RESUMEN
A human cDNA of 2166 bp encoding a novel cathepsin B-related protein was isolated and characterized. The amino acid sequence of the predicted protein of 467 aa was 46% identical with that of human tubulointerstitial nephritis antigen (TIN-ag), and therefore, the protein was tentatively designated as the TIN-ag-related protein (TIN-ag-RP). The amino acid sequence of TIN-ag-RP is composed of a 21 aa long signal sequence, a 181 aa long N-terminal domain containing two epidermal growth factor-like domains, a follistatin motif, and a 265 aa long cathepsin B-like domain. Interestingly, a serine residue has replaced the active site cysteine residue in the cathepsin B-like domain, resulting in a proteolytically inactive protein. Evolutionary analysis revealed that a distinct family of "TIN-ag-like" proteins had evolved in vertebrates. Northern blot analysis revealed a single TIN-ag-RP transcript of 2.4 kb in various tissues with the highest transcript levels detected in aorta, heart, placenta, skeletal muscle, kidney, and a colorectal adenocarcinoma cell line. Using a polyclonal anti-TIN-ag-RP antibody, TIN-ag-RP expression was predominantly seen in vascular smooth muscle (VSM) cells, but also in cardiac and skeletal muscle cells as well as in kidney. Interestingly, uterine smooth muscle cells completely lacked TIN-ag-RP expression, implying a regulated gene expression. Localization studies in HeLa cells stably transfected with TIN-ag-RP cDNA showed that TIN-ag-RP is glycosylated and actively secreted, a finding in line with its proposed function as a structural or regulatory protein similar to TIN-ag.
Asunto(s)
Catepsina B/química , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Factor de Crecimiento Epidérmico/química , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Nefritis Intersticial/metabolismo , Proteínas de Unión a Telómeros , Adulto , Secuencia de Aminoácidos , Animales , Antígenos de Superficie , Secuencia de Bases , Catálisis , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Línea Celular , ADN Complementario/química , Activación Enzimática , Proteínas de la Matriz Extracelular , Células HeLa , Humanos , Lipocalinas , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Proteínas Musculares/biosíntesis , Proteínas Musculares/química , Proteínas Musculares/genética , Músculo Liso Vascular/química , Músculo Liso Vascular/citología , Especificidad de Órganos/genética , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Transfección , Células Tumorales CultivadasRESUMEN
The human gene encoding a novel tubulointerstitial nephritis antigen (TIN-ag)-related protein (TIN-ag-RP) was isolated, and its genomic organization was determined. BLAST searches revealed the highest degree of homology to several mammalian TIN-ag orthologues, and a weak homology to cathepsin B-like proteases. The 12 kb gene was mapped by fluorescence in situ hybridization to chromosome 1p34.2-3, a locus neither related to that of the human TIN-ag (6p11.2-12) nor to that of cathepsin B (8p22-23.1). The TIN-ag-RP is encoded in ten exons with introns ranging from 83 bp to 4 kb. In addition, the gene contained one exon in the 5'UTR, but none in the 3'UTR. Five of the 10 splice sites of the TIN-ag-RP gene were fully conserved when compared to a related gene of C. elegans, whereas only one splice site was identical to those found in cathepsin B genes. Furthermore, human TIN-ag-RP tagged with the T7-epitope, was expressed in HeLa cells, and was found to be localized in vesicular compartments as well as secreted into the medium suggesting the involvement of the endosomal trafficking pathway. Based on the high degree of homology of the amino acid sequences and genomic organization between TIN-ag-RP and TIN-ag, we suggest that both molecules may form a distinct group or family of TIN-ag-like proteins.
Asunto(s)
Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Matriz Extracelular/química , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Proteínas de Unión a Telómeros , Secuencia de Aminoácidos , Antígenos de Superficie , Western Blotting , Catepsina B/química , Cromosomas Humanos Par 1 , Clonación Molecular , Exones , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Hibridación Fluorescente in Situ , Intrones , Modelos Genéticos , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
We have localized cathepsin K in rat osteoclasts and within exposed resorption pits by immuno-fluorescence microscopy. Intracellular staining using an antibody raised against recombinant mouse cathepsin K was vesicular and uniformly distributed throughout the cell. Confocal microscopy analysis did not reveal an accumulation of cathepsin K containing vesicles opposing the ruffled border and the resorption lacuna. Exposed resorption pits exhibited a uniform distribution of cathepsin K, and no differences were observed between the edges and the centers of the pits. The immunostaining of resorption pits with anti-cathepsin K antibodies demonstrates that the protease is secreted into the sub-osteoclastic compartment. Cathepsin K-specific inhibition using peptidyl vinyl sulfones as selective cysteine protease inactivators reduced bone resorption by 80% in a dose-dependent manner at sub-micromolar concentrations. No reduction of bone resorption was observed at those low concentrations using a potent cathepsin L, S, B-specific inhibitor. That the inhibition of bone resorption can be attributed to cathepsin K-like protease inhibition was corroborated by the selective inhibition of the osteoclastic Z-Gly-Pro-Arg-MbetaNA hydrolyzing activity by the cathepsin K, L, S, B-inhibitor, but not by the cathepsin L, B, and S inhibitor. Z-Gly-Pro-Arg-MbetaNA is efficiently hydrolyzed by cathepsin K but only poorly by cathepsins L, S, and B. On the contrary, the intracellular hydrolysis of the cathepsin B-specific substrate, Z-Arg-Arg-MbetaNA, was prevented by both types of inhibitors. The identification of cathepsin K in resorption pits and the inhibition of bone resorption and intracellular cathepsin K activity by selective vinyl sulfone inhibitors indicate the critical role of the protease in osteoclastic bone resorption.