Centre de Biophysique Moléculaire, CNRS UPR 4301, affiliated with INSERM and the University of Orléans, F 45071 Orléans Cédex 2, France
1 To whom correspondence should be addressed at: Centre de Biophysique Moléculaire, Bât. B, rue Charles Sadron, F45071 Orléans Cédex 2, France. E-mail: pillere{at}cnrs-orleans.fr
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: comparative molecular modelling/glycosyltransferases/mutagenesis/polypeptide N-acetylgalactosaminyltransferases/UDP-GalNAc
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sequence analysis of the cDNAs of the different isoforms of the family and of the corresponding proteins have shown that these enzymes are type II membrane proteins (Figure 1A). They are anchored in the Golgi and contain a short (619 amino acids) N-terminal cytosolic domain, a unique transmembrane anchor (1527 amino acids) followed by a luminal region which can be subdivided into three parts: a stem region of variable length (55416 amino acids) which projects the catalytic entity (about 350 amino acids) fully accessible into the lumen of the Golgi; this catalytic domain is followed by a lectin-like domain of 130150 amino acids at the C-terminus of all functional ppGaNTases. The comparison of the entire sequence of ppGaNTases shows that cytosolic, transmembrane, stem and lectin-like regions present very little sequence similarity, the regions of higher amino acid identity being situated in the catalytic domains (Figure 1B). The lectin-like domains show only structural similarities: molecular modelling studies have led to the conclusion that this part of the enzyme could adopt the same fold as the lectin domain of ricin (Imberty et al., 1997; Hagen et al., 1999
). On the other hand, a careful examination of the catalytic domains of mammalian ppGaNTases emphasizes numerous sequence identities in all isoforms since a number of residues are perfectly conserved (Figure 1B). A majority of them is also found at the same position in the ppGaNTase isoforms of Drosophila melanogaster (Schwientek et al., 2002
) or of Caenorhabditis elegans (Hagen and Nehrke, 1998
), which strongly suggests that they may be important for enzyme activity. A comparison of the peptide sequence of the catalytic domain among the different members of the family allows one to distinguish two highly conserved regions: a glycosyltransferase 1 or GT1 pattern and a Gal/GalNAcT pattern. The GT1 pattern comprises about 110 amino acids in the N-terminal half of the catalytic domain and contains a DXH motif, thought to correspond to the DXD motif present in many glycosyltransferases of different specificities (Breton et al., 1998
; Busch et al., 1998
; Kapitonov and Yu, 1999
). In all glycosyltransferases of known three-dimensional (3D) structure which present this motif, it has been shown to bind the sugar-nucleotide donor via an associated divalent metal ion. The Gal/GalNAcT pattern comprises about 40 amino acids in the C-terminal portion of the catalytic domain of ppGaNTases and contains a motif WXXE which has been suggested, in four of the glycosyltransferases of known 3D structure, to participate in the transfer of Gal or GalNAc (Gastinel et al., 1999
; Boix et al., 2002
; Patenaude et al., 2002
), although the available data do not allow firm conclusions. Until now, the atomic coordinates of only 18 crystallized glycosyltransferases have been published (Vrielink et al., 1994
; Charnock and Davies, 1999
; Gastinel et al., 1999
, 2001
; Ha et al., 2000
; Pedersen et al., 2000
, 2003
; Unligil et al., 2000
; Mulichak et al., 2001
, 2003
, 2004
; Persson et al., 2001
; Boix et al., 2002
; Gibbons et al., 2002
; Gibson et al., 2002
; Patenaude et al., 2002
; Chiu et al., 2004
; Lobsanov et al., 2004
). Most of these enzymes present no or very little amino acid sequence similarity among each other. However, they all share some similitude in their overall protein fold. Bioinformatic analysis and 3D structure comparison have led to the proposal that glycosyltransferases adopt principally two different topologies: the nucleotide-diphospho-sugar transferase from Bacillus subtilis or SpsA fold and the DNA-modifying ß-glucosyltransferase from bacteriophage T4 or BGT fold (Unligil and Rini, 2000
; Zhang et al., 2003
and references cited therein). The SpsA fold consists of a ß/
/ß Rossmann-like fold (Rossmann et al., 1974
) organized in such a way that central parallel ß-sheets are flanked by
-helices and arranged in two tightly associated subdomains, one involved in the nucleotide binding and one in the acceptor binding. The BGT fold is characterized by two similar ß/
/ß fold subdomains less tightly associated than in the SpsA fold and with the binding pocket of the donor and acceptor substrates situated in the cleft between the two subdomains. Until now, all enzymes of characterized 3D structure with a SpsA fold have shown a DXD motif involved in the binding of a divalent cation itself coordinated to the dinucleotide sugar. Among the 18 crystallized enzymes of known 3D structure, half are retaining glycosyltransferases (same anomeric linkage of donor substrate and product) while the other half are inverting (different anomeric linkage of donor substrate and product). Six of them adopt the BGT fold while 12 adopt the SpsA fold that is the type of fold predicted for ppGaNTases (Hagen et al., 1999
; Breton et al., 2002
). The overall fold of the six enzymes which function with a retention of the transferred sugar configuration and present a SpsA topology is similar especially in the active-site pocket where the UDP-sugar is bound. However, although great efforts have been made to understand the enzymatic mechanism involved in the sugar transfer reaction for inverting as well as retaining glycosyltransferases (Unligil and Rini, 2000
; Ly et al., 2002
) there are no data yet available on the precise enzymatic mechanism involved for any of the enzymes of known 3D structure.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The amino acid sequences of the bovine ppGaNTase-T1 and of 13 human ppGaNTases plus the ppGaNTase-T5 cloned from rat, were aligned using the ClustalW method (Thompson et al., 1994) and refined manually using GeneDoc (Nicholas et al., 1997
). This multiple alignment of ppGaNTases was employed to carry out secondary structure predictions of the bovine ppGaNTase-T1 with two programs available on WWW servers: the PHDsec methods (Rost and Sander, 1993
, 1994
; http://www.EMBL-Heidelberg.DE/services/index.html), using a neural network algorithm based on position-specific conservation weight and the quadratic-logistic method (Di Francesco et al., 1995
; http://abs.cit.nih.gov/ql2/), operating according to a logistic discriminating function methodology. Other programs, which only work with unique sequences were run with the ppGaNTase-T1 sequence: the NPS method (http://pbil.ibcp.fr/NPSA/npsa_prediction.html) which provides a consensus secondary prediction using different algorithms and PREDATOR (Frishman and Argos, 1997
; http://bioweb.pasteur.fr/seqanal/interfaces/predator.html) which uses information from related proteins in order to predict a secondary structure.
Fold prediction
Fold recognition was used to propose a structural model for the catalytic domain of the ppGaNTase-T1. Several fold recognition programs were run using the putative catalytic domain of the bovine ppGaNTase-T1 (amino acids 84426) as query sequence: Superfamily (Gough et al., 2001; http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/), mGenTHREADER (Jones, 1999
; McGuffin et al., 2000
; http://bioinf.cs.ucl.ac.uk/psipred/), Hybrid Fold Recognition (Fischer, 2000
; http://www.cs.il/~bioinbgu/) and LIBELLULA (Juan et al., 2003
; http://www.pdg.cnb.uam.es/servers/libellula.html), that uses and improves the results of two other methods (SAMT99 and 3DPSSM). These threading processes (Lemer et al., 1995
) allow one to align one sequence with one structure.
Comparative modelling
The region comprised between amino acids 96 and 227 of the bovine ppGaNTase-T1 was aligned on the nucleotide-sugar binding segment of bovine 1,3galactosyltransferases (
3GalT) and the corresponding region of human blood group A transferase or
1,3N-acetylgalactosaminyltransferase (GTA). Because of the very low sequence identity between ppGaNTase-T1 and the other transferases
3GalT and GTA, the alignment was carried out according to the hydrophobic cluster analysis (HCA) program (Gaboriaud et al., 1987
; http://www.lmcp.jussieu.fr/~soyer/www-hca/hca-form.html). This graphical method allows to easily visualize
-helices and ß-strands and to align the conserved hydrophobic clusters.
The models of the [96227] fragment of ppGaNTase-T1 were built using MODELLER 6.2 software (Sali and Blundell, 1993). MODELLER is a program based on the satisfaction of spatial restraints generated on the target sequence from its alignment with the 3D structure of the templates [
3GalT in complex with UDP (1K4V; Boix et al., 2001
) and GTA in complex with UDP and the H antigen (1LZI; Patenaude et al., 2002
)] and also from the statistical analysis of a data base including 416 protein structures. The PROCHECK program (Laskowski et al., 1993
) was used to assess the stereochemical quality of the structures. Additional evaluations were completed by various profile programs: PROSA II (Sippl, 1993
) and VERIFY3D (Luthy et al., 1992
; http://www.doe-mbi.ucla.edu/Services/Verify_3D/) and also by examination of the structure on a graphic display. Using these assessments, a representative model was selected and energy minimised with the SYBYL software (TRIPOS Inc., St Louis, MO). The secondary structures of the model were analysed with the PROMOTIF program (Hutchinson and Thornton, 1996
).
Docking of UDP-GalNAc
UDP-GalNAc was built using the geometry of UDP-2FGalMn2+ in complex with the 1,4 galactosyltransferase from Neisseria meningitis or LgtC (Persson et al., 2001
). The fluorine atom was changed into a N-acetyl group and the coordinates of the molecule were refined by energy minimization within SYBYL. The ligand was then docked into the active site of ppGaNTase-T1. In a first step, the UDP moiety of UDP-GalNAc was placed in a position similar to the position of UDP ligands in
3GalT and GTA X-ray structures. In a second step, the geometry of the whole ligand and of the protein side chains in the vicinity of the active site were energy minimized within SYBYL.
Site-directed mutagenesis
Site-directed mutagenesis was performed by PCR using the QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The plasmid pcDNA3 (Invitrogen, San Diego, CA) containing an insert encoding a soluble form of the bovine ppGaNTase-T1 (S.Duclos, N.Bureaud, F.Piller and V.Piller, manuscript in preparation) was used as template with the following couples of primers: W129A, 5'-GTTTTCCACAATGAGGCTGCCAGCACACTTCTGCGAAC-3' upstream and 5'-GTTCGCAGAAGTGTGCTGGCAGCCTCATTGTGGAAAAC-3' downstream; W129F: 5'-GATTGTTTTCCACAATGAGGCTTTCAGCACACTTCTGCGAACTGTC-3' upstream and 5'-GACAGTTCGCAGAAGTGTGCTGAAAGCCTCATTGTGGAAAACAATC-3'downstream; W129R: 5'-GGTGATTGTTTTCCACAATGAGGCTCGGAGCACACTTCTGCGAACTGTCCATAGC-3' upstream and 5'-GCTATGGACAGTTCGCAGAAGTGTGCTCCGAGCCTCATTGTGGAAAACAATCACC-3' downstream; N126A: 5'-CCTACAACCAGTGTGGTGATTGTTTTCCACGCTGAGGCTTGGAGCACACTTCTGCGAAC-3' upstream and 5'-GTTCGCAGAAGTGTGCTCCAAGCCTCAGCGTGGAAAACAATCACCACACTGGTTGTAGG-3' downstream; F124A: 5'-CCTTCCTACAACCAGTGTGGTGATTGTTGCCCACAATGAGGCTTGGAGCACACTTCTGC-3' upstream and 5'-GCAGAAGTGTGCTCCAAGCCTCATTGTGGGCAACAATCACCACACTGGTTGTAGGAAGG-3' downstream; L189A: 5'-CGAATGGAGCAGCGTTCTGGAGCGATCAGAGCTAGGTTAAAAGG-3' upstream and 5'-CCTTTTAACCTAGCTCTGATCGCTCCAGAACGCTGCTCCATTCG-3' downstream; R193A: 5'-CGTTCTGGATTGATCAGAGCTGCTTTAAAAGGTGCTGCTGTGTCTAAAGG-3' upstream and 5'-CCTTTAGACACAGCAGCACCTTTTAAAGCAGCTCTGATCAATCCAGAACG-3' downstream; D209A: 5'-AAGTGATCACCTTTTTAGCCGCGCACTGTGAG-3' upstream and 5'-CTCACAGTGCGCGGCTAAAAAGGTGATCACTT-3' downstream. Nucleotides mutated to convert one amino acid into another residue are underlined and in bold characters. All mutations were confirmed by sequencing (MWG Biotech, Germany). Each mutated cDNA was excised from the pcDNA3 vector by BamHI and NotI and cloned in frame with the alpha-factor signal peptide into the YEpFLAGTM-1 yeast expression vector (Sigma, St Louis, MO) opened by the same enzymes.
Expression of wild type and mutant ppGaNTase-T1 in Saccharomyces cerevisiae
Saccharomyces cerevisiae (strain BJ5465) cells were transformed with the YEpFLAGTM-1 vector containing either the wild type cDNA or a mutated cDNA, using the lithium acetate transformation method (Ito et al., 1983). The selection method is based on the Trp auxotrophy of the BJ5465 strain. Transformants were selected on Trp-deficient synthetic complete agar (0.67% yeast nitrogen base without amino acids, 0.074% complete supplement mixture minus Trp, 2% dextrose and 2% agar) since the TRP1 gene of the vector YEpFLAGTM-1 complements the mutation of the BJ5465 strain and restores the Trp-prototrophy. Selected clones were grown at 30°C in SC minus Trp liquid medium for 48 h (OD600nm = 810). Starter cultures were diluted to OD600nm = 0.4 in the expression medium (1% yeast extract, 8% peptone, 1% glucose, 3% glycerol and 1 mM CaCl2) for induction of protein expression. The expression cultures were grown at 30°C for 72 h.
Purification of recombinant ppGaNTase-T1 and mutants
Ten millilitres of expression culture were centrifuged for 5 min at 10 000 g and 4°C, and the supernatant was dialysed against 50 mM sodium phosphate buffer, pH 8, at 4°C. The supernatant was adjusted to 300 mM NaCl and 5 mM imidazole and then applied onto a 1 ml nickel-agarose column (Qiagen, Germany) equilibrated with the 50 mM sodium phosphate buffer, pH 8, 300 mM NaCl and 5 mM imidazole. The column was washed once with 5 ml of the same buffer and once with 5 ml of the same buffer but 10 mM in imidazole. The bound enzyme was eluted in 500 µl fractions of 250 mM imidazole, pH 7 and analysed by SDSPAGE, western blot and enzymatic activity.
SDSPAGE and immunoblotting
Eluted enzymes (5 µl) were analysed by SDSPAGE (Laemmli, 1970) using an 8% (w/v) polyacrylamide separation gel. Proteins were transferred onto nitrocellulose membrane (Schleicher & Schuell, Germany) and blocking was carried out for 30 min in Tris-buffered saline (TBS) containing 0.5% (v/v) Tween-20 (TBS-T). The membrane was first incubated for 30 min with a mouse anti-FLAGTM antibody (Sigma) at 10 µg/ml TBS. After three washings in TBS, the second antibody, a goat anti-mouse antibody coupled to alkaline phosphatase (Bio-Rad, Hercules, CA) was used at a dilution of 1:2500 in TBS-T. The colour reaction was developed with the Western BlueTM detection reagent (Promega, Madison, WI) and the amount of ppGaNTase-T1 or mutant enzymes was determined by densitometry analysis (Image Quant; Molecular Dynamics, Sunnyvale, CA) by comparison to 50 and 100 ng of FLAGTM-tagged inactive bacterial alkaline phosphatase (BAP-FLAGTM; Sigma).
Determination of ppGaNTase activity and kinetic parameters
PpGaNTase-T1 activity was assayed by measuring the synthesis of [3H]GalNAc-substituted peptide in 25 µl final volume reactions. Standard reaction mixtures contained 50 mM MES, pH 6.5, 15 mM MnCl2, 1 mg/ml BSA, 20 µM UDP-[3H]GalNAc (American Radiolabeled Chemicals, St Louis, MO) with a specific activity of 50 000 c.p.m./nmol, 0.7 mM acceptor peptide STPSTPSTPSTPSTP (STP5, Sigma) and an appropriate amount of enzyme such as <10% of UDP-[3H]GalNAc were consumed at the end of the reaction (about 0.3 µU enzyme per test). After 30 min incubation at 37°C and separation by Dowex-1X8 (Serva, Germany) chromatography, the products were quantified by scintillation counting. All reactions were done in triplicates.
To determine the apparent Km for UDP-GalNAc, the concentration of UDP-[3H]GalNAc was varied from 3.5 to 100 µM (and up to 250 µM for mutants N126A and W129A) in the presence of 0.7 mM STP5. The apparent Km for the acceptor peptide was obtained by varying the concentration of STP5 from 0.15 to 1.5 mM in the presence of 100 µM UDP-[3H]GalNAc. Use of double reciprocal plots (1/V versus 1/[S]) allowed the calculation of kinetic parameters.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sequence alignment, secondary structure prediction and fold recognition of ppGaNTases
Most ppGaNTase isoforms that have been cloned in mammals are of human origin; however, whenever a corresponding enzyme of another species has been cloned (bovine, mouse, rat or porcine) and functionally expressed, it showed only differences in a few amino acids with the human enzymes (9099% identity among orthologues). For instance, among the 559 amino acids that constitute the entire protein sequence of the bovine ppGaNTase-T1 used for this study, only four residues differ from the human ppGaNTase-T1. Thus, 13 human isoform sequences (ppGaNTases-T1T4 and -T6T14) and the primary structure of ppGaNTase-T5 from rat (because there is no human equivalent cloned to date) were compared in this study. The sequence of the bovine ppGaNTase-T1 is reported above the corresponding human sequence in Figure 1B. Due to low sequence identity in the cytosolic, transmembrane, stem and lectin domains, only the putative catalytic ppGaNTase domains (corresponding to residues 84426 of the bovine ppGaNTase-T1) are aligned. The multiple alignment thus obtained and the sequence of the bovine ppGaNTase-T1 were submitted to several secondary structure prediction programs. The resulting predicted topology found for the ppGaNTase-T1 fold is characteristic of a Rossmann fold with alternating -helixes and ß-strands. In the N-terminal half of the considered sequence (corresponding to residues 117242 of the bovine ppGaNTase-T1) the different methods agree perfectly, suggesting a high reliability of the predictions. On the other hand, the predictions are more variable in the second half (residues 243426 in the bovine ppGaNTase-T1) of the catalytic domain.
The 84426 amino acid sequence of ppGaNTase-T1 was also submitted to a fold recognition analysis in order to search for proteins that share the same fold with the bovine ppGaNTase-T1. The secondary structures given by fold recognition programs were compared to those obtained by the secondary structure prediction programs and this allowed one to select the templates according to the highest scores of the structural prediction. Threading calculations aligned the ppGaNTase-T1 sequence with X-ray structures of glycosyltransferases which adopt a SpsA fold: SpsA (Charnock and Davies, 1999), LgtC (Persson et al., 2001
), GTA and human blood group B transferase (GTB) (Patenaude et al., 2002
) and
3GalT (Gastinel et al., 2001
; Boix et al., 2002
). The first two are bacterial enzymes. SpsA is believed to be an inverting glycosyltransferase, a member of the family 2 of glycosyltransferases (GT-2 family, CAZY database), able to bind UDP as well as TDP (Tarbouriech et al., 2001
), although its specificity for both donor and acceptor substrates is still unknown. LgtC is a retaining glycosyltransferase which transfers Gal from UDP-Gal in an
-linkage to a terminal Gal of lipopolysaccharides, forming a Gal
1,4 Gal linkage, and is assigned on the basis of sequence similarity to the GT-8 family (CAZY database). Human ABO blood group transferases, GTA and GTB are enzymes showing high sequence identity (only four different residues on a total of 354 amino acids); GTA transfers GalNAc from UDP-GalNAc and GTB transfers Gal from UDP-Gal to the same acceptor [Fuc
1,2] Gal, both in
configuration, forming the blood group A and B antigens: GalNAc
[Fuc
1,2] Gal and Gal
[Fuc
1,2] Gal, respectively. Based on sequence similarity, they are classified in the retaining GT-6 family as well as
3GalT which is a bovine enzyme transferring Gal from UDP-Gal in
to Galß1,4GlcNAc structures, forming the xenoantigen Gal
1,3Galß1,4GlcNAc responsible for the hyperacute rejection of xenografts in humans. GTA, GTB and
3GalT are all involved in the biosynthesis of Gal(NAc)
1,3Gal-containing antigens; they display many amino acid similarities and exhibit roughly the same fold: two subdomains separated by the catalytic pocket, one N-terminal binding UDP-Gal or UDP-GalNAc, adopting the Rossmann fold characteristic of nucleotide binding regions and one C-terminal involved in the acceptor substrate recognition. They are enzymes of mammalian origin, resident in the Golgi apparatus and present a type II membrane protein orientation. We finally retained as templates only the GTA and the
3GalT and discarded all the other potential candidates, either because their primary structure or their substrate specificities were too far from those of ppGaNTases (SpsA and LgtC) or because, in the case of GTB, the identity with GTA is so high that including both GTA and GTB would strongly bias the modelling procedure.
Alignment of 3GalT, GTA and bovine ppGaNTase-T1 and molecular modelling of the UDP-GalNAc binding domain
An alignment of the putative catalytic domain of ppGaNTase-T1 and of the identified catalytic domains of 3GalT and GTA was undertaken using the hydrophobic cluster analysis (HCA). The HCA plot obtained for the first N-terminal part of the putative catalytic domain of ppGaNTase-T1 (amino acids 96227) appeared closely related to the HCA plots of the N-terminal region of the catalytic domains of both
3GalT and GTA corresponding to the UDP binding sites in the two enzymes. An alignment of the three HCA plots was thus possible in this region and the hydrophobic clusters were adjusted manually in order to take into account the secondary structure data which coincide particularly well in this region showing for the templates as well as for ppGaNTase-T1 a regular alternation of
-helices and ß-strands (Figure 2). The resulting sequence alignment is given in Figure 3. On the other hand, the C-terminal part of the putative catalytic domain of ppGaNTase-T1 is longer than the corresponding C-terminal catalytic domains of
3GalT and GTA. The large loop (amino acids 242274) separating the aligned stretches (GT1 and Gal/GalNAc-T, Figure 1) hinders the modelling process, therefore only the sequence comprised between amino acids 96 and 227 of bovine ppGaNTase-T1 was modelled from the corresponding sequences (amino acids 93227 for GTA and 116241 for
3GalT) of the templates. A best-fitted superimposition of the two templates in blue and green (Figure 4A) shows that the structures are very similar, although the coordinates of the 177195 segment of GTA are missing in the PDB file. The r.m.s.d. between the C
coordinates of the aligned residues (107 in total) is 2.6 Å.
|
|
|
Ten models of the segment 96227 of bovine ppGaNTase-T1 were built according to the procedure described in the Materials and methods. After evaluation with PROCHECK, VERIFY-3D and PROSA II, one model was selected and considered as representative of the 3D structure of this part of the ppGaNTase-T1. As expected, the modelled enzyme subdomain adopts the /ß fold characteristic of nucleotide binding regions (Figure 4A). The structure starts at R96, with the
2 helix (
3GalT nomenclature) from E104 to Y111 followed by the ß2 strand (V121H125). This strand is connected to the ß3 strand (E149D155) by the long
3 helix (S130S143), which is preceded by a 310 helical turn from N126 to W129. The two chain reversals between the ß3 and the ß4 strands are ensured by a type I ß turn between A157 and R160 which replaces the 310 helical turn found in
3GalT and by a long loop between L167 and K175 corresponding to a large insertion compared to the sequences of the two templates. This ß4 strand is very short (I181M183), but residues 172185 are found in an extended conformation. The
4 helix which is missing in the density map of GTA runs from R186 to V204; the ß5 and ß6 short strands which frame the DXD motif in
3GalT structures are not found although residues T206A210 and G217K223 are in an extended conformation and the 206210 segment is parallel to the three parallel strands ß2, ß3 and ß4 while the fragment 217223 runs in an antiparallel direction. The presence of an insertion after the DXH motif in the ppGaNTase-T1 sequence leads to the formation of a six-residue turn between these two pseudo strands. This turn is much longer than that observed for the two templates but after G127, the backbones of ppGaNTase-T1,
3GalT and GTA are well superimposed. Globally, the proposed 3D structure for ppGaNTase-T1 is rather close to the structure of the templates in its N-terminal part. The r.m.s.d. between the coordinates of the C
atoms of the secondary structure elements of residues 120186 of
3GalT and of the modelled structure is only 1.89 Å. However, the modelled structure differs more from that of the templates in the helix
4 position and in the loops and turns which link the secondary structure elements in the C-terminal region.
UDP-GalNAc was docked into the active site which appears as a deep cleft located between the parallel ß-sheets and helix 4 (Figure 4B). The catalytic pocket includes the DXH motif that replaces the DXD motif common to a wide range of glycosyltransferases. This motif was shown to be involved in the coordination of the Mn2+ divalent cation that is assumed to stabilize the UDP leaving group during bond cleavage (Pedersen et al., 2000
; Tarbouriech et al., 2001
). In our model, the the Mn2+ ion shows seven interactions of coordination. It involves the two O
oxygens of D209 and the N
of H211 (Figure 4C and Table I) but also the four phosphate oxygens of UDP. The characteristic distances of coordination range between 2.1 and 2.35 Å. The binding of UDP-GalNAc is stabilized by a number of specific interactions (Figure 4C and Table I). The uracil base is well stacked on the ring of W129, and its O2 carbonyl is involved in an hydrogen bond with the NH group of the N126 backbone, while the ribose is in van der Waals contacts with F124 and L189 and the N
H2 of N126 forms a H-bond with the O2 of the ribose. The galactosyl ring adopts a 4C1 chair conformation similar to that found for other UDP-Gal(NAc) molecules (Persson et al., 2001
; Pedersen et al., 2003
). The O3' atom of the sugar is hydrogen bonded to the NH
atom of the guanidinium group of R193. A hydrogen bond is also found between the N
H2 of R193 and the oxygen atom of the acetyl group. Moreover, the GalNAc moiety makes favourable van der Waals contacts with L194 and L208.
|
Molecular modelling of the most N-terminal part of the putative ppGaNTase-T1 catalytic domain points out several possible interactions between the donor substrate UDP-GalNAc and amino acids conserved in this subdomain for all ppGaNTases. In order to test if these interactions are really important for the expression and/or activity of the ppGaNTase-T1, we carried out a series of mutagenesis experiments on several amino acids present in this region and expressed the corresponding mutant enzymes. For that purpose, cDNA constructs have been designed for the expression of a soluble and tagged form of the ppGaNTase-T1 in the culture medium of the yeast S.cerevisiae, allowing one to readily obtain rather large amounts of pure enzyme after a fast and simple purification step (Figure 5). All the different forms of the enzyme were not grossly misfolded since they were expressed and secreted into the medium. The wild-type and mutant forms obtained therein were used for Km and kcat determinations with both the donor and the acceptor substrates. The results are reported in Table II. For several mutants, the low activity of the enzyme did not allow kinetic constant determination (F124A, L189A, R193A and D209A).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
UDP-GalNAc appears in the model described here to lie in a deep cleft containing the DXD-like motif and composed by a central sheet of ß-strands surrounded by -helices. Thus, the modelled catalytic subdomain of this enzyme adopts a typical SpsA fold as already predicted for this enzyme family (Breton et al., 1998
; Kikuchi et al., 2003
; Liu and Mushegian, 2003
) and as expected from modelling carried out on the basis of two template sequences adopting a SpsA fold (GTA and
3GalT).
Our model displays several well-defined interactions between the substrate and the enzyme, which can be directly compared to the same type of interactions found in both templates (see Table I). The amino acids suspected to be involved in those interactions have been mutated in the ppGaNTase-T1 and the results strongly support the model. First, the DXD sequence is a motif found in most prokaryotic and eukaryotic glycosyltransferases and in other nucleotide binding proteins as well. It has been shown to interact with the phosphate groups of the nucleotide through a divalent cation. If we consider the different glycosyltransferases of known 3D structure, the DXD sequence does not seem always to participate in the same manner in substrate binding. As a matter of fact the conservation of this short sequence is not absolute in glycosyltransferases since it can be either DXD, DDD, DXX or XDD. In the ppGaNTase family this sequence is a DXH motif, well conserved for all the members, except for the ppGaNTase-T4, for which the D209 residue is replaced by a Y. In the model, a change from D to Y is possible since the OH of the tyrosine can easily replace the COO of the aspartic acid in the interaction with the Mn2+. However, Y being much bulkier than D, this replacement probably alters the folding of the adjacent peptide chain which may in part account for the unique substrate specificity of the ppGaNTase-T4 as compared to the other members of the ppGaNTase family (Hassan et al., 2000). The interaction of DXH with the Mn2+ ion involved in the donor substrate binding, as shown in the model, is characteristic of the type of interaction described for retaining glycosyltransferases, i.e. with both conserved residues of the DXH motif in direct contact with the cation, a feature which probably is of relevance for the catalytic action of these enzymes (Patenaude et al., 2002
). Compared to other glycosyltransferases, the presence of a histidine interacting with the Mn2+ ion has also been found in the LgtC binding site (H244) (Persson et al., 2001
) and in rabbit glycogenin (H211) (Gibbons et al., 2002
). On the other hand, replacement of H211 by a D in the ppGaNTase-T1 (Hagen et al., 1999
) abolished the enzyme activity, indicating that D could not functionally substitute for a H. However, in the model presented here, this substitution is possible without impairment of the donor substrate recognition (data not shown). In this context it should be emphasized that our model focuses only on a segment of the UDP-GalNAc binding domain and the H211D mutation could be of importance in interactions not taken into account by the present study.
Secondly, in our model the uracil base is stabilized in the binding pocket through a stacking interaction with W129. The equivalent is found not only in the templates GTA (Y126) and 3GalT (Y139) but also in glycosyltransferases like LgtC with Y11 (Persson et al., 2001
), SpsA with Y11 (Charnock and Davies, 1999
), the bovine ß1,4 galactosyltransferase 1 (ß4Gal-T1) with F226 and R191 (Gastinel et al., 1999
), GlcAT-1 with Y84 (Pedersen et al., 2002
), glycogenin with Y14 (Gibbons et al., 2002
) and EXTL2 with Y74 (Pedersen et al., 2003
). W129 is not conserved in all ppGaNTases, but replaced by R (in ppGaNTases-T2 and -T4) or by L (in ppGaNTases-T8 and -T9), two amino acids which can be involved in N-H/p or C-H/p interaction, respectively, with an aromatic ring. Our mutation experiments show unambiguously that an R can replace W129 in the ppGaNTase-T1 without significant change in enzyme activity and even the W129A mutation conserves some activity. One can also predict that a leucine will make a hydrophobic interaction in this region as well, as it happens in glycogenin with a valine.
Thirdly, the N126 of the modelled ppGaNTase-T1 subdomain interacts via its NH and NH2 groups through hydrogen bonds with the O2 of the uracil base and the O2 of the ribose, respectively. N126 is changed to D in the rat ppGaNTase-T5 isoform and otherwise conserved in all known mammalian members of the family. However, in the model an aspartic acid could interact with the uridine as well. On the other hand, it is most probable that a change from N to A does not allow any interaction with the ribose, which explains the low activity of the alanine mutant. In GTA (I123, F121, V212 and D213) (Patenaude et al., 2002
),
3GalT (F134, V136 and D225) (Gastinel et al., 2001
), glycogenin (L8, N11 and D103) (Gibbons et al., 2002
), EXTL2 (Q72, N101, N130 and D152) (Pedersen et al., 2003
), GlcAT-1 (D113 and D195) (Pedersen et al., 2002
) and LgtC (A6, D8, N10 and I104) (Persson et al., 2001
), the same types of interaction have been observed in the crystals.
Fourthly, the model suggests the hydrogen bonding of the guanidinium group of R193 with the GalNAc of the donor sugar. Again, the equivalent is seen in the templates used for the present modelling with R202 binding to the Gal moiety of UDP-Gal in the 3GalT X-ray structure (Boix et al., 2002
) and most probably R188 binding to the GalNAc of UDP-GalNAc in the GTA binding site. This R188 is present in a disordered loop adjacent to the active site of the GTA but could be shown to form contacts with the donor substrate modelled into the binding site (Patenaude et al., 2002
). Interactions of a guanidinium group with the donor sugar were also found in LgtC where R86 binds to the Gal of UDP-2FGal (Persson et al., 2001
), in GlcAT-I where R161 forms a hydrogen bond with the GlcUA of the donor sugar (Pedersen et al., 2002
) and in EXTL2 where R135 is hydrogen bonded to the GalNAc of UDP-GalNAc (Pedersen et al., 2003
). It should be also mentioned that R193 is invariant in all the ppGaNTases cloned so far. Furthermore, a point mutation of R193 to W in a fruitfly ppGaNTase is lethal for the insect and, when expressed in COS cells, the resulting protein is inactive (Ten Hagen and Tran, 2002
). Indeed in our model, the presence of a tryptophan instead of R193 would induce profound changes in the structure of the catalytic pocket. It is also noteworthy that R193 contributes notably to the folded back conformation of the UDP-sugar and may thus be important for the catalytic mechanism of the transferase.
Finally, the hydrophobic pocket formed by F124, L189, L194 and L208 in our model is found also in the GTA (F121 and V184) (Patenaude et al., 2002), in the
3GalT (F134 and I198) (Boix et al., 2001
), in LgtC (A6 and I104) (Persson et al., 2001
) and in glycogenin (L8 and V82) (Gibbons et al., 2002
). F124 and L189 are two strictly conserved amino acids in the ppGaNTases and mutagenesis confirmed their importance for enzyme activity. L208 is conserved in all ppGaNTases except in the ppGaNTase-T9 where it is conservatively replaced by a F. On the other hand, L194 is less well conserved since it is changed in V, I, M, N or S for six ppGaNTases of the 14 analysed. It is likely that in this region the contact with the substrate is looser.
Since we used GTA and 3GalT as templates to model a subdomain of the ppGaNTase-T1, it may not be surprising, a priori, to find in the model the same type of interactions which are seen in the crystals between the enzymes and their substrates. However, these enzymes do not have the same function, even if they show common enzymatic features, and the primary sequence of the ppGaNTase-T1 is quite different from that of the GTA/GTB and of the
3GalT. It is thus interesting to note that the global structure of the nucleotide binding site is similar to such an extent for the three enzymes. More restricted structural similarities could also be revealed between the model, the templates and three other retaining glycosyltransferases: LgtC, glycogenin and EXTL2. These observations strengthen our model and suggest that retaining glycosyltransferases may use very similar mechanisms.
Recently, derivatives of UDP-GalNAc O-methylated on C3, C4 or C6 of the GalNAc ring have been tested as substrates or inhibitors of the bovine ppGaNTase-T1 (Busca et al., 2003). None of the UDP(3-OMe)GalNAc, UDP(4-OMe)GalNAc or UDP(6-OMe)GalNAc structures are substrates and they are only weak inhibitors with Kis similar to those for UDP or UDP-GlcNAc. Furthermore, synthesis of UDP-GalNAc biotinylated on the C6 of the GalNAc moiety gave rise to a compound which was not transferred by the ppGaNTase-T1 (Bulter et al., 2001
). These results indicate that the GalNAc portion of UDP-GalNAc fits tightly into the donor binding site which cannot accommodate OMe or biotinyl groups. This is in agreement with the model presented here, with the GalNAc bound in the site by hydrogen bonds with R193 as well as by hydrophobic interactions with L residues.
Although the model presented here has been tested by mutagenesis experiments, the final confirmation has to await X-ray crystallography of one of the members of the ppGaNTases family. The model, however, may contribute to our understanding of nucleotide sugar binding in glycosyltransferases and help with the design and development of new substrate analogues or inhibitors of the ppGaNTases.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Boix,E., Zhang,Y., Swaminathan,G.J., Brew,K. and Acharya,K.R. (2002) J. Biol. Chem., 277, 2831028318.
Breton,C., Bettler,E., Joziasse,D.H., Geremia,R.A. and Imberty,A. (1998) J. Biochem. (Tokyo), 123, 10001009.[Abstract]
Breton,C., Heissigerova,H., Jeanneau,C., Moravcova,J. and Imberty,A. (2002) Biochem. Soc. Symp., 69, 2332.[Medline]
Bulter,T., Schumacher,T., Namdjou,D.J., Gutierrez Gallego,R., Clausen,H. and Elling,L. (2001) Chembiochem, 2, 884894.[CrossRef][ISI][Medline]
Busca,P., Piller,V., Piller,F. and Martin,O.R. (2003) Bioorg. Med. Chem. Lett., 13, 18531856.[CrossRef][ISI][Medline]
Busch,C., Hofmann,F., Selzer,J., Munro,S., Jeckel,D. and Aktories,K. (1998) J. Biol. Chem., 273, 1956619572.
Charnock,S.J. and Davies,G.J. (1999) Biochemistry, 38, 63806385.[CrossRef][ISI][Medline]
Chiu,C.P., Watts,A.G., Lairson,L.L., Gilbert,M., Lim,D., Wakarchuk,W.W., Withers,S.G. and Strynadka,N.C. (2004) Nat. Struct. Mol. Biol., 11, 163170.[CrossRef][ISI][Medline]
de Vries,T., Knegtel,R.M., Holmes,E.H. and Macher,B.A. (2001) Glycobiology, 11, 119R128R.
Di Francesco,V., Munson,P.J. and Garnier,J. (1995) 28th Hawaii Int. Conf. Syst. Sci., 5, 285291.
Fischer,D. (2000) Pac. Symp. Biocomput., 119130.
Frishman,D. and Argos,P. (1997) Proteins, 27, 329335.[CrossRef][ISI][Medline]
Gaboriaud,C., Bissery,V., Benchetrit,T. and Mornon,J.P. (1987) FEBS Lett., 224, 149155.[CrossRef][ISI][Medline]
Gastinel,L.N., Cambillau,C. and Bourne,Y. (1999) EMBO J., 18, 35463557.
Gastinel,L.N., Bignon,C., Misra,A.K., Hindsgaul,O., Shaper,J.H. and Joziasse,D.H. (2001) EMBO J., 20, 638649.
Gibbons,B.J., Roach,P.J. and Hurley,T.D. (2002) J. Mol. Biol., 319, 463477.[CrossRef][ISI][Medline]
Gibson,R.P., Turkenburg,J.P., Charnock,S.J., Lloyd,R. and Davies,G.J. (2002) Chem. Biol., 9, 13371346.[CrossRef][ISI][Medline]
Gibson,R.P., Tarling,C.A., Roberts,S., Withers,S.G. and Davies,G.J. (2004) J. Biol. Chem., 279, 19501955.
Gough,J., Karplus,K., Hughey,R. and Chothia,C. (2001) J. Mol. Biol., 313, 903919.[CrossRef][ISI][Medline]
Gulberti,S., Fournel-Gigleux,S., Mulliert,G., Aubry,A., Netter,P., Magdalou,J. and Ouzzine,M. (2003) J. Biol. Chem., 278, 3221932226.
Ha,S., Walker,D., Shi,Y. and Walker,S. (2000) Protein Sci., 9, 10451052.[Abstract]
Hagen,F.K. and Nehrke,K. (1998) J. Biol. Chem., 273, 82688277.
Hagen,F.K., Hazes,B., Raffo,R., deSa,D. and Tabak,L.A. (1999) J. Biol. Chem., 274, 67976803.
Hassan,H., Benett,E.P., Mandel,U., Hollingworth,M.A. and Clausen H. (2000) In: Ernst,B., Hart,G., and Sinaÿ,P. (eds), Carbohydrates in Chemistry and Biology. Wiley-VCH, pp. 273292.
Heissigerova,H., Breton,C., Moravcova,J. and Imberty,A. (2003) Glycobiology, 13, 377386.
Hutchinson,E.G. and Thornton,J.M. (1996) Protein Sci., 5, 212220.
Imberty,A., Piller,V., Piller,F. and Breton,C. (1997) Protein Eng., 10, 13531356.[CrossRef][ISI][Medline]
Imberty,A., Monier,C., Bettler,E., Morera,S., Freemont,P., Sippl,M., Flockner,H., Ruger,W. and Breton,C. (1999) Glycobiology, 9, 713722.
Ito,H., Fukuda,Y., Murata,K. and Kimura,A. (1983) J. Bacteriol., 153, 163168.[ISI][Medline]
Jones,D.T. (1999) J. Mol. Biol., 287, 797815.[CrossRef][ISI][Medline]
Juan,D., Grana,O., Pazos,F., Fariselli,P., Casadio,R. and Valencia,A. (2003) Proteins, 50, 600608.[CrossRef][ISI][Medline]
Kapitonov,D. and Yu,R.K. (1999) Glycobiology, 9, 961978.
Kikuchi,N., Kwon,Y.D., Gotoh,M. and Narimatsu,H. (2003) Biochem. Biophys. Res. Commun., 310, 574579.[CrossRef][ISI][Medline]
Laemmli,U.K. (1970) Nature, 227, 680685.[ISI][Medline]
Laskowski,R.A., MacArthur,M.W., Moss D.S.A. and Thornton,J.M. (1993) J. Appl. Crystallogr., 26, 283291.[CrossRef][ISI]
Lazarus,B.D., Milland,J., Ramsland,P.A., Mouhtouris,E. and Sandrin,M.S. (2002) Glycobiology, 12, 793802.
Lemer,C.M., Rooman,M.J. and Wodak,S.J. (1995) Proteins, 23, 337355.[ISI][Medline]
Liu,J. and Mushegian,A. (2003) Protein Sci., 12, 14181431.
Lobsanov,Y.D., Romero,P.A., Sleno,B., Yu,B., Yip,P., Herscovics,A. and Howell,P.L. (2004) J. Biol. Chem., 279, 1792117931.
Luthy,R., Bowie,J.U. and Eisenberg,D. (1992) Nature, 356, 8385.[CrossRef][ISI][Medline]
Ly,H.D., Lougheed,B., Wakarchuk,W.W. and Withers,S.G. (2002) Biochemistry, 41, 50755085.[CrossRef][ISI][Medline]
McGuffin,L.J., Bryson,K. and Jones,D.T. (2000) Bioinformatics, 16, 404405.[Abstract]
Mulichak,A.M., Losey,H.C., Walsh,C.T. and Garavito,R.M. (2001) Structure (Camb.), 9, 547557.[Medline]
Mulichak,A.M., Losey,H.C., Lu,W., Wawrzak,Z., Walsh,C.T. and Garavito,R.M. (2003) Proc. Natl Acad. Sci. USA, 100, 92389243.
Mulichak,A.M., Lu,W., Losey,H.C., Walsh,C.T. and Garavito,R.M. (2004) Biochemistry, 43, 51705180.[CrossRef][ISI][Medline]
Negishi,M., Dong,J., Darden,T.A., Pedersen,L.G. and Pedersen,L.C. (2003) Biochem. Biophys. Res. Commun., 303, 393398.[CrossRef][ISI][Medline]
Nicholas,K.B., Nicholas,H.B. and Deerfield,D.W. (1997) EMBnet News, 4, 1417.
Ouzzine,M., Gulberti,S., Levoin,N., Netter,P., Magdalou,J. and Fournel-Gigleux,S. (2002) J. Biol. Chem., 277, 2543925445.
Patenaude,S.I., Seto,N.O., Borisova,S.N., Szpacenko,A., Marcus,S.L., Palcic,M.M. and Evans,S.V. (2002) Nat. Struct. Biol., 9, 685690.[CrossRef][ISI][Medline]
Pedersen,L.C., Tsuchida,K., Kitagawa,H., Sugahara,K., Darden,T.A. and Negishi,M. (2000) J. Biol. Chem., 275, 3458034585.
Pedersen,L.C., Darden,T.A. and Negishi,M. (2002) J. Biol. Chem., 277, 2186921873.
Pedersen,L.C., Dong,J., Taniguchi,F., Kitagawa,H., Krahn,J.M., Pedersen,L.G., Sugahara,K. and Negishi,M. (2003) J. Biol. Chem., 278, 1442014428.
Persson,K., Ly,H.D., Dieckelmann,M., Wakarchuk,W.W., Withers,S.G. and Strynadka,N.C. (2001) Nat. Struct. Biol., 8, 166175.[CrossRef][ISI][Medline]
Rao,M. and Tvaroska,I. (2001) Proteins, 44, 428434.[CrossRef][ISI][Medline]
Rossmann,M.G., Moras,D. and Olsen,K.W. (1974) Nature, 250, 194199.[ISI][Medline]
Rost,B. and Sander,C. (1993) Protein Eng., 6, 831836.[ISI][Medline]
Rost,B. and Sander,C. (1994) Proteins, 20, 216226.[ISI][Medline]
Sali,A. and Blundell,T.L. (1993) J. Mol. Biol., 234, 779815.[CrossRef][ISI][Medline]
Schonbrun,J., Wedemeyer,W.J. and Baker,D. (2002) Curr. Opin. Struct. Biol., 12, 348354.[CrossRef][ISI][Medline]
Schwientek,T. et al. (2002) J. Biol. Chem., 277, 2262322638.
Sippl,M.J. (1993) Proteins, 17, 355362.[ISI][Medline]
Tarbouriech,N., Charnock,S.J. and Davies,G.J. (2001) J. Mol. Biol., 314, 655661.[CrossRef][ISI][Medline]
Ten Hagen,K.G. and Tran,D.T. (2002) J. Biol. Chem., 277, 2261622622.
Ten Hagen,K.G., Fritz,T.A. and Tabak,L.A. (2003) Glycobiology, 13, 116.
Thompson,J.D., Higgins,D.G. and Gibson,T.J. (1994) Nucleic Acids Res., 22, 46734680.[Abstract]
Tsai,Y.W., Chia,J.S., Shiau,Y.Y., Chou,H.C., Liaw,Y.C. and Lou,K.L. (2000) FEMS Microbiol. Lett., 188, 7579.[CrossRef][ISI][Medline]
Unligil,U.M. and Rini,J.M. (2000) Curr. Opin. Struct. Biol., 10, 510517.[CrossRef][ISI][Medline]
Unligil,U.M., Zhou,S., Yuwaraj,S., Sarkar,M., Schachter,H. and Rini,J.M. (2000) EMBO J., 19, 52695280.
Vrielink,A., Ruger,W., Driessen,H.P. and Freemont,P.S. (1994) EMBO J., 13, 34133422.[Abstract]
Zhang,Z., Kochhar,S. and Grigorov,M. (2003) Protein Sci., 12, 22912302.
Received July 13, 2004; revised September 6, 2004; accepted September 10, 2004.
Edited by Stephen Withers