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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.

We have previously produced mAb against angiotensin II (AII), a phylogenetically conserved vasopressive octapeptide, and shown that they identify four distinct epitopes on the AII molecule. In addition we used internal image bearing polyclonal antiidiotypic antibodies raised against rabbit anti AII to produce mAb3. In this study we analyze the segregation of the idiotypic and paratopic repertoires of the mAb1 and mAb3. Analysis of mAb1 carried out with polyclonal Ab2 raised against the four distinct paratopes permitted classification of the mAb1 into four categories: (p+, id+) comprises antibodies with shared paratopic and idiotypic specificities: (p+, id-) is made up of antibodies that fail to express the Id defined by Ab2 raised against other antibodies pertaining to the same paratopic group; (p-, id+) includes antibodies that express cross-reactive Id on distinct paratopes; (p-, id-) refers to antibodies unrelated by their paratopes and Id mAb2 confirmed these results and showed expression of identical or closely related Id on clearly distinct paratopes. At the Ab3 level, using polyclonal Ab4, there was a higher degree of Id cross-reactivity between the two paratopes available. These data suggest that the parallel set concept may apply to the immune response to a natural peptidic Ag and its internal image. Comparison of idiotypic repertoires of mAb1 and mAb3 (using Ab2 and Ab4 antibodies) confirmed the lack of public Id and showed the predominance on mAb3 of "new" idiotypes absent from mAb1 molecules, as expected for internal image-induced antibodies. Cross-reactive idiotypes defined on mAb1 and conserved on mAb3 were expressed on the two paratopes defined at the Ab3 level. They were located on the H chain of the homologous paratope and required the association of H and L chains on the heterologous paratope. Our analysis suggests that, in the AII system, the idiotypic and paratopic repertoires segregate at least in part independently. The paratopic repertoire is limited to a small number of phylogenetically conserved specificities and may be encoded by germline genes. In contrast, the idiotypic repertoire is broader with respect to specificities, species, and localization on H and L chains. This extended diversity may be generated by somatic mutations or use of various combinations of H and L chains and/or V, D, J segments.

Uptake of vitamin B12 (cyanocobalamin) is facilitated by the cobalamin-binder gastric intrinsic factor (IF), which recognizes a 460-kD receptor, cubilin, present in the epithelium of intestine and kidney. Surface plasmon resonance analysis of ligand-affinity-purified human cubilin demonstrated a high-affinity calcium- and cobalamin-dependent binding of IF-cobalamin. Complete cDNA cloning of the human receptor showed a 3597 amino acid peripheral membrane protein with 69% identity to rat cubilin. Amino-terminal sequencing of the receptor indicates that the cDNA sequence encodes a precursor protein undergoing proteolytic processing due to cleavage at a recognition site (Arg7-Glu8-Lys9-Arg) for the trans-Golgi proteinase furin. Using fluorescence in situ hybridization, radiation hybrid mapping, and screening of YAC clones, the human cubilin gene was mapped between the markers D10S1661 and WI-5445 on the short arm of chromosome 10. This is within the autosomal recessive megaloblastic anemia (MGA1) 6-cM region harboring the unknown recessive-gene locus of juvenile megaloblastic anemia caused by intestinal malabsorption of cobalamin (Imerslund-Gräsbeck's disease). In conclusion, the present molecular and genetic information on human cubilin now provides circumstantial evidence that an impaired synthesis, processing, or ligand binding of cubilin is the molecular background of this hereditary form of megaloblastic anemia.

Epithelial structures lining the proximal tubule and the yolk sac are characterized by a high rate of internalization followed by degradation of the proteins exposed to their apical pole. This function implies the expression by these epithelia of specialized proteins which have the ability to bind numerous ligands and/or lysosomal targeting properties. An improved knowledge of these molecules is needed since their expression in a limited number of epithelia may account for the specificity of some pathologies induced in particular by toxins. This paper deals with two such "candidate" proteins, gp330/megalin and gp280 which have been expressed in all species studied. gp280 is the target of teratogenic antibodies and is identified here as the receptor for intrinsic factor-cobalamin complexes but in all likelihood also binds other ligands. Antibodies to gp280 markedly stimulate the fusion of renal endosomes suggesting that it may play a role in targeting processes. gp330/megalin has been recently identified as the ligand of polybasic compounds such as gentamicin and is a key component in the internalization of the drug by tubular cells leading to renal toxicity. gp330/megalin also intervenes in endosomal fusion and some of its toxic properties may be mediated by this route.

One of the functions of the proximal tubule cells is to internalize and degrade proteins that have not been retained by the glomerulus. The magnitude of the amounts of protein reabsorbed (several hundreds of milligrams per day) imply specific structures that favor endocytosis and recycling of plasma membrane components as well as the expression of specific molecules that play a role in the endocytic traffic and/or act as multiligand receptors. In this study we review recent data that link 2 high molecular weight glycoproteins, gp330/megalin (600 kDa) and gp280 (460 kDa) to the endolytic process in proximal tubule cells.

To analyze the influence of epithelial cell differentiation and the effects of hormones on the characteristics of cell-associated and secreted proteoglycans (PGs), we studied their distribution, synthesis, and biochemical features in a model of renal proximal tubule cells in primary culture in which cell differentiation could be controlled by medium composition. In cells cultured in serum-free, hormonally defined medium supplemented with insulin and dexamethasone that exhibited a high degree of morphological and functional proximal differentiation (Ronco et al., 1990), cell-associated PGs were similar to those extracted in vivo by their size estimated by Sepharose CL-6B chromatography (Kav = 0.27, vs. 0.26), composition (heparan-sulfate), and localization in a continuous basal layer of extra-cellular matrix (ECM). In contrast, major quantitative and qualitative anomalies of cell-associated PGs were observed in poorly differentiated cells grown in 1% fetal calf serum-supplemented medium (FCS). PGs alterations included: (1) reduced and irregular expression of PGs at the cell basal pole, (2) a 2.8-fold decrease in [35S]-sulfate incorporation into cell-associated PGs, (3) a 3.1-fold increase in trypsin-releasable PGs, and (4) the emergence of a high MW PG composed exclusively of chondroitin-sulfate (CS) (Kav = 0.09 on Sepharose CL-6B) as well as of putative free CS-glycosaminoglycan (GAG) chains (Kav = 0.49 on Sepharose CL-6B). The same alterations were identified in the basal defined medium devoid of hormones but were partially or totally abolished by addition of insulin and dexamethasone, respectively. At variance with cell-associated PGs, production and biochemical features of secreted PGs were not influenced by cell differentiation status and medium composition.

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.

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)

mAb-131 is a monoclonal antibody that binds with high affinity (K alpha = 7.4 x 10(9) M-1) to the 8-residue peptide hormone angiotensin II, the major effector of the renin/angiotensin system. mAb-131 is a member of a well characterized idiotypic antibody network since it was raised as an anti-anti-idiotype of an antibody raised against angiotensin II. mAb-131 Fabs prepared with papain contain four major charge isoforms that can be separated by pH gradient elution from an anion-exchange column. Diffraction quality isomorphous crystals of two of the isoforms and of the Fab.peptide complexes have been grown. The crystals diffract to 3.5 A resolution, are tetragonal, space group P4(1) (or P4(3] with cell dimensions a = b = 78.6 A, c = 125.2 A, and have two Fab molecules per asymmetric unit. By using a different buffer, a second crystal form has been grown which diffracts to 3.3 A. It also belongs to space group P4(1) (or P4(3] but has cell dimensions of a = b = 109.6 A and c = 125.2 A. Knowledge of the three-dimensional structure of this Fab and of the peptide.Fab complex will give insight into two problems: 1) the recognition of small peptide hormones (which exist as random coils in solution) with high affinity by proteins, and 2) the nature of conservation of antibody combining sites in idiotypic networks.

The sera of six patients infected with Trichinella spiralis were studied for the presence of circulating immune complexes (CIC). CIC were present in all six patients studied 1 month after infection. In two patients in whom serial serum samples were available, as clinical improvement occurred there was a decrease in the level of CIC and an increase in the fluorescent antibody titres.

Significant levels of EAC-rosette inhibition compared to control subjects were found in the sera of patients with focal and segmental hyalinosis (FSH), membranoproliferative glomerulonephritis (MPGN) and extra-membranous glomerulonephritis (EGN). In patients with IgA disease, although some sera produced high levels of inhibition, the group as a whole did not differ significantly from the controls. Evidence was obtained suggesting that the rosette inhibitory activity was due to immune complexes (IC) bearing C3 rather than C3 fragments. Firstly, the inhibitory activity was precipitable by 4% PEG, a concentration which does not precipitate the C3 fragments. Secondly, the inhibitory activity was selectively removed from the PEG precipitates by an anti-human immunoglobulin G immunoabsorbent. Finally, since it had been suggested that in some instances an unknown serum factor could inhibit EAC-rosette formation and activation of the alternative pathway of complement, the latter was studied and found to be normal in all the sera studied. Taken together, these results suggest that the inhibition of EAC-rosette formation obtained with the sera of the patients studied was due to the presence in these sera of some material behaving as IC. No clear-cut association was, however, seen between rosette inhibition and the presence or absence of Ig or C3 deposits in the kidney.

Four patients with thrombotic microangiopathy had evidence of intravascular C3 activation on presentation which persisted after bilateral nephrectomy. In several serum samples, C3-splitting activity was not associated with the presence of circulating immune complexes, which were detected in all four patients before nephrectomy and in three after nephrectomy.