Identification of interleukin-8 converting enzyme as cathepsin L.
IL-8 is produced by various cells, and the NH(2)-terminal amino acid sequence of IL-8 displays heterogeneity among cell types. The mature form of IL-8 has 72 amino acids (72IL-8), while a precursor form (77IL-8) of IL-8 has five additional amino acids to the 72IL-8 NH(2)-terminal. However, it has been unclear how IL-8 is processed to yield the mature form. In this study, converting enzyme was purified as a single 31-kDa band on silver-stained polyacrylamide gel from 160 l of cultured fibroblast supernatant by sequential chromatography. NH(2)-terminal amino acid sequence analysis revealed a sequence, EAPRSVDWRE, which was identified as a partial sequence of cathepsin L. Polyclonal antibodies raised against cathepsin L recognized the purified converting enzyme on Western blot. Moreover, human hepatic cathepsin L cleaved 77IL-8 between Arg(5) and Ser(6), which is the same cleavage site as the putative converting enzyme, resulting in 72IL-8 formation. These data indicate that the converting enzyme of the partially purified fraction of the human fibroblast culture supernatant was cathepsin L. Furthermore, 72IL-8 was sevenfold more potent than 77IL-8 in a neutrophil chemotaxis assay. These results show that cathepsin L is secreted from human fibroblasts in response to external stimuli and plays an important role in IL-8 processing in inflammatory sites.
Ohashi
Kensaku
K
Pharmaceutical Research Laboratories, Toray Industries, Inc, 1111 Tebiro, Kanagawa Kamakura 248-8555, Japan. Kenasaku_Ohashi@tmc.toray.co.jp
Naruto
Masanobu
M
Nakaki
Toshio
T
Sano
Emiko
E
D015153:Blotting, Western; D056668:Cathepsin L; D002403:Cathepsins / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003470:Culture Media; D003546:Cysteine Endopeptidases; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism* / Q000494:pharmacology; D008099:Liver / Q000201:enzymology; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational
D003470:Culture Media; D016209:Interleukin-8; D011498:Protein Precursors; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L