged OST

• Used MBP to tag and identify some of the proteins

Currently solved structures

Glycosylation and ER quality

control

Ruddock & Molinari (2006) J Cell Science 119:4373

N-glycan processing

Lehle et al (2006) Angew Chem Int Ed 45:6802

Humanizing a fungiCurrently only Pichia pastoris has been engineered to make human like glycans.

Reviewed in Hamilton & Gerngross (2007) Curr Opin Biotech 18:387

O-GlcNAcasasignaling

modifica<on

ReviewedinLance..Hart(2003)BBRC302:435

TetheredMucins

ReviewedinHa[rup&Gendler(2008)AnnuRevPhysiol70:431

•Themucuslayeriscomposedofproteoglycans•Essen<alasaprotec<vebarrier•Problemsleadtocys<cfibrosisamongstotherthings

Mucinsoftherespiratorysystem

ReviewedinHa[rup&Gendler(2008)AnnuRevPhysiol70:431

Molecularweightinthemillions!

ProteoglycanoftheExtracellularMatrix

Bacterialglycans• Glycolipids• Pep<doglycan• Polysaccharide• Glycopro<ens

Essen)alsofGlycobiology

SecondEdi<onChapter20,Figure1&2

©2017TheConsortiumofGlycobiologyEditors,LaJolla,California

SymbolNomenclatureforGlycans(SNFG)Buy the Book

Fig.21.1

Conceptual Organization of the Cell Envelopes of Gram-Negative Bacteria,

Gram-Positive Bacteria, and Mycobacteria

Endotoxin

• Lipopolysaccharide• BoundstructuretoFhuA(irontransport)

Fergusonetal(1998)Science282:2215(2fcp)FromWikipedia

LPSBiosynthesis

KEGGPathwayMapsh[p://www.genome.jp

Pep<doglycanHansMar<n–AquaspirillumserpensSacculi

TheSecretsoftheSacculush[p://schaechter.asmblog.org/schaechter/

Pep<doglycanstructure

VollmeretalFEMSMicroRev(2008)32:149

Varia<onsinthetheme

VollmeretalFEMSMicroRev(2008)32:149

Vancomycin & Oritavancin

Patti et al (2009) JMB 392:1178

Discovered in 1956

Secreted polysaccharide• EPS – extracellular

(slime)• CPS – capsular (discrete

layer)• Thick, mucus-like layer• Cloaks antigenic proteins• MW in the range of

100-1000kDa• Linear consisting of up to

six monosaccharides• Extremely diverse (E.

coli produces ~200)• Make great vaccines

Campylobacter jejuniE. coli K30

Review: Cuthbertson et al (2001) Infection & Immunity 69:5921

Fig. 39.4

Review: Cuthbertson et al (2001) Infection & Immunity 69:5921

Polysaccharide synthesis

Wza structure

2j58; Dong, Beis..Whitfield & Naismith (2006) Nature

New class of membrane protein!

• First discovered in 1999 (Szymanski et al Mol Micro)

• C jejuni Major gut pathogen - only one OST component

• N-linked glycosylation required for infection

Bacterial N-linked protein glycosylation

Archael S-layer

M. voltae• First found in H. salinarium

– Mescher & Strominger (1976) JBC 251:2005

• Also demonstrated in H. volcanii

• Not essential• Role?

Chaban et al (2006) Mol Micro 61:259

Engineering glycans

Schwartz et al (2010) Nat Chem Biol 6(4):264

O-linked glycosylation

Review: Nothoft & Szymanski (2010) Nature Rev Micro