Degradation and invasion of basement membrane by
tumor cells involves the cooperative hydrolysis of
proteoglycans,
collagens, and
glycoproteins mediated by a number of
enzymes including
proteases,
collagenases, and
glycosidases. In order to study these processes in vitro, a tissue culture system was developed in which bovine corneal endothelial cell extracellular matrix (ECM) serves as a substrate for attachment and degradation by human ovarian
carcinoma cells. Using this system, a correlation was observed between solubilization of
glycoconjugates present in ECM and extracellular levels of
beta-N-acetylglucosaminidase (EC 3.2.1.30). To determine the role of individual
isoenzymes of
beta-N-acetylglucosaminidase (beta-NAG) in ECM degradation, the cellular and secreted forms of the
enzyme were fractionated and characterized. Three intracellular
isoenzyme forms A, I, and B, were isolated from invasive human ovarian
carcinoma cell line
A-121. In cell homogenate, forms A and B corresponded to 65 and 33% of total beta-NAG activity, respectively. Form I was found to be localized in the plasma membrane fraction of these cells. Two secreted forms of beta-NAG (As and Bs) were detected in serum-free medium. The separated intracellular and secreted
isoenzymes demonstrated similar Km values, ranging from 1 to 5 mM, with p-nitro-B-N-acetylglucosaminide substrate. Treatment of [3H]
glucosamine-labeled ECM with the separated
isoenzymes of beta-NAG resulted in time- and concentration-dependent releases of radioactivity with potency of 1 greater than B much greater than A. These results suggest that human ovarian
carcinoma cell
beta-N-acetylglucosaminidase isoenzymes (forms B and A) contribute to ECM degradation as secreted
enzymes and form I as a membrane
enzyme.