Isolation, partial characterization, and localization of a rat renal tubular glycoprotein antigen. Antibody-induced birth defects.

Abstract

A glycoprotein with an apparent 340,000 mol wt (gp 340K) was isolated from rat kidney saline-soluble extract by ammonium sulfate precipitation, DE 52 ion-exchange cellulose chromatography, concanavalin A affinity column, Sephacryl S-300 gel filtration, and discontinuous polyacrylamide gel electrophoresis (PAGE). The relative purity of gp 340K was examined by double immunodiffusion analysis, disc PAGE, and immunoelectrophoresis. Injection of rabbit gp 340K antiserum into pregnant rats during the organogenetic period induced abnormal embryonic development, fetal growth retardation, and embryonic death. Antiserum against the immunocomplexes isolated by immobilized protein A also produced the same embryotoxic effects. The biologic effects of the antisera appeared to be dose dependent. Defects such as anophthalmia, hydrocephaly, exencephaly, cleft palate, cleft lip, and some cardiovascular anomalies were observed. The most frequently observed anomaly was anophthalmia. Immunofluorescent localization studies indicated that gp 340K antibodies localized in vivo in the visceral yolk-sac endodermal cells and the embryonic endoderm. In vitro immunofluorescent localization studies revealed that gp 340K was a component of the renal tubular cells that cross-reacted with antigen in the visceral yolk-sac endodermal cells and embryonic endoderm. The underlying mechanism whereby gp 340K antibodies induce birth defects is not known. Three hypotheses were discussed.

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.

“…The induction of birth defects by antikidney antibodies was described by Brent (9) and confirmed by others (10,11) . Our observations provide new data in two domains : (a) Leung (11) isolated a 340-kD BB antigen, reported as diffuse on BB microvilli, which elicited teratogenic antibodies reactive with epithelial cells of the VYS, but the antigen isolated from the latter structure (12) consisted of two peptides of 30 and 60 kD .…”

Teratogenic antibodies are directed against a coated‐pit glycoprotein

“…Teratogenic rabbit immunoglobulins G (IgG) against gp340 were isolated from teratogenic antiserum (Leung, 1982). Briefly, rabbit IgG molecules were precipitated at 33% ammonium sulfate saturation, pH 7.8, and subsequently isolated by DE 52 ion-exchange cellulose equilibrated with 0.0175 M phosphate buffer, pH 6.3.…”

“…Our laboratory isolated a renal tubular glycoprotein antigen with an apparent 340,000 molecular weight (designated as gp340) and showed that rabbit antibodies against gp340 were teratogenic (Leung, 1982). Later, two small antigenic peptides (60,000 and 30,000) from VYS endodermal cells were identified and shown to be in complete identity with gp340 (Leung et al, 1985).…”

Teratogenic antibody internalization by rat visceral yolk‐sac endoderm in vitro: An ultrastructural colloidal gold tracer study

“…The VYS was then cut into pieces of about 5 mm on a side and subsequently incubated at 4°C in MEM containing teratogenic rabbit immunoglobulins G (IgG) against gp340 for 30 min with slight agitation or in heat-inactivated normal rabbit serum (1:200 dilution) serving as a control. The teratogenic IgGs were isolated from teratogenic antiserum (Leung, 1982). Briefly, rabbit IgG molecules were precipitated at 33% ammonium sulfate saturation, pH 7.8, and subsequently isolated by DE 52 ion-exchange cellulose equilibrated with 0.0175 M phosphate buffer, pH 6.3.…”

“…Our laboratory isolated the responsible antigen from both rat kidney homogenate (Leung, 1982) and from extract of VYS endoderm (Leung et al, 1985). Freeman and Brown (1986) also identified a low molecular weight VYS peptide as the responsible antigen.…”