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

Neurosin (also known as zyme or protease M) is a trypsin-like serine protea se dominantly expressed in the human brain. According to the official nomen clature, this gene is now designated as human kallikrein 6 (KLK6) and the p rotein is designated hK6. To investigate the metabolism of neurosin in huma n brain, neurosin contained in the human cerebrospinal fluid (CSF) was anal yzed. 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 mot. 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 hav e no enzyme activity. CSF-neurosin neither showed any enzyme activity to Bo c-Ph-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 usin g antibodies specific to the pro- or mature enzyme was carried out. The ant ibody against the mature neurosin fragment did not react with CSF-neurosin. Only the antibody against the pro-enzyme fragment detected CSF-neurosin. T hus, our results suggest that neurosin is present as an inactive pro-enzyme in the human CSF. NeuroReport 12:1345-1350. (C) 2001 Lippincott Williams & Wilkins.