PURIFICATION AND CLONING OF CARP NEPHROSIN, A SECRETED ZINC ENDOPEPTIDASE OF THE ASTACIN FAMILY

We have purified a secreted proteinase of 23 kDa from carp head kidney by sequential column chromatography on a Reactive Blue 72-agarose dye affinity column and an FPLC Mono-P column. The secretion of this prot einase from carp head kidney can be stimulated by high concentrations of potassium. Since the carp proteinase is present mainly in the head kidney, kidney, and spleen (all of which are lymphohematopoietic organ s), it is named nephrosin. The carp nephrosin is most sensitive to met al chelators, but not to inhibitors specific for other classes of prot einases. A cDNA clone has been isolated from a carp head kidney cDNA l ibrary by immunoscreening with a polyclonal antiserum raised against p urified nephrosin. The cloned cDNA is 1086 base pairs in length and ha s an open reading frame encoding a protein of 273 amino acids, includi ng a 19-amino acid signal peptide and 56-amino acid propeptide. The de duced amino acid sequence shows moderate levels of identity to medaka HCE1 (52.5%), medaka LCE (50.7%), crayfish astacin (33.2%), murine mep rin-alpha (34%), and murine meprin-beta (33.5%), all members of the as tacin family of zinc endopeptidases. Nephrosin is the first member of the astacin family found in lymphohematopoietic tissues.