Open Access

Fold-recognition and comparative modeling of human α2,3-sialyltransferases reveal their sequence and structural similarities to CstII from Campylobacter jejuni

BMC Structural Biology20066:9

DOI: 10.1186/1472-6807-6-9

Received: 28 November 2005

Accepted: 19 April 2006

Published: 19 April 2006

Abstract

Background

The 3-D structure of none of the eukaryotic sialyltransferases (SiaTs) has been determined so far. Sequence alignment algorithms such as BLAST and PSI-BLAST could not detect a homolog of these enzymes from the protein databank. SiaTs, thus, belong to the hard/medium target category in the CASP experiments. The objective of the current work is to model the 3-D structures of human SiaTs which transfer the sialic acid in α2,3-linkage viz., ST3Gal I, II, III, IV, V, and VI, using fold-recognition and comparative modeling methods. The pair-wise sequence similarity among these six enzymes ranges from 41 to 63%.

Results

Unlike the sequence similarity servers, fold-recognition servers identified CstII, a α2,3/8 dual-activity SiaT from Campylobacter jejuni as the homolog of all the six ST3Gals; the level of sequence similarity between CstII and ST3Gals is only 15–20% and the similarity is restricted to well-characterized motif regions of ST3Gals. Deriving template-target sequence alignments for the entire ST3Gal sequence was not straightforward: the fold-recognition servers could not find a template for the region preceding the L-motif and that between the L- and S-motifs. Multiple structural templates were identified to model these regions and template identification-modeling-evaluation had to be performed iteratively to choose the most appropriate templates. The modeled structures have acceptable stereochemical properties and are also able to provide qualitative rationalizations for some of the site-directed mutagenesis results reported in literature. Apart from the predicted models, an unexpected but valuable finding from this study is the sequential and structural relatedness of family GT42 and family GT29 SiaTs.

Conclusion

The modeled 3-D structures can be used for docking and other modeling studies and for the rational identification of residues to be mutated to impart desired properties such as altered stability, substrate specificity, etc. Several studies in literature have focused on the development of tools and/or servers for the large-scale/automated modeling of 3-D structures of proteins. In contrast, the present study focuses on modeling the 3-D structure of a specific protein of interest to a biochemist and illustrates the associated difficulties. It is also able to establish a sequence/structure relationship between sialyltransferases of two distinct families.

Background

Sialyltransferases (SiaTs) catalyze the transfer of sialic acid from CMP-Neu5Ac donor substrate to the terminal non-reducing saccharide of glycoproteins or glycolipids [14]. They are type II transmembrane proteins with a short, cytoplasmic N-terminal domain followed by a transmembrane domain, a flexible stem region of variable length and a catalytic domain. SiaTs use a variety of glycoconjugates as acceptor substrates in vivo; they can also use mono-, di- or oligo-saccharides as acceptor substrates in vitro. The sialic acid residue can be transferred in α2,3-linkage to Gal, in α2,6-linkage to Gal, GlcNAc or GalNAc and in α2,8-/α2,9-linkage to another sialic acid. SiaTs constitute a superfamily and have been further classified as ST3 (α2,3), ST6Gal (α2,6 to Gal), ST6GalNAc (α2,6 to GalNAc) and ST8 (α2,8/9) families on the basis of the linkage in which sialic acid is transferred [5]. Further classification viz., ST3Gal I, ST3Gal II, etc., is based on acceptor specificity and amino acid sequence.

Eukaryotic SiaTs share four sequence motifs in their catalytic domain; these are L- (large), S- (small), and VS- (very small) motifs [6] and motif III [7]. The roles of conserved residues found within the sialylmotifs have been investigated by site-specific mutation analyses in ST6Gal I. Residues in the L-motif have been implicated in binding donor substrate [8] whereas those in the S-motif have been implicated in binding both the donor and acceptor substrates [8, 9]. Mutation of the conserved His residue in the VS-motif to Lys led to loss of activity [10]. Mutating the conserved histidine in VS-motif to alanine gave rise to an enzyme with no activity. Similarly, mutations of histidine and tyrosine residues in motif III to alanine in ST3Gal I also resulted in complete loss of enzyme activity [7]. These motifs are common to all SiaTs and are thus expected to be involved in shared functions such as donor substrate binding, folding and maintaining proper 3-D structure, and catalysis.

The residues that are not conserved across the families are expected to generate differential acceptor specificity, oligomerization, protein-protein interaction, etc. A recent sequence analysis study identified linkage- (family-) specific sequence motifs [11]. Two motifs were found to be unique to the ST3Gal family: 185TTx(4)YPE193 and 209FKxxDxxW216 (human ST3Gal I numbering; accession no. AAA36612). The former motif is contiguous to the L-motif. These motifs, being specific to the ST3 family, are expected to contribute to the characteristic linkage- and acceptor substrate-specificities of the family members [11].

Knowledge of the 3-D structure of SiaTs is crucial to understand the origin of the substrate specificity and to rationalize the site-specific mutation data on the conserved residues in sialylmotifs. This knowledge will also help in establishing the structure-function relationship in this family of proteins and thereby in generating SiaTs with modified substrate specificity for chemo-enzymatic synthesis of oligosaccharides. However, the 3-D structure of none of the eukaryotic SiaTs is known to date. In view of this, the 3-D structures of six human SiaTs belonging to ST3Gal family have been modeled using fold-recognition and comparative modeling methods. Six different ST3Gals were considered for modeling since their pair-wise sequence similarity ranges from 41 to 66% and they are expected to share the same fold because of their biochemical functional similarities.

Fold-recognition servers identified CstII, a α2,3/8 dual-activity SiaT from Campylobacter jejuni as the homolog of all the six ST3Gals. The generated 3-D models have acceptable stereochemistry. It was also possible to provide a structure-based rationalization for the functional behavior of many of the site-specific mutants. Independent modeling of the six ST3Gals leading to the similar structures enhanced the confidence levels in the generated models. The results also establish that the GT29 and GT42 family SiaTs share sequence and structural similarities.

Results and discussion

Sequence similarity between ST3Gals

Pairwise sequence similarity between ST3Gal I, II, III, IV, V and VI ranges from 41 to 66% (see Additional file 1). The similarity is higher (45–80%) in the region from the L-motif up to the C-terminus. ST3Gal I and II are more similar to each other than they are to other four ST3s as has been noted previously [5]. The six ST3Gal sequences were also multiply aligned using the TCoffee server (Figure 1). The level of confidence in the alignment is quite high, as judged by the confidence scores generated by the TCoffee algorithm, except in regions encompassing the stem, transmembrane and N-terminal cytoplasmic domains (Figure 1). The motifs of the SiaT superfamily (L-, S- and VS-motifs and motif III) and linkage-specific motifs of the ST3 family align with each other. A cysteine residue in the stem region is conserved in all the six ST3Gals (Figure 1).
Figure 1

Multiple sequence alignment of ST3Gals (Table 1) obtained from the Tcoffee server [41]. A hyphen (" - ") indicates a one-residue gap. The ruler at the top is for the entire alignment and has no reference to any individual sequence; the latter are numbered on the right. The residues constituting the various motifs are marked below: L-motif (alignment position 173–216, denoted by *), linkage-specific motifs (219–227, denoted by & and 248–255, denoted by #), S-motif (321–343, denoted by @), motif III (356–359, denoted by %) and VS-motif (373–378, denoted by +). The structural and functionally important residues identified by mutation studies (Table 2) have been highlighted in red with bold font. Confidence is the confidence score given by Tcoffee. The regions having the same secondary structure in all the ST3Gals are also shown (marked cons-sec for consensus secondary structure). The names of the various strands and helices, indicated below the consensus secondary structure, are the same as those of the corresponding regions in CstII [13]. Residues highlighted in yellow and cyan constitute α-helices and β-strands, respectively, (as identified by SwissPDBviewer/RasMol) in at least one of the top models. The conserved cysteine residue in the stem region is highlighted in pink.

Table 1

Accession numbers and modelled regions of human SiaTs

Name

NCBI accession number

Length (number of residues)

Transmembrane domain

Modeled region§

Reference

ST3Gal I

AAA36612

340

12–35

66–332

[63]

ST3Gal II

NP_008858

350

7–29

76–347

[64]

ST3Gal III

AAA35778

375

4–26

92–369

[65]

ST3Gal IV

AAA16460

332

5–20

51–329

[66]

ST3Gal V

Q9UNP4

362

15–32

70–354

[67]

ST3Gal VI

Q9Y274

331

10–32

51–323

[19]

Predicted using the TMHMM server [42].

§All the residues of the protein except those in the N-terminal cytoplasmic, transmembrane domains and last few residues of the catalytic region were considered for 3-D structure modeling. However, part of the neck/stem region could not be modeled for reasons detailed in the text.

Table 2

Roles deduced for some of the residues which are conserved in the eukaryotic SiaT superfamily and whose mutations have been experimentally characterized#

Mutant

Equivalent residue in ST3Gal I

Km(μM) Donor/Acceptor

Activity

Role deduced for the mutated residue from the modeled 3-D structures

Mutations in L motif

Wild type

 

50/331

100%

 

C181A

C142

-/-

< 5%

Structural role: involved in disulphide bridge

V184A

V145

300/372

~45%

Structural role: part of hydrophobic core

L190A

L151

597/188

~28%

Structural role: part of hydrophobic core

R207A

R168

-/-

< 5%

Structural role: is buried and hydrogen bonds with side chains of N147 and E178 (ST3Gal I numbering). N147 is replaced by Ser in ST3Gal V, but Ser does not form hydrogen bond with Arg

V220A

V181

343/260

30%

Structural role: part of the hydrophobic core

S222A

T183

-/-

< 5%

Structural role: These are at beginning of strand β4 and are solvent exposed

K223A

K184

330/400

32%

 

T225A

T186

160/200

27%

 

Mutations in S motif§

Wild type

 

50/330

100

 

P318A

P267

-/-

very low

Structural role: helix J nucleator

S319A

S268

-/-

< 5%

Functional role: close to ribose

C332A

C281

-/-

< 5%

Structural role: forms disulphide bridge

V335L

V284

80/322

79%

Structural role: part of the hydrophobic core

V335A

V284

89/947

83%

 

Mutations in motif-III*

H299A

 

-/-

loss of activity

Functional role: close to phosphate

Y300A

 

-/-

  

#Residues that are either strictly conserved or have conservative replacements in the eukaryotic SiaT superfamily are from [11].

Data corresponds to mutants of ST6Gal I [8]. Hyphen (" - ") indicates values could not be determined.

§Data corresponds to mutants of ST6Gal I [9]. Hyphen (" - ") indicates values could not be determined.

*Data corresponds to mutants of ST3Gal I [7]. Hyphen (" - ") indicates values could not be determined.

The mutation is considered structural if it destabilizes the structure and is far from functional site and it is termed functional if it is part of the substrate binding site.

Are part of the same hydrophobic core.

Secondary structure prediction

A consensus secondary structure was derived for each SiaT based on the results from eight secondary structure prediction servers (see Additional file 2). The region predicted by the TMHMM server as the transmembrane domain is predicted to be helical in all the ST3Gals. The sequence and length of the region between the transmembrane domain and L-motif in the six ST3Gals are different; this region has only helices but the number of helices varies between 3 and 5. The significance of this variability and its relevance (if any) to differences in acceptor substrate specificity are as yet unknown. The order of occurrence of the secondary structural elements from the L-motif onwards is very nearly the same in all the ST3Gals. The L-motif region is made of coils and strands. The S-motif begins with a helix, immediately followed by a strand. The six-residue-long VS-motif is partly helical. The region between the L- and S-motifs has a mixture of strands and helices. Of the two ST3 family-specific motifs, TTx(4)YPE is part of a strand and FKxxDxxW is in coil conformation.

Overall, 25–32% of residues are in helices and 9–12% residues in strands. The conservation of the nature and order of occurrence of secondary structural elements is strongly suggestive of the conservation of the overall fold in these ST3Gals. It can be inferred from the predicted secondary structures that ST3Gals belong to the α/β class, as defined in the SCOP database [12]. Other glycosyltransferases (GlyTs) whose 3-D structures have been determined so far also belong to the same class. Within this class, there are three fold types designated as nucleotide-diphospho-sugar transferases, UDP-glycosyltransferase/glycogen phosphorylase and α-2,3/8-sialyltransferase CstII.

Template identification by fold-recognition servers

Two approaches were employed to identify the potential templates: (1) Submitting a multiple sequence alignment (MSA) of all the six ST3Gals and (2) Submitting each of the six ST3Gal sequences individually. In the former, MSA for the entire sequence from N- to C-terminus (Figure 1) was submitted to the FUGUE server; the templates that were identified had very low confidence levels (Z-score for the top hit = 2.54; guess). Even the GeneSilico metaserver identifies templates with very low confidence levels (pcons5 score for the top hit = 0.15; unreliable); the α2,3/8 dual-activity sialyltransferase CstII from Campylobacter jejuni (PDB id 1RO7 [13]; referred to as CstII henceforth) has a pcons5 score of 0.09. However, the alignment with CstII began from only the L-motif onwards of ST3Gals; no template was identified for the region preceding the L-motifs, most likely due to the very low sequence similarity in this region of the ST3Gals. In view of these, MSA starting from the L-motif onwards up to the C-terminus was submitted to these servers. Both the servers identify CstII as the top hit (Z-score = 5.2; likely and pcons5 score = 0.32; unreliable).

In the second approach, complete sequence from N- to C-terminus of the six ST3Gals was used separately as query to search for homologs in the PDB database using BLAST and PSI-BLAST. No significant hits were obtained. Among the fold-recognition servers, only FFAS03 and the GeneSilico metaserver identified CstII as a hit and the alignment began from the L-motif region of ST3Gals. However, if only the sequence from L-motif onwards is used as query, then even FUGUE and SAM-T02 servers identify CstII as the possible template with a high level of confidence (see Additional file 3). The template-target alignments generated for motif regions (Figure 1) when ST3Gal sequences were submitted individually were same as that obtained by submitting the multiple sequence alignment. In all the cases, the secondary structures of the target and template residues in the alignment regions 250–290 (Figure 1) were entirely different (Figure 2).
Figure 2

Target (ST3Gal I) – template (CstII, PDB id 1RO7) sequence alignment generated by the fold-recognition servers. The alignments were generated by the indicated servers when ST3Gal I sequence from the L-motif region till the C-terminus was used as query. Regions of alignment that are same for the four servers are highlighted. The alignments for the region spanning residues 215 to 254 of ST3Gal I generated by the four servers are different. The mismatch in alignment of secondary structures of target -- template are also seen in these regions. The helices are highlighted as yellow and the strands are shown in blue (as in Figure 1). Such a disagreement for this region is seen in the case of other ST3Gals also.

The alignments generated by different servers do not agree with each other in some regions. The disagreement was resolved based on secondary structure states of the residues at some regions. For example, the residues 215–254 of ST3Gal I are aligned differently with CstII by the four fold-recognition servers (Figure 2); even the secondary structure states of the aligned residues are different (Figure 2). A similar mismatch was found for the corresponding region of other ST3Gals also. For such regions, other template(s) that would satisfy the predicted secondary structure in that region were identified by submitting only the relevant part of the sequence to the fold-recognition servers and/or PSI-BLAST (see Additional file 4). Thus, the use of pair-wise target-template alignment seems to be more appropriate than deriving templates based on multiple sequence alignment [14].

Sequence alignment for regions preceding L-motif in ST3Gals

The membrane-association region in CstII is at the C-terminus [13] unlike the human SiaTs, which have the transmembrane domain at the N-terminus (see Additional file 5). Consequently, the N-terminus of ST3Gals (~150 residues containing the cytoplasmic and transmembrane domains and the stem region) and the C-terminus of CstII (~90 residues; containing the membrane-association region) are left out of alignment generated by the fold-recognition servers. The alignment begins with the N-terminus of CstII and L-motif of ST3Gals; specifically, Lys2 of CstII aligns with Arg140 (ST3Gal I numbering), the second residue of the L-motif (Figure 2). Reversing the directionality of the polypeptide chain in the C-terminus of CstII (i.e., from residue 210 onwards) sets the transmembrane domain of ST3Gal in a position equivalent to the membrane-association region of CstII. The N-terminal region preceding the L-motif of ST3Gals was thus modeled following the Cα-trace of the CstII C-terminus in reverse direction. A considerable amount of similarity in secondary structures was also observed in these regions.

Modeling 3-D structures starting from alignments

The 3-D structure of CstII (PDB id 1RO7; A chain) is the main template for modeling the 3-D structures of all the ST3Gals. Additional templates have been used for regions, which do not have a match in CstII by separately submitting the sequence of these regions to fold-recognition servers and PSI-BLAST (see Additional file 6). Even after this step, suitable templates could not be found for some regions immediately following the transmembrane domain; these regions were not modeled (Table 1). The combined sequence alignments (see Additional file 6) were used to model the 3-D structures of ST3Gals. Only the backbone conformation of the template is taken, and side chains are modeled independently, in regions where the template – target sequences disagree. Modeller uses a loop algorithm to model regions for which no template is specified. Twenty-five models were generated for each ST3Gals. The different structures vary in their backbone conformation, especially in regions that did not have a template, and in side chain conformations.

Stereochemical evaluation of the predicted models

The stereochemical properties and quality of all the models were evaluated by MODELLER, PROCHECK and Verify3D (see Additional file 7). Three to four models were selected for each ST3Gal based on these evaluations. For all the selected models, the value of the objective function, reported as current energy by MODELLER, is in the same range as that if the template is aligned with its own sequence. On an average, 87% of the residues are found in the allowed region of Ramachandran map; PROCHECK considers the model to be very good if it has 90% of the residues in the most favored region. The inter-atomic distances are within acceptable range. Verify3D score is greater than zero for the region from the L-motif onwards but the score drops below 0 for certain regions preceding the L-motif. The models were also evaluated using Colorado3D server, which facilitates the change of amino acid window size when calculating the overall score. Two window sizes, 5 and 21, were used to calculate the average Verify3D and ProsaII score per residue for each of the top models and 25 models generated for the template. The scores calculated using these two window sizes were found to be very similar (see Additional file 7). The template and target models were rendered with the residues color-coded based on ProsaII (see Additional file 8) and verify3D (see Additional file 9) scores. With ProsaII score-based coloring, most of the residues are green and yellow (i.e., average score) in both the target and template proteins (see Additional file 8). With verify3D score-based coloring, even the template proteins has residues in red color (i.e., bad score) although the number of such residues are more in the targets (see Additional file 9).

Characterization and comparison of modeled ST3Gal structures

The ST3Gal fold is characterized by a six-stranded (β7, β1, β2, β4, β5 and β6; Figure 3) parallel β-sheet flanked on the two sides by strands β8 and β5' in an antiparallel orientation; strand β8 is present in only some ST3Gals (Figure 1). Helices E, F and I share a common interface and are in spatial proximity of strands β1, β2, β4 and β5 (Figure 4). Helices A and B are very small i.e., 3 to 4 residue long. Helices B and K' are found in only some ST3Gals.
Figure 3

Topology diagrams for CstII (PDB ID: 1RO7) and modeled ST3Gals. Triangles and circles represent strands and helices, respectively. Strands 5' and 8 are antiparallel to other strands in ST3Gals and are hence inverted. Certain helices and strands are not found in all the modeled ST3Gals (Figure 1) and these are represented by dotted lines. The nomenclature used to identify strands and helices in CstII are the same as those used by Chiu et al. [13]. The equivalent secondary structure elements in ST3Gals are identified by the same names. Strand 9 and helices D, G and H are absent in ST3Gals; strand 5' and helix K' are absent in CstII. Note that the region of the polypeptide from strand 8 till helix K is at the C-terminus in CstII but they are at the N-terminus in ST3Gals; hence, the direction of the polypeptide chain is opposite to one another in this region.

Figure 4

Cartoon diagram of human ST3Gal I modeled using the structure of CstII (PDB id 1RO7). Helices (A, B, C, E, F, I, J, K and K'; in yellow) and strands (β1, β2, β4, β5, β5', β6, β7 and β8; in cyan) have been given the same names as those of corresponding helices and strands in CstII. Residues constituting these helices and strands are given in Figure 1. The location of CMP-3-fluoro-NeuNAc (stick representation; carbon, green; oxygen, red; nitrogen, blue) has been derived by superposition of the modeled structure on that of the CstII- CMP-3-fluoro-NeuNAc complex. The structure was rendered using PyMol.

The 3-D structures of ST3Gals compare well with each other to a large extent. Strands β7, β1, β2, β4, β5 andβ6 and helices E, F and I in various ST3Gals superpose well on each other (Figure 5). The length of the loop region between helices E and F is variable (Figure 1): it is shorter in ST3Gal I and II compared to that in the other four ST3Gals. It has been reported that ST3Gal I and II do not bind substrates that contain GlcNAc attached to terminal galactose whereas the other four do bind such substrates, albeit with varying affinities [1520]. The relationship between the size of H6-H7 loop and the observed differences in the acceptor substrate specificities needs experimental validation. The conformation of the region from helix C to strand β6 also varies in different ST3Gals. This difference is due to differences in the amino acid sequences, which, in turn, required the use of different templates for modeling these regions.
Figure 5

3-D rendering of the backbone traces of ST3Gal I, II, III, IV, V and VI superposed on each other using SwissPDBViewer. Helices A, E, F and I (all in cyan) and strands β1, β2, β4, β5, β5', β6 and β7 (all in yellow) superpose well on each other. Residues constituting these helices and strands are given in Figure 1. Loops and other helices/strands have different conformations in the six proteins and hence have been shown for only ST3Gal I (dark gray). PyMol was used for rendering.

Comparison of the modeled structures with CstII structure

The modeled 3-D structures of ST3Gals are similar to, but not exactly same as, that of CstII (Figure 3). The similarity is to be expected since CstII was the main template for deriving the models. Helix B is 8–10 residues long in CstII; in ST3Gals, it is only a helical loop formed by a few residues in the alignment region 226–231 (Figure 1). Helix J is not as prominent in CstII as it is in the modeled ST3Gals. The average RMS deviation between the target (ST3Gals) and template (CstII) structures is calculated to be 1.9 Å by the SSM server and 2.4 Å by the DALI server (see Additional file 10). The 3-D structure of no other protein was found to be similar to that of ST3Gals by the SSM and DALI servers.

Residues involved in binding to CMP-Neu5Ac, the donor substrate

CstII and ST3Gals are both sialyltransferases and use the same donor substrate, CMP-Neu5Ac. The crystal structure of CstII has been determined in complex with the donor substrate analog, CMP-3-fluoro-NeuNAc (PDB id 1RO7) [13]. The modeled ST3Gal structures were superposed on the structure of CstII; for this purpose, the backbone atoms of the residues constituting the L-, S- and VS-motifs were used as reference atoms. This enabled the identification of residues that are likely to interact with CMP-Neu5Ac in ST3Gals. The residues that are found within 5 Å from CMP-Neu5Ac were found to be part of the L-, S- and VS-motifs, motif III and one of the ST3Gal family-specific motifs viz., TTx(4)YPE (Figure 6A). The second family-specific motif FKxxDxxW is in spatial proximity of TTx(4)YPE and seems to have a role in binding the acceptor substrate (Figure 6A). In this putative binding mode, the loop between β7 and helix I is near cytosine, beginning of L-motif is near ribose, Tyr300 (ST3Gal I numbering) is close to phosphate, middle of L-motif is close to phosphate and sialic acid, and Tyr191 (ST3Gal I numbering), beginning of S-motif, His of VS-motif are close to sialic acid (Figure 6B).
Figure 6

3-D renderings of ST3Gal I model highlighting the various functionally and structurally important residues and regions. The location of the ligand CMP-3-fluoro-NeuNAc as shown in these renderings has been derived by superposition of the modeled structure on that of the CstII- CMP-3-fluoro-NeuNAc complex. (A, top left) 3-D rendering showing the L-motif (magenta), S-motif (purple), VS-motif (burgundy), motif III (yellow), linkage-specific motifs TTx(4)YPE (orange) and FKxxDxxW (green) and (stick diagram). (B, top right) Location of some of the amino acid residues (colored green) whose roles have been investigated by site-directed mutagenesis studies (Table 2). The loop proposed in this study as important for nucleotide binding is shown in blue. The nomenclature used for identifying helices (cyan) and strands (magenta) is as that in Figure 1. (C, bottom left) The interactions of the conserved Arg168 with Asn147 and Glu178. All three residues are buried within the protein. The disulphide bridge (colored red) proposed as required for structural stability has also been displayed. The ligand is in pink. (D, bottom right) 3-D rendering showing the cluster of apolar residues proposed as important for maintaining the 3-D structure (Table 2). Note that not all these residues are in direct contact with the ligand.

Location of residues whose functional importance has been studied by site-specific mutations

Site-directed mutagenesis has been used to investigate the role of several residues conserved in SiaT superfamily [710, 21]. Quantitative analysis of rat ST6Gal I indicated the presence of only one disulphide bond although the enzyme has seven cysteine residues [21]. All the modeled ST3Gals have one disulphide bond between two conserved cysteine residues, one present at the beginning of the L-motif and the other in the middle of the S-motif (Figure 6C). These two cysteine residues come in spatial proximity of each other when no specific constraints were used for the purpose of bringing them together. This disulfide bridge holds the β-strand of L-motif and the helix of S-motif together and is away from the putative CMP-Neu5Ac binding site (Figure 6C). Hence, mutation of either of these two residues is expected to destabilize the enzyme and consequently, lead to loss of activity. Structural/functional roles have also been deduced for other residues that are conserved in the SiaT superfamily based on the modeled 3-D structures; these deductions are in consonance with the results of experimental site-specific mutation studies (Table 2; Figure 6D).

Relationship between family GT29 and family GT42 SiaTs

Eukaryotic [35] and prokaryotic [2227] SiaTs have been classified into four families based on sequence similarity in the CAZy database [28]: (a) family GT29 contains viral and eukaryotic SiaTs; these enzymes have α2,3-, α2,6-, and α2,8-activities; (b) family GT38 contains bacterial polySiaTs mainly from Escherichia coli and Neisseria meningitides; (c) family GT42 contains SiaTs from Campylobacter jejuni and Haemophilus influenzae and (d) family GT52 contains α2,3-SiaT from Neisseria gonorrhoeae, Neisseria meningitides and few hypothetical SiaTs from Haemophilus influenzae. No sequence-based evolutionary relationship among these SiaT families has been established till date. Surprisingly, CstII was identified as the template for modeling the 3-D structures of human ST3Gals by fold-recognition servers; CstII belongs to family GT42 whereas human ST3Gals belong to family GT29. The modeled 3-D structures were found to be stereochemically acceptable and also were able to provide qualitative explanations for some of the site-specific mutagenesis data.

The L-, S- and VS-motifs characteristic of mammalian SiaTs are thought to be absent in prokaryotic SiaTs [5]. The residues in CstII which correspond to these motif regions were identified by the structure-based sequence alignment generated by fold-recognition servers. A multiple sequence alignment of 14 experimentally characterized ST3Gal sequences (same as those in [11]) was submitted to the FUGUE server, which aligned these to CstII (Z score = 5.35). Using this alignment, multiple sequence alignments of experimentally characterized ST3Gals and family GT42 SiaTs were merged (see Additional file 11) and sequence logos were generated (Figure 7). Several residues in the L-, S- and VS-motif regions were found to be either strictly conserved or have conservative replacements in GT42 family SiaTs. This suggests that family GT42 SiaTs also have the L-, S- and VS-motifs (alignment positions 17–59, 165–189 and 225–230, respectively, in see Additional file 11). Conserved residues are found in other regions also (see Additional file 11). One such is the proline residue immediately after the L-motif (corresponding to position 54 in Figure 7); this residue is conserved in ST8 family also [11].
Figure 7

Sequence logos of (a) extended L-motif (b) S-motif (c) VS-motif. The conserved regions for generating the logos were extracted from the multiple sequence alignment of 23 experimentally characterized only α2,3-SiaTs from GT29 families and GT42 families (see Additional file 11). The multiple sequence alignment was generated using the FUGUE server. The colors used for the logos are as follows: blue, Lys, Arg and His; green, Cys, Ser, Gly, Thr and Tyr; red, Asp and Glu; pink, Asn and Gln; black, Ala, Val, Leu, Phe, Ile, Met, Pro and Trp.

Family GT29 is actually a superfamily consisting of ST3Gal, ST6Gal, ST6GalNAc and ST8Sia families [5]. CstII was identified as the top hit by the fold-recognition server FFAS03 even for the human ST6Gal, ST6GalNAc and ST8Sia family members; the E-value in these cases is comparable to that obtained for ST3Gals. This suggests that other members of the GT29 family also share the CstII fold and thereby establish the structural similarities between GT29 and GT42 family members. On the contrary, CstII was not identified as a potential template when representative members of GT38 and GT52 families were submitted to FFAS03 server. This indicates the absence of any detectable structural similarities of GT38 and GT52 families with GT29 and GT42 family SiaTs.

Conclusion

The knowledge of the 3-D structures of glycosyltransferases is important to better understand their biological function and to delineate structure-function relationships, as borne out, for example, in the case of galactosyltransferases [2931]. This latter aspect is especially beneficial for the chemoenzymatic synthesis of carbohydrates and in turn, for glycomics (see, for example, [32]). SiaTs are another equally important class of glycosyltransferases but the 3-D structure for none of the human SiaTs is available till date. In light of these, the 3-D structure models of ST3Gals obtained in this study can be used to identify mutations that are likely to alter the donor and/or acceptor substrate specificities, thereby facilitating their use in the chemoenzymatic synthesis of complex carbohydrates and also to refine the predicted structures in the present study. This study has also provided another example of sequentially divergent proteins sharing a common fold to perform the same biochemical function.

Methods

Databases

The amino acid sequences of the experimentally characterized, human SiaTs belonging to the ST3Gal family (Table 1) were retrieved from the protein sequence database at NCBI http://www.ncbi.nlm.nih.gov. The 3-D structures of proteins were obtained from the protein data bank [33]. The fold classification of proteins is from the SCOP database [12, 34].

Servers

Protein sequence databases were searched using BLAST [35] or PSI-BLAST [36] servers at NCBI. FFAS03 [37], FUGUE [38], PHYRE (successor of 3D-PSSM, [39]), SAM-T02 [40] and GeneSilico Metaserver [41] were used for fold-recognition. Multiple sequence alignments were obtained using the TCoffee server [42, 43]. Transmembrane helices were predicted using the TMHMM server v. 2.0 [44]. Secondary structures were predicted using the APSSP [45], JPRED [46], NNPREDICT [47], PROF [48], PSIPRED [49], SAM-T99 [50], SOPMA [51] and SSPRO [52] servers. Verify3D [53, 54] and Colorado3D [55] were used to evaluate the models. DALI [56] and SSM [57] servers were used for 3-D structure comparisons. Sequence logos were created using WebLogo (version 2.8.1) [58]. All the servers were used with default values for the various parameters, except where mentioned otherwise.

Software and hardware

BioEdit [59] was used for display and manipulation of sequences. SwissPDBviewer [60], Rasmol [61] and PyMol [62] were used to visualization and/or rendering. Modeller6v1, a homology modeling software, was used for modeling the 3-D structures [63, 64]. The stereochemical quality of the generated model was assessed using PROCHECK [65, 66]. All the software were run on an Intel Pentium IV desktop personal computer, except for modeller6v1, which was run on a SGI octane workstation. Default values were used for all the parameters, unless specified otherwise.

Secondary structure prediction

The secondary structures of each of the six ST3Gals were predicted separately using eight prediction servers mentioned earlier. The secondary structures were predicted as three states, helix (H), strand (E) and coil (C). A consensus secondary structure was obtained by comparing the predictions of the eight servers. If different secondary structure states are predicted for a residue by the servers, the state that has been predicted by at least five of eight servers was taken as the consensus state; in other cases, it was marked as U (uncertain).

Template-target sequence alignment

The ST3Gal sequences were submitted to fold-recognition servers separately. All the servers provide alignment of the submitted ST3Gal sequence (target) with the sequence of the potential hits (templates). Inspection of the template-target alignments generated by these fold-recognition servers revealed that certain regions of ST3Gals either did not have a template or the template-target secondary structures did not match. Such regions of ST3Gals were separately submitted to PSI-BLAST and fold-recognition servers. The best hits identified from these were then used as additional templates to model the target sequences.

Validation of predicted 3-D structures

The stereochemical properties of predicted 3-D structures were assessed by PROCHECK and the residue environments by Verify3D and Colorado3D. Regions that are found by these servers as poorly modeled were improved by iterative manual adjustment of alignments and re-modeling. In the second stage of structure validation, the ability of the predicted structures to rationalize the results from the site-specific mutagenesis experiments reported in literature was investigated.

Web links

Declarations

Acknowledgements

Authors thank Professor Andrej Sali for providing Modeller6v1. Authors also thank Mr. Ronak Y Patel for sharing his experimental database of ST3Gals and the anonymous referee for his/her useful comments. MSS is grateful to Indian Institute of Technology Bombay for teaching assistantship. This work was supported by a grant from the Council for Scientific and Industrial Research, India to PVB (Grant No. 37(1110)/02/EMR-II).

Authors’ Affiliations

(1)
School of Biosciences and Bioengineering, Indian Institute of Technology Bombay

References

  1. Angata K, Fukuda M: Polysialyltransferases: major players in polysialic acid synthesis on the neural cell adhesion molecule. Biochimie 2003, 85: 195–206. 10.1016/S0300-9084(03)00051-8View ArticlePubMedGoogle Scholar
  2. Dall'Olio F, Chiricolo M: Sialyltransferases in cancer. Glycoconj J 2001, 18: 841–850. 10.1023/A:1022288022969View ArticlePubMedGoogle Scholar
  3. Harduin-Lepers A, Vallejo-Ruiz V, Krzewinski-Recchi MA, Samyn-Petit B, Julien S, Delannoy P: The human sialyltransferase family. Biochimie 2001, 83: 727–737. 10.1016/S0300-9084(01)01301-3View ArticlePubMedGoogle Scholar
  4. Tsuji S: Molecular cloning and functional analysis of sialyltransferases. J Biochem 1996, 120: 1–13.View ArticlePubMedGoogle Scholar
  5. Harduin-Lepers A, Mollicone R, Delannoy P, Oriol R: The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach. Glycobiology 2005, 15: 805–817. 10.1093/glycob/cwi063View ArticlePubMedGoogle Scholar
  6. Datta AK, Paulson JC: Sialylmotifs of sialyltransferases. Indian J Biochem Biophys 1997, 34(1–2):157–165.PubMedGoogle Scholar
  7. Jeanneau C, Chazalet V, Auge C, Soumpasis DM, Harduin-Lepers A, Delannoy P, Imberty A, Breton C: Structure-function analysis of the human sialyltransferase ST3Gal I: role of N-glycosylation and novel conserved sialylmotif. J Biol Chem 2004, 279: 13461–13468. 10.1074/jbc.M311764200View ArticlePubMedGoogle Scholar
  8. Datta AK, Paulson JC: The sialyltransferase "sialylmotif" participates in binding the donor substrate CMP-NeuAc. J Biol Chem 1995, 270: 1497–1500. 10.1074/jbc.270.4.1497View ArticlePubMedGoogle Scholar
  9. Datta AK, Sinha A, Paulson JC: Mutation of the sialyltransferase S-sialylmotif alters the kinetics of the donor and acceptor substrates. J Biol Chem 1998, 273: 9608–9614. 10.1074/jbc.273.16.9608View ArticlePubMedGoogle Scholar
  10. Kitazume-Kawaguchi S, Kabata S, Arita M: Differential biosynthesis of polysialic or disialic acid structure by ST8Sia II and ST8Sia IV. J Biol Chem 2001, 276: 15696–15703. 10.1074/jbc.M010371200View ArticlePubMedGoogle Scholar
  11. Patel RY, Balaji PV: Identification of linkage-specific sequence motifs in sialyltransferases. Glycobiology 2006. DOI 101093/glycob/cwj046.Google Scholar
  12. Murzin AG, Brenner SE, Hubbard T, Chothia C: SCOP: a structural classification of proteins database for the investigation of sequences and structures. J Mol Biol 1995, 247: 536–540. 10.1006/jmbi.1995.0159PubMedGoogle Scholar
  13. Chiu CPC, Watts AG, Lairson LL, Gilbert M, Lim D, Wakarchuk WW, Withers SG, Strynadka NCJ: Structural analysis of the sialyltransferase CstII from Campylobacter jejuni in complex with substrate analog. Nat Struct Mol Biol 2004, 11: 163–170. 10.1038/nsmb720View ArticlePubMedGoogle Scholar
  14. Venclovas C, Thelen MP: Structure-based predictions of Rad1, Rad9, Hus1 and Rad17 participation in sliding clamp and clamp-loading complexes. Nucleic Acids Res 2000, 28: 2481–2493. 10.1093/nar/28.13.2481PubMed CentralView ArticlePubMedGoogle Scholar
  15. Fukuda M, Marth JD: ST3Gal-I. In Handbook of glycosyltransferases and related genes. Edited by: Taniguchi N, Honke K, Fukuda M. Springer-Verlag, Tokyo; 2002:267–273.View ArticleGoogle Scholar
  16. Hamamoto T, Tsuji S: ST3Gal-II (SAT-IV). In Handbook of glycosyltransferases and related genes. Edited by: Taniguchi N, Honke K, Fukuda M. Springer-Verlag, Tokyo; 2002:274–278.View ArticleGoogle Scholar
  17. Kitazume-Kawaguchi S, Tsuji S: ST3Gal-III. In Handbook of glycosyltransferases and related genes. Edited by: Taniguchi N, Honke K, Fukuda M. Springer-Verlag, Tokyo; 2002:279–283.View ArticleGoogle Scholar
  18. Kitazume-Kawaguchi S, Tsuji S: ST3Gal-IV. In Handbook of glycosyltransferases and related genes. Edited by: Taniguchi N, Honke K, Fukuda M. Springer-Verlag, Tokyo; 2002:284–288.View ArticleGoogle Scholar
  19. Saito S, Ishii A: ST3Gal-V (GM3 synthase, SAT-I). In Handbook of glycosyltransferases and related genes. Edited by: Taniguchi N, Honke K, Fukuda M. Springer-Verlag, Tokyo; 2002:289–294.View ArticleGoogle Scholar
  20. Okajima T, Fukumoto S, Miyazaki H, Ishida H, Kiso M, Furukawa K, Urano T, Furukawa K: Molecular cloning of a novel α2,3-sialyltransferase (ST3Gal VI) that sialylates type II lactosamine structures on glycoproteins and glycolipids. J Biol Chem 1999, 274: 11479–11486. 10.1074/jbc.274.17.11479View ArticlePubMedGoogle Scholar
  21. Datta AK, Chammas R, Paulson JC: Conserved cysteines in the sialyltransferase sialylmotifs form an essential disulphide bond. J Biol Chem 2001, 276: 15200–15207. 10.1074/jbc.M010542200View ArticlePubMedGoogle Scholar
  22. Gilbert M, Watson DC, Cunningham AM, Jennings MP, Young NM, Wakarchuk WW: Cloning of the lipooligosaccharide α2,3-sialyltransferase from the bacterial pathogens Neisseria meningitidis and Neisseria gonorrhoeae . J Biol Chem 1996, 271: 28271–28276. 10.1074/jbc.271.45.28271View ArticlePubMedGoogle Scholar
  23. Gilbert M, Brisson JR, Karwaski MF, Michniewicz J, Cunningham AM, Wu Y, Young NM, Wakarchuk WW: Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384. Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-mhz (1)h and (13)c NMR analysis. J Biol Chem 2000, 275: 3896–3906. 10.1074/jbc.275.6.3896View ArticlePubMedGoogle Scholar
  24. Hood DW, Cox AD, Gilbert M, Makepeace K, Walsh S, Deadman ME, Cody A, Martin A, Mansson M, Schweda EK, Brisson JR, Richards JC, Moxon ER, Wakarchuk WW: Identification of a lipopolysaccharide alpha-2,3-sialyltransferase from Haemophilus influenzae. Mol Microbiol 2001, 39: 341–350. 10.1046/j.1365-2958.2001.02204.xView ArticlePubMedGoogle Scholar
  25. Jones PA, Samuels NM, Phillips NJ, Munson RS Jr, Bozue JA, Arseneau JA, Nichols WA, Zaleski A, Gibson BW, Apicella MA: Haemophilus influenzae type b strain A2 has multiple sialyltransferases involved in lipooligosaccharide sialylation. J Biol Chem 2002, 277: 14598–14611. 10.1074/jbc.M110986200View ArticlePubMedGoogle Scholar
  26. Shen GJ, Datta AK, Izumi M, Koeller KM, Wong CH: Expression of alpha2,8/2,9-polysialyltransferase from Escherichia coli K92. Characterization of the enzyme and its reaction products. J Biol Chem 1999, 274: 35139–35146. 10.1074/jbc.274.49.35139View ArticlePubMedGoogle Scholar
  27. Yamamoto T, Nakashizuka M, Terada I: Cloning and expression of a marine bacterial beta-galactoside alpha2,6-sialyltransferase gene from Photobacterium damsela JT0160. J Biochem 1998, 123: 94–100.View ArticlePubMedGoogle Scholar
  28. Coutinho PM, Deleury E, Davies GJ, Henrissat B: An evolving hierarchical family classification of glycosyltransferases. J Mol Biol 2003, 328: 307–317. 10.1016/S0022-2836(03)00307-3View ArticlePubMedGoogle Scholar
  29. Qasba PK, Ramakrishnan B, Boeggeman E: Substrate-induced conformational changes in glycosyltransferases. Trends Biochem Sci 2005, 30: 53–62. 10.1016/j.tibs.2004.11.005View ArticlePubMedGoogle Scholar
  30. Ramakrishnan B, Boeggeman E, Ramasamy V, Qasba PK: Structure and catalytic cycle of β1,4-galactosyltransferase. Curr Opin Struct Biol 2004, 14: 593–600. 10.1016/j.sbi.2004.09.006View ArticlePubMedGoogle Scholar
  31. Zhang Y, Deshpande A, Xie Z, Natesh R, Acharya KR, Brew K: Roles of active site tryptophans in substrate binding and catalysis by α1,3-galactosyltransferase. Glycobiology 2004, 14: 1295–1302. 10.1093/glycob/cwh119View ArticlePubMedGoogle Scholar
  32. Khidekel N, Arndt S, Lamarre-Vincent N, Lippert A, Poulin-Kerstien KG, Ramakrishnan B, Qasba PK, Hsieh-Wilson LC: A chemoenzymatic approach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications. J Am Chem Soc 2003, 125: 16162–16163. 10.1021/ja038545rView ArticlePubMedGoogle Scholar
  33. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE: The protein data bank. Nucl Acids Res 2000, 28: 235–242. 10.1093/nar/28.1.235PubMed CentralView ArticlePubMedGoogle Scholar
  34. Andreeva A, Howorth D, Brenner SE, Hubbard TJP, Chothia C, Murzin AG: SCOP database in 2004: refinements integrate structure and sequence family data. Nucl Acids Res 2004, 32: D226-D229. 10.1093/nar/gkh039PubMed CentralView ArticlePubMedGoogle Scholar
  35. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 1990, 215: 403–410. 10.1006/jmbi.1990.9999View ArticlePubMedGoogle Scholar
  36. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acids Res 1997, 25: 3389–3402. 10.1093/nar/25.17.3389PubMed CentralView ArticlePubMedGoogle Scholar
  37. Jaroszewski L, Rychlewski L, Li Z, Li W, Godzik A: FFAS03: a server for profile-profile sequence alignments. Nucl Acids Res 2005, 33: W284-W288. 10.1093/nar/gki418PubMed CentralView ArticlePubMedGoogle Scholar
  38. Shi J, Blundell TL, Mizuguchi K: FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure- dependent gap penalties. J Mol Biol 2001, 310: 243–257. 10.1006/jmbi.2001.4762View ArticlePubMedGoogle Scholar
  39. Kelley LA, MacCallum RM, Sternberg MJE: Enhanced Genome Annotation using Structural Profiles in the Program 3D-PSSM. J Mol Biol 2000, 299: 499–520. 10.1006/jmbi.2000.3741View ArticlePubMedGoogle Scholar
  40. Karplus K, Karchin R, Draper J, Casper J, Mandel-Gutfreund Y, Diekhans M, Hughey R: Combining local-structure, fold-recognition, and new-fold methods for protein structure prediction. Proteins 2003, 53: 491–496. 10.1002/prot.10540View ArticlePubMedGoogle Scholar
  41. Kurowski MA, Bujnicki JM: GeneSilico protein structure prediction meta-server. Nucleic Acids Res 2003, 31: 3305–3307. 10.1093/nar/gkg557PubMed CentralView ArticlePubMedGoogle Scholar
  42. Notredame C, Higgins D, Heringa J: T-Coffee: a novel method for multiple sequence alignments. J Mol Biol 2000, 302: 205–217. 10.1006/jmbi.2000.4042View ArticlePubMedGoogle Scholar
  43. Poirot O, Suhre K, Abergel C, O'Toole E, Notredame C: 3DCoffee: a web server for mixing sequences and structures into multiple sequence alignments. Nucl Acids Res 2004, 32: W37–40. 10.1093/nar/gnh031PubMed CentralView ArticlePubMedGoogle Scholar
  44. Krogh A, Larsson B, von Heijne G, Sonnhammer ELL: Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 2001, 305: 567–580. 10.1006/jmbi.2000.4315View ArticlePubMedGoogle Scholar
  45. Raghava GPS: Protein secondary structure prediction using nearest neighbor and neural network approach. CASP4 2000, 75–76.Google Scholar
  46. Cuff JA, Barton GJ: Application of enhanced multiple sequence alignment profiles to improve protein secondary structure prediction. Proteins 2000, 40: 502–511. 10.1002/1097-0134(20000815)40:3<502::AID-PROT170>3.0.CO;2-QView ArticlePubMedGoogle Scholar
  47. Kneller DG, Cohen FE, Langridge R: Improvements in protein secondary structure prediction by an enhanced neural network. J Mol Biol 1990, 214: 171–182. 10.1016/0022-2836(90)90154-EView ArticlePubMedGoogle Scholar
  48. Ouali M, King RD: Cascaded multiple classifiers for secondary structure prediction. Protein Sci 2000, 9: 1162–1176.PubMed CentralView ArticlePubMedGoogle Scholar
  49. McGuffin LJ, Bryson K, Jones DT: The PSIPRED protein structure prediction server. Bioinformatics 2000, 16: 404–445. 10.1093/bioinformatics/16.4.404View ArticlePubMedGoogle Scholar
  50. Karplus K, Hu B: Evaluation of protein multiple alignments by SAM-T99 using the BAliBASE multiple alignment test set. Bioinformatics 2001, 17: 713–720. 10.1093/bioinformatics/17.8.713View ArticlePubMedGoogle Scholar
  51. Geourjon C, Deleage G: SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 1995, 11: 681–684.PubMedGoogle Scholar
  52. Pollastri G, Przybylski D, Rost B, Baldi P: Improving the prediction of protein secondary structure in three and eight classes using recurrent neural networks and profiles. Proteins 2002, 47: 228–235. 10.1002/prot.10082View ArticlePubMedGoogle Scholar
  53. Bowie JU, Luthy R, Eisenberg D: A method to identify protein sequences that fold into a known three-dimensional structure. Science 1991, 253: 164–170.View ArticlePubMedGoogle Scholar
  54. Luthy R, Bowie JU, Eisenberg D: Assessment of protein models with three-dimensional profiles. Nature 1992, 356: 83–85. 10.1038/356083a0View ArticlePubMedGoogle Scholar
  55. Sasin JM, Bujnicki JM: COLORADO3D, a web server for the visual analysis of protein structures. Nucleic Acids Res 2004, 32(Web Server):W586-W589.PubMed CentralView ArticlePubMedGoogle Scholar
  56. Holm L, Sander C: Mapping the protein universe. Science 1996, 273: 595–602.View ArticlePubMedGoogle Scholar
  57. Krissinel E, Henrick K: Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr D Biol Crystallogr 2004, 60(Pt 12 Pt 1):2256–2268. 10.1107/S0907444904026460View ArticlePubMedGoogle Scholar
  58. Crooks GE, Hon G, Chandonia JM, Brenner SE: WebLogo: a sequence logo generator. Genome Res 2004, 14: 1188–1190. 10.1101/gr.849004PubMed CentralView ArticlePubMedGoogle Scholar
  59. Hall TA: BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Series 1999, 41: 95–98.Google Scholar
  60. Guex N, Peitsch MC: SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 1997, 18: 2714–2723. 10.1002/elps.1150181505View ArticlePubMedGoogle Scholar
  61. Sayle R: RASMOL molecular visualization program. Biomolecular Structure Group, Glaxo Research and Development, Greenford, Middlesex, UK; 1994.Google Scholar
  62. DeLano WL: The PyMOL molecular graphics system. DeLano Scientific, San Carlos, CA, USA; 2002.Google Scholar
  63. Marti-Renom MA, Stuart A, Fiser A, Sanchez R, Melo F, Sali A: Comparative protein structure modeling of genes and genomes. Annu Rev Biophys Biomol Struct 2000, 29: 291–325. 10.1146/annurev.biophys.29.1.291View ArticlePubMedGoogle Scholar
  64. Sali A, Blundell TL: Comparative protein modeling by satisfaction of spatial restraints. J Mol Biol 1993, 234: 779–815. 10.1006/jmbi.1993.1626View ArticlePubMedGoogle Scholar
  65. Laskowski RA, MacArthur MW, Moss DS, Thornton JM: PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst 1993, 26: 283–291. 10.1107/S0021889892009944View ArticleGoogle Scholar
  66. Morris AL, MacArthur MW, Hutchinson EG, Thornton JM: Stereochemical quality of protein structure coordinates. Proteins 1992, 12: 345–364. 10.1002/prot.340120407View ArticlePubMedGoogle Scholar
  67. Kitagawa H, Paulson JC: Differential expression of five sialyltransferase genes in human tissues. J Biol Chem 1994, 269: 17872–17878.PubMedGoogle Scholar
  68. Kim YJ, Kim KS, Kim SH, Kim CH, Ko JH, Choe IS, Tsuji S, Lee YC: Molecular cloning and expression of human Galβ1,3GalNAcα2,3-sialytransferase (hST3Gal II). Biochem Biophys Res Commun 1996, 228: 324–327. 10.1006/bbrc.1996.1660View ArticlePubMedGoogle Scholar
  69. Kitagawa H, Paulson JC: Cloning and expression of human Galβ1,3(4)GlcNAcα2,3-sialyltransferase. Biochem Biophys Res Commun 1993, 194: 375–382. 10.1006/bbrc.1993.1830View ArticlePubMedGoogle Scholar
  70. Kitagawa H, Paulson JC: Cloning of a novel α2,3-sialyltransferase that sialylates glycoprotein and glycolipid carbohydrate groups. J Biol Chem 1994, 269: 1394–1401.PubMedGoogle Scholar
  71. Ishii A, Ohta M, Watanabe Y, Matsuda K, Ishiyama K, Sakoe K, Nakamura M, Inokuchi J, Sanai Y, Saito M: Expression cloning and functional characterization of human cDNA for ganglioside GM3 synthase. J Biol Chem 1998, 273: 31652–31655. 10.1074/jbc.273.48.31652View ArticlePubMedGoogle Scholar

Copyright

© Sujatha and Balaji; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.