Purification and structural characterization of intact and fragmented nidogen obtained from a tumor basement membrane

Abstract

Extraction of a basement‐membrane‐producing mouse tumor with 6 M guanidine/HCl in the presence of protease inhibitors allowed the purification of the genuine form of the matrix protein nidogen (M_r= 150000) and, in addition, two defined fragments (M_r= 130000 and 100000). Smaller fragments (M_r= 80000 and 40000) were obtained under conditions with less stringent control of endogenous proteolysis. Intact nidogen and the larger fragments were similar in amino acid and carbohydrate (about 5%) composition, the presence of a single polypeptide chain, conformational features as revealed by CD spectroscopy and all shared major epitopes located on the M_r= 80000 fragment. Additional epitopes were found on intact nidogen and the M_r= 130000 fragment. Nidogen and the various fragments possess different N‐terminal amino acid sequences indicating a stepwise degradation from the N‐terminal end of the molecule. Electron microscopical and hydrodynamic studies of the M_r= 80000 fragment demonstrated a structure consisting of a globular head connected to a thin tail. Intact nidogen appears to contain a somewhat larger globule but the same tail, which is terminated at its opposite end by a second, smaller globular structure. The data suggest a multidomain structure for nidogen containing sites highly susceptible to proteolytic cleavage.