Identification of Sequences in the Polysialyltransferases ST8Sia II and ST8Sia IV That Are Required for the Protein-specific Polysialylation of the Neural Cell Adhesion Molecule, NCAM

Abstract

The polysialyltransferases ST8Sia II and ST8Sia IV polysialylate the glycans of a small subset of mammalian proteins. Their most abundant substrate is the neural cell adhesion molecule (NCAM). An acidic surface patch and a novel ␣-helix in the first fibronectin type III repeat of NCAM are required for the polysialylation of N-glycans on the adjacent immunoglobulin domain. Inspection of ST8Sia IV sequences revealed two conserved polybasic regions that might interact with the NCAM acidic patch or the growing polysialic acid chain. One is the previously identified polysialyltransferase domain (Nakata, D., Zhang, L., and Troy, F. A. (2006) Glycoconj. J. 23, 423-436). The second is a 35-amino acid polybasic region that contains seven basic residues and is equidistant from the large sialyl motif in both polysialyltransferases. We replaced these basic residues to evaluate their role in enzyme autopolysialylation and NCAM-specific polysialylation. We found that replacement of Arg . Replacement of these residues with alanine largely inactivated ST8Sia IV, whereas reversing these residues enhanced enzyme autopolysialylation but significantly reduced NCAM polysialylation. In sum, we have identified selected amino acids in this conserved polysialyltransferase polybasic region that are critical for the protein-specific polysialylation of NCAM.Polysialic acid is a linear homopolymer of ␣2,8-linked sialic acid that is added to a small subset of mammalian glycoproteins by the polysialyltransferases (polySTs) 3 ST8Sia II (STX) and ST8Sia IV (PST) (1-4). Substrates for the polySTs include the neural cell adhesion molecule (NCAM) (5, 6), the ␣-subunit of the voltage-dependent sodium channel (7, 8), CD36, a scavenger receptor found in milk (9), neuropilin-2 expressed by dendritic cells (10), and the polySTs themselves, which can polysialylate their own N-glycans in a process called autopolysialylation (11,12). This small number of polysialylated proteins and other evidence from our laboratory (13-15) suggest that polysialylation is a protein-specific modification that requires an initial protein-protein interaction between the polySTs and their glycoprotein substrates. The most abundant polysialylated protein is NCAM. The three major NCAM isoforms consist of five Ig domains, two fibronectin type III repeats, and a transmembrane domain and cytoplasmic tail (NCAM140 and NCAM180) or a glycosylphosphatidylinositol anchor (NCAM120) (16). Polysialylation takes place primarily on two N-linked glycans in the Ig5 domain (17). We have previously shown that a truncated NCAM140 protein consisting of Ig5, the first fibronectin type III repeat (FN1), the transmembrane region, and cytoplasmic tail is fully polysialylated (13). However, a protein consisting of Ig5, the transmembrane region, and cytoplasmic tail is not polysialylated (13). This suggests that the polySTs recognize and bind the FN1 domain to polysialylate N-glycans on the adjacent Ig5 domain. We subsequently identified an acidic patch unique to NCAM FN1, consisting of Asp 497 , Asp 5...

Sequences Prior to Conserved Catalytic Motifs of Polysialyltransferase ST8Sia IV Are Required for Substrate Recognition

“…Interestingly, Arg 82 appears to play a greater role in the recognition and polysialylation of these two proteins than does Arg 93 . This is in contrast to PST recognition of NCAM, where Arg 82 and Arg 93 seem to play relatively equivalent roles (61). Collectively, our work has identified two residues in a polybasic region prior to the conserved sialyl motifs that are essential for substrate recognition and polysialylation.…”

“…We identified a conserved polybasic region (PBR) we thought might function as the complementary binding region for the FN1 acidic patch (residues 71-105 in PST and 86 -120 in STX). Mutation of specific basic residues (Arg 82 /Arg 93 in PST and Arg 97 /Lys 108 in STX) within this region substantially decreased NCAM polysialylation without similarly decreasing enzyme autopolysialylation, suggesting that the PBR is important for recognition of the NCAM substrate and not general catalytic activity (61). Unfortunately, we were unable to consistently detect decreases in the interaction of PST PBR mutants and NCAM in co-immunoprecipitation experiments (data not shown).…”

“…They postulated that the PSTD facilitates processivity of the polysialylation process by interacting with the growing, negatively charged polySia chain (60). However, our analysis of these critical PSTD residues suggested that this region is important for polyST catalytic activity (61). We identified a conserved polybasic region (PBR) we thought might function as the complementary binding region for the FN1 acidic patch (residues 71-105 in PST and 86 -120 in STX).…”

“…The polybasic region (PBR) is found between residues 71 and 105 in PST. Mutation of Arg 82 and Arg 93 in the PBR was previously shown to greatly reduce NCAM polysialylation (61). The PSTD is a stretch of 32 amino acids (residues 246 -277 in PST) that is contiguous with the SMS and is conserved in the two polySTs (60).…”

“…The PSTD is a stretch of 32 amino acids (residues 246 -277 in PST) that is contiguous with the SMS and is conserved in the two polySTs (60). Previous work demonstrated that selected residues in the PSTD are required for NCAM polysialylation (60,61).…”