Abstract
Seven monoclonal antibodies (mAbs) were raised against a rabbit renal brush-border glycoprotein (molecular mass, 63-66 kDa), presumably involved in Na+/Pi cotransport, which we had previously purified and reconstituted in active form in proteoliposomes (Debiec, H., Lorenc, R., and Ronco, P. M. (1992) Biochem. J. 286, 97-102). Antibody specificity for the 63-66-kDa protein was analyzed by enzyme-linked immunosorbent assay and confirmed by Western blotting and immunoaffinity chromatography of solubilized brush-border membranes (BBM), which both yielded a single 63-66-kDa band. Enzyme-linked immunosorbent assay and immunoblotting of renal cortical cell subfractions localized the immunoreactive protein to the brush-border membrane. This location was confirmed by indirect immunofluorescence of kidney cortex sections. Binding of two of the seven mAbs (63A20 and 206A126) to native BBM only occurred when the related epitope was exposed in the presence or absence of Na+, respectively; the other mAbs did not react with native BBM probably because of intramembranous orientation of the epitopes. mAb 63A20 inhibited dose-dependently Na+/Pi cotransport when preincubation of BBM was carried out in the presence of Na+ but did not affect Na+/D-glucose cotransport. Proteoliposomes formed from BBM proteins depleted of the 63-66-kDa protein by affinity chromatography with mAb 63A20 showed an 85% reduction in Na+/Pi cotransport, whereas Na+/D-glucose cotransport was not modified. These results thus establish that the 63-66-kDa BBM protein is the essential component of the Na+/Pi cotransport system. The present study also provides the first immunologic tools available for immunohistochemical localization of the Na+/Pi cotransporter. Finally, the identification of a functional epitope by mAb 63A20 opens up new ways to explore the molecular aspects of Pi uptake.
Highlights
By indirect immunofluorescence of kidney cortex sec- Membrane transport proteinshave been difficult to purify, tions
Reformed fromBBM proteins depletedof the 63-66-kDa cently, using a different approach based on expression cloning, protein by affinity chromatography with mAb 63A20 a presumed component of the rabbit kidney Na+/Pi cotransshowed an 85% reductioninNa+/Pi cotransport, porter was identified as a 61-kDa glycoprotein with a protein whereas Na+/D-glucosecotransport was not modified
The results described above indicate that the seven mAbs produced were specific preincubating the mAbs with the 63-66-kDa protein, for the 63-66-kDa protein and that this antigenwas specifi- indicating nonspecific bindingtoBLMand mitochondria cally identified in thesolubilized BBM preparation
Summary
Renal Na+/PiCotranspPorrottein three additional washes in T-K buffer, the filters were dried and the RESULTS. The uptakes at time 0 were measured by adding BBMV suspension and theincubation medium directly to therespecantibodies TEPC183 and 50A5 did not show any reactivity with the 63-66-kDa protein (Fig. 1). In the same experiment the reactivity of the two mAbs pre- 66-kDa protein (Fig. 5, lane 7)and by replacing them by the viously tested in Fig. 1 (63A20 and 85A127) with the same control IgM TEPC183 (Fig. 5, lane 6 ) which both yielded amount (20 pg) of BBM, BLM, and mitochondria proteins negative results. The five mAbs all failed to react with BLM adsorbed to the bottoomf plastic wells Both antibodies bound (Fig. 5, lane 8 ) and mitochondria (Fig. 5, lane 9) under the dose-dependently to the BBM preparation;specificity of the same experimentalconditions. Replaced by the irrelevant IgM TEPC183, or pretreated with an excess of antigen (data not shown)
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