Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M311764200 on January 13, 2004

J. Biol. Chem., Vol. 279, Issue 14, 13461-13468, April 2, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/14/13461    most recent
M311764200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jeanneau, C.
Right arrow Articles by Breton, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jeanneau, C.
Right arrow Articles by Breton, C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Structure-Function Analysis of the Human Sialyltransferase ST3Gal I

ROLE OF N-GLYCOSYLATION AND A NOVEL CONSERVED SIALYLMOTIF*

Charlotte Jeanneau{ddagger}, Valérie Chazalet{ddagger}, Claudine Augé§, Dikeos Mario Soumpasis¶, Anne Harduin-Lepers||, Philippe Delannoy||, Anne Imberty{ddagger}, and Christelle Breton{ddagger}**

From the {ddagger}Centre de Recherches sur les Macromolécules Végétales (affiliated to Joseph Fourier University), GDR CNRS n° 2590, F-38041 Grenoble, France, the §UMR CNRS 8614, Paris-Sud University, F-91405 Orsay, France, the Center for Biological Sequence Analysis, Technical University of Denmark, DK-2800 Lyngby, Denmark, and the ||UMR CNRS 8576, USTL, F-59655 Villeneuve d'Ascq, France

Received for publication, October 27, 2003 , and in revised form, December 24, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
All eukaryotic sialyltransferases have in common the presence in their catalytic domain of several conserved peptide regions (sialylmotifs L, S, and VS). Functional analysis of sialylmotifs L and S previously demonstrated their involvement in the binding of donor and acceptor substrates. The region comprised between the sialylmotifs S and VS contains a stretch of four highly conserved residues, with the following consensus sequence (H/y)Y(Y/F/W/h)(E/D/q/g). (Capital letters and lowercase letters indicate a strong or low occurrence of the amino acid, respectively.) The functional importance of these residues and of the conserved residues of motif VS (HX4E) was assessed using as a template the human ST3Gal I. Mutational analysis showed that residues His299 and Tyr300 of the new motif, and His316 of the VS motif, are essential for activity since their substitution by alanine yielded inactive enzymes. Our results suggest that the invariant Tyr residue (Tyr300) plays an important conformational role mainly attributable to the aromatic ring. In contrast, the mutants W301F, E302Q, and E321Q retained significant enzyme activity (25–80% of the wild type). Kinetic analyses and CDP binding assays showed that none of the mutants tested had any significant effect in nucleotide donor binding. Instead the mutant proteins were affected in their binding to the acceptor and/or demonstrated lower catalytic efficiency. Although the human ST3Gal I has four N-glycan attachment sites in its catalytic domain that are potentially glycosylated, none of them was shown to be necessary for enzyme activity. However, N-glycosylation appears to contribute to the proper folding and trafficking of the enzyme.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The sialyltransferase (ST)1 gene family represents a group of enzymes that transfer sialic acid from CMP-Neu5Ac to carbohydrate groups of various glycoproteins and glycolipids. To date, 20 distinct protein members have been cloned and characterized (for a recent review, see Ref. 1). They are classically split into 4 groups, depending on the type of linkage formed and the nature of the sugar acceptor (ST6Gal, ST6GalNAc, ST3Gal, and ST8Sia) (2). The sialyltransferases are localized in the Golgi apparatus and they share with the other Golgi-resident glycosyltransferases a typical type II architecture consisting in a short N-terminal cytoplasmic tail, a transmembrane domain followed by a stem region, and a large C-terminal catalytic domain facing the luminal side (3). Comparison of peptide sequences strongly indicates that the length and amino acid composition of catalytic domains are relatively well conserved and variations in protein sizes are generally attributable to differences in the length of the stem region. The stem region can be defined as the peptide portion after the transmembrane domain that can be removed without altering the activity. The most striking differences are observed in the ST6GalNAc subfamily where ST6GalNAc I exhibits the longest stem region (about 200 amino acids) and ST6GalNAc III appears to be devoid of stem region. In this group, it appears that the broader the acceptor specificity, the longer the stem region (1). The stem region often displays high variability in amino acid composition and little secondary organization and was therefore predicted to be flexible (4). However, this peptide portion often contains cysteine residues as well as several N- and O-glycosylation sites, which could contribute to a local conformation. Recent data suggested that the stem portion could also modulate the in vivo acceptor specificity (5).

All eukaryotic sialyltransferases share a unique feature, the presence, in their catalytic domain, of several conserved peptide regions referred to as sialylmotifs L and S (6) and VS (7). The functional significance of the sialylmotifs L and S has been assessed by site-directed mutagenesis, using ST6Gal I as a model. The mutagenesis of the most conserved residues led to the conclusion that the L-sialylmotif is mainly involved in donor substrate binding (8), whereas mutations in the S-sialylmotif were shown to affect both donor and acceptor binding (9). Considering all known ST sequences, the number of invariant residues in sialylmotifs L and S is 5 and 2, respectively, one cysteine residue being conserved in each region. Mutation of the two conserved cysteines yielded inactive enzymes (8–10) and recent data suggest that they participate in the formation of an intramolecular disulfide linkage that is essential for maintaining an active conformation of the enzyme (11). Similar observations were made with the polysialyltransferase ST8Sia IV (12). In the latter case, a second intramolecular disulfide bond, which brings the sialylmotifs and the C terminus within proximity, has been evidenced. Formation of dimer through disulfide bonds has also been demonstrated in the case of ST6Gal I (13). This dimer comprises ~20–30% of the enzyme found in liver Golgi and exhibits reduced catalytic activity because of its lower affinity for the sugar nucleotide donor, CMP-Neu5Ac. Recent data demonstrated that the mutation of a single Cys residue (Cys24) in the transmembrane domain of ST6Gal I abolishes dimerization of the enzyme (14). Two other Cys residues located downstream the S-sialylmotif could also be critical for in vivo enzyme activity (14).

The enzymatic properties of sialyltransferases have been extensively studied in terms of substrate specificities toward synthetic acceptors as well as their glycoprotein and glycolipid acceptor preference, which revealed the exquisite specificity of some of them. However, at the present time, there is no structural information for sialyltransferases and the detailed mechanism of action remains unclear. The crystal structures of sixteen glycosyltransferases belonging to distinct sequence-based families have been recently solved and among them eight are of mammalian origin. Although belonging to different glycosyltransferase families showing no primary sequence identity, these protein structures fall into only two different structural superfamilies named GT-A (or SpsA fold) and GT-B (or BGT fold) (recently reviewed in Refs. 15 and 16). In addition, both topologies exhibit the same class of fold; that is the threelayer {alpha}/{beta}/{alpha} sandwich that resembles the "Rossmann fold." These structural data have begun to shed light on the role played by short conserved peptide motifs, such as the DXD motif. This motif has been identified in many different glycosyltransferase families (17–19) and was shown, in several crystal structures, to interact mainly with the phosphate groups of nucleotide donor through the coordination of a metal cation. It is always flanked by apolar amino acids and located in a loop ({beta}-turn) connecting two {beta}-strands, and enzymes sharing this motif have an absolute requirement of divalent cation to be active. In the absence of crystal structures, alternative methods can be used to get insight in the folding properties of other GT families. The use of "fold recognition" or "threading" methods suggests that many other GT families could fall into one of these two structural families (15, 20). The sialyltransferase family was among those families for which it was not possible to predict with a high level of confidence which of the two currently known folds they could adopt. However, threading analyses favored the existence of a Rossmann fold (15).

In the present study, we performed an extensive sequence analysis of all the known animal STs to gain further insight into the structure/function relationships of this large and biologically important glycosyltransferase family. During this work we evidenced a new peptide motif located between the sialylmotifs S and VS. To address the importance of this new motif and of the VS-sialylmotif in the enzymatic properties of STs, a site-directed mutagenesis was performed using the human ST3Gal I, as a protein model. ST3Gal I transfers Neu5Ac to the galactose residue of type 3 disaccharide found on glycolipids or O-glycosyl proteins. Kinetic studies have shown that ST3Gal I exhibits high transfer efficiency and high affinity (Km of 51 µM) toward the core 1 mucin type disaccharide Gal{beta}1–3GalNAc{alpha}- (21). Therefore, this enzyme was considered as a good candidate for structure-function studies and for further crystallization trials. Our results contributed to delineate more precisely the nucleotide sugar and acceptor binding regions in the protein. Additional experiments allowed to determine the impact of each of the four predicted N-glycan attachment sites present in the catalytic domain of hST3Gal I on the glycosylation status and enzyme activity of the protein.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Cytidine 5'-monophosphono-N-acetyl neuraminic acid (CMP-Neu5Ac), peroxidase-conjugated goat anti-mouse secondary antibody, gentamicin, L-glutamine, fetal bovine serum, and, AEC staining kit were obtained from Sigma. CMP-[14C]Neu5Ac (289 mCi/mmol) was obtained from Amersham Biosciences (Orsay, France), Gal{beta}1–3Gal-NAc{alpha}-sp-biotin was from Lectinity Holdings, Inc (Moscow, Russia). Oligonucleotides for PCR were purchased from MWG Biotech (Courtaboeuf, France). Anti-Xpress antibody, restriction enzymes, insect cells and culture media were from Invitrogen (Cergy Pontoise, France), Escherichia coli XL1-Blue cells and the QuickChange site directed mutagenesis kit from Stratagene (Amsterdam-Zuidoost, The Netherlands). CDP-fractogel (15 µmol of CDP/ml of gel) was from Calbiochem and His-Bind Resin from Novagen, both purchased from VWR international S. A. S (Fontenay-sous-Bois, France). Pfu DNA polymerase was from Promega (Charbonnières-les-Bains, France) and BaculoGold expression system from BD Pharmingen. The nitrocellulose membranes were from Pall Gelman Laboratory (St Germain-en-Laye, France) and C18 SepPak cartridges from Millipore Corp. CDP-hexanolamine-Sepharose (4 µmol of CDP/ml of gel) was a gift from Dr C. Augé (University of Paris-Sud, France).

Overexpression of hST3Gal I in Insect Cells—Baculovirus-mediated insect cell expression was used to express native and mutant soluble forms of human hST3Gal I with an N-terminal His6 tag and X-PresssTM epitope in order to facilitate the detection and further purification of the recombinant protein. Two cDNA fragments lacking the first 25 (ST3G-{Delta}25) or 56 (ST3G-{Delta}56) amino acids were generated by PCR using as template the plasmid pFlagST3G harboring the human ST3Gal I gene (22). The 951-bp and 860-bp coding regions corresponding to the desired truncated soluble forms of hST3Gal I were obtained using the forward primers (5'-AGACGCGGCCGCTAACTACTCCCACACCATGG-3') for hST3G-{Delta}25 and (5'-AAGGGAATTCGCGTCATCTCCCCTTGAAG-3') for hST3G-{Delta}56 and the same reverse primer (5'-ACTGGCGGCCGCCAGGCCTTGCACCTGCACCC-3'). Primers were designed to create NotI and EcoRI restriction sites at each end of the gene. The PCR products were obtained by using the Pfu DNA polymerase in 30 cycles, with each cycle comprising 45 s at 94 °C, 1 min of annealing at 50 °C, and 3 min of elongation at 70 °C. The PCR fragments were excised with NotI and EcoRI and cloned into the NotI/EcoRI sites of the baculovirus transfer vector, pVTBac-His (23) to give the plasmids pVT-ST3G-{Delta}25 and pVT-ST3G-{Delta}56. Recombinant proteins encoded by these vectors will contain a melittin cleavable signal peptide at their N terminus and be secreted from baculovirus-infected cells. Spodoptera frugiperda cells (Sf9) were used for the production and amplification of recombinant baculoviruses. The cells were cultured at 27 °C in Grace's medium supplemented with 10% fetal bovine serum, and 50 µg/ml gentamicin. The co-transfection of Sf9 cells with the transfer vectors and the BaculoGold linear DNA was done according to the manufacturer's instructions. Viral stocks of 108 plaque-forming units (pfu)/ml were prepared by repeated amplification. Trichoplusia ni (High FiveTM) cells grown at 27 °C in the protein-free Express FiveTM SFM medium, supplemented with 1 mM L-glutamine and 50 µg/ml gentamicin, were generally used for the production of recombinant native and mutant forms of human ST3Gal I. Recombinant baculoviruses were used to infect High FiveTM cells at a multiplicity of infection of 5 pfu per cell. Medium was collected 96 h after infection, clarified by centrifugation, and the supernatants were stored at –70 °C until they were used.

Site-directed Mutagenesis—Mutant forms of hST3Gal I were prepared by using the plasmid pVT-ST3G-{Delta}56 as the template. PCR-based mutagenesis was used for all mutations. The primers used to create mutants of hST3Gal I are described in Table I. All of the recombinant plasmids were propagated into E. coli XL1-Blue cells. Mutants were systematically checked by sequencing.


View this table:
[in this window]
[in a new window]
  		TABLE I
Sequence of the primers used for site-directed mutagenesis

 
Western Blot Analysis—Aliquots (1 ml) of crude High FiveTM supernatants were incubated for 1 h at 4 °C with 50 µl of affinity adsorbent (CDP-Fractogel, or CDP-Agarose or His-Bind) in order to trap the recombinant proteins. Beads were then washed twice with phosphatebuffered saline, resuspended into 50 µl of Laemmli denaturing buffer. Protein samples were separated on sodium dodecyl sulfate-10% polyacrylamide gels and electrotransferred to a nitrocellulose membrane. The blot was developed by adsorption of the anti-Xpress antibody (1: 4000) followed by peroxidase-conjugated goat anti-mouse secondary antibody (1:2000), and the protein bands were visualized using the AEC staining kit.

Sialyltransferase Assay—Standard reactions were conducted at 37 °C for 20 min in a final volume of 50 µl in the presence of 50 µM of donor substrate CMP-Neu5Ac, 110,000 cpm of CMP-[14C]Neu5Ac, 50 µM of acceptor substrate Gal{beta}1–3GalNAc{alpha}-sp-biotin in 0.1 M cacodylate buffer, pH 6.5. The reaction was initiated by addition of the recombinant enzyme source and stopped by addition of 450 µl of cold water. Reaction products were applied on C18 SepPak cartridges and eluted with methanol. The radioactivity was measured by scintillation counting. The apparent Km value for CMP-Neu5Ac was obtained using 1–200 µM of CMP-Neu5Ac with 0.5 mM of acceptor, and for the acceptor, using 2–500 µM of Gal{beta}1–3GalNAc{alpha}-sp-biotin with 200 µM of CMP-Neu5Ac. For comparison of mutant and wild type activity, ST activity was normalized for protein expression (relative enzyme mass) assessed by Western blotting of wild-type and mutant proteins. All enzyme assays were done in triplicate.

Sequence Analysis—Protein sequences were retrieved from Gen-Pept or SwissProt and analyzed using BLAST (24), LALIGN (25), and ClustalW (26) programs. The sensitive Hydrophobic Cluster Analysis method (HCA) was used to compare protein sequences with very low level of sequence identity (27).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Sequence Analysis of Sialyltransferases—All eukaryotic sialyltransferases share a unique feature, the presence in their catalytic domain of several conserved peptide regions commonly referred to as sialylmotifs L, S, and VS. Except for these peptide motifs there are few sequence similarities between the various groups of STs (ST6Gal, ST6GalNAc, ST3Gal, and ST8Sia). Examination of all the known ST protein sequences reveals another conserved motif located between the the sialylmotifs S and VS (Fig. 1). The sequence consensus of this motif, rich in aromatic residues, is as follows (H/y)Y(Y/W/F/h)(D/E/q/g) (capital letters and lowercase letters indicate a strong or low occurrence of the amino acid, respectively).



View larger version (60K):
[in this window]
[in a new window]
  		FIG. 1.
Conserved motifs of sialyltransferases. Sequence alignment of the most conserved regions present in all sialyltransferases. Strictly invariant residues in all sialyltransferase sequences are indicated in white on a black background. The other most conserved amino acid positions are shaded in gray. Motifs are numbered (1–4), and the correspondence with the names of sialylmotifs (L, S, and VS) is shown. All the sequences are of human origin (human ST8Sia VI.2

 

Of striking interest is the presence of the invariant Tyr residue at the second position. Taken together, the sialylmotifs S, VS, and the new one cover a large part of the C terminus of the catalytic domain (55–70 amino acids). For better clarity in the text, the sialylmotifs will be numbered as indicated in Fig. 1. The 20 distinct ST sequences that have been cloned to date have in common the presence of 10 invariant residues and about 30 conserved or semiconserved positions located in these motifs. The function of the most conserved residues in sialylmotifs L and S (motifs 1 and 2) has already been investigated, using ST6Gal I as a model (8, 9). To elucidate the functional relevance of residues of motifs 3 and 4, we constructed a series of mutants of the human ST3Gal I. Each residue of motif 3 was mutated (His299-Tyr300-Trp301-Glu302) as well as the two invariant amino acids of motif 4 (His316 and Glu321).

Expression of hST3Gal I and Mutants in Insect Cells—For expression of the wild-type human ST3Gal I and its mutants, a baculoviral expression plasmid containing an N-terminal signal sequence, (His)6 tag and X-Press tag was used for expression of a soluble form of the cDNA for human ST3Gal I. Two different soluble forms of the native enzyme were generated: ST3G-{Delta}25 (truncated just after the transmembrane domain after the residue 25) and ST3-{Delta}56 (deleted of the first 56 amino acids), which was recently shown to be the minimal catalytically active form when expressed in COS-7 cells (22). The level of protein expression in the culture medium of insect cells was monitored by Western blotting using an anti-X-Press antibody and upon trapping of the recombinant proteins secreted in the culture supernatants with different affinity gels (CDP-beads and His-Bind resin). At this stage, we noticed that CDP-Fractogel was the most efficient affinity system to catch the recombinant proteins since almost 100% of enzyme activity was recovered (only 80–90% for the other affinity sorbents) (data not shown). However, we also observed a tight, almost irreversible, and nonspecific binding of the recombinant proteins on CDP-Fractogel beads (binding was only poorly inhibited with a large excess of CDP, and the enzyme binds with the same efficiency to GDP-Fractogel beads). In contrast, a specific and reversible binding was obtained using homemade CDP-agarose beads. Therefore, CDP-Fractogel beads were used to quantify the amount of secreted recombinant proteins whereas CDP-Agarose beads were used for the functional tests (i.e. capability of mutants to bind to CDP). The two isoforms ST3G-{Delta}25 and ST3G-{Delta}56 were equally produced in the culture medium upon transfection of Sf9 or Hi-5TM cells with the recombinant baculoviruses. Although they differ in the number of N-glycosylation sites (5 for ST3G-{Delta}25 and 4 for ST3G-{Delta}56) they display an apparent similar pattern of glycosylation since 3 major polypeptide bands with diffuse borders (glycoforms) can be distinguished by Western blotting whatever the construct and the cell line utilized (Fig. 2). As well, enzyme activity measurements did not reveal major differences between the two constructs, a slightly higher specific activity being observed for ST3-{Delta}56 expressed in Hi-5TM cells (data not shown). Therefore, the ST3-{Delta}56 construct overexpressed in Hi-5 cells was selected to further explore the function of the most conserved residues of motifs 3 and 4.



View larger version (75K):
[in this window]
[in a new window]
  		FIG. 2.
Expression of two truncated forms of hST3Gal I in two different insect cell lines. The level of recombinant protein expression of ST3G-{Delta}25 (lanes 1 and 2) and ST3G-{Delta}56 (lanes 3 and 4) secreted in the culture medium of transfected Sf9 cells (lanes 1 and 3) or High-FiveTM cells (lanes 2 and 4) was determined by Western blotting after SDS-PAGE as described under "Experimental Procedures."

 
Characterization of Native and Mutant Enzymes—As seen in Fig. 3A, eight of the nine mutants were efficiently secreted in the culture medium and most of them are produced at protein levels roughly equivalent to the native enzyme. Their similar pattern of migration (molecular mass and glycosylation status) indicated that the introduced point mutation did not affect the overall structure of the enzyme. However one mutant (Y300A) was repeatedly poorly expressed in the culture medium of insect cells. The mutation of this Tyr residue in Phe resulted in a better expression level.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 3.
Expression and activity of wild-type ST3G-{Delta}56 and mutants. A, enzyme activity and expression levels of wild-type and mutant proteins. Expression levels of mutant proteins were compared with the wild-type (WT) enzyme by Western blotting after SDS-PAGE (lower panel), as described under "Experimental Procedures." Enzyme samples were adjusted to give comparable amounts of proteins for activity assays. Sialyltransferase activity of mutants is expressed relative to the wild-type enzyme. Enzyme assays were conducted as described under "Experimental Procedures," and values are the average of at least three determinations. B, binding of wild-type ST3G-{Delta}56 and mutants to CDP-agarose beads. CDP-beads were incubated with samples of culture supernatants of infected cells (volumes were adjusted to give comparable amounts of recombinant proteins). Bound proteins were separated from unbound proteins by centrifugation of CDP-beads and visualized by imunoblotting. Results were qualitatively evaluated by comparison to wild-type enzyme as follows: binding similar to the WT (+++), binding estimated in the range 40–80% (++) or less than 30% (+) of the WT, no significant binding (–).

 
The relative amounts of proteins were determined for a comparative activity assay. Medium from cells mock-transfected using only the vector (pVTBac) showed no detectable sialyltransferase activity (data not shown). The native ST3G-{Delta}56 construct yielded an average transferase activity of 400 nmol·min–1·mg protein–1. As shown in Fig. 3A, the substitution in motif 3 of the highly conserved His residue (H299A) and of the invariant Tyr residue in Ala (Y300A) resulted in a complete loss of enzyme activity. The mutation H299Y was dictated by the fact that ST6Gal I is the only ST displaying a Tyr instead of a His residue at this amino acid position (see Fig. 1). A low but detectable activity (2% relative to the wild-type) is observed for the mutant H299Y, thus suggesting a possible role of the aromatic ring at this position. Interestingly, if the mutation Y300A yielded a completely inactive enzyme, the mutation Y300F restored substantial enzyme activity (30% activity relative to the native enzyme). These results combined to the expression levels of the mutants strongly suggest that the invariant Tyr residue of motif 3 plays an important conformational role mainly attributable to the aromatic ring. The third position of motif 3 is a conserved aromatic residue (Tyr, Phe, Trp, or His). The substitution W301A in ST3G-{Delta}56 reduced enzyme activity to a low but significant level of activity (2%) whereas the conservative mutation W301F had little effect on enzyme activity (30%). The fourth position of motif 3 is less conserved and the mutation of this residue in Gln (E302Q) did not really affect enzyme activity (80% of the wild type). These results demonstrated the importance of aromatic amino acids in motif 3 in the catalytic activity. Motif 4 comprised 2 invariant residues (His316 and Glu321), separated by four residues. The mutation H316A yielded a totally inactive enzyme, but the conservative substitution E321Q only slightly reduced enzyme activity.

Effect of Mutations on Binding to CDP-beads—To determine the role of conserved residues in motifs 3 and 4 in nucleotide binding, we compared the ability of the wild type and ST3Gal I mutants to bind to CDP-beads (Fig. 3B). Four mutant proteins that were shown to be inactive or very poorly active (H299A, H299Y, W301A, and H316A) demonstrated the same or similar binding capacity relative to the wild type enzyme. The mutant proteins that still retained significant enzyme activity (W301F, E302Q, and E321Q) also demonstrated significant binding to CDP-beads. For all of these mutants the binding was inhibited upon addition of free CDP (data not shown). Only the mutation of the invariant residue Tyr300 strongly impaired CDP binding, but as mentioned above, a conformational role is postulated for this residue and therefore it could play a central role in maintaining the active conformation of the enzyme without establishing any contact with the nucleotide donor. Altogether, these results suggest that none of the conserved amino acid positions in motifs 3 and 4 plays a crucial role in nucleotide binding.

Kinetic Analysis of the Native and Mutant Enzymes—Kinetic parameters for the donor and acceptor were measured for the native enzyme and for the four mutants that retained enough enzyme activity (Y300F, W301F, E302Q, and E321Q).

The apparent Kms for CMP-Neu5Ac and the acceptor Gal{beta}1–3GalNAc{alpha}-sp-biotin of native hST3Gal I are 8.5 µM and 15 µM, respectively (Table II). These values are in good agreement with the data previously obtained with the same enzyme expressed in COS cells (21, 22). Rather similar apparent Km values toward the donor substrate have been obtained for the four mutants tested thus confirming the behavior of these mutants in CDP binding tests (Fig. 3B). In contrast, the Km values for the acceptor substrate were significantly altered for the E302Q and E321Q mutants with increases in the range of 3–7-fold. Taken together, these results strongly suggest that the conserved sialylmotifs 3 and 4 are mostly involved in the binding of the acceptor molecule. The decrease in activity observed for the Y300F mutant can be attributed to a Vmax effect, thus altering the catalytic efficiency of the enzyme. This residue does not directly participate in donor and acceptor substrate binding, but presumably it has a more complex function in maintaining an active enzyme conformation as suggested above.


View this table:
[in this window]
[in a new window]
  		TABLE II
Kinetic analysis of wild type and mutants of hST3Gal I

Kinetic constants were determined for the wild type enzyme and mutants that retained significant enzyme activity, as described under "Experimental Procedures."

 
The Role of N-Glycans in Activity and Stability of hST3Gal I—Analysis of the primary sequence of hST3Gal I indicates the presence of five potential N-glycosylation sites (Fig. 4A). Three of them (Asn27, Asn79, and Asn323) are conserved in all of the known ST3Gal I sequences. The first N-glycan attachment site located just after the transmembrane domain (Asn27) is not crucial for activity since the truncated ST3-{Delta}56 soluble form demonstrated roughly the same specific activity as the longest soluble form (ST3-{Delta}25).



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 4.
Influence of N-glycosylation on protein expression and activity of ST3G-{Delta}56. A, schematic representation of the topology of human ST3Gal I. The positions of potential N-glycosylation sites are shown by arrowheads and those that were mutated in the present study were numbered (1–4). Black arrowheads indicate the N-glycan attachment sites conserved in all ST3Gal I sequences. The amino acid substitution is as indicated in Table I. B, expression levels of wild type ST3G-{Delta}56 and mutants. The labeling {Delta}x indicates which N-glycosylation site has been deleted. Arrowhead on the right of panel shows the position of the fully deglycosylated isoform. C, enzyme activity of mutants lacking one, two, three or the four N-glycan attachment sites. Enzyme assays were conducted as described under "Experimental Procedures." Results were qualitatively evaluated (++, enzyme activity similar to WT; +, reduced activity).

 
The importance of each of the four remaining N-glycan attachment sites in enzyme function was further evaluated. N-glycosylation sites were destroyed by substituting asparagine in the NX(S/T) motifs by the amino acids indicated in Table I. This choice was dictated by the conservative nature of the substitution (N79D) or by the presence of the amino acid in the other ST3Gal I sequences (N114S and N201Q) or in the closest ST3Gal II sequences (N323H). These mutations were expected to have less drastic effects on the overall three-dimensional conformation of the recombinant protein. A panel of 6 mutants with single and multiple N-glycosylation defects has been generated by site-directed mutagenesis using the ST3-{Delta}56 construct. The mutations of one of the two conserved N-glycan sites ({Delta}1 and {Delta}4), as well as the double mutation ({Delta}1,4) do not affect the protein level expression or the activity (Fig. 4). All mutant proteins can be detected by Western blots in the extracellular culture medium (Fig. 4B). However, the fully deglycosylated form ({Delta}1,2,3,4) and the mutant lacking the three first N-glycan sites, Asn79, Asn114, and Asn201 ({Delta}1,2,3), were repeatedly very poorly expressed (Fig. 4B). Both mutants were converted to a 36.8 kDa form, which corresponds to the hST3G-{Delta}56 core protein plus the N-terminal tag. Despite their low levels of expression, these mutants still retain significant enzyme activity. These results suggest that N-glycosylation is not necessary for enzyme function but that it probably greatly contributes to the proper folding and to the stability of the protein. Comparison of the electrophoretic mobility indicates a variable glycosylation status of the secreted polypeptides, which suggests that each of the N-glycan site attachment predicted in the catalytic domain, is potentially glycosylated.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The aim of the present study was to get further insights into structure/function relationships in the large ST family. STs appear to cluster into seven subfamilies (named A to G), which better reflect the sequence similarities between the different members of this large family (Fig. 5A). Such divergence, which occurred during evolution by tandem duplication from an ancestral gene, was dictated by the need to transfer sialic acid to a large repertoire of acceptor molecules with different types of linkage.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 5.
Clustering of sialyltransferase sequences and schematic representation of the catalytic domain of sialyltransferases. A, the dendrogram describes the approximate groupings of the sequences by similarity using Clust-alW alignments (26). B, schematic representation of the catalytic domain of a mammalian sialyltransferase. The gray regions correspond to the location of motifs 1–4. The double head arrows indicate the regions of the catalytic domain that correspond (solid lines) to the nucleotide binding domain (NBD) or acceptor binding domain (ABD), and their possible extensions (dotted lines).

 
Comparison of the peptide sequences showed that there is little sequence similarities between the different ST subgroups with the exception of three consensus sequences, the so-called sialylmotifs L, S, and VS (6, 7). We here described the presence of an additional motif located between sialylmotifs S and VS with the general peptide signature as follows (H/y)Y(Y/W/F/h)(D/E/q/g). The presence of this motif (motif 3 in Fig. 1) in sialyltransferases has already been described but with a different signature (H/C/R)(Y/H)(Y/W/F)(D/E/H/Y) thus suggesting that the authors got a slightly different sequence alignment (not shown in the article) in this region (28). Using the human ST3Gal I as a template, our results clearly demonstrated the importance of aromatic amino acids in this region of its catalytic domain. Kinetic measurements suggested their possible involvement in acceptor recognition and contribution to optimal catalytic efficiency. Particularly, an important structural role is postulated for the invariant Tyr residue (Tyr300). In a recent study, a series of mutants of the human ST6Gal I was generated by random mutagenesis (29) and analyzed using a high throughput solid-phase enzyme assay with asialofetuin as acceptor. Two of these mutants were shown to be affected in residues of the motif 3 (354YYYQ in hST6Gal I, see Fig. 1). ST6Gal I is the only enzyme to display a tyrosine at the first position of motif 3. The following substitutions Y354D and Y355N in hST6Gal I resulted in mutated enzymes, which displayed significant residual activity toward asialofetuin (5–20% and 20–40%, respectively) as well as a good capability to bind to the glycoprotein acceptor (in surface plasmon resonance experiments). These results do not support our hypothesis that aromatic residues are needed at these two positions for optimal catalytic efficiency. Nevertheless, the strong conservation of aromatic residues in motif 3 in the whole ST family together with our detailed mutational analysis strongly suggest an important functional and/or structural role of these residues in enzyme function. The invariant His and Glu residues of sialylmotif VS (motif 4 in Fig. 1) were previously thought to be important for catalytic activity (7). The functional role of His residue has been recently examined in the polysialyltransferases ST8Sia II and IV (30). In both enzymes the replacement of this His by lysine was shown to be critical for their catalytic activity since they exhibited no detectable enzyme activity. However the mutated proteins showed similar binding to CDP-hexanolamine beads as wild type enzyme. In the present study, similar results were obtained with the mutation of the invariant His residue of motif 4 in hST3Gal I thus reinforcing the hypothesis of its involvement in catalysis. In contrast, the replacement of the conserved Glu residue by glutamine did not inactivate the enzyme (25% residual activity compared with the wild type). However the strict conservation of HX4E motif in all the vertebrate STs characterized to date, as well as its predicted occurrence as a N-capping substructure of an {alpha}-helix (data not shown), strongly suggest that both residues are necessary for optimal catalytic efficiency of the glycosyl transfer reaction. From these observations and kinetic data, it is thus conceivable that motif 4 (VS) is part of the active site, mainly located on the acceptor side or at the vicinity of both donor and acceptor sugar substrates. From the present work and previous mutagenesis studies (8, 9, 29, 30) it is now clear that the C-terminal part of the catalytic domain (including motifs 3 and 4 and part of motif 2) is primarily dedicated to the recognition of the acceptor substrate whereas the N-terminal part is mostly involved in nucleotide sugar binding (Fig. 5B). However, we cannot rule out the possibility that residues upstream of sialylmotif L (motif 1) could be involved in acceptor recognition (or modulate the acceptor specificity) as suggested in previous studies (5). Recent data indicated the presence of an intramolecular disulfide bridge connecting the two conserved cysteine of motifs 1 and 2 (11, 12). Whether the occurrence of disulfide bridge is a common feature to all STs remains to be established, but this would bring in close proximity sialylmotifs L and S and possibly residues at the N terminus of the catalytic domain near the catalytic center. The minimal catalytic domain has been defined experimentally for a few STs, or alternatively by sequence comparison (4, 22, 31). The proposed boundaries to delineate the stem region from the catalytic domain generally coincide, for each subfamily, with the end of a hypervariable region in the N-terminal ends of the polypeptides (Ref. 4).3 The catalytic domain is therefore assumed to start ~70–90 residues upstream from motif 1 (40–45 residues for the sub-family B which corresponds to the ST6GalNAc III to VI). Therefore, we can estimate the average size of the catalytic domain around 300 (± 20) residues, with the sialylmotifs located as depicted in Fig. 5B.

Glycosyltransferases are very often themselves glycosylated. The role of N-glycosylation sites and their glycosylation has been studied in several glycosyltransferases and it seems that it varies from protein to protein. Concerning the sialyltransferases, it was shown for ST6Gal I that N-glycosylation may stabilize the protein but is not required for activity of the full-length enzyme in vivo (31). In the case of GD3 synthase (ST8Sia I), the deglycosylated forms were enzymatically more unstable than the native form and N-glycosylation and proper trimming appear to be critical for proper trafficking of GD3 synthase to the Golgi complex (32). Here we addressed this question for the case of human ST3Gal I whose primary sequence predicts five potential N-glycosylation sites. Recent data demonstrated that each of the N-glycan attachment sites is potentially glycosylated in the recombinant enzyme transiently expressed in COS cells (22). We here demonstrated, using the minimal active soluble form of hST3GalI, that none of the attached N-glycans is essential for enzyme activity and that the totally deglycosylated mutant still retains enzyme activity. However these mutants are not processed and secreted as efficiently than the wild type thus suggesting a probable role of N-glycosylation in the proper folding and trafficking of the enzyme as shown for other sialyltransferases.

Despite progress in deciphering the location and identities of some of the active site residues, no structure has yet been identified for a member of the ST family. Because STs share the presence of four conserved motifs dispersed throughout the catalytic domain, a similar fold is expected for the whole ST family. The sialylmotifs are highly specific for this family of eukaryotic STs and we were unable to find any sequence or structural similarities (using Psi-BLAST and the HCA method) with other protein families, including bacterial STs and also CMP-KDO transferases. In favorable cases, correct structural and functional information about a protein can be predicted by detection of remote homologs (i.e. using PSI-BLAST programs) or through the use of fold recognition methods. In the past few years, evidence has accumulated that the majority of glycosyltransferase families fall only into two structural families called GT-A (or SpsA fold) and GT-B (or BGT fold) (15, 16, 20). These two superfamilies have different folds and different active sites, but in both cases, the nucleotide binding domain was shown to adopt a Rossmann (GT-B) or Rossmann-like (GT-A) fold, a structural motif commonly found in nucleotide binding proteins. Recent threading analyses also favored the existence of at least one Rossmann domain in the catalytic domain of STs (15).

It is now highly desirable to determine the three-dimensional structure of ST by crystallization to confirm the various hypotheses and work is in progress to get crystals of hST3Gal I. Getting a three-dimensional structure would be invaluable for detailed mechanistic studies and for deciphering the molecular bases responsible for donor and acceptor substrate specificities.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed: CERMAV-CNRS, BP 53, F-38041 Grenoble cedex 9, France. Tel.: 33-4-7603-7635; Fax: 33-4-7654-7203; E-mail: Christelle.Breton{at}cermav.cnrs.fr.

1 The abbreviations used are: ST, sialyltransferase; CMP-Neu5Ac, CMP-N-acetylneuraminic acid; ST3Gal I, CMP-Neu5Ac:Gal{beta}1–3GalNAc {alpha}2,3-sialyltransferase I (EC 2.4.99.4 [EC] ). Back

2 M. Teintenier-Lelierie and A. Harduin-Lepers, unpublished data. Back

3 C. Breton, unpublished data. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Harduin-Lepers, A., Vallejo-Ruiz, V., Krzewinski-Recchi, M. A., Samyn-Petit, B., Julien, S., and Delannoy, P. (2001) Biochimie (Paris) 83, 727–737
  2. Tsuji, S., Datta, A. K., and Paulson, J. C. (1996) Glycobiology 6, v-vii[Medline] [Order article via Infotrieve]
  3. Paulson, J. C., and Colley, K. J. (1989) J. Biol. Chem. 264, 17615–17618[Free Full Text]
  4. Donadio, S., Dubois, C., Fichant, G., Roybon, L., Guillemot, J. C., Breton, C., and Ronin, C. (2003) Biochimie (Paris) 85, 311–321
  5. Legaigneur, P., Breton, C., El Battari, A., Guillemot, J. C., Auge, C., Malissard, M., Berger, E. G., and Ronin, C. (2001) J. Biol. Chem. 276, 21608–21617[Abstract/Free Full Text]
  6. Datta, A. K., and Paulson, J. C. (1997) Ind. J. Biochem. Biophys. 34, 157–165[Medline] [Order article via Infotrieve]
  7. Geremia, R. A., Harduin-Lepers, A., and Delannoy, P. (1997) Glycobiology 7, v-vii[Medline] [Order article via Infotrieve]
  8. Datta, A. K., and Paulson, J. C. (1995) J. Biol. Chem. 270, 1497–1500[Abstract/Free Full Text]
  9. Datta, A. K., Sinha, A., and Paulson, J. C. (1998) J. Biol. Chem. 273, 9608–9614[Abstract/Free Full Text]
  10. Drickamer, K. (1993) Glycobiology 3, 2–3[Free Full Text]
  11. Datta,. A. K., Chammas, R., and Paulson, J. C. (2001) J. Biol. Chem. 276, 15200–15207[Abstract/Free Full Text]
  12. Angata, K., Yen, T. Y., El-Battari, A., Macher, B. A., and Fukuda, M. (2001) J. Biol. Chem. 276, 15369–15377[Abstract/Free Full Text]
  13. Ma, J., and Colley, K. J. (1996) J. Biol. Chem. 271, 7758–7766[Abstract/Free Full Text]
  14. Qian, R., Chen, C., Colley, K. J. (2001) J. Biol. Chem. 276, 28641–28649[Abstract/Free Full Text]
  15. Breton, C., Heissigerova, H., Jeanneau, C., Moravcova, J., and Imberty, A. (2002) Biochem. Soc. Symp. 69, 23–32
  16. Coutinho, P. M., Deleury, E., Davies, G. J., and Henrissat, B. (2003) J. Mol. Biol. 328, 307–317[CrossRef][Medline] [Order article via Infotrieve]
  17. Breton, C., Bettler, E., Joziasse, D. H., Geremia, R. A., and Imberty, A. (1998) J. Biochem. 123, 1000–1009[Abstract/Free Full Text]
  18. Breton, C., and Imberty, A. (1999) Curr. Opin. Struct. Biol. 9, 563–571[CrossRef][Medline] [Order article via Infotrieve]
  19. Wiggins, C. A., and Munro, S. (1998) Proc. Natl. Acad. Sci., U. S. A. 95, 7945–7950[Abstract/Free Full Text]
  20. Wrabl, J. O., and Grishin, N. V. (2001) J. Mol. Biol. 314, 365–374[CrossRef][Medline] [Order article via Infotrieve]
  21. Kono, M., Ohyama, Y., Lee, Y. C., Hamamoto, T., Kojima, N., and Tsuji, S. (1997) Glycobiology 7, 469–479[Abstract/Free Full Text]
  22. Vallejo-Ruiz, V., Haque, R., Mir, A-M., Schwientek, T., Mandel, U., Cacan, R., Delannoy, P., and Harduin-Lepers, A. (2001) Biochim. Biophys. Acta 1549, 161–173[CrossRef][Medline] [Order article via Infotrieve]
  23. Sarkar, M., Pagny, S., Unligil, U., Joziasse, D., Mucha, J., Glossl, J., and Schachter, H. (1998) Glycoconj. J. 15, 193–197[CrossRef][Medline] [Order article via Infotrieve]
  24. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389–3402[Abstract/Free Full Text]
  25. Huang, X., and Miller, W. (1991) Adv. Appl. Math. 12, 337–357[CrossRef]
  26. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673–4680[Abstract/Free Full Text]
  27. Gaboriaud, C., Bissery, V., Benchetrit, T., and Mornon, J. P. (1987) FEBS Lett. 224, 149–155[CrossRef][Medline] [Order article via Infotrieve]
  28. Kapitonov, D., Bieberich, E., and Yu, R. K. (1999) Glycoconj. J. 16, 337–350[CrossRef][Medline] [Order article via Infotrieve]
  29. Laroy, W., Ameloot, P., and Contreras, R. (2001) Glycobiology 11, 175–182[Abstract/Free Full Text]
  30. Kitazume-Kawaguchi, S., Kabata, S., and Arita, M. (2001) J. Biol. Chem. 276, 15696–15703[Abstract/Free Full Text]
  31. Chen, C., and Colley, K. J. (2000) Glycobiology 11, 531–538
  32. Martina, J. A., Daniotti, J. L., and Maccioni, H. J. (1998) J. Biol. Chem. 273, 3725–3731[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
D. A. Foley, K. G. Swartzentruber, and K. J. Colley
Identification of Sequences in the Polysialyltransferases ST8Sia II and ST8Sia IV That Are Required for the Protein-specific Polysialylation of the Neural Cell Adhesion Molecule, NCAM
J. Biol. Chem., June 5, 2009; 284(23): 15505 - 15516.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
J. Cheng, H. Yu, K. Lau, S. Huang, H. A Chokhawala, Y. Li, V. K. Tiwari, and X. Chen
Multifunctionality of Campylobacter jejuni sialyltransferase CstII: Characterization of GD3/GT3 oligosaccharide synthase, GD3 oligosaccharide sialidase, and trans-sialidase activities
Glycobiology, September 1, 2008; 18(9): 686 - 697.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
Y. Kakuta, N. Okino, H. Kajiwara, M. Ichikawa, Y. Takakura, M. Ito, and T. Yamamoto
Crystal Structure of Vibrionaceae Photobacterium sp. JT-ISH-224 {alpha}2,6-Sialyltransferase in a Ternary Complex With Donor Product CMP and Acceptor Substrate Lactose: Catalytic Mechanism and Substrate Recognition
Glycobiology, January 1, 2008; 18(1): 66 - 73.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
S. DeFrees, Z.-G. Wang, R. Xing, A. E. Scott, J. Wang, D. Zopf, D. L. Gouty, E. R. Sjoberg, K. Panneerselvam, E. C. M. Brinkman-Van der Linden, et al.
GlycoPEGylation of recombinant therapeutic proteins produced in Escherichia coli
Glycobiology, September 1, 2006; 16(9): 833 - 843.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. K. Altheide, T. Hayakawa, T. S. Mikkelsen, S. Diaz, N. Varki, and A. Varki
System-wide Genomic and Biochemical Comparisons of Sialic Acid Biology Among Primates and Rodents: EVIDENCE FOR TWO MODES OF RAPID EVOLUTION
J. Biol. Chem., September 1, 2006; 281(35): 25689 - 25702.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
S. Uemura, T. Kurose, T. Suzuki, S. Yoshida, M. Ito, M. Saito, M. Horiuchi, F. Inagaki, Y. Igarashi, and J.-i. Inokuchi
Substitution of the N-glycan function in glycosyltransferases by specific amino acids: ST3Gal-V as a model enzyme
Glycobiology, March 1, 2006; 16(3): 258 - 270.
[Abstract] [Full Text] [PDF]

Home page
 J BiochemHome page
S. Takashima, T. Abe, S. Yoshida, H. Kawahigashi, T. Saito, S. Tsuji, and M. Tsujimoto
Analysis of Sialyltransferase-Like Proteins from Oryza sativa
J. Biochem., February 1, 2006; 139(2): 279 - 287.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
R. Y. Patel and P. V. Balaji
Identification of linkage-specific sequence motifs in sialyltransferases
Glycobiology, February 1, 2006; 16(2): 108 - 116.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
A. Harduin-Lepers, R. Mollicone, P. Delannoy, and R. Oriol
The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach
Glycobiology, August 1, 2005; 15(8): 805 - 817.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
D. Yu, A. Petermann, U. Kunter, S. Rong, S. J. Shankland, and J. Floege
Urinary Podocyte Loss Is a More Specific Marker of Ongoing Glomerular Damage than Proteinuria
J. Am. Soc. Nephrol., June 1, 2005; 16(6): 1733 - 1741.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/14/13461    most recent
M311764200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jeanneau, C.
Right arrow Articles by Breton, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jeanneau, C.
Right arrow Articles by Breton, C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement