Characterization of a brain-related serine protease, neurosin (human kaillikrein 6), in human cerebrospinal fluid.

ABSTRACT

Neurosin (also known as zyme or protease M) is a trypsin-like serine protease dominantly expressed in the human brain. According to the official nomenclature, this gene is now designated as human kallikrein 6 (KLK6) and the protein is designated hK6. To investigate the metabolism of neurosin in human brain, neurosin contained in the human cerebrospinal fluid (CSF) was analyzed. Neurosin was detected in the all CSFs tested by Western blot analysis using an anti-neurosin monoclonal antibody. We purified neurosin from CSF (CSF-neurosin) using an immunoaffinity chromatography and an anion-exchange chromatography. SDS-PAGE revealed that the purified protein has a relative mol. mass (Mr) of 25,000 Da. The observed sequence of the N-terminal amino acids, Glu-Glu-Gln-Asn-Lys, of the purified CSF-neurosin was identical to the sequence of N-terminal of the pro-enzyme form, which is presumed to have no enzyme activity. CSF-neurosin neither showed any enzyme activity to Boc-Phe-Ser-Arg-4-methylcoumaryl-7-amide, which is known to be degraded by the mature neurosin, nor cleaved gelatin. To confirm that the major portion of CSF-neurosin is present in the pro-enzyme form, Western blot analysis using antibodies specific to the pro- or mature enzyme was carried out. The antibody against the mature neurosin fragment did not react with CSF-neurosin. Only the antibody against the pro-enzyme fragment detected CSF-neurosin. Thus, our results suggest that neurosin is present as an inactive pro-enzyme in the human CSF.