IDENTIFICATION OF PROBASIN-RELATED ANTIGEN AS CYSTATHIONINE GAMMA-LYASE BY MOLECULAR-CLONING

We reported previously that a monoclonal antibody against probasin (ra t prostatic secretory protein) recognizes a 40-kDa protein localized i n rat liver and kidney. The protein (probasin-related antigen, PRB-RA) may participate in a specific differentiated function of these tissue s. To clarify the molecular nature of PRB-RA, a series of cDNA clones coding for the protein were isolated from a rat liver expression libra ry using an affinity-purified polyclonal antibody. The amino acid sequ ence deduced from the determined cDNA sequence included sequences iden tical with those of proteolytic fragments of PRB-RA, which covered abo ut 70% of the deduced sequence. Northern blot hybridization of poly(A) + RNA isolated from rat tissues showed the presence of predominant and minor mRNA species of about 2.0 and 4.3 kilobases, respectively, in t he liver and kidney. A sequence homology search revealed that PRB-RA i s almost completely identical to rat cystathionine gamma-lyase (cystat hionase) and that it does not show overall homology with probasin. Thr ee candidates for an epitope common to probasin and PRB-RA were found on close examination of the amino acid sequences of the two proteins. A synthetic peptide, TYFRRI, corresponding to one of the candidates, n eutralized the reactivity of the anti-probasin monoclonal antibody to both probasin and PRB-RA on Western blot analysis. These results show that PRB-RA/cystathionase is neither structurally nor functionally rel ated to probasin except for a common epitope and that cystathionase, a cystein-producing enzyme, is localized in urinary tubular epithelial cells in a highly restricted region of the kidney in addition to in li ver parenchymal cells.