Polysialic Acid on Neuropilin-2 Is Exclusively Synthesized by the Polysialyltransferase ST8SiaIV and Attached to Mucin-type O-Glycans Located between the b2 and c Domain

• Published in: The Journal of biological chemistry 2013-08, Vol.288 (32), p.22880-22892
• Main Authors: Rollenhagen, Manuela, Buettner, Falk F.R., Reismann, Marc, Jirmo, Adan Chari, Grove, Melanie, Behrens, Georg M.N., Gerardy-Schahn, Rita, Hanisch, Franz-Georg, Mühlenhoff, Martina
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Animals; Bone Marrow Cells - cytology; Bone Marrow Cells - metabolism; Chlorocebus aethiops; COS Cells; Dendritic Cells; Dendritic Cells - cytology; Dendritic Cells - metabolism; Glycobiology and Extracellular Matrices; Glycoprotein; Glycosylation; Glycosyltransferases; Humans; Mice; Mice, Knockout; Neuropilin-2 - genetics; Neuropilin-2 - metabolism; O-Glycan Analysis; Polysialic Acid; Post-translational Modification; Protein Processing, Post-Translational - physiology; Protein Structure, Tertiary; Sialic Acids - genetics; Sialic Acids - metabolism; Sialyltransferases - genetics; Sialyltransferases - metabolism; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.463927

Neuropilin-2 (NRP2) is well known as a co-receptor for class 3 semaphorins and vascular endothelial growth factors, involved in axon guidance and angiogenesis. Moreover, NRP2 was shown to promote chemotactic migration of human monocyte-derived dendritic cells (DCs) toward the chemokine CCL21, a function that relies on the presence of polysialic acid (polySia). In vertebrates, this posttranslational modification is predominantly found on the neural cell adhesion molecule (NCAM), where it is synthesized on N-glycans by either of the two polysialyltransferases, ST8SiaII or ST8SiaIV. In contrast to NCAM, little is known on the biosynthesis of polySia on NRP2. Here we identified the polySia attachment sites and demonstrate that NRP2 is recognized only by ST8SiaIV. Although polySia-NRP2 was found on bone marrow-derived DCs from wild-type and St8sia2−/− mice, polySia was completely lost in DCs from St8sia4−/− mice despite normal NRP2 expression. In COS-7 cells, co-expression of NRP2 with ST8SiaIV but not ST8SiaII resulted in the formation of polySia-NRP2, highlighting distinct acceptor specificities of the two polysialyltransferases. Notably, ST8SiaIV synthesized polySia selectively on a NRP2 glycoform that was characterized by the presence of sialylated core 1 and core 2 O-glycans. Based on a comprehensive site-directed mutagenesis study, we localized the polySia attachment sites to an O-glycan cluster located in the linker region between b2 and c domain. Combined alanine exchange of Thr-607, -613, -614, -615, -619, and -624 efficiently blocked polysialylation. Restoration of single sites only partially rescued polysialylation, suggesting that within this cluster, polySia is attached to more than one site. Background: Polysialylated neuropilin-2 mediates CCL21-driven chemotactic migration of dendritic cells. Results: Deletion of either ST8SiaIV or O-glycosylation sites located between b2 and c domain abrogates polysialylation of neuropilin-2. Conclusion: Polysialylation of neuropilin-2 occurs in the same linker region as GAGylation of neuropilin-1. Significance: Defining enzyme and acceptor site requirements is crucial for understanding how polysialylation of neuropilin-2 is regulated.