Sequence Requirements for Neuropilin-2 Recognition by ST8SiaIV and Polysialylation of Its O-Glycans

Abstract

Polysialic acid is an oncofetal glycopolymer, added to the glycans of a small group of substrates, that controls cell adhesion and signaling. One of these substrates, neuropilin-2, is a VEGF and semaphorin co-receptor that is polysialylated on its O-glycans in mature dendritic cells and macrophages by the polysialyltransferase ST8SiaIV. To understand the biochemical basis of neuropilin-2 polysialylation, we created a series of domain swap chimeras with sequences from neuropilin-1, a protein for which polysialylation had not been previously reported. To our surprise, we found that membrane-associated neuropilin-1 is polysialylated at ϳ50% of the level of neuropilin-2 but not polysialylated when it lacks its cytoplasmic tail and transmembrane region and is secreted from the cell. This was not the case for neuropilin-2, which is polysialylated when either membraneassociated or soluble. Evaluation of the soluble chimeric proteins demonstrated that the meprin A5 antigen-tyrosine phosphatase (MAM) domain and the O-glycan-containing linker region of neuropilin-2 are necessary and sufficient for its polysialylation and serve as better recognition and acceptor sites in the polysialylation process than those regions of neuropilin-1. In addition, specific acidic residues on the surface of the MAM domain are critical for neuropilin-2 polysialylation. Based on these data and pull-down experiments, we propose a model where ST8SiaIV recognizes and docks on an acidic surface of the neuropilin-2 MAM domain to polysialylate O-glycans on the adjacent linker region. These results together with those related to neural cell adhesion molecule polysialylation establish a paradigm for the process of protein-specific polysialylation. Polysialic acid (polySia)2 is a glycopolymer consisting of 8 to Ͼ100 ␣2,8-linked sialic acid residues (1). It is synthesized on the N-linked or O-linked glycans of a very specific set of cell surface proteins by two Golgi-localized polysialyltransferases (polySTs), ST8SiaII and ST8SiaIV (2, 3). Polysialylated proteins include the neural cell adhesion molecule (NCAM) (4), neuropilin-2 (NRP-2) (5), synaptic cell adhesion molecule 1 (6), the CD36 scavenger receptor in human milk (7), the ␣ subunit of the voltage-dependent sodium channel (8), and the polySTs themselves (9).PolySia has been shown to be crucially important for the development of the nervous system, synaptic plasticity and cell migration in the adult nervous system, and the regeneration of damaged nerves and tissues, and it is also up-regulated in many types of late stage cancers, where it is suggested to promote cancer metastasis (reviewed in Refs. 2, 3, and 10). Notably, work by Tanaka et al. (11) demonstrates that a substantial proportion of late stage non-small cell lung cancers express polySia, but not NCAM, suggesting that other substrates are polysialylated in these cancers. These functions of polySia are due to its abilities to serve as an anti-adhesive, a reservoir for biologically significant ligands, and a signaling modulator (reviewed ...

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

“…Detection of proteins by indirect immunofluorescence microscopy was performed as described previously. 30 Briefly, fixed and permeabilized cells were incubated with mouse monoclonal anti-V5 antibody (Thermo-Fisher) in blocking buffer (5% normal goat serum in PBS) and stained with FITC-conjugated goat anti-mouse IgG and 4′,6-diamidino-2-phenylindol (DAPI) to stain the nucleus. Cells were visualized and imaged using a Zeiss LSM 700 inverted confocal microscope equipped with an AxioCam digital microscope camera using a 100× oil-immersion lens.…”

“…25,29,30 Mutating surface acidic patch residues (Asp 520 , Glu 521 , and Glu 523 in NCAM and Asp 652 and Asp 653 in NRP-2) reduces the level of polysialylation of these proteins. 25,30 A similar two-domain paradigm is likely and has been previously suggested for the SynCAM 1 Ig2 domain (recognition) and Ig1 domain (polysialylation). 31 …”

Sialylation of N-glycans: mechanism, cellular compartmentalization and function

“…Using NCAM as a model substrate, and subsequent studies with NRP-2, our laboratory has determined that polysialylation is indeed protein specific in that the pol-ySTs must recognize the substrate through an initial protein-protein interaction that allows it to dock and then modify glycans in an adjacent domain [reviewed in Colley (2010), Colley et al (2014) and Bhide et al (2016)] (Fig. 3).…”

Crystal Structure of the Neuropilin-1 MAM Domain: Completing the Neuropilin-1 Ectodomain Picture

“…Although the molecular basis of this interaction is unknown, it is possible that GAL-1 associates with the glycans upstream of the NRP1 MAM, thereby bridging the NRP ectodomains. In a recent report (Bhide et al., 2016), two glutamate residues in the NRP2 MAM domain were identified as the recognition site for the polysialyltransferase (an enzyme that specifically modifies NRP2 but not NRP1). In our molecular model of the NRP2 MAM domain these residues are located on the unique protrusion of the molecular surface (Figure 4A), further supporting our suggestion that, in NRPs, the MAM domains interact with the specific regulatory proteins by utilizing distinct molecular surfaces.…”

“…NRP2 MAM was modeled based on the structure of NRP1 reported here. For the surface of NRP2, the green arrow points to the region, recently described as being engaged in interaction with polysialyltransferase (Bhide et al., 2016). See also Figure S1.…”