Cloning and preliminary characterization of a calcium-binding protein closely related to nucleolin on the apical surface of inner medullary collecting duct cells.
Calcium stone crystal attachment to the urinary epithelium plays an essential role in the development of kidney stones by allowing small crystals to be retained in the kidney until they become macroscopic. We among others have described attachment of stone crystals to cultured renal epithelia (Wiessner, J. H., Kleinman, J. G., Blumenthal, S. S., Garancis, J. C., and Mandel, G. S. (1987) J. Urol. 138, 640-643). To isolate protein(s) that may participate in crystal attachment, apical membranes of cultured renal inner medullary collecting duct were biotinylated, the cells were lysed with detergent, the lysate was subjected to hydroxyapatite chromatography, and fractions were incubated with calcium oxalate monohydrate. Electrophoresis of material solubilized from the crystals showed several selectively adsorbed protein bands. A 110-kDa band stained positively for biotin and for glycosides and bound (45)Ca. The amino acid sequence of this band was determined to be that of a protein closely related to rat nucleolin (nucleolin-related protein; NRP). NRP was cloned and sequenced and was 83% homologous with the previously sequenced nucleolar protein nucleolin. Using temperature-induced phase partitioning with Triton X-114, NRP was associated with both the insoluble membrane skeleton pellet and the soluble aqueous phase but not the soluble detergent phase. This association with the membrane skeleton was increased in the presence of calcium. Thus, NRP is associated with the apical membranes of cultured renal tubular cells and is bound to membrane skeletal elements in a calcium-dependent fashion. The physiological role of NRP remains to be determined; however, a pathophysiological role may be that of mediating the attachment to the renal tubular epithelium of calcium stone crystals.
Sorokina
E A
EA
Department of Veterans Affairs Medical Center, Nephrology Section, Milwaukee, Wisconsin 53295, USA.
Kleinman
J G
JG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002645:Chickens; D003001:Cloning, Molecular; D016384:Consensus Sequence; D006224:Cricetinae; D017931:DNA Primers; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007679:Kidney Medulla / Q000166:cytology / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010750:Phosphoproteins / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D000097796:Nucleolin
D002118:Calcium; D002135:Calcium-Binding Proteins; D017931:DNA Primers; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000097796:Nucleolin; C121937:Ncl-ps1 protein, rat