The Polysialyltransferases Interact with Sequences in Two Domains of the Neural Cell Adhesion Molecule to Allow Its Polysialylation

• Published in: The Journal of biological chemistry 2013-03, Vol.288 (10), p.7282-7293
• Main Authors: Thompson, Matthew G., Foley, Deirdre A., Colley, Karen J.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites - genetics; Carbohydrate Processing; Chlorocebus aethiops; COS Cells; Fibronectins - genetics; Fibronectins - metabolism; Glycobiology; Glycobiology and Extracellular Matrices; Glycoprotein Biosynthesis; Glycosylation; Immunoblotting; Immunoglobulins - genetics; Immunoglobulins - metabolism; Microscopy, Fluorescence; Models, Molecular; Molecular Sequence Data; Mutation; Neural Cell Adhesion Molecule; Neural Cell Adhesion Molecules - chemistry; Neural Cell Adhesion Molecules - genetics; Neural Cell Adhesion Molecules - metabolism; Polysaccharide; Polysaccharides - metabolism; Polysialic Acid; Polysialyltransferase; Protein Binding; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Sialic Acids - metabolism; Sialyltransferase; Sialyltransferases - genetics; Sialyltransferases - metabolism; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.438374

The neural cell adhesion molecule (NCAM) is the major substrate for the polysialyltransferases (polySTs), ST8SiaII/STX and ST8SiaIV/PST. The polysialylation of NCAM N-glycans decreases cell adhesion and alters signaling. Previous work demonstrated that the first fibronectin type III repeat (FN1) of NCAM is required for polyST recognition and the polysialylation of the N-glycans on the adjacent Ig5 domain. In this work, we highlight the importance of an FN1 acidic patch in polyST recognition and also reveal that the polySTs are required to interact with sequences in the Ig5 domain for polysialylation to occur. We find that features of the Ig5 domain of the olfactory cell adhesion molecule (OCAM) are responsible for its lack of polysialylation. Specifically, two basic OCAM Ig5 residues (Lys and Arg) found near asparagines equivalent to those carrying the polysialylated N-glycans in NCAM substantially decrease or eliminate polysialylation when used to replace the smaller and more neutral residues (Ser and Asn) in analogous positions in NCAM Ig5. This decrease in polysialylation does not reflect altered glycosylation but instead is correlated with a decrease in polyST-NCAM binding. In addition, inserting non-conserved OCAM sequences into NCAM Ig5, including an “extra” N-glycosylation site, decreases or completely blocks NCAM polysialylation. Taken together, these results indicate that the polySTs not only recognize an acidic patch in the FN1 domain of NCAM but also must contact sequences in the Ig5 domain for polysialylation of Ig5 N-glycans to occur. Background: Recognition of the protein substrate is the first step in polysialylation of its glycans. Results: Residues in the OCAM Ig5 domain are non-permissive for its polysialylation. Conclusion: The polysialyltransferases interact with residues in both the Ig5 and FN1 domains of NCAM to allow its polysialylation. Significance: A two-domain polysialyltransferase recognition site may be required for all polysialylated proteins.