Abstract
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 proteinase 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 organs), it is named nephrosin. The carp nephrosin is most sensitive to metal chelators, but not to inhibitors specific for other classes of proteinases. A cDNA clone has been isolated from a carp head kidney cDNA library by immunoscreening with a polyclonal antiserum raised against purified nephrosin. The cloned cDNA is 1086 base pairs in length and has an open reading frame encoding a protein of 273 amino acids, including a 19-amino acid signal peptide and 56-amino acid propeptide. The deduced amino acid sequence shows moderate levels of identity to medaka HCE1 (52.5%), medaka LCE (50.7%), crayfish astacin (33.2%), murine meprin-alpha (34%), and murine meprin-beta (33.5%), all members of the astacin family of zinc endopeptidases. Nephrosin is the first member of the astacin family found in lymphohematopoietic tissues.
Highlights
Teleost head kidney consists of interrenal tissue [1,2,3] homologous to the adrenal cortex, chromaffin cells [4, 5] responsible for epinephrine and norepinephrine secretion, immune cells (6 –9) responsible for immunoglobulin M secretion, and hematopoietic cells [6, 10, 11]
We have demonstrated that secretion of nephrosin can be stimulated by high concentrations of extracellular potassium
Nephrosin is a new member of the astacin family of metalloproteinases based on the following observations: First, the activity of nephrosin is inhibited by metal chelators but not by inhibitors of other classes of proteinases, suggesting that it is a metalloproteinase
Summary
Teleost head kidney consists of interrenal tissue [1,2,3] homologous to the adrenal cortex, chromaffin cells [4, 5] responsible for epinephrine and norepinephrine secretion, immune cells (6 –9) responsible for immunoglobulin M secretion, and hematopoietic cells [6, 10, 11]. In addition to the head kidney, spleen and kidney are lymphohematopoietic organs in teleost fish [6]. The presence of four different types of cells (neurons, endocrine cells, immune cells, and hematopoietic cells) in the head kidney prompted us to study the secreted proteins from head kidney since these cells are all active in secretion. We attempted to purify the secreted proteins from carp head kidney and eventually to understand the functions of the secreted molecules. We report the purification and cloning of one secreted protein, nephrosin. Nephrosin is a zinc metalloproteinase and is present mainly in the gill, kidney, head kidney, and spleen. Our evidence suggests that nephrosin is probably not a hatching enzyme
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