Phosphacan, a soluble nervous tissue-specific chondroitin sulfate proteoglycan, is an alternative splicing product representing the entire extracellular domain of a transmembrane receptor-type protein-tyrosine phosphatase (RPTP/) that also occurs as a chondroitin sulfate proteoglycan in brain. We have previously demonstrated that phosphacan binds with high affinity to neural cell adhesion molecules (Ng-CAM/L1 and N-CAM) and to the extracellular matrix protein tenascin and that it is a potent inhibitor of cell adhesion and neurite outgrowth. Tryptic digests of I-labeled phosphacan contain two glycopeptides that bind to Ng-CAM/L1, N-CAM, and tenascin. The larger of these (17 kDa) begins at Gln-209 near the end of the carbonic anhydrase-like domain of phosphacan/RPTP/, whereas a 13-kDa glycopeptide begins at His-361 located in the middle of the fibronectin type III-like domain. Treatment of phosphacan with peptide N-glycosidase under nondenaturing conditions reduced its binding to the neural cell adhesion molecules and tenascin by 65-75%, whereas endo--N-acetylglucosaminidase H had no effect, and peptide N-glycosidase treatment both decreased the molecular sizes of the tryptic peptides to 11 kDa and abolished their binding. Based on the amino acid sequence of phosphacan, it can be concluded that each of the tryptic peptides contains one potential N-glycosylation site (at Asn-232 and Asn-381), and analyses of the isolated glycopeptides demonstrated the presence of sialylated complex-type oligosaccharides. Our results therefore indicate that the interactions of phosphacan/RPTP/ with neural cell adhesion molecules and tenascin are mediated by asparagine-linked oligosaccharides present in their carbonic anhydrase- and fibronectin type III-like domains.

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