Characterization of a protease responsible for truncated actin increase inneutrophils of patients with Behcet's disease

As described previously (Yamashita S. et al., Biol. Pharm. Bull., 23, 519-5 22 (2000)), high levels of a truncated actin with an N-terminus of Met-44 w ere detected in neutrophils of patients,vith Behcet's disease. Since the in crease of the truncated actin in neutrophils of patients may be important f or understanding the pathology of Behcet's disease, the mechanism of the tr uncated actin formation was studied. First, to investigate the presence of a specific protease, which cleaves the actin at the site between Val-43 and Met-44, a peptide with a partial amino acid sequence of actin from the N-t erminal Pro-38 to Asp-51 was synthesized as the protease substrate. The syn thesized peptide was digested with cytosolic fractions of neutrophils from patients and healthy volunteers, and digestion products were analyzed by C1 8-reverse phase HPLC. The chromatograms of these samples showed that an end oprotease, which cleaved the peptide at a specific site, was present in cyt osolic fractions of neutrophils from patients with Behcet's disease. Then, the effects of various kinds of protease inhibitors on the digestion of the peptide were investigated in order to identify the responsible endoproteas e. The digestion of the peptide was suppressed by 4-(2-aminoethyl) benzenes ulfonylfluoride (AEBSF, a serine protease inhibitor) and N-methoxysuccinyl- Ala-Ala-Pro-Val chloromethylketone (CMK, a polymorphonuclear (PMN)-elastase inhibitor) in the presence of EDTA. Furthermore, PMN-elastase was found to cleave the substrate peptide and actin at the site between Val-43 and Met- 44. These results lead to the conclusion that the PMN-elastase is responsib le for cleavage of actin at the N-terminal site between Val-43 and Met-44 i n neutrophils from patients with Behcet's disease.