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 ∼50% 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 membrane-associated or soluble. Evaluation of the soluble chimeric proteins demonstrated that the meprin A5 antigen-μ 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.
Highlights
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 up-regulated in many types of late stage cancers, where it is suggested to promote cancer metastasis
To determine the sequence requirements for NRP-2 polysialylation, we created a series of domain deletion mutants, chimeric proteins, and point mutants and evaluated their polysialylation when co-expressed with ST8SiaIV (Fig. 1)
Rollenhagen et al [35] showed that NRP-2 is polysialylated on O-glycans located in the linker region between the second factor 5/8 homology (F5/8) (F5/8-2) and MAM domains
Summary
Tissue culture media and reagents, including DMEM, FBS, and penicillin and streptomycin, were purchased from Fisher. Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA). To insert the signal sequence, the first 22 amino acids of NRP-2 were amplified to include a HindIII site at the N terminus and a KpnI site at the C terminus using primer set 2 (Table 1), and the amplified fragment was ligated into the pcDNA3.1 NRP-2⌬CF vector between the HindIII and KpnI sites. For the construction of the NRP-2⌬LCF construct, NRP-2 sequences from the MAM domain to the cytoplasmic tail were amplified with a KpnI site on the N terminus and a XbaI site on the C terminus using primer set 3 (Table 1). The EcoRV and NheI restriction sites flanking the MAM domains were removed from both the chimeric mutants using primer sets 8 –11 (Table 1) by site-directed mutagenesis. During the removal of the restriction sites from NRP-2⌬1, a APRIL 29, 2016 VOLUME 291 NUMBER 18
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
