Characterization of L-CAM, a major cell adhesion molecule from embryonic liver cells

Abstract

We have developed a method for purifying L-CAM, the cell adhesion molecule from embryonic chicken liver cells, and have compared its properties with those of N-CAM, the neural cell adhesion molecule. L-CAM was released from membranes with trypsin, purified by a series of chemical techniques, and used to generate monoclonal antibodies which allowed the identification of the intact L-CAM molecule from membranes. The monoclonal antibodies were used to isolate trypsin-released L-CAM in a single step by affinity chromatography. Material purified by either technique was predominantly a component of M r 81,000 on NaDodSO 4 /polyacrylamide gel electrophoresis with a pI of 4.0-4.5. Rabbit antibodies to this component and to the M r 81,000 species that had been further purified on NaDodSO 4 /polyacrylamide gel electrophoresis displayed all of the activities of anti-L-CAM. Some of the trypsin-released L-CAM bound specifically to lentil lectin, suggesting that L-CAM is a glycoprotein. The apparent molecular weight of material having L-CAM antigenic determinants depended upon the procedures used to extract membranes; this appears to account for the various values reported previously in the literature. Both the rabbit serum antibodies and the monoclonal antibodies detected the M r 81,000 species on immunoblots of unfractionated trypsin-released material. Immunoblots of whole liver cell membranes with the same antibodies revealed a major M r 124,000 component, with minor components of M r 94,000 and 81,000. Active L-CAM derivatives released by trypsin in the presence of EGTA were detected as a species of M r 40,000. L-CAM derivatives obtained by extraction of membranes with EDTA alone appeared as species of M r 53,000, 62,000, and 81,000. The combined results suggest that L-CAM on the cell surface is an acidic glycoprotein of M r 124,000. In the presence of calcium, the molecule can be released from membranes by trypsin as a soluble M r 81,000 fragment; in the absence of calcium, it is released by either endogenous proteases or by trypsin as a variety of smaller fragments.