Characterization of a protease responsible for truncated actin increase in neutrophils of patients with Behçet's disease.
As described previously (Yamashita S. et al., Biol. Pharm. Bull., 23, 519-522 (2000)), high levels of a truncated actin with an N-terminus of Met-44 were detected in neutrophils of patients with Behcet's disease. Since the increase of the truncated actin in neutrophils of patients may be important for understanding the pathology of Behçet's disease, the mechanism of the truncated 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-terminal Pro-38 to Asp-51 was synthesized as the protease substrate. The synthesized peptide was digested with cytosolic fractions of neutrophils from patients and healthy volunteers, and digestion products were analyzed by C18-reverse phase HPLC. The chromatograms of these samples showed that an endoprotease, which cleaved the peptide at a specific site, was present in cytosolic fractions of neutrophils from patients with Behçet'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 endoprotease. The digestion of the peptide was suppressed by 4-(2-aminoethyl) benzenesulfonylfluoride (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 responsible for cleavage of actin at the N-terminal site between Val-43 and Met-44 in neutrophils from patients with Behçet's disease.
Yamashita
S
S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hoshi University, Tokyo, Japan.
Suzuki
A
A
Yanagita
T
T
Hirohata
S
S
Toyoshima
S
S
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000595:Amino Acid Sequence; D001528:Behcet Syndrome / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology
D000199:Actins; D000590:Amino Acid Chloromethyl Ketones; D004791:Enzyme Inhibitors; D013450:Sulfones; C058167:methoxysuccinyl-alanyl-alanyl-prolyl-alanine chloromethyl ketone; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D019272:Leukocyte Elastase