Abstract
Polysialylated neural cell adhesion molecule (NCAM) is thought to play a critical role in neural development. Polysialylation of NCAM was shown to be achieved by two alpha2,8-polysialyltransferases, ST8Sia IV (PST) and ST8Sia II (STX), which are moderately related to another alpha2,8-sialyltransferase, ST8Sia III. Here we describe that all three alpha2,8-sialyltransferases can utilize oligosaccharides as acceptors but differ in the efficiency of adding polysialic acid on NCAM. First, we found that ST8Sia III can form polysialic acid on the enzyme itself (autopolysialylation) but not on NCAM. These discoveries prompted us to determine if ST8Sia IV and ST8Sia II share the property of ST8Sia III in utilizing low molecular weight oligosaccharides as acceptors. By using a newly established method, we found that ST8Sia IV, ST8Sia II, and ST8Sia III all add oligosialic and polysialic acid on various sialylated N-acetyllactosaminyl oligosaccharides, including NCAM N-glycans, fetuin N-glycans, synthetic sialylated N-acetyllactosamines, and on alpha(2)-HS-glycoprotein. Our results also showed that monosialyl and disialyl N-acetyllactosamines can serve equally as an acceptor, suggesting that no initial addition of alpha2,8-sialic acid is necessary for the action of polysialyltransferases. Polysialylation of NCAM by ST8Sia IV and ST8Sia II is much more efficient than polysialylation of N-glycans isolated from NCAM. Moreover, ST8Sia IV and ST8Sia II catalyze polysialylation of NCAM much more efficiently than ST8Sia III. These results suggest that no specific acceptor recognition is involved in polysialylation of low molecular weight sialylated oligosaccharides, whereas the enzymes exhibit pronounced acceptor specificities if glycoproteins are used as acceptors.
Highlights
Polysialic acid is a unique carbohydrate of a linear homopolymer of ␣2,8-linked sialic acid, which may contain as many as 55 sialic acid residues per polymer [1, 2]
We demonstrated that ST8Sia IV and ST8Sia II are much more efficient in polysialylation on neural cell adhesion molecule (NCAM) than ST8Sia III. These results strongly suggest that ST8Sia IV and ST8Sia II apparently evolved to participate in NCAM polysialylation from a primordial enzyme that adds ␣2,8-sialic acid to low molecular weight acceptors
These results combined together indicate that ST8Sia II and ST8Sia IV add polysialic acid to N-glycans attached to NCAM with a much better efficiency than N-glycans isolated from NCAM
Summary
Isolation of a Full-length cDNA Encoding Human ST8Sia III—A cDNA harboring a catalytic domain of human ST8Sia III was obtained as follows. PCR was carried out to obtain a catalytic domain using the formed cDNA as a template and 5Ј-primer and 3Ј-nested primer Both primers were adapted from the mouse ST8Sia III sequence (nucleotides 113–130 and 1125–1147, respectively) [24], and BglII and XhoI sites are included in these primers. The PCR product obtained was digested by HindIII and XbaI, and this fragment was appended to the 5Ј-end XbaI site (nucleotides 201–206) of a large cDNA fragment (encompassing nucleotides 113–1145), producing a full-length cDNA encoding human ST8Sia III. Upstream and downstream primers for this PCR correspond to nucleotides 890 –911 and nucleotides 1110 –1130 of the human ST8Sia III sequence, respectively. Sialyltransferase Assays and Product Characterization—HeLa cells expressing a soluble chimeric form of ST8Sia II or ST8Sia III or ST8Sia IV were established as described previously [4].
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