The Polybasic Region of the Polysialyltransferase ST8Sia-IV Binds Directly to the Neural Cell Adhesion Molecule, NCAM

Abstract

Polysialic acid (polySia) is a unique post-translational modification found on a small set of mammalian glycoproteins. Composed of long chains of α2,8-linked sialic acid, this large, negatively charged polymer attenuates protein and cell adhesion and modulates signaling mediated by its carriers and proteins that interact with these carriers. PolySia is crucial for the proper development of the nervous system and is upregulated during tissue regeneration and in highly invasive cancers. Our laboratory has previously shown that the neural cell adhesion molecule, NCAM, has an acidic surface patch in its first fibronectin type III repeat (FN1) that is critical for the polysialylation of N-glycans on the adjacent immunoglobulin domain (Ig5). We have also identified a polysialyltransferase (polyST) polybasic region (PBR) that may mediate substrate recognition. However, a direct interaction between the NCAM FN1 acidic patch and the polyST PBR has yet to be demonstrated. Here, we have probed this interaction using isothermal titration calorimetry and nuclear magnetic resonance (NMR) spectroscopy. We observe direct and specific binding between FN1 and the PBR peptide that is dependent upon acidic residues in FN1 and basic residues of the PBR. NMR titration experiments verified the role of the FN1 acidic patch in the recognition of the PBR and suggest a conformational change of the Ig5–FN1 linker region following binding of the PBR to the acidic patch. Finally, mutation of residues identified by NMR titration experiments impacts NCAM polysialylation, supporting their mechanistic role in protein-specific polysialylation.

Molecular Interactions of the Polysialytransferase Domain (PSTD) in ST8Sia IV with CMP-Sialic Acid and Polysialic Acid Required for Polysialylation of the Neural Cell Adhesion Molecule Proteins: An NMR Study

“…In our recent molecular modeling study, we found that the PBR and PSTD domains are remarkably close, the shortest distance between them being 4-6 Å (Figure 1) [27]. This finding is in accord with the previous report of Colley and colleagues [30]. Therefore, it is a reasonable supposition that an intramolecular interaction exists between PBR and PSTD in ST8Sia IV, based on the short distance between PBR and PSTD.…”

“…In summary, results from our current NMR studies, and previous findings on the DP of polySia chains and the cooperative interactions between PSTD-CMP-Sia, PSTD-polySia, PBR-NCAM, and PBR-PSTD [26][27][28][29][30]63,78] now provide greater insight into key molecular processes regulating the importance of these interactions in modulating NCAM polysialylation, which underlie many of the neuronal and immune system development and cancer metastatic events in human health and disease.…”

“…The interaction between PBR and NCAM was confirmed in NMR studies by Colley and colleagues in which the basic amino acid residues in the PBR of ST8Sia IV were found to be bind directly to the acidic patch in NCAM [30]. In our present studies, we initiated structural studies of the interactive binding of PSTD and CMP-Sia, TriSia (DP 3) and polySia using NMR and molecular modeling approaches [31].…”

“…Volkers et al [61] proposed that PSTD and PBR together form an extended basic groove for substrate recognition for the processive synthesis of the growing polySia chain. Bhide et al [30] further suggested that a possible PBR-PSTD interaction might relate to the basic surface of the polySTs for substrate recognition and/or polySia chain elongation. In our recent molecular modeling study, we found that the PBR and PSTD domains are remarkably close, the shortest distance between them being 4-6 Å (Figure 1) [27].…”

Autopolysialylation of polysialyltransferases is required for polysialylation and polysialic acid chain elongation on select glycoprotein substrates

“…These results suggested that these basic residues are required for ST8Sia-IV recognition of NCAM (33). Recently, we confirmed this notion using biophysical techniques to demonstrate a direct interaction between the isolated PBR region and NCAM FN1 that depended upon PBR residues Arg 82 and Arg 93 and FN1 residues Asp 520 , Glu 521 , and Glu 523 (34).…”