Unique Disulfide Bond Structures Found in ST8Sia IV Polysialyltransferase Are Required for Its Activity

Abstract

NCAM polysialylation plays a critical role in neuronal development and regeneration. Polysialylation of the neural cell adhesion molecule (NCAM) is catalyzed by two polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST), which contain sialylmotifs L and S conserved in all members of the sialyltransferases. The members of the ST8Sia gene family, including ST8Sia II and ST8Sia IV are unique in having three cysteines in sialylmotif L, one cysteine in sialylmotif S, and one cysteine at the COOH terminus. However, structural information, including how disulfide bonds are formed, has not been determined for any of the sialyltransferases. To obtain insight into the structure/function of ST8Sia IV, we expressed human ST8Sia IV in insect cells, Trichoplusia ni, and found that the enzyme produced in the insect cells catalyzes NCAM polysialylation, although it cannot polysialylate itself ("autopolysialylation"). We also found that ST8Sia IV does not form a dimer through disulfide bonds. By using the same enzyme preparation and performing mass spectrometric analysis, we found that the first cysteine in sialylmotif L and the cysteine in sialylmotif S form a disulfide bridge, whereas the second cysteine in sialylmotif L and the cysteine at the COOH terminus form a second disulfide bridge. Site-directed mutagenesis demonstrated that mutation at cysteine residues involved in the disulfide bridges completely inactivated the enzyme. Moreover, changes in the position of the COOH-terminal cysteine abolished its activity. By contrast, the addition of green fluorescence protein at the COOH terminus of ST8Sia IV did not render the enzyme inactive. These results combined indicate that the sterical structure formed by intramolecular disulfide bonds, which bring the sialylmotifs and the COOH terminus within close proximity, is critical for the catalytic activity of ST8Sia IV.In the development of the nervous system, various cell-typespecific carbohydrates presented by glycoproteins, proteoglycans, and glycolipids function in signal transduction, neurite outgrowth, neuronal plasticity, and synapse formation (1, 2). Among these carbohydrates, polysialic acid is a linear homopolymer of ␣2,8-linked sialic acid, primarily attached to the neural cell adhesion molecule (NCAM) 1 (3-5). Polysialylated NCAM is abundant in embryonic brain, whereas most NCAM in adult brain does not contain polysialic acid. However, polysialylated NCAM is continuously present in hippocampus and the olfactory bulb, where neuronal regeneration persists in the adult (6). Studies using endoneuraminidase showed that cell migration and synaptic plasticity are dependent on the presence of polysialic acid (7,8). In contrast with vertebrate NCAM, NCAM homologues in Aplysia and Drosophila, apCAM and FasII, respectively, are not polysialylated. In these species, their activity in neuronal regeneration and axon guidance is modulated by removal of the protein from the cell surface by endocytosis (9) or expression of an anti-adhesive protein (10). These results indicate that ...

Structure-Function Analysis of the Human Sialyltransferase ST3Gal I

“…Mutation of the two conserved cysteines yielded inactive enzymes (8 -10) and recent data suggest that they participate in the formation of an intramolecular disulfide linkage that is essential for maintaining an active conformation of the enzyme (11). Similar observations were made with the polysialyltransferase ST8Sia IV (12). In the latter case, a second intramolecular disulfide bond, which brings the sialylmotifs and the C terminus within proximity, has been evidenced.…”

“…However, we cannot rule out the possibility that residues upstream of sialylmotif L (motif 1) could be involved in acceptor recognition (or modulate the acceptor specificity) as suggested in previous studies (5). Recent data indicated the presence of an intramolecular disulfide bridge connecting the two conserved cysteine of motifs 1 and 2 (11,12). Whether the occurrence of disulfide bridge is a common feature to all STs remains to be established, but this would bring in close proximity sialylmotifs L and S and possibly residues at the N terminus of the catalytic domain near the catalytic center.…”

Highly Conserved Cysteines of Mouse Core 2 β1,6-N-Acetylglucosaminyltransferase I Form a Network of Disulfide Bonds and Include a Thiol That Affects Enzyme Activity

“…3 Interestingly, the disulfide linkage formed between Cys 59 and Cys 413 brings the amino acid residues located near the N and C termini of the catalytic domain within close proximity. This distinctive arrangement is similar to that occurring in ␣(1,3/1,4) fucosyltransferase III (51) and polysialyltransferase ST8Sia IV (52), where site-directed mutagenesis demonstrated that such steric conformation may be associated with acceptor substrate specificity (53,54) and catalytic efficiency (52), respectively. Similarly, the catalytic activity of GM2 synthase depends on the formation of disulfide-bonded homodimers, whose antiparallel orientation also brings the N and C termini in close proximity, most likely to establish a catalytic domain (55).…”

“…2ϩ . These fragments were generated from preferential cleavage of the amide bonds of Pro 52 and Pro 408 , respectively, and correspond to those predicted to occur in Pro-containing peptides, as indicated in studies reported previously (41). Frag- FIG.…”

Molecular evolution and expression of zebrafish St8SiaIII, an alpha‐2,8‐sialyltransferase involved in myotome development

“…A polysialyltransferase domain (PSTD), located immediately upstream of SM-L in polysialyltransferases, is obligatorily required for polysialylation of target molecules (Nakata et al, 2006). The catalytic activity of STs depends on the formation of two disulfide bonds, through which these motifs are arranged in close proximity of each other (Angata et al, 2001; reviewed in Harduin- Lepers et al, 2005;Patel andBalaji, 2006, Nakata et al, 2006).…”