Immunohistochemical localization and characterization of a protein from the basolateral membrane of rat small intestine epithelium using monoclonal antibody GZ-1.

The proteins of the basolateral membrane (BLM) of small intestine epithelial cell in rat have been less precisely described than those of the microvillus membrane (MVM). To identify BLM-specific proteins, Balb/c mice were immunized with isolated intestinal epithelial cells and monoclonal antibodies (MAb) to their cell membrane, produced with the hybridoma technique. One of the MAb so obtained (GZ-1), a class 1 IgG, is specifically directed to a surface membrane protein of intestinal epithelium (GZ-1-Ag). The MAb served to characterize the protein as follows. Light microscopic immunohistochemical FITC labeling and, still more clearly, electron microscopic labeling with colloidal gold on Lowicryl sections of small intestinal tissue, show that the GZ-1-Ag occurs only in BLM of the absorptive cell and the goblet cell. It is not present in the MVM, the tight-junction area, and probably in the desmosomal sections of the membrane. The crypt cells are more markedly labeled with GZ-1 than are the villus cells; the villus cells are also more clearly labeled from the duodenum to the ileum. Gross analysis of the position of the gold marker on the BLM indicates that GZ-1-Ag is probably integrated into the lipid bilayer. With immunoblotting (with HRP as marker), a single band of MW 42,000 D can be identified as the corresponding GZ-1-Ag from the protein band pattern obtained with SDS-PAGE from BLM isolated in the presence of protease inhibitors (PI). In BLM fractions isolated without protease inhibition, a band of MW 30,000 D can be labeled with GZ-1. These results are interpreted as follows: GZ-1-Ag is a protein of MW 42,000 D. On isolation of the BLM without PI, a piece of this protein is broken off by proteolysis. The larger piece of the molecule (30,000 D) is not accessible to the proteolytic enzyme owing to its localization in the BLM, and therefore remains intact (and recognizable by the Ab). The preferred position of the gold marker on the BLM is in agreement with this explanation.