Mapping of three carbohydrate attachment sites in embryonic and adult forms of the neural cell adhesion molecule.

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Abstract
The sialic-rich carbohydrate moiety of the neural cell adhesion molecule (N-CAM) undergoes major structural changes during development and plays a significant role in altering the homophilic binding of the molecule. To understand the mechanism of these changes, a cyanogen bromide (CNBr) fragment that contained 90% of the sialic acid of N-CAM was isolated and characterized according to the number of carbohydrate attachment sites and reactivity with specific monoclonal antibodies. The CNBr sialopeptide migrated on SDS-PAGE [sodium dodecyl sulfate-polyacrylamide gel electrophoresis] as a broad zone of MW 42,000-60,000. Upon treatment with neuraminidase, it was converted to a single component of MW 42,000, and subsequent, limited treatment with endoglycosidase F gave 4 evenly spaced components of MW 35,000-42,000, suggesting that it contained 3 attachment sites for N-linked oligosaccharides. The fragment reacted with monoclonal antibody 15G8, which detects the sialic acid in embryonic N-CAM, and with a monoclonal antibody, anti-(N-CAM) no. 2. Treatment with neuraminidase or with endoglycosidase F destroyed reactivity with 15G8 but not with anti-(N-CAM) no. 2. A similar CNBr sialopeptide was obtained from adult N-CAM; it contained sialic acid, had 3 N-linked oligosaccharides and reacted with anti-(N-CAM) no. 2, but not with 15G8 monoclonal antibodies. A peptide fragment, Fr2, comprising the NH2 terminal and middle regions of the molecule yielded a CNBr fragment closely similar to the fragment obtained from the whole molecule. The CNBr fragment from Fr2 reacted with monoclonal antibody anti-(N-CAM) no. 2. Fr1, comprising the NH2 terminal region alone, failed to react. The majority of the sialic acid evidently is localized in the middle region of the N-CAM molecule. Embryonic to adult conversion of N-CAM apparently is the result of differences in sialidase or sialyltransferase activity.