Mapping of three carbohydrate attachment sites in embryonic and adult forms of the neural cell adhesion molecule.
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. In order 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 as a broad zone of Mr 42,000-60,000. Upon treatment with neuraminidase, it was converted to a single component of Mr 42,000, and subsequent, limited treatment with endoglycosidase F gave four evenly spaced components of Mr 35,000-42,000, suggesting that it contained three 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 three 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. These data confirm that the majority of the sialic acid is localized in the middle region of the N-CAM molecule and support the hypothesis that embryonic to adult conversion of N-CAM is the result of differences in sialidase or sialytransferase activity.
D000375:Aging; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D050260:Carbohydrate Metabolism; D015815:Cell Adhesion Molecules; D002642:Chick Embryo; D002645:Chickens; D003488:Cyanogen Bromide; D007158:Immunologic Techniques; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid; D009439:Neuraminidase / Q000494:pharmacology; D010446:Peptide Fragments / Q000302:isolation & purification; D012794:Sialic Acids / Q000032:analysis
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D015815:Cell Adhesion Molecules; D010446:Peptide Fragments; D012794:Sialic Acids; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid; D003488:Cyanogen Bromide