Myeloma light chains are ligands for cubilin (gp280).

Although myeloma light chains are known to undergo receptor-mediated endocytosis in the kidney, the molecular identity of the receptor has not been characterized. We examined the interaction between cubilin (gp280) and four species of light chains isolated from the urine of patients with multiple myeloma. Four lines of evidence identify cubilin, a giant glycoprotein receptor, which is restricted in distribution to endocytic scavenger pathways and which has potent effects on endosomal trafficking, as a potentially physiologically relevant binding site for light chains: 1) light chains coeluted during immunoaffinity purification of cubilin; 2) polyclonal antisera to cubilin but not control sera, displaced human light chain binding from rat renal brush-border membranes; 3) cubilin bound to multiple species of light chains during surface plasmon resonance; 4) anti-cubilin antiserum interfered with light chain endocytosis by visceral yolk sac epithelial cells. However, both binding of light chains to brush-border membranes and endocytosis of light chains by yolk sac epithelial cells were only partially inhibited by anticubilin antibodies, suggesting presence of additional or alternate binding sites for light chains. Excess light chain had a potent inhibitory effect on endosomal fusion in vitro. Binding showed dose and time-dependent saturability with low-affinity, high-capacity equilibrium binding parameters. These data demonstrate that cubilin plays a role in the endocytosis and trafficking of light chains in renal proximal tubule cells.

Membrane potential mediates H(+)-ATPase dependence of "degradative pathway" endosomal fusion.

In some epithelial cell lines, the uptake and degradation of proteins is so pronounced as to be regarded as a specialized function known as "degradative endocytosis." The endosomal pathways of the renal proximal tubule and the visceral yolk sac share highly specialized structures for "degradative endocytosis." These endosomal pathways also have a unique distribution of their H(+)-ATPase, predominantly in the subapical endosomal pathway. Previous studies provide only indirect evidence that H(+)-ATPases participate in endosomal fusion events: formation of vesicular intermediates between early and late endosomes is H(+)-ATPase dependent in baby hamster kidney cells, and H(+)-ATPase subunits bind fusion complex proteins in detergent extracts of fresh rat brain. To determine directly whether homotypic endosomal fusion is H(+)-ATPase dependent, we inhibited v-type H(+)-ATPase during flow cytometry and cuvette-based fusion assays reconstituting endosomal fusion in vitro. We report that homotypic fusion in subapical endosomes derived from rat renal cortex, and immortalized visceral yolk sac cells in culture, is inhibited by the v-type H(+)-ATPase specific inhibitor bafilomycin A1. Inhibition of fusion by H(+)-ATPase is mediated by the membrane potential as collapsing the pH gradient with nigericin had no effect on homotypic endosomal fusion, while collapsing the membrane potential with valinomycin inhibited endosomal fusion. Utilizing an in vitro reconstitution assay this data provides the first direct evidence for a role of v-type H(+)-ATPase in mammalian homotypic endosomal fusion.

The intrinsic factor-vitamin B12 receptor and target of teratogenic antibodies is a megalin-binding peripheral membrane protein with homology to developmental proteins.

The present report shows the molecular characterization of the rat 460-kDa epithelial glycoprotein that functions as the receptor facilitating uptake of intrinsic factor-vitamin B12 complexes in the intestine and kidney. The same receptor represents also the yolk sac target for teratogenic antibodies causing fetal malformations in rats. Determination of its primary structure by cDNA cloning identified a novel type of peripheral membrane receptor characterized by a cluster of eight epidermal growth factor type domains followed by a cluster of 27 CUB domains. In accordance with the absence of a hydrophobic segment, the receptor could be released from renal cortex membranes by nonenzymatic and nonsolubilizing procedures. The primary structure has no similarity to known endocytic receptors but displays homology to epidermal growth factor and CUB domain proteins involved in fetal development, e.g. the bone morphogenic proteins. Electron microscopic immunogold double labeling of rat yolk sac and renal proximal tubules demonstrated subcellular colocalization with the endocytic receptor megalin, which is expressed in the same epithelia as the 460-kDa receptor. Furthermore, megalin affinity chromatography and surface plasmon resonance analysis revealed a calcium-dependent high affinity binding of the 460-kDa receptor to megalin, which thereby may mediate its vesicular trafficking. Due to the high number of CUB domains, accounting for 88% of the protein mass, we propose the name cubilin for the novel receptor.

Gentamicin inhibits rat renal cortical homotypic endosomal fusion: role of megalin.

Megalin, a giant glycoprotein receptor heavily concentrated in the early endosomal pathway of renal proximal tubular cells, binds gentamicin with high affinity and delivers the drug to lysosomes. Utilizing an in vitro reconstitution assay we tested whether gentamicin-induced vacuolation is associated with inhibition of early endosomal fusion, as well as whether megalin plays a role in mediating these effects. Pretreatment of rats with gentamicin inhibited rat renal proximal tubular homotypic endosomal fusion. Administered simultaneously, gentamicin and polymers of polyaspartic acid, which protect against the hemodynamic effects of gentamicin nephrotoxicity, had no net effect on fusion. Polyaspartic acid alone had no effect on fusion. Antisera to the tail of the megalin/gentamicin receptor inhibited fusion, whereas non-specific controls had no effect. Peptides matching homologous NPXY repeat sequence motifs in the cytosolic tail stimulated endosomal fusion, whereas reverse sequence control peptides had no effect. These data suggest that gentamicin inhibition of endosomal fusion in the renal proximal tubule is a damage mechanism mediated by specific peptide sequences in the cytosolic tail of the giant gentamicin-binding receptor megalin and that receptors can effect the fusion properties of membranes in which they reside.

Immunofunctional properties of a yolk sac epithelial cell line expressing two proteins gp280 and gp330 of the intermicrovillar area of proximal tubule cells: inhibition of endocytosis by the specific antibodies.

The apical domain of epithelial cells lining the proximal tubule and the yolk sac is characterized by the development of extensive microvilli which limit intermicrovillar spaces backed on their cytoplasmic aspect by a coat of clathrin. These membrane areas which give rise to endocytic vesicles are characterized by the expression on their outer aspect of two high molecular weight glycoproteins: gp330 and gp280. In this study we report on an epithelial cell line, BN/MSV, derived from a yolk sac carcinoma which expresses these two glycoproteins. By indirect immunofluorescence, gp330 and gp280 were detectable on the cell surface and after permeabilization in intracytoplasmic vesicles. At the ultrastructural level they were concentrated in clathrin-coated membrane areas and although gp280 could also be detected in non-coated areas. The two proteins were synthesized independently in the form of high molecular weight polymers by biosynthetically labeled BN/MSV cells. Both were released in the supernatant, but, in spite of previously reported similarities by peptide mapping, only gp330 coprecipitated with a 45 kDa protein comigrating with the alpha 2-macroglobulin receptor-associated protein (MRAP). Culture of the cells in the presence of antibodies to gp280 and to a lesser extent of antibodies to gp330 inhibited the internalization of [14C]sucrose and peroxidase. When followed intracellularly at the ultrastructural level, the compartments containing peroxidase in the presence of anti-gp280 or gp330 antibodies were morphologically distinct from those observed under control conditions: vesicles were of smaller size and irregular shape and accumulation in lysosomes was delayed.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II (AII)-related idiotypic network. I. Common occurrence of AII internal image on rabbit anti-idiotypic antibodies.

Internal images of foreign Ag have been demonstrated in a variety of systems as anticipated by the idiotypic network theory formulated by Jerne. However, they seem to be of rare occurrence. In order to estimate the actual frequency of antibodies bearing internal images (Ab2-beta) of angiotensin II (AII), a phylogenetically conserved peptide made up of eight amino acids, nine rabbits were immunized with affinity or protein A purified anti-AII antibodies (Ab1) from allotype-matched rabbits. Four of nine antiidiotypic antibodies (Ab2) exhibited internal image-like reactivity. They recognized all the polyclonal Ab1 tested, whatever the species (rabbit, mouse, guinea pig). In addition, they were strongly reactive with three mAb specific for a carboxy terminus epitope on AII (mAb 110, 199, and 211) and with a fourth monoclonal Ab1 (133) identifying a more central epitope. Advantage was taken of this reactivity with mAb1 to purify Ab2-beta by affinity chromatography of Ab2 on Sepharose 4B covalently linked to the three monoclonal Ab1 specific for the carboxy terminus epitope. The eluate displayed typical internal image properties: 1) it reacted with all the polyclonal Ab1 tested, 2) this reaction was completely abolished by AII, and 3) rabbits and mice immunized with the eluate all produced Ab1. The AII related idiotypic network is thus characterized by high frequency and immunogenicity of AII internal images. In addition, reactivity of the latter with monoclonal Ab1 indicates variable expression on Ab2-beta of the epitopes defined by the mAb on the nominal Ag.

Angiotensin II (AII)-related idiotypic network. II. Heterogeneity and fine specificity of AII internal images analyzed with monoclonal antibodies.

Although the structural basis of internal images borne by beta type monoclonal anti-idiotypic antibody (Ab2) begins to be elucidated, there is little information on the repertoire of epitopes which make up the internal images expressed by polyclonal Ab2. We addressed this question by using a two-way approach in the angiotensin II (AII)-related idiotypic network, a system characterized by common occurrence of internal images on rabbit Ab2. First, two sets of internal images were purified in parallel by affinity chromatography on Sepharose 4B covalently linked to either mAb 110 (S4B-110), a mAb specific for a phenylalanine requiring carboxy terminus epitope (Phe8) on AII, or mAb 133 (S4B-133), reactive with a more central epitope also expressed on Phe8 substituted peptide analogs. The respective eluates, EL1 110 and E11 133, exhibited only partially overlapping reactivity, as demonstrated by 1) a different pattern of inhibition by various AII peptide analogues of EL1 110 and E11 133 binding to the same anti-AII antibody (Ab1) (either the homologous polyclonal Ab1 102 or mAb 133), 2) and a distinct profile of EL1 110 and EL1 133 binding to 12 biotinylated monoclonal Ab1 identifying a variety of epitopes on AII. To analyze further the respective distribution of mAb 110 and mAb 133 defined epitopes on Ab2-beta molecules, Ab2 were submitted to sequential affinity chromatography on S4B-110 followed by S4B-133, and the fractionated internal images were characterized by the pattern of binding to the various monoclonal Ab1. It was thus possible to purify two Ab2-beta subpopulations that exclusively imaged the determinant identified by mAb 110 (ii 110) or that identified by mAb 133 (ii 133). A third subpopulation which was successively retained on S4B-110 and S4B-133 expressed both internal images (ii 110 + 133), and was additionally reactive with all the other monoclonal Ab1 tested. In any case, monoclonal Ab1 binding to the different sets of internal images was totally inhibited by an excess of AII. These results indicate that the repertoire of internal epitopes is similar to that of the nominal Ag, but is scattered over distinct subpopulations of Ab2-beta molecules that can be fractionated by affinity chromatography. Some of the latter seem to bear several epitopes and resemble the whole nominal Ag, whereas others appear to image only one determinant. Second, we raised 7 anti-anti-idiotypic mAb (monoclonal Ab3) against affinity-purified Ab2-beta and analyzed their fine specificity for AII.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of monoclonal antibodies specific for human Tamm-Horsfall protein.

Fifteen monoclonal antibodies have been produced to human Tamm-Horsfall protein (THP), identifying at least seven distinct epitopes. The antibodies have been used to isolate from serum an immunoreactive protein which comigrates with urinary THP. In addition, the antibodies may prove useful to set up an immunoenzymoassay for urinary THP as well as for immunoaffinity purification.

A monoclonal antibody to brush border and passive Heymann nephritis.

An IgG2a monoclonal antibody (MoAb) reacting with the brush border of the renal proximal tubule and glomerular capillary wall was produced by fusion of NS1 myeloma cells with spleen cells from BALB/c mice immunized with renal brush border preparations from rat kidney cortex. This antibody reacts with a 90,000 mol. wt protein which can be isolated by immunoprecipitation of radiolabelled brush border or glomerular preparations and localized on these structures by immunoperoxidase electron microscopy, thus demonstrating the presence of common antigenic determinants. Survey of various organs showed that the MoAb reacted with the brush border of the gut, but also with antigens associated with the distal vascular system. In the liver antigenic determinants were located along the sinusoid walls but mainly on bile canaliculi. Specific glomerular binding could be demonstrated in vivo by immunofluorescence after an intravenous injection of 2 mg of antibody or by paired label methodology using tracer amounts. Kinetics however were dramatically different from those observed in classical passive Heymann nephritis since glomerular binding was transient during the first hours after injection. Binding was also found in tubular structures, as well as in lung, liver, spleen and heart. These results identify a well defined antigen-antibody system responsible for the formation of transient extramembranous glomerular deposits and may be relevant to some human cases of glomerulonephritis. They may also provide new models to study glomerular and tubular transfer of membrane bound antibodies.