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

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 α-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 Arg²⁷⁶/Arg²⁷⁷ or Arg²⁶⁵ in the polysialyltransferase domain of ST8Sia IV decreased both NCAM polysialylation and autopolysialylation in parallel, suggesting that these residues are important for catalytic activity. In contrast, replacing Arg⁸²/Arg⁹³ in ST8Sia IV with alanine substantially decreased NCAM-specific polysialylation while only partially impacting autopolysialylation, suggesting that these residues may be particularly important for NCAM polysialylation. Two conserved negatively charged residues, Glu⁹² and Asp⁹⁴, surround Arg⁹³. 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 α2,8-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 α-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 Asp⁴⁹⁷, Asp⁵¹¹, Glu⁵¹², and Glu⁵¹⁴ (15).⁴ When three of these residues (Asp⁵¹¹, Glu⁵¹², and Glu⁵¹⁴) are mutated to alanine or arginine, NCAM polysialylation is reduced or abolished, suggesting that the acidic patch is part of a larger recognition region. We anticipate that within this putative recognition region there will be amino acids required for mediating polyST-NCAM binding, and those that do not mediate binding per se but instead are required for correct positioning of the enzyme-substrate complex for polysialylation. For example, we have identified a novel α-helix in the FN1 domain that when replaced leads to polysialylation of O-glycans found on the FN1 domain rather than N-glycans on the Ig5 domain (14). This helix may mediate an interdomain interaction that positions the Ig5 N-glycans for polysialylation by an enzyme bound to the FN1 domain (14). Alternatively, the helix could act as a secondary interaction site that positions the polyST properly on the substrate.The expression of the polySTs is developmentally regulated with high levels of STX and moderate levels of PST expressed throughout the developing embryo (2, 18, 19). STX levels decline after birth, although PST expression persists in specific regions of the adult brain where polysialylated NCAM is involved in neuronal regeneration and synaptic plasticity (18–23). The large size and negative charge of polysialic acid disrupt NCAM-dependent and NCAM-independent interactions, thereby negatively modulating cell adhesion (24–26). Simultaneous disruption of both PST and STX in mice results in severe neuronal defects and death usually within 4 weeks after birth (27). Interestingly, when NCAM expression is also eliminated in these mice, they have a nearly normal phenotype, suggesting the main function of polysialic acid is to modulate NCAM-mediated cell adhesion during development (27). In addition, re-expression of highly polysialylated NCAM has been associated with several cancers, including neuroblastomas, gliomas, small cell lung carcinomas, and Wilms tumor. The presence of polysialic acid is thought to promote cancer cell growth and invasiveness (28–35).Sialyltransferases, including the polySTs, have three motifs required for catalytic activity (36–38) (see Fig. 1A). Sialyl motif Large (SML) is thought to bind the donor substrate CMP-sialic acid (39), whereas sialyl motif Small (SMS) is believed to bind both donor and carbohydrate acceptor substrates (40). The sialyl motif Very Small (SMVS) has a conserved His residue that is required for catalytic activity (38, 41). However, the precise function of this motif is unknown. An additional 4-amino acid motif, motif III, is conserved in the sialyltransferases (42–44). It was suggested that this motif, and particularly His and Tyr residues within its sequence, may be required for optimal activity and acceptor recognition (42).Open in a separate windowFIGURE 1.PST and STX polybasic regions and mutants generated for this study. A, representation of the polySTs and their polybasic regions and sialyl motifs. The PBR is a 35-amino acid region present in both PST and STX, equidistant from the SML of each enzyme and rich in conserved positively charged amino acids. The PSTD is a region identified by Nakata et al. (47) that is 32 amino acids in length, rich in basic residues, and contiguous with the SMS of the enzymes. The sialyl motifs (SML, SMS, SMVS, and motif III) are regions of homology found in all sialyltransferases that are believed to be involved in substrate and donor interactions. B, PSTD of PST and the mutants made in this region that are used in this study. C, PBR of PST and STX and the mutants made in this region that are used in this study.Angata et al. (45) used chimeric enzymes to identify regions within the polySTs required for catalytic activity and NCAM polysialylation. Sequences from PST, STX, and ST8Sia III were used to construct the chimeric proteins. ST8Sia III is an α2,8-sialyltransferase that typically adds one or two sialic acid residues to glycoprotein or glycolipid substrates, can autopolysialylate its own glycans, but cannot polysialylate NCAM (46). Deletion analysis showed that amino acids 62–356 are required for PST catalytic activity. Replacement of segments of this region with corresponding STX or ST8Sia III sequences led to the suggestion that amino acids 62–127 and possibly 194–267 of PST may be required for NCAM recognition (45).Recently, Troy and co-workers (47, 48) identified a stretch of basic residues, termed the polysialyltransferase domain (PSTD), which is only observed in the two polySTs and not in other sialyltransferases. The PSTD is contiguous with SMS and extends from amino acids 246–277 in PST and 261–292 in STX. Mutation analysis demonstrated that the overall positive charge of this motif is important for activity and identified specific residues required for NCAM polysialylation (Arg²⁵², Ile²⁷⁵, Lys²⁷⁶, and Arg²⁷⁷) (47).In this study, we have scanned the critical polyST regions identified by the work of Angata et al. (45) for sequences that may be involved in protein-protein recognition and NCAM polysialylation. We identified a second polybasic motif that we named the polybasic region (PBR). The PBR is conserved in PST and STX and is located equidistant from the SML of each enzyme. It consists of 35 amino acids of which 7 are the basic amino acids Arg and Lys. We found that the replacement of two specific residues within the PBR (Arg⁸² and Arg⁹³ of PST and Arg⁹⁷ and Lys¹⁰⁸ of STX) have a greater negative effect on NCAM polysialylation than on autopolysialylation. Replacement of acidic residues surrounding PST Arg⁹³ led to a similar disparate effect on these processes. Comparison of the critical residues in both the PSTD and PBR demonstrated that the replacement of PSTD residues had an equally negative impact on both NCAM polysialylation and enzyme autopolysialylation, whereas replacement of selected PBR residues more severely impacted NCAM polysialylation, suggesting that the PBR residues may play important roles in NCAM-specific polysialylation.