Characterization of a 280-kD protein restricted to the coated pits of the renal brush border and the epithelial cells of the yolk sac. Teratogenic effect of the specific monoclonal antibodies.

Abstract

Within the brush border (BB) of proximal tubule cells (PTC), the intermicrovillar area connecting the base of the microvilli constitutes a distinct microdomain characterized by the presence on the cytoplasmic side of the membrane of an extensive clathrin coat (1). Kerjaschki and Farquhar (2) and Chatelet et al. (3) have shown that a 330-kD glycoprotein (gp), initially described as the nephritogenic antigen of Heymann's nephritis, is concentrated on the luminal aspect of these areas, at variance with other BB proteins such as maltase (4), which are distributed over the entire surface of the microvilli. To our knowledge, gp330 is the only membrane gp confined to the intermicrovillar domain (IMVD) . It is also expressed in coated pits of glomerular epithelial cells, pneumocytes type II, epididymis, and epithelial cells of the visceral yolk sac (VYS) (5, 6), but its function remains unknown . This paper describes a 280-kD BB protein, defined by mAbs raised against rat renal BB, which is only found in the IMVD of the PTC and on the apical domain of epithelial cells of the VYS . When injected to pregnant rats, the mAbs induce embryonic resorption and/or fetal abnormalities, suggesting that the 280-kD protein plays a key role in endocytosis-related cell function. Materials and MethodsAntibodies. The two mAbs reported in this study (F5/75, an IgGI with a pl of 6.8-7.6, and F5/46, an IgGI with a pl of 8.1-8.5) were obtained by immunization of mice against rat BB (7) . They were analyzed in relation to mAb F1/12 and polyclonal antibodies specific for gp330 (8), and mAb F2/180 reactive with the 300-kD maltase .Characterization of the 280-kD Antigen. The antigen identified by mAb F5/75 and F5/46 on tubular BB was identified by immunoprecipitation ofradiolabeled BB membrane vesicles (BBMV) (7) and immunoadsorption techniques . Purified mAbs F5/75 and F5/46, as well as mAbs F1/12 and F2/180, were individually coupled to Sepharose 4B (Pharmacia Fine Chemicals, Velizy, France) . All steps involving antigenic preparations and immunoadsorption were carried out in the presence of 1 mM PMSF and 2 mM benzamidine . BBMV (7,8) and yolk sacs dissected at 19 d of gestation were washed extensively in PBS and solubilized in 1 % Triton X100 . 1-2 ml of each immunoabsorbent was incubated with either 20 mg of solubilized BBMV, or with the material from 10 yolk sacs.

Light chains are a ligand for megalin

“…These antibodies, used as whole serum preserved with azide, were monospecific by immunoblotting on whole brush border preparations and by immunoprecipitation of biosynthetically labeled yolk sac epithelial cells in culture (17,18). For a control antiserum, available antisera unrelated to cubilin or megalin but of the same species and isotype were surveyed by flow cytometry to find a serum with the same peak binding as the cubilin and megalin antisera.…”

“…Except as noted, experiments were conducted using immortalized yolk sac cells from the Brown Norway rat (BN-16) (17). An apical brush border and a specialized endosomal pathway similar to the renal proximal tubule, including abundant expression of megalin and cubilin, characterize these cells.…”

“…Residual free dye was separated from light chain conjugated dye by dialysis into a suitable binding buffer using a Slidalyzer cassette (Pierre, Rockford, IL). Binding of light chains was investigated in presence of 1,000-to 2,000-fold dilutions of anti-cubilin or anti-megalin polyclonal antibodies recognizing the holoprotein (2,10,17,18). Binding of FluorX conjugated light chains to brush-border membrane vesicles, with and without antibody or ligand interference, was analyzed by flow cytometry using a FACStar Plus flow cytometer to collect data files of 2,000 observations per sample.…”

“…Polyclonal antibodies against cubilin and megalin were raised in rabbits against proteins purified by immunoaffinity chromatography using previously reported monoclonal antibodies coupled to Sepharose 4B (2,10,17,18). These antibodies, used as whole serum preserved with azide, were monospecific by immunoblotting on whole brush border preparations and by immunoprecipitation of biosynthetically labeled yolk sac epithelial cells in culture (17,18).…”

Cubiline : rôle physiopathologique et relations avec la mégaline

“…Le cas du rein est particulier, car la mégaline exprimée au pôle apical des cellules du TCP permet l'internalisation des complexes TCII-B12 présents dans le fluide tubulaire, et la sauvegarde de quantités importantes de vitamine B12, probablement du même ordre de grandeur que les quantités absorbées dans l'intestin. résultats peuvent être rapportés à la fixation des anticorps sur le sac vitellin et à une inhibition des méca-nismes d'endocytose [22,23] qui assurent les transferts de la mère vers le foetus (Figure 4). L'effet des anticorps anti-cubiline pourrait être expliqué par une diminution de la quantité de protéines internalisées, ce qui conduirait à une diminution de la quantité d'acides aminés transférés vers l'embryon.…”