Biomedical subjects

Philippe Delannoy

Publications and source records attributed to Philippe Delannoy.

10 recordsLinked to original sources

PubMed2006

Probing the substrate specificity of four different sialyltransferases using synthetic beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O) (CH(2))7CH3 analogues general activating effect of replacing N-acetylglucosamine by N-propionylglucosamine.

The acceptor specificities of ST3Gal III, ST3Gal IV, ST6Gal I and ST6Gal II were investigated using a panel of beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH(2))(7)CH(3) analogues. Modifications introduced at either C2, C3, C4, C5, or C6 of terminal D-Gal, as well as N-propionylation instead of N-acetylation of subterminal D-GlcN were tested for their influence on the alpha-2,3- and alpha-2,6-sialyltransferase acceptor activities. Both ST3Gal enzymes displayed the same narrow acceptor specificity, and only accept reduction of the Gal C2 hydroxyl function. The ST6Gal enzymes, however, do not have the same acceptor specificity. ST6Gal II seems less tolerant towards modifications at Gal C3 and C4 than ST6Gal I, and prefers beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc (LacdiNAc) as an acceptor substrate, as shown by replacing the Gal C2 hydroxyl group with an N-acetyl function. Finally, a particularly striking feature of all tested sialyltransferases is the activating effect of replacing the N-acetyl function of subterminal GlcNAc by an N-propionyl function.

Acetylglucosamine↗

PubMed2005

Molecular cloning and expression of a human hST8Sia VI (alpha2,8-sialyltransferase) responsible for the synthesis of the diSia motif on O-glycosylproteins.

Based on BLAST analysis of the human and mouse genome databases using the human CMP sialic acid; alpha2,8-sialyltransferase cDNA (hST8Sia I; EC 2.4.99.8), a putative sialyltransferase gene, was identified on human chromosome 10. The genomic organization was found to be similar to that of hST8Sia I and hST8Sia V. Transcriptional expression analysis showed that the newly identified gene was constitutively expressed at low levels in various human tissues and cell lines. We have isolated a full-length cDNA clone from the breast cancer cell line MCF-7 that encoded a type II membrane protein of 398 amino acid residues with the conserved motifs of sialyltransferases. We have established a mammary cell line (MDA-MB-231) stably transfected with the full-length hST8Sia VI and the analysis of sialylated carbohydrate structures expressed at the cell surface clearly indicated the disappearance of Neu5Acalpha2-3-sialylated structures. The transient expression of a truncated soluble form of the enzyme in either COS-7 cells or insect Sf-9 cells led to the production of an active enzyme in which substrate specificity was determined. Detailed substrate specificity analysis of the hST8Sia VI recombinant enzyme in vitro, revealed that this enzyme required the trisaccharide Neu5Acalpha2-3Galbeta1-3GalNAc (where Neu5Ac is N-acetylneuraminic acid and GalNAc is N-acetylgalactosamine) to generate diSia (disialic acid) motifs specifically on O-glycans.

Amino Acid Sequence↗

PubMed2005

The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach.

The animal sialyltransferases are Golgi type II transmembrane glycosyltransferases. Twenty distinct sialyltransferases have been identified in both human and murine genomes. These enzymes catalyze transfer of sialic acid from CMP-Neu5Ac to the glycan moiety of glycoconjugates. Despite low overall identities, they share four conserved peptide motifs [L (large), S (small), motif III, and motif VS (very small)] that are hallmarks for sialyltransferase identification. We have identified 155 new putative genes in 25 animal species, and we have exploited two lines of evidence: (1) sequence comparisons and (2) exon-intron organization of the genes. An ortholog to the ancestor present before the split of ST6Gal I and II subfamilies was detected in arthropods. An ortholog to the ancestor present before the split of ST6GalNAc III, IV, V, and VI subfamilies was detected in sea urchin. An ortholog to the ancestor present before the split of ST3Gal I and II subfamilies was detected in ciona, and an ortholog to the ancestor of all the ST8Sia was detected in amphioxus. Therefore, single examples of the four families (ST3Gal, ST6Gal, ST6GalNAc, and ST8Sia) have appeared in invertebrates, earlier than previously thought, whereas the four families were all detected in bony fishes, amphibians, birds, and mammals. As previously hypothesized, sequence similarities among sialyltransferases suggest a common genetic origin, by successive duplications of an ancestral gene, followed by divergent evolution. Finally, we propose predictions on these invertebrates sialyltransferase-related activities that have not previously been demonstrated and that will ultimately need to be substantiated by protein expression and enzymatic activity assays.

Amino Acid Sequence↗

PubMed2005

Stable expression of sialyl-Tn antigen in T47-D cells induces a decrease of cell adhesion and an increase of cell migration.

Sialyl-Tn is a carbohydrate antigen overexpressed in several epithelial cancers including breast cancer, and usually associated with poor prognosis. Sialyl-Tn is synthesized by a CMP-Neu5Ac: GalNAc alpha2,6-sialyltransferase: ST6GalNAc I, which catalyzes the transfer of a sialic acid residue in alpha2,6-linkage to the GalNAcalpha1-O-Ser/Thr structure. The resulting disaccharide (Neu5Acalpha2-6GalNAcalpha1-O-Ser/Thr) cannot be further elongated and sialyl-Tn expression results therefore in a shortening of the O-glycan chains. However, usual breast cancer cell lines express neither ST6GalNAc I nor sialyl-Tn antigen. We have previously shown that stable transfection of MDA-MB-231 cells with the hST6GalNAc I cDNA induces the sialyl-Tn antigen expression at the cell surface and leads to a decreased cell growth and an increased cell migration. We describe herein the generation of new T47-D clones expressing sialyl-Tn antigen after hST6GalNAc I cDNA stable transfection. sialyl-Tn antigen is carried by several high molecular weight membrane bound O-glycoproteins, including MUC1. We show that sialyl-Tn expression induces a decrease of cell growth and adhesion, and an increase of cell migration in sialyl-Tn positive clones compared to mock transfected cells. These observations show that the alteration of the O-glycans pattern is sufficient to modify the biological features of cancer cells. These T47-D sialyl-Tn expressing clones might allow further in vivo investigation to determine precisely the impact of such O-glycosylation modifications on breast cancer development.

Antigens, Tumor-Associated, Carbohydrate↗

PubMed2004

Structure-function analysis of the human sialyltransferase ST3Gal I: role of n-glycosylation and a novel conserved sialylmotif.

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 (HX(4)E) was assessed using as a template the human ST3Gal I. Mutational analysis showed that residues His(299) and Tyr(300) of the new motif, and His(316) of the VS motif, are essential for activity since their substitution by alanine yielded inactive enzymes. Our results suggest that the invariant Tyr residue (Tyr(300)) 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.

Amino Acid Sequence↗

PubMed2003

Molecular cloning, gene organization and expression of the human UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4-N-acetylgalactosaminyltransferase responsible for the biosynthesis of the blood group Sda/Cad antigen: evidence for an unusual extended cytoplasmic domain.

The nucleotide sequence of the short and long transcripts of beta1,4- N -acetylgalactosaminyltransferase have been submitted to the DDBJ, EMBL, GenBank(R) and GSDB Nucleotide Sequence Databases under accession nos AJ517770 and AJ517771 respectively. The human Sd(a) antigen is formed through the addition of an N -acetylgalactosamine residue via a beta1,4-linkage to a sub-terminal galactose residue substituted with an alpha2,3-linked sialic acid residue. We have taken advantage of the previously cloned mouse cDNA sequence of the UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4- N -acetylgalactosaminyltransferase (Sd(a) beta1,4GalNAc transferase) to screen the human EST and genomic databases and to identify the corresponding human gene. The sequence spans over 35 kb of genomic DNA on chromosome 17 and comprises at least 12 exons. As judged by reverse transcription PCR, the human gene is expressed widely since it is detected in various amounts in almost all cell types studied. Northern blot analysis indicated that five Sd(a) beta1,4GalNAc transferase transcripts of 8.8, 6.1, 4.7, 3.8 and 1.65 kb were highly expressed in colon and to a lesser extent in kidney, stomach, ileum and rectum. The complete coding nucleotide sequence was amplified from Caco-2 cells. Interestingly, the alternative use of two first exons, named E1(S) and E1(L), leads to the production of two transcripts. These nucleotide sequences give rise potentially to two proteins of 506 and 566 amino acid residues, identical in their sequence with the exception of their cytoplasmic tail. The short form is highly similar (74% identity) to the mouse enzyme whereas the long form shows an unusual long cytoplasmic tail of 66 amino acid residues that is as yet not described for any other mammalian glycosyltransferase. Upon transient transfection in Cos-7 cells of the common catalytic domain, a soluble form of the protein was obtained, which catalysed the transfer of GalNAc residues to alpha2,3-sialylated acceptor substrates, to form the GalNAcbeta1-4[Neu5Acalpha2-3]Galbeta1-R trisaccharide common to both Sd(a) and Cad antigens.

Amino Acid Sequence↗

PubMed2003

Recombinant (2-->3)-alpha-sialyltransferase immobilized on nickel-agarose for preparative synthesis of sialyl Lewis(x) and Lewis(a) precursor oligosaccharides.

The specificity of recombinant (2-->3)-alpha-sialyltransferase (ST3Gal-III), expressed in baculovirus-infected insect cells, has been determined with various oligosaccharide acceptors and sugar-nucleotide donors using a fluorescence based assay. Recombinant ST3Gal-III tagged with a polyhistidine tail was immobilized on Ni(2+)-NTA-Agarose as an active enzyme for use in the synthesis of three sialylated oligosaccharides: (i) the divalent molecule [alpha-Neu5Ac-(2-->3)-D-Galp-(1-->4)-beta-D-GlcpNAc-O-CH(2)](2)-C-(CH(2)OBn)(2) (12); (ii) the dansylated derivative, alpha-Neu5Ac-(2-->3)-D-Galp-(1-->3)-beta-D-GlcpNAc-O-(CH(2))(6)-NH-dansyl and; (iii) the tetrasacharide alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-O-CH(3). Compound 12 was itself prepared from the divalent N-acetyllactosamine molecule built on pentaerythritol by a chemo-enzymatic route.

Animals↗

PubMed2003

Identification and functional expression of a second human beta-galactoside alpha2,6-sialyltransferase, ST6Gal II.

BLAST analysis of the human and mouse genome sequence databases using the sequence of the human CMP-sialic acid:beta-galactoside alpha-2,6-sialyltransferase cDNA (hST6Gal I, EC2.4.99.1) as a probe allowed us to identify a putative sialyltransferase gene on chromosome 2. The sequence of the corresponding cDNA was also found as an expressed sequence tag of human brain. This gene contained a 1590 bp open reading frame divided in five exons and the deduced amino-acid sequence didn't correspond to any sialyltransferase already known in other species. Multiple sequence alignment and subsequent phylogenic analysis showed that this new enzyme belonged to the ST6Gal subfamily and shared 48% identity with hST6Gal-I. Consequently, we named this new sialyltransferase ST6Gal II. A construction in pFlag vector transfected in COS-7 cells gave raise to a soluble active form of ST6Gal II. Enzymatic assays indicate that the best acceptor substrate of ST6Gal II was the free disaccharide Galbeta1-4GlcNAc structure whereas ST6Gal I preferred Galbeta1-4GlcNAc-R disaccharide sequence linked to a protein. The alpha2,6-linkage was confirmed by the increase of Sambucus nigra agglutinin-lectin binding to the cell surface of CHO transfected with the cDNA encoding ST6Gal II and by specific sialidases treatment. In addition, the ST6Gal II gene showed a very tissue specific pattern of expression because it was found essentially in brain whereas ST6Gal I gene is ubiquitously expressed.

Amino Acid Sequence↗

PubMed2003

Comparative analysis of the site-specific N-glycosylation of human lactoferrin produced in maize and tobacco plants.

We have compared the site-by-site N-glycosylation status of human lactoferrin (Lf) produced in maize, a monocotyledon, and in tobacco, used as a model dicotyledon. Maize and tobacco plants were stably transformed and recombinant Lf was purified from both seeds and leaves. N-glycopeptides were generated by trypsin digestion of recombinant Lf and purified by reverse-phase HPLC. The N-glycosylation pattern of each site was determined by mass spectrometry. Our results indicated that the N-glycosylation patterns of recombinant Lf produced in maize and tobacco share common structural features. In particular, both N-glycosylation sites of each recombinant Lf are mainly substituted by typical plant paucimannose-type N-glycans, with beta1,2-xylose and alpha1,3-linked fucose at the proximal N-acetylglucosamine. However, tobacco Lf shows a significant amount of processed N-glycans with one or two beta1,2GlcNAc linked to the trimannose core, which are weakly expressed in maize Lf. Finally, no Lewisa epitope was observed on tobacco Lf.

Asparagine↗

PubMed2002

The unfolded protein response in a dolichyl phosphate mannose-deficient Chinese hamster ovary cell line points out the key role of a demannosylation step in the quality-control mechanism of N-glycoproteins.

The CHO (Chinese hamster ovary) glycosylation mutant cell line, B3F7, transfers the truncated glycan Glc(3)Man(5)GlcNAc(2) on to nascent proteins. After deglucosylation, the resulting Man(5)GlcNAc(2) glycan is subjected to two reciprocal enzymic processes: the action of an endoplasmic-reticulum (ER) kifunensine-sensitive alpha1,2-mannosidase activity to yield a Man(4)GlcNAc(2) glycan, and the reglucosylation involved in the quality-control system which ensures that only correctly folded glycoproteins leave the ER. We show that the recombinant secreted alkaline phosphatase (SeAP) produced in stably transfected B3F7 cells, is co-immunoprecipitated with the GRP78 (glucose-regulated protein 78), a protein marker of the unfolded protein response (UPR). The level of GRP78 transcription has been evaluated by reverse transcription-PCR (RT-PCR) and we demonstrate that B3F7 cells present a constitutively higher level of UPR in the absence of inductors, compared with Pro(-5) cells. Interestingly, a decrease was observed in the UPR and an increase in SeAP secretion in the kifunensine-treated B3F7 cells. Altogether, these data highlight the relationships between the glycan structure, the quality control system and the UPR. Moreover, they support the idea that a specific demannosylation step is a key event of the glycoprotein quality control in B3F7 cells.

Actins↗