Differential biosynthesis of polysialic or disialic acid Structure by ST8Sia II and ST8Sia IV.
ST8Sia II (STX) and ST8Sia IV (PST) are polysialic acid (polySia) synthases that catalyze polySia formation of neural cell adhesion molecule (NCAM) in vivo and in vitro. It still remains unclear how these structurally similar enzymes act differently in vivo. In the present study, we performed the enzymatic characterization of ST8Sia II and IV; both ST8Sia II and IV have pH optima of 5.8-6.1 and have no requirement of metal ions. Because the pH dependence of ST8Sia II and IV enzyme activities and the pK profile of His residues are similar, we hypothesized that a histidine residue would be involved in their catalytic activity. There is a conserved His residue (cf. His(348) in ST8Sia II and His(331) in ST8Sia IV, respectively) within the sialyl motif VS in all sialyltransferase genes cloned to date. Mutant ST8Sia II and IV enzymes in which this His residue was changed to Lys showed no detectable enzyme activity, even though they were folded correctly and could bind to CDP-hexanolamine, suggesting the importance of the His residue for their catalytic activity. Next, the degrees of polymerization of polySia in NCAM catalyzed by ST8Sia II and IV were compared. ST8Sia IV catalyzed larger polySia formation of NCAM than ST8Sia II. We also analyzed the (auto)polysialylated enzymes themselves. Interestingly, when ST8Sia II or IV itself was sialylated under conditions for polysialylation, the disialylated compound was the major product, even though polysialylated compounds were also observed. These results suggested that both ST8Sia II and IV catalyze polySia synthesis toward preferred acceptor substrates such as NCAM, whereas they mainly catalyze disialylation, similarly to ST8Sia III, toward unfavorable substrates such as enzyme themselves.
Kitazume-Kawaguchi
S
S
Glycobiology Research Group, Frontier Research Program, Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan.
Kabata
S
S
Arita
M
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002122:Calcium Chloride / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D002522:Chlorocebus aethiops; D004187:Disaccharides / Q000096:biosynthesis; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D017895:Manganese Compounds / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D012794:Sialic Acids / Q000096:biosynthesis*; D012799:Sialyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
D002712:Chlorides; D004187:Disaccharides; D017895:Manganese Compounds; D011134:Polysaccharides; D011994:Recombinant Proteins; D012794:Sialic Acids; C021319:polysialic acid; D015636:Magnesium Chloride; C046778:CMP-N-acetylneuraminate-poly-alpha-2,8-sialosyl sialyltransferase; D012799:Sialyltransferases; D002122:Calcium Chloride; C025340:manganese chloride