glycoconjugates glycobiology

Glycoconjugates

Structural diversity

Diversity of functions

• Structural / modulation

• Recognition

• Glycoproteins

• Glycolipids

• Proteoglycans

Altered glycosylation and glycosylation

defects are observed in many pathological

situations (inflammation, cancer, congenital

disorders of glycosylation, �)

GLYCOBIOLOGYStudy of structure, synthesis and biology of

sugars, particularly the sugar chains that are

attached to proteins and lipids

1

:

Although proteins comprise the largest fraction of a cell's dry mass, it is

estimated that more than half are modified with glycans, lipids or other

metabolites.

Prescher and Bertozzi, 2005, Nat. Chem. Biol. 1, 13-21

Post-translational modifications of proteins are important regulators

of protein function

2

ER

Golgi

M.E. Taylor & K. Drickamer (Introduction to Glycobiology -2003- Oxford University Press)

Location of glycoconjugates in multicellular organisms

3

.

BIOSYNTHESIS OF GLYCANS : FOCUS ON THE

LARGE GLYCOSYLTRANSFERASE FAMILY

Structural and Functional aspects

Prof. Christelle BRETON

4

Photo credit : Barbieri, Chenu, Francillon, Vinçon, D.R. AEPI

This slide shoud always be used with the AEPI logo. Any modification of the slide has to be expressly notified to the AEPI

Grenoble

5

Centre de Recherches sur les

Macromolécules Végétales

Glycosciences

6

Structure-Function

Studies of Lectins and

Glycosyltransferases

Molecular Glycobiology at CERMAV

7

.

1- General features

• General reaction

• Cellular localization & topology

• Glycosylation events in the Golgi

• Take home message (1)

2- Classification

• Processivity / stereochemistry

• Classification

• GT repertoire


3- Structure

• Structural information (current picture)

• GT-A fold / DxD motif

• GT-B fold

• Other folds


8

GLYCOSYLTRANSFERASE course : Contents

4- Reaction mechanisms

• Inverting/retaining reaction mechanisms

• Ordered binding in GT-A enzymes

• additional difficulties


5- Future directions

.UDP-sugars

Glucose

Galactose

N-acetylglucosamine

N-acetylgalactosamine

Glucuronic acid

Xylose

...

CMP-sugars

Sialic acids

GDP-sugars

Fucose

Mannose

XDP-sugar + OH-Acceptor sugar-O-Acceptor + XDP

GT

GLYCOSYLTRANSFERASES (Leloir enzymes*)

specificity for both the donor sugar and acceptor substrates

* In honor of Luis L. Leloir who discovered the first nucleotide sugar (Nobel Prize in Chemistry in 1970)9

Examples of “activated” glycosyl donors.

Nucleotide-sugar donors, sugar phosphate

donors, and lipid-phosphate donors.

90% GTs use Nucleotide Donors

Reaction catalyzed by a

ββββ1,3-galactosyltransferase

(ββββ3-GalT1)

10

.

� ER, Golgi (membrane proteins )

� cytosol and nucleus (soluble forms)

� biological fluids (soluble forms) ??

� plasma membrane (membrane proteins)

Eukaryotes

Cellular localization and topology

GLYCOSYLTRANSFERASES

Secretory pathway

Most of the complex glycans are

built into the ER and GOLGI

11

.

Topologies of glycosyltransferases in the ER and Golgi

RE lumen

Cytoplasm

GT using a Dolichol-P-sugar

integral membrane protein

TMD

NH2

Cytoplasm

Golgi lumenstem

catalytic domain

Typical Golgi GT using a nucleotide-sugar

Type II membrane protein

12

.

TMD stem

Add-on domain

(i.e. Lectin domain)

lumen

N-

N-

N-

Catalytic domain

Topology of Golgi glycosyltransferases

Stem variable length

may modulate in vivo GT activity

« cleavable »

Catalytic domain globular, minimal active structure,

typically ~300-350 aa

Breton et al. (2000) Biochimie

��

13

퀀

Glycosylation events in Golgi

Nucleotide-sugars are

synthesized in the cytosol

and then transported into

the Golgi by specific

transporters

(CMP-NeuAc synthesized in

the nucleus)

GT

Cytoplasme

14

.

Glycosylation events in Golgi

GT localization ?

Supramolecular organization ?

De Graffenreid & Bertozzi, 2004Brockhausen, 2006

O-glycans N-glycans

15

.

Although the mechanisms of Golgi enzyme

localization are still under debate, both the

distribution of these enzymes among the Golgi

cisternae and specific association among

glycosylation enzymes can dictate the overall glycan

structures (glycome) produced by a cell.

TAKE HOME MESSAGE (1)

16

.

1- General features





2- Classification


• Classification

• GT repertoire


3- Structure



• GT-B fold

• Other folds


17








.

Processive enzymes : transfer multiple sugar residues to the acceptor

(polysaccharide synthases,.....)

Non-processive enzymes : transfer a single sugar residue to the

acceptor

PROCESSIVITY

GLYCOSYLTRANSFERASES N

18

. O

OOHHO

HO

HOP

OP

O

O O

O O

ON

NH

HO OH

O

O

OOHHO

HO

HO

RRRReeeettttaaaaiiiinnnniiiinnnngggg GGGGllllyyyyccccoooossssyyyyllllttttrrrraaaannnnssssffffeeeerrrraaaasssseeee

IIIInnnnvvvveeeerrrrttttiiiinnnnggggGGGGllllyyyyccccoooossssyyyyllllttttrrrraaaannnnssssffffeeeerrrraaaasssseeee

OOHHO

HOOH

O

O

HO

OH

OH

OH

HO

O

HO

OH

OH

OH

HO

O

HO

OH

OH

OH

O

O

HO

OH

OH

OH

Transfer of a monosaccharide

from a donor to an acceptor:

Reaction can occur with retention

or inversion of configuration of

the transferred sugar

STEREOCHEMISTRY OF THE

GLYCOSIDIC LINKAGE

19

.

Galactosyltransferases (αααα3-GalTs, ββββ4-GalTs, ...)

Fucosyltransferases (αααα2-FucTs, αααα3-FucTs, αααα6-FucTs, ...)

Sialyltransferases (ST6Gal, ST3Gal, ST8Sia, ....)

.........

There is no or limited sequence homology among the different GT families

or even, within one family, among members catalyzing different reactions

They are primarily classified according to the type of sugar that they transfer

GLYCOSYLTRANSFERASES N

20

.

Classification based on amino acid sequence similarity, correlates with

(i) protein folding

(ii) enzyme mechanism

Glycosyltransferases .... CAZy classification

▪ Two very large families (~half of total number of GT entries)

inverting GT2 and retaining GT4 – considered as the ancestral

families

▪ Many families are polyspecific

~ 100,000 known and

putative GT sequences

distributed over 90

families

http://www.cazy.org/

21

October 2012

훀

� New GT families are exclusively created based on the

availability of at least one biochemically-characterized protein

member

Discovery of new GT families

� Experimental investigation (i.e. Rumi GT90)

� Bioinformatics and biochemical characterization

(i.e. plant XylTs, GT77)

� The number of GT families in CAZy is continually increasing

with the discovery of new GT genes (27 families in 1997, more

than 90 in 2012)

22

훀

235 GT genes

43 families

~ 80 % annotated genes

The most populated families

GT1 : UGTs

GT7 : ββββ4-GTs

GT27 : ppGalNAcTs

GT29 : SiaTs

GT31 : ββββ3-GTs

A large repertoire of genes is required for glycan assembly and function in

multicellular organism : 1-2% of the genes of an organism code for GTs

GT repertoire ....

23

.

▪ Only 1 plant specific GT family (GT37)

~ 460 GT genes

42 families

20 % are annotated

▪ Several highly populated GT families

GT1 : UGT-family (glycosylation of secondary metabolites)

GT2, 8, 31, 47 : biosynthesis of cell wall PS

Plants tend to have far more GT genes than

any other organism sequenced to date

24

�Ϟ

� A huge number of putative GT genes have been identified

through large genome sequencing projects (currently ~100,000)

� Large number of sequence-based GT families (> 90)

� The vast majority of these sequences (more than 90%) are

uncharacterized open-reading frames (particularly bacterial and

plant genomes)

� This is a challenging task for glycobiologists to assign

functions to the many uncharacterized GT sequences


25

�Ϟ

1- General features





2- Classification


• Classification

• GT repertoire


3- Structure



• GT-B fold

• Other folds


8








.

�� Structural information for 39 CAZY families

�� Only two general folds, termed GT-A and GT-B (and variants), have

been observed for all structures of nucleotide-sugar-dependent GTs

solved to date

GT-A fold GT-B fold

Glycosyltransferases .... 3D structures

26

�Ϟ

GT-A and variants

2, 6, 7, 8, 13, 14, 15, 27,

29, 31, 42, 43, 44, 55,

64, 78, 81, 88

12, 16, 17, 21, 24, 25, 32,

34, 40, 45, 49, 54, 60, 62,

67, 69, 71, 73, 74, 75, 77,

82, 84, 92

GT-B and variants

1, 3, 4, 5, 9, 10, 20, 23

28, 30, 35, 41, 52, 63,

65, 68, 70, 72, 80

11, 18, 19, 33, 37, 38,

47, 56, 61, 90

~ 80 % of the current GT families are known, or predicted, to adopt a GT-A or GT-B fold (or variants)

3D

Predicted*

Breton et al. Biochem. Soc. Symp. (2002)

Breton et al. Glycobiology (2006)

Breton et al. COSB (2012)* Based on 3D-PSSM and PHYRE programs

(Bennett-Lovsey et al., 2008; Kelley et al., 2000)

(37)

(34)

27

͵

There is no correlation between the overall

fold of the GT and its mechanism of action

since both inverting and retaining

enzymes of both fold types (GT-A and GT-

B) are known.

28

�Ϟ

GT-A fold

a single domain

αααα/ββββ/αααα structure

mixed ββββ-sheet (3214657)

All of the strictly metal ion-dependent GTs for which structures have

been determined to date have been of this type and include a DxD

Metal Binding motif (Mg2+, Mn2+)* One exception (GT14)

29

眐Ϛ

Ubiquity and structural basis of the DxD motif in the GT-A fold

Variations in the DxD sequence : DVD, DID, DDD, EDD, EED, TDD, DxH, EPD, N.

cation-dependent GTs (Mn2+, Mg2+,…)

30

禠Ϝ

Cst II - sialyltransferase

Campylobacter jejuni

// ββββ-sheet (8712456)

no DxD motif

Canonical GT-A

mixed ββββ-sheet (3214657)

DxD motif

GT-A like

GT-A variant

GT42

New type of fold !31

禠Ϝ

GT-B fold

2 Rossmann-type

domain assembly

αααα/ββββ/αααα structure

// β-sheet (3214567)

The catalytic site is located in a cleft between the two domains

Metal ion-independent GTs

32

禠Ϝ

GT-B variant

Coiled coil region

(N-term)

SH3 domain

(Cterm)

Catalytic domain

Rossmann

domain in the C-

term of catalytic

domain

GT23

Human αααα6-Fucosyltransferase (FucT-VIII)

33

禠Ϝ

GT-B variant

Human O-ββββ-GlcNAcT (OGT)

Int-D

TPR

GT41• Unique and reversible

modification

• Found on a myriad of nuclear and

cytoplasmic proteins

• Extensive cross-talk with

phosphorylation

Hart et al., (2007) Nature, 446: 1017−1022

Lazarus et al., (2011) Nature, 469:564-56734

禠Ϝ

New folds are observed for GTs using lipid-phosphate sugar donors

No Rossmann fold !!

Peptidoglycan glycosyltransferase(Lovering et al., 2007, Science)

GT51

GT domain: bacteriophage λλλλ-lysozyme-like fold !!

Staphylococcus aureus

35

㐐

The process of N-linked protein glycosylation

Schwarz and Aebi (2011) COSB, 21:576-582 36

㐐

| N AT U R E | V O L 4 7 4 | 1 6 J U N E 2 0 1 1

GT66

The first structure of a membrane-embedded GT !

37

㐐

3D structure of PglB (C. jejuni) GT66

Lizak et al., (2011) Nature, 474, 350-355.38

㐐

Virtually nothing is known about the structures and mechanisms of

these large membrane-embedded GTs. The successful crystallization of

PglB now opens new avenues to fill the gap.

� The large GT family is characterized by a conserved 3D architecture

(GT-A, GT-B folds and variants)

The small variety of folds observed for GTs (constraint in nucleotide-sugar

binding ?) is compensated by a large structural variability in the acceptor

binding domain: «Functional plasticity» which allows fine-tuning with

respect to the acceptor.

� Novel folds are expected for GTs that use a lipid-phosphate donor


39

㐐

1- General features





2- Classification


• Classification

• GT repertoire


3- Structure



• GT-B fold

• Other folds


8








㐐

Reaction mechanisms of GTs

In contrast to the well-characterized

catalytic mechanisms used for glycosidic

bond hydrolysis (glycosidases), the

mechanisms for glycoside bond formation

remain less clear.

For a review, see Lairson et al., (2008) Ann. Rev. Biochem., 77:521-555

Breton et al., (2012) COSB40

㐐

Inverting Glycosyltransferase Mechanism

A direct-displacement SN2-like reaction via a single oxycarbenium ion

Catalytic base (i.e. Asp, Glu, His) deprotonates acceptor OH

From Lairson et al. (2008) Ann Rev Biochem41

負Ϝ

Retaining Glycosyltransferase Mechanism: currently two hypotheses

SN2-like double displacement

mechanism with formation of a

covalently bound glycosyl-enzyme

intermediate (by analogy to

glycosylhydrolases)

Soya et al., (2011) Glycobiology, 21 (5)

From Lairson et al. (2008) Ann Rev Biochem

SNi-like mechanism

Front-face nucleophilic attack

involving hydrogen bonding between

leaving group and the acceptor

nucleophile

Lee et al., (2011) Nature Chem. Biol., 7

42

負Ϝ

The « closed » active conformation

of glycosyltransferases

LgtC

ααααGalT

Persson et al, Nature Struct. Biol., 8 (2001) 166

Base of UDP-Gal

Acceptordisaccharide

Reaction mechanism for GTs

of the GT-A family

Ordered binding of donor and

acceptor substrates, linked to

a donor substrate-induced

conformational change

Binding of a distorted conformation

of nucleotide sugar may be important

for catalysis

Ret Inv Inv

43

負Ϝ

Superimposition of open (free

enzyme) and closed (upon UDP-Gal

binding) forms

openclose

Additional difficulties for glycosyltransferases

Loop movement domain movementGT-A GT-B

Superimposition of open state

(solid) and closed conformation

(transparent)

Buschiazzo et al. (2004)Gastinel et al. (2001); Boix et al., (2001)

αααα3-GalT Glycogen synthase GT5 GT6

44

負Ϝ

Crystallographic and modelling studies should

allow for a better understanding of molecular

bases responsible for substrate specificityN

The blood group synthases as a case of study !

GlycosyltransferasesN still puzzling

45

負Ϝ

OOHHO

OO

OR

OOHHO

HOOH

O

HOOH

OH

OOHHO

OO

OR

OOHHO

HONH

O

HOOH

OH

OOHHO

HOO

OR

O

HOOH

OH

OH3C

O

OOHHO

HONH

OH3C

P

O

OO

P

O

OO

ON

NH

O

O

OHHO

O

OOHHO

HOOH P

O

OO

P

O

OO

ON

NH

O

O

OHHO

UDP-GalNAc UDP-Gal

GTA GTB

O(H) Precursor

Blood Group A Blood Group B

Blood Group A & B Structures Made by Closely Related GTs

Yamamoto, Hakomori (Nature, 1990)Both enzymes adopt a GT-A fold

46

負Ϝ

– Highly homologous enzymes differing at only four critical amino acids

out of a total of 354 residues

– Alteration of these four crucial amino acid residues converts the

specificity from GTA to GTB

AAAA

BBBB

Blood group synthases GTA/GTB

Molecular basis for nucleotide sugar specificity ?

UDP-Gal vs UDP-GalNAc

47

負Ϝ

L266

E303

D302

G268

M266

E303

D302

A268

GTA GTB

S185 S185

αααα3-GalNAcT αααα3-GalT

Homology Modelling


Only 2 of the 4 critical residues in binding site (L266M, G268A)

M266 + A268 excludes UDP-GalNAc (steric conflict)

G268 accomodation of N-acetamido group

GTA should utilize UDP-Gal ???

Heissigerova et al. Glycobiology, 13 (2002) 377

GT6 (GT-A fold)

48

負Ϝ


Still unclear !!!

What can we learn about substrate binding and catalysis

of blood group GTs ?

Crystallographic studies

Modelling studies

Natural mutations in blood banks

Mutagenesis (chimeric enzymes)

Flexibles enzymes

Mutagenesis unpredictable

Much work to be done to understand catalysis

Monica Palcic and coll (in Alberta and Copenhagen)49

.A better understanding of catalytic mechanisms of GTs together with

much more structural information is needed, for the rational design of

specific inhibitors as well as for engineering purposes to expand the

biotechnological uses of GTs.

� Catalytic mechanisms are still poorly understood

• Movements of loops and/or domains involved in the binding

• Flexibility


50

負Ϝ

Glycosyltransferases

“in vivo”

- Functional organization in Golgi

* supramolecular organisation

- Spatial and temporal expression of

GTs in physiological and pathological

situations (Glycomics)

“in vitro”

- “Structure/function” relationships

* new 3D structures

* mechanism of action, inhibitors

- “ORFeome” of GTs

* heterologous expresssion

* library of acceptors

Biotechnological applications

- Enzymatic synthesis of bioactive oligosaccharides

- Production of recombinant GP for therapeutic use

- GTs and medicine (diagnostic tools, new therapies )

- GT engineering (new catalysts with improved capabilities)

- Oligo/polysaccharide engineering (modification of functional

properties)

.....

Future directions N

51

負ϜTHE END

負Ϝ

Database development at Cermav : Glyco3D

�ҕ

Collaborations

Eric Maréchal

(CEA-Grenoble)

Emmanuel Bettler

(IBCP Lyon)

Jan Mucha

(Bratislava)

Soren B. Engelsen

(Univ. Copenhagen)

Monica Palcic & Ole Hindsagaul

(CRL- Copenhagen)

CERMAV - GT team

Christelle Breton

Anne Imberty

Aline Thomas

Olivier Lerouxel

Valerie Chazalet

Post-doc and PhDs

Magali Audry

Gaelle Batot

Peter Both

Sara Fasmer Hansen

Charlotte Jeanneau

Joana Rocha

Funding

French ANR

CEE

�ҕ

Test Arabidopsis proteome

HMMSearch

(1D level)

PSI-BLAST

+

Structural overlap

(2D level)

ProFit

(3D level)

> 150 new candidate sequences

Hansen et al. (2009) J. Proteome Res.

Arabidopsis proteome

30690 sequences

1- Removal of large

sequences(>1000 aa)

29457 sequences

2- Removal of

sequences with a cTP or

mTP (TargetP)

21755 sequences

3- Removal of proteins

with no TMD (TMHMM)

5666 sequences

4- Removal of CAZy

accessions (460 seqs)

Test Arabidopsis proteome

(5315 protein sequences)

Bioinformatics strategy

Use of several remote homology detection methods to search the

« test Arabidopsis proteome » for new candidate GT genes

+ Chemometrics

Hansen et al., (2010) Mol. Biosystems

�ҕ

Evaluation of candidate GT sequences (~150)…

Close examination of each sequence

� Annotation in databases

� Blast and Psi-BLAST

� TMHMM

� Phyre (fold recognition)

� Hydrophobic Cluster Analysis method (HCA)

~ 20 strong candidate genes*

harbouring a clear GT signature

* Distantly related to GT4, GT14, GT23, GT65

In any case, the validation step requires the critical expertise of the biologist

犐ҕ

The populations of the various GT families show

wide variations, with two large families, the

inverting GT2 and retaining GT4, accounting for

about half of the total number of GT entries, and

which can be considered as the most ancestral

families for which enzymes of both

stereochemistries have evolved

(Martinez-Fleites et al., 2006).

犐ҕ

number of sequences

GT2 (> 13,000 seqs)

GT4 (> 10,000 seqs)

GT63 (1 seq)

origin and function

GT2 Cellulose synthase, chitin synthase, Dol-P-Man/Glc synthase

GlcTs, GalTs, GalfTs, GnTs, RhaT, ...

animal, plant, yeast, bacteria,Q

GT27 pp-αααα-GalNAcTs (mucin-type glycosylation)

animal

Glycosyltransferases …. CAZy

Large differences among GT families

8 families have > 1000 sequences

13 families have < 20 sequences

50% of total seqs

犐ҕ

GLYCOSYLTRANSFERASES and biosynthesis of glycans

- Structure-function studies and engineering of GTs

- Biogenesis of PCW polysaccharides

Specific recognition of carbohydrates by LECTINS

- Lectins and bacterial infections

- Development of anti-adhesive drugs

- Lectins and biodiversity

New tools in Glycosciences

- Functionalized surfaces, gold nanoparticles

- Glyco-bioinformatics

- Modelling sulfated polysaccharides

Group Leader

A. ImbertyMOLECULAR GLYCOBIOLOGY at CERMAV

�Ϣ

A better understanding of catalytic mechanisms of GTs together with much more

structural information is needed, however, for the rational design of specific inhibitors as

well as for engineering purposes to expand the biotechnological uses of GTs.

the limiting factor in developing carbohydrate-based compounds for clinical application

was the high cost and complexity of producing glycans in large quantities.

the use of metabolically engineered bacteria that overexpress heterologous GTs has

become a powerful method for the large-scale production of complex glycans at low

cost (Endo and Koizumi, 2000; Fierfort and Samain, 2008). Glycobiology research will

greatly benefit from these major advances in cost-effective technologies for

carbohydrate synthesis.

� Catalytic mechanisms are still poorly understood

Movements of loops and/or domains involved in the binding

glycoconjugates glycobiology

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