title: Generation of globular fragment of adiponectin by leukocyte elastase secreted by monocytic cell line THP-1.
abstract: Previous studies revealed that carboxyl-terminal fragment containing the globular domain of adiponectin exists in human plasma. Although it is proposed that the globular fragment is generated by proteolytic cleavage, the place and responsible enzyme of the cleavage are still unclear. In this study, we evaluated the activity to cleave adiponectin in culture medium of several cell lines in vitro. Adiponectin cleavage into several carboxyl-terminal fragments containing the globular domain was observed in the medium of phorbol 12-myristate 13-acetate-stimulated monocytic cell lines THP-1 and U937. The molecular masses of the major products were 25, 20, and 18 kDa. The cleavage was thought to be mediated by leukocyte elastase (also known as neutrophil elastase) based on the following observations. First, the cleavage was inhibited by serine-protease inhibitors [phenylmethylsulfonylfluoride, Pefabloc SC (Roche Diagnostics, Basel, Switzerland) and aprotinin] and by the leukocyte elastase-specific peptide inhibitor MeOSuc-AAPV-CMK. Second, no activity was detected after THP-1 cells had fully differentiated into macrophages. Third, purified leukocyte elastase cleaved adiponectin with the same cleavage pattern as THP-1 cells. Finally, leukocyte elastase secreted by activated neutrophils cleaved adiponectin into the globular fragments. Amino-terminal sequence analysis revealed that cleavage sites of adiponectin by purified leukocyte elastase were between 38Thr and 39Cys, 40Ala and 41Gly, 44Ala and 45Gly, 91Ala and 92Glu, and 110Ala and 111Ala (the numbering of the positions of the amino acids starts at the signal sequence), suggesting that the cleavage occurs in the collagenous domain. These data indicate that the cleavage of adiponectin by leukocyte elastase secreted from activated monocytes and/or neutrophils could be a candidate for the mechanism of the generation of the globular fragment of adiponectin.
authors.affiliation: Department of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan.
authors.forename: Hironori
authors.initials: H
authors.lastname: Waki
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Yamauchi
authors.forename: Junji
authors.initials: J
authors.lastname: Kamon
authors.forename: Shunbun
authors.initials: S
authors.lastname: Kita
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Ito
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Hada
authors.forename: Shoko
authors.initials: S
authors.lastname: Uchida
authors.forename: Atsushi
authors.initials: A
authors.lastname: Tsuchida
authors.forename: Sato
authors.initials: S
authors.lastname: Takekawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Kadowaki
mesh_terms: D052242:Adiponectin; D000595:Amino Acid Sequence; D002454:Cell Differentiation; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D019272:Leukocyte Elastase / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000378:metabolism; D020298:U937 Cells
chemical_list: D052242:Adiponectin; D036341:Intercellular Signaling Peptides and Proteins; D012697:Serine Endopeptidases; D019272:Leukocyte Elastase