Download GT-B fold

GLYCOBIOLOGY
Study of structure, synthesis and biology of
sugars, particularly the sugar chains that are
attached to proteins and lipids
Glycoconjugates
Structural diversity
• Glycoproteins
• Glycolipids
• Proteoglycans
Diversity of functions
• Structural / modulation
• Recognition
Altered glycosylation and glycosylation
defects are observed in many pathological
situations (inflammation, cancer, congenital
disorders of glycosylation, )
1
Post-translational modifications of proteins are important regulators
of protein function
:
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
2
Location of glycoconjugates in multicellular organisms
ER
Golgi
M.E. Taylor & K. Drickamer (Introduction to Glycobiology -2003- Oxford University Press)
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
Grenoble
This slide shoud always be used with the AEPI logo. Any modification of the slide has to be expressly notified to the AEPI
5
Centre de Recherches sur les
Macromolécules Végétales
Glycosciences
6
Molecular Glycobiology at CERMAV
Structure-Function
Studies of Lectins and
Glycosyltransferases
7
GLYCOSYLTRANSFERASE course : Contents
1- General features
• General reaction
• Cellular localization & topology
• Glycosylation events in the Golgi
• Take home message (1)
2- Classification
• Processivity / stereochemistry
• Classification
• GT repertoire
• Take home message (2)
.
3- Structure
• Structural information (current picture)
• GT-A fold / DxD motif
• GT-B fold
• Other folds
• Take home message (3)
4- Reaction mechanisms
• Inverting/retaining reaction mechanisms
• Ordered binding in GT-A enzymes
• additional difficulties
• Take home message (4)
5- Future directions
8
GLYCOSYLTRANSFERASES (Leloir enzymes*)
GT
XDP-sugar + OH-Acceptor
sugar-O-Acceptor + XDP
specificity for both the donor sugar and acceptor substrates
GDP-sugars
UDP-sugars
CMP-sugars
Fucose
Mannose
Glucose
Galactose
N-acetylglucosamine
N-acetylgalactosamine
Glucuronic acid
Xylose
...
Sialic acids
.
* 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.
Reaction catalyzed by a
β1,3-galactosyltransferase
(β
β3-GalT1)
90% GTs use Nucleotide Donors
10
Eukaryotes
GLYCOSYLTRANSFERASES
Cellular localization and topology
ER, Golgi (membrane proteins )
cytosol and nucleus (soluble forms)
biological fluids (soluble forms) ?? .
plasma membrane (membrane proteins)
Most of the complex glycans are
built into the ER and GOLGI
Secretory pathway
11
Topologies of glycosyltransferases in the ER and Golgi
Type II membrane protein
integral membrane protein
.
catalytic domain
RE lumen
stem
Golgi lumen
TMD
Cytoplasm
NH2
Typical Golgi GT using a nucleotide-sugar
Cytoplasm
GT using a Dolichol-P-sugar
12
Topology of Golgi glycosyltransferases
stem
TMD
N-
Catalytic domain
Add-on domain
(i.e. Lectin domain)
N-
N-
.
lumen
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
Glycosylation events in Golgi
Cytoplasme
Nucleotide-sugars are
synthesized in the cytosol
and then transported into
the Golgi by specific
transporters
퀀
GT
(CMP-NeuAc synthesized in
the nucleus)
14
Glycosylation events in Golgi
GT localization ?
Supramolecular organization ?
N-glycans
O-glycans
.
Brockhausen, 2006
De Graffenreid & Bertozzi, 2004
15
TAKE HOME MESSAGE (1)
Although
localization
the
are
mechanisms
still
under
of
Golgi
debate,
enzyme
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.
16
GLYCOSYLTRANSFERASE course : Contents
1- General features
• General reaction
• Cellular localization & topology
• Glycosylation events in the Golgi
• Take home message (1)
2- Classification
• Processivity / stereochemistry
• Classification
• GT repertoire
• Take home message (2)
.
3- Structure
• Structural information (current picture)
• GT-A fold / DxD motif
• GT-B fold
• Other folds
• Take home message (3)
4- Reaction mechanisms
• Inverting/retaining reaction mechanisms
• Ordered binding in GT-A enzymes
• additional difficulties
• Take home message (4)
5- Future directions
17
GLYCOSYLTRANSFERASES N
PROCESSIVITY
Processive enzymes :
transfer multiple sugar residues to the
acceptor
.
(polysaccharide synthases,.....)
Non-processive enzymes :
transfer a single sugar residue to the
acceptor
18
HO
OH
O
OH
HO
STEREOCHEMISTRY OF THE
GLYCOSIDIC LINKAGE
HO
HO
O
OH
O
OH
OH
Retaining
Glycosyltransferase
Transfer of a monosaccharide
from a donor to an acceptor:
OH
O
O
OH
HO
OH
O
HO
.
Reaction can occur with retention
or inversion of configuration of
the transferred sugar
HO
HO
HO O
NH
O
O
P
P
O
O
N
O
O
OH
HO
Inverting
Glycosyltransferase
O
O
OH
HO
HO
O
OH
OH
HO
HO
OH
OH
O
OH
O
HO
O
OH
OH
19
GLYCOSYLTRANSFERASES N
They are primarily classified according to the type of sugar that they transfer
Galactosyltransferases
(α
α3-GalTs, β4-GalTs, ...)
Fucosyltransferases
(α
α2-FucTs, α3-FucTs, α6-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
20
Glycosyltransferases ....
CAZy classification
Classification based on amino acid sequence similarity, correlates with
(i) protein folding
(ii) enzyme mechanism
http://www.cazy.org/
October 2012
~ 100,000 known and
putative GT sequences
.
distributed over 90
families
▪ 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
21
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)
New GT families are exclusively created based on the
availability of at least one biochemically-characterized protein
훀ͮ
member
Discovery of new GT families
Experimental investigation (i.e. Rumi GT90)
Bioinformatics and biochemical characterization
(i.e. plant XylTs, GT77)
22
GT repertoire ....
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
훀ͮ
235 GT genes
43 families
~ 80 % annotated genes
The most populated families
GT1 : UGTs
GT7 : β4-GTs
GT27 : ppGalNAcTs
GT29 : SiaTs
GT31 : β3-GTs
23
Plants tend to have far more GT genes than
any other organism sequenced to date
.
~ 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
▪ Only 1 plant specific GT family (GT37)
24
TAKE HOME MESSAGE (2)
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
�Ϟ
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
GLYCOSYLTRANSFERASE course : Contents
1- General features
• General reaction
• Cellular localization & topology
• Glycosylation events in the Golgi
• Take home message (1)
2- Classification
• Processivity / stereochemistry
• Classification
• GT repertoire
• Take home message (2)
�Ϟ
3- Structure
• Structural information (current picture)
• GT-A fold / DxD motif
• GT-B fold
• Other folds
• Take home message (3)
4- Reaction mechanisms
• Inverting/retaining reaction mechanisms
• Ordered binding in GT-A enzymes
• additional difficulties
• Take home message (4)
5- Future directions
8
Glycosyltransferases ....
3D structures
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
26
~ 80 % of the current GT families are known, or predicted, to
adopt a GT-A or GT-B fold (or variants)
GT-A and variants
3D
(37)
2, 6, 7, 8, 13, 14, 15, 27,
29, 31, 42, 43, 44, 55,
64, 78, 81, 88
GT-B and variants
1, 3, 4, 5, 9, 10, 20, 23
28, 30, 35, 41, 52, 63,
65, 68, 70, 72, 80
�Ϟ
Predicted*
(34)
12, 16, 17, 21, 24, 25, 32,
34, 40, 45, 49, 54, 60, 62,
67, 69, 71, 73, 74, 75, 77,
82, 84, 92
* Based on 3D-PSSM and PHYRE programs
(Bennett-Lovsey et al., 2008; Kelley et al., 2000)
11, 18, 19, 33, 37, 38,
47, 56, 61, 90
Breton et al. Biochem. Soc. Symp. (2002)
Breton et al. Glycobiology (2006)
Breton et al. COSB (2012)
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 GTB) are known.
28
GT-A fold
a single domain
α/β
β/α
α structure
�Ϟ
mixed β-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
Ubiquity and structural basis of the DxD motif in the GT-A fold
眐Ϛ
cation-dependent GTs
(Mn2+, Mg2+,…)
Variations in the DxD sequence : DVD, DID, DDD, EDD, EED, TDD, DxH, EPD, N.
30
GT-A variant
GT-A like
Canonical GT-A
GT42
禠Ϝ
mixed β-sheet (3214657)
DxD motif
// β-sheet (8712456)
no DxD motif
Cst II - sialyltransferase
Campylobacter jejuni
New type of fold !
31
GT-B fold
2 Rossmann-type
domain assembly
α/β
β/α
α structure
禠Ϝ
// β-sheet (3214567)
The catalytic site is located in a cleft between the two domains
Metal ion-independent GTs
32
GT-B variant
Human α6-Fucosyltransferase (FucT-VIII)
GT23
Catalytic domain
禠Ϝ
SH3 domain
(Cterm)
Coiled coil region
(N-term)
Rossmann
domain in the Cterm of catalytic
domain
33
GT-B variant
Human O-β
β-GlcNAcT (OGT)
GT41
• Unique and reversible
modification
TPR
禠Ϝ
• Found on a myriad of nuclear and
cytoplasmic proteins
• Extensive cross-talk with
phosphorylation
Hart et al., (2007) Nature, 446: 1017−1022
Int-D
Lazarus et al., (2011) Nature, 469:564-567
34
New folds are observed for GTs using lipid-phosphate sugar donors
No Rossmann fold !!
GT51
Peptidoglycan glycosyltransferase
(Lovering et al., 2007, Science)
禠Ϝ
Staphylococcus aureus
GT domain: bacteriophage λ-lysozyme-like fold !!
35
The process of N-linked protein glycosylation
㐐ऀ
Schwarz and Aebi (2011) COSB, 21:576-582
36
| N AT U R E | V O L 4 7 4 | 1 6 J U N E 2 0 1 1
GT66
㐐ऀ
The first structure of a membrane-embedded GT !
37
3D structure of PglB (C. jejuni)
GT66
㐐ऀ
Lizak et al., (2011) Nature, 474, 350-355.
38
TAKE HOME MESSAGE (3)
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.
㐐ऀ
Novel folds are expected for GTs that use a lipid-phosphate donor
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.
39
GLYCOSYLTRANSFERASE course : Contents
1- General features
• General reaction
• Cellular localization & topology
• Glycosylation events in the Golgi
• Take home message (1)
2- Classification
• Processivity / stereochemistry
• Classification
• GT repertoire
• Take home message (2)
㐐ऀ
3- Structure
• Structural information (current picture)
• GT-A fold / DxD motif
• GT-B fold
• Other folds
• Take home message (3)
4- Reaction mechanisms
• Inverting/retaining reaction mechanisms
• Ordered binding in GT-A enzymes
• additional difficulties
• Take home message (4)
5- Future directions
8
Reaction mechanisms of GTs
In
contrast
to
the
well-characterized
catalytic mechanisms used for glycosidic
bond
hydrolysis
㐐ऀ
(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) COSB
40
Inverting Glycosyltransferase Mechanism
㐐ऀ
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 Biochem
41
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
負Ϝ
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
The « closed » active conformation
of glycosyltransferases
Reaction mechanism for GTs
of the GT-A family
Ordered binding of donor and
acceptor substrates, linked to
a donor substrate-induced
conformational change
LgtC
αGalT
負Ϝ
Base of
UDP-Gal
Binding of a distorted conformation
of nucleotide sugar may be important
for catalysis
Acceptor
disaccharide
Persson et al, Nature Struct. Biol., 8 (2001) 166
Ret
Inv
Inv
43
Additional difficulties for glycosyltransferases
GT-A
Loop movement
GT-B
domain movement
close
open
負Ϝ
α3-GalT
GT6
Superimposition of open (free
enzyme) and closed (upon UDP-Gal
binding) forms
Gastinel et al. (2001); Boix et al., (2001)
Glycogen synthase
GT5
Superimposition of open state
(solid) and closed conformation
(transparent)
Buschiazzo et al. (2004)
44
GlycosyltransferasesN still puzzling
Crystallographic and modelling studies should
allow for a better understanding of molecular
bases responsible for substrate specificityN
負Ϝ
The blood group synthases as a case of study !
45
Blood Group A & B Structures Made by Closely Related GTs
HO
OH
O
OR
HO
O
O
OH
HO
OH
O(H) Precursor
HO
O
OH
HO
O
NH
O
O
NH O P O P O
O
O
O
HO
H3C
OH
O
HO
N
OH
O
O
OHO P O P O
O
O
HO
O
GTA
HO
HO
OH
NH
H3C
O
O
OH
HO
O
HO
OR
OH
O
O
O
O
O
O
OH
OH
OH
Blood Group A
Yamamoto, Hakomori (Nature, 1990)
OH
O
OR
HO
N
OH
O
HO
O
UDP-Gal
OH
O
NH
HO
GTB
負Ϝ
UDP-GalNAc
HO
O
O
HO
OH
Blood Group B
Both enzymes adopt a GT-A fold
46
Blood group synthases GTA/GTB
–
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
負Ϝ
BBBB
Molecular basis for nucleotide sugar specificity ?
UDP-Gal vs UDP-GalNAc
47
Homology Modelling
GT6 (GT-A fold)
UDP-Gal vs UDP-GalNAc
α3-GalNAcT
α3-GalT
S185
D302
S185
D302
E303
E303
負Ϝ
G268
A268
M266
L266
GTA
GTB
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
48
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)
負Ϝ
UDP-Gal vs UDP-GalNAc
Still unclear !!!
Flexibles enzymes
Mutagenesis unpredictable
Much work to be done to understand catalysis
Monica Palcic and coll (in Alberta and Copenhagen)
49
TAKE HOME MESSAGE (4)
Catalytic mechanisms are still poorly understood
• Movements of loops and/or domains involved in the binding
• Flexibility
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.
50
Glycosyltransferases
Future directions N
“in vivo”
“in vitro”
- Functional organization in Golgi
* supramolecular organisation
- Spatial and temporal expression of
GTs in physiological and pathological
situations (Glycomics)
- “Structure/function” relationships
* new 3D structures
* mechanism of action, inhibitors
負Ϝ
- “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)
.....
51
THE END
負Ϝ
Database development at Cermav : Glyco3D
負Ϝ
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
Collaborations
�ҕ
Eric Maréchal
(CEA-Grenoble)
Emmanuel Bettler
(IBCP Lyon)
Jan Mucha
(Bratislava)
Soren B. Engelsen
(Univ. Copenhagen)
Monica Palcic & Ole Hindsagaul
(CRL- Copenhagen)
Funding
French ANR
CEE
Bioinformatics strategy
Arabidopsis proteome
30690 sequences
Use of several remote homology detection methods to search the
« test Arabidopsis proteome » for new candidate GT genes
1- Removal of large
sequences
(>1000 aa)
29457 sequences
Test Arabidopsis proteome
2- Removal of
sequences with a cTP or
mTP (TargetP)
21755 sequences
HMMSearch
(1D level)
PSI-BLAST
+
Structural overlap
(2D level)
�ҕ
ProFit
(3D level)
3- Removal of proteins
with no TMD (TMHMM)
5666 sequences
4- Removal of CAZy
accessions (460 seqs)
> 150 new candidate sequences
+ Chemometrics
Hansen et al. (2009) J. Proteome Res.
Hansen et al., (2010) Mol. Biosystems
Test Arabidopsis proteome
(5315 protein sequences)
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)
�ҕ
~ 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
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
犐ҕ
which can be considered as the most ancestral
families
for
which
enzymes
stereochemistries have evolved
(Martinez-Fleites et al., 2006).
of
both
Glycosyltransferases ….
CAZy
Large differences among GT families
number of sequences
GT2
(> 13,000 seqs)
GT4
(> 10,000 seqs)
GT63
(1 seq)
50% of total seqs
8 families have > 1000 sequences
13 families have < 20 sequences
犐ҕ
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-α
α-GalNAcTs
animal
(mucin-type glycosylation)
MOLECULAR GLYCOBIOLOGY at CERMAV
Group Leader
A. Imberty
GLYCOSYLTRANSFERASES and biosynthesis of glycans
- Structure-function studies and engineering of GTs
- Biogenesis of PCW polysaccharides
Specific犐recognition
of carbohydrates by LECTINS
ҕ
- 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
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
�Ϣ
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