Structure and localization of O- and N-linked oligosaccharide chains on basement membrane protein nidogen.

The carbohydrate content of mouse nidogen predicts the occupation of two N- and about seven O-linked acceptor sites. The corresponding oligosaccharides were examined by sequential exoglycosidase digestions. The data indicate N-linked substitutions by several bi-, tri- and tetraantennary complex types of oligosaccharides which are further modified by additional lactosamines and terminal alpha-galactose and/or sialic acid. Mannose-rich oligosaccharides were of low abundance. O-linked structures included a di- and tetrasaccharide core structure that were in addition sialylated and may be similar to structures found in fetuin. Evidence is provided that the two sequence-predicted asparagine acceptors are almost fully substituted. Sequence analysis of tryptic peptides identified Thr-271, Ser-303, Thr-309, Thr-317, Thr-320, Thr-892 and Thr-905 as the most likely sites for galactosamine substitutions. These residues are located in the flexible link connecting the N-terminal globular domains G1 and G2 of nidogen and at the border between the rod and the C-terminal globe G3. Four of them showed Pro in the -1 or +3 position. All these Ser, Thr and Pro residues but not the N-linked attachment sites are identical in human nidogen.