title: Recognition of cell surface acceptors by two human alpha-2,6-sialyltransferases produced in CHO cells.
abstract: The action of sialyltransferases (STs) on cell surface glycoconjugates is a key process in shaping cell phenotype in a variety of cells mostly involved in migratory and adhesive pathways. The factors determining cell-specific pattern of glycosylation are so far poorly understood. Most STs are resident proteins of the Golgi apparatus, where acceptors are sialylated while they are in transit to the cell surface. To identify putative structural features that may account for their acceptor preference, we analyzed 53 cloned animal and human STs. We could identify conserved regions and peptide motifs representative of ST subfamilies, located at the C-terminal end of the hypervariable region upstream from the L-sialyl motif. Residues 93-100 in human ST6Gal I (hST6Gal I) were shown to be crucial for enzymatic activity when deleted and expressed in CHO cells. The Delta100 hST6Gal I mutant protein was fully recognized by polyclonal anti-hST6Gal I antibodies and followed the intracellular secretory pathway. This indicated that the conserved QVWxKDS sequence is essential for the whole catalytic domain to acquire a biologically active conformation. When full-length epitope-tagged hST6Gal I and hST6GalNAc I constructs were transfected in CHO cells, the alpha-2,6 sialylated glycotope was found to be largely restricted to intracellular resident acceptors and enzymatic activity based on fluorescent lectin staining. In contrast, both enzymes deprived of their membrane anchor and part of the hypervariable region but still possessing the conserved domains exhibited a very efficient transfer of sialic acid to cell surface glycoconjugates. Colocalization of the ST6Gal I mutant proteins with early and late Golgi markers such as giantin or rab6 proteins confirmed that soluble STs migrate forward in these subcompartments where they can act upon newly synthesized acceptors and follow the secretory pathway. It is thus concluded that downstream from the transmembrane domain, native STs possess peptide sequences that allow them to sialylate glycoprotein acceptors selectively along their transit within Golgi stacks.
authors.affiliation: Laboratoire de Neuroglycobiologie, Institut du Cerveau, UMR 6149 CNRS et Université de Provence, GLM-CNRS, IFR du Cerveau, Marseille, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Donadio
authors.forename: Christophe
authors.initials: C
authors.lastname: Dubois
authors.forename: Gwennaele
authors.initials: G
authors.lastname: Fichant
authors.forename: Laurent
authors.initials: L
authors.lastname: Roybon
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Guillemot
authors.forename: Christelle
authors.initials: C
authors.lastname: Breton
authors.forename: Catherine
authors.initials: C
authors.lastname: Ronin
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D020134:Catalytic Domain / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D006001:Glycoconjugates / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
chemical_list: D018076:DNA, Complementary; D006001:Glycoconjugates; D011994:Recombinant Proteins; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase