Microdomains of distinctive glycoprotein composition in the kidney proximal tubule brush border.

Abstract

Two membrane proteins, maltase and gp330 (the pathogenic antigen of Heymann nephritis), present in the proximal tubule brush border have recently been independently purified and found to be large glycoproteins of similar molecular weight (Mr = 300,000) by SDS PAGE . To determine the relationship between the two, monoclonal antibodies raised against the purified proteins were used for comparative immunochemical analyses and immunocytochemical localization . When a detergent extract of [35S] methionine-labeled rat renal cortex was used for immunoprecipitation with monoclonal antimaltase IgG, a single band of -300 kdaltons was precipitated, whereas a single 330-kdalton band was precipitated with monoclonal anti-gp330 IgG . Monoclonal antimaltase (gp300) IgG also immunoprecipitated maltase activity from solubilized renal maltase preparations, whereas monoclonal antigp330 IgG failed to do so . When cyanogen bromide-generated peptide maps of the two proteins were compared, there were many similar peptides, but some differences . When maltase and gp330 were localized by indirect immunofluorescence and by indirect immunoperoxidase and immunogold techniques at the electron microscope level, they were found to be differently distributed in the brush border of the initial (S1 and S2) segments of the proximal tubule : maltase was concentrated (-90%) on the microvilli, and gp330 was concentrated (-90%) in the clathrin-coated apical invaginations located at the base of the microvilli . We conclude that maltase (gp300) and the Heymann nephritis antigen (gp330) are structurally related membrane glycoproteins with a distinctive distribution in the proximal tubule brush border which may serve as markers for the microvillar and coated microdomains, respectively, of the apical plasmalemma .The luminal aspect of the kidney proximal tubule consists of a brush border that is differentiated into two distinct types of structures-microvilli and intermicrovillar apical invaginations (1) . The microvilli appear to closely resemble those of the intestine in their absorptive function and supporting cytoskeletal elements (2, 3). The apical invaginations are known to function in endocytosis (1) and have recently been shown to have extensive clathrin coats on their cytoplasmic surface (4), thus resembling clathrin-coated pits in other locations . At present there is relatively little information available on the comparative protein composition of these two proximal tubule structures. Microvillar fractions (which consist mainly ofvesicles derived from microvilli along with some THE JOURNAL OF CELL BIOLOGY " VOLUME 98 APRIL 1984 1505-1513 C The Rockefeller University Press -0021-9525/84/04/1505/09 $1 .00 contaminating intermicrovillar membranes), have been isolated and analyzed (5) and found to contain numerous proteins, some ofwhich are cytoskeletal and others are transmembrane glycoproteins identified as hydrolytic enzymes (aminopeptidases, disaccharidases, etc .) (5) or transport systems (e.g., for glucose and amino acids) (6) . Evide...

gp330 associates with a 44-kDa protein in the rat kidney to form the Heymann nephritis antigenic complex.

“…1, lanes F and G). The larger protein was previously shown to be gp330 by immunoprecipitation (5,6,16) and Western blot analysis (17). These results suggest that the HN antigen is a dimer consisting of gp330 and an associated 44-kDa subunit.…”

“…Immunocytochemical and immunoblotting data indicate that gp330 is an abundant protein in the brush border of rat kidney proximal tubule (6,16). To facilitate biochemical characterization of gp330, microvillar fractions were prepared from perfused rat kidneys to enrich for membrane proteins associated with the brush border (Fig.…”

Membrane-bound and fluid-phase macromolecules enter separate prelysosomal compartments in absorptive cells of suckling rat ileum.

“…In kidney proximal tubule cells, for example, an extensive system of apical tubules is involved in the uptake of protein from glomerular filtrate (14). Although these tubules may accomplish the recycling of endocytosed membrane (4), the tubular membranes differ antigenically from the intermicrovillar, endocytic microdomains of the apical plasma membrane (19). In hepatocytes (44) and hepatoma cells (10,24), peripheral tubulocisternae function as an intermediate prelysosomal compartment through which ligands pass during receptor-mediated endocytosis.…”

Heparan sulfate proteoglycans are concentrated on the sinusoidal plasmalemmal domain and in intracellular organelles of hepatocytes.

“…Immunoperoxidase: Procedures used for indirect immunoperoxidasc staining were given elsewhere (21)(22)(23). In brief, cryostat sections (15-20 urn) were prepared from periodate-lysine-paraformaldehyde-fixed liver and incubated in (a) normal rabbit or immune serum (diluted Ys) or IgG (3 #g/ml) for 12-16 h and (b) peroxidase-conjugated sheep anti-rabbit Fab (2 h).…”