PMID:14722111

Citation	Jeanneau, C, Chazalet, V, Augé, C, Soumpasis, DM, Harduin-Lepers, A, Delannoy, P, Imberty, A and Breton, C (2004) Structure-function analysis of the human sialyltransferase ST3Gal I: role of n-glycosylation and a novel conserved sialylmotif. J. Biol. Chem. 279:13461-8
Abstract	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.
Links	PubMed Online version:10.1074/jbc.M311764200
Keywords	Amino Acid Sequence; Animals; Catalytic Domain; Cell Line; Gene Expression Regulation, Enzymologic; Glycosylation; Humans; Insects; Kinetics; Molecular Sequence Data; Mutagenesis; N-Acetylneuraminic Acid/metabolism; Protein Folding; Protein Structure, Tertiary; Sialyltransferases/chemistry; Sialyltransferases/genetics; Sialyltransferases/metabolism; Structure-Activity Relationship
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
HUMAN:SIA4A		GO:0003836: beta-galactoside (CMP) alpha-2,3-sialyltransferase activity	ECO:0000314:		F		Figure 3	complete CACAO 10333
HUMAN:SIA4A	enables	GO:0003836: beta-galactoside (CMP) alpha-2,3-sialyltransferase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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