Generation of globular fragment of adiponectin by leukocyte elastase secreted by monocytic cell line THP-1
Generation of globular fragment of adiponectin by leukocyte elastase secreted by monocytic cell line THP-1
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DOI：
10.1210/en.2004-1096
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发表时间：
2005-02-01
期刊：
影响因子：
4.8
通讯作者：
Kadowaki, T
中科院分区：
文献类型：
--
作者：
Waki, H;Yamauchi, T;Kadowaki, T
以往的研究表明，人血浆中存在脂联素的羧基端含有球状结构域的片段。虽然有人提出球状片段是由蛋白水解切割产生的，但切割的位置和负责的酶仍然不清楚。在这项研究中，我们在体外评估了几种细胞系在培养基中切割脂联素的活性。在佛波醇12-肉豆蔻酸酯13-乙酸酯刺激的单核细胞系THP-1和U937的培养基中观察到脂联素裂解成几个含有球状结构域的羧基末端片段。主要产物的分子量为25、20和18 kDa。基于以下观察结果，认为裂解由白细胞弹性蛋白酶（也称为中性粒细胞弹性蛋白酶）介导。首先，通过丝氨酸蛋白酶抑制剂[苯甲基磺酰氟，Pefabloc SC（Roche Diagnostics，巴塞尔，瑞士）和抑肽酶]和白细胞弹性蛋白酶特异性肽抑制剂MeOSuc-AAPV-CMK抑制切割。第二，在THP-1细胞完全分化成巨噬细胞后没有检测到活性。第三，纯化的白细胞弹性蛋白酶切割脂联素与THP-1细胞相同的切割模式。最后，由活化的中性粒细胞分泌的白细胞弹性蛋白酶将脂联素切割成球状片段。氨基末端序列分析表明，脂联素酶切位点位于38个Thr和39个Cys之间，40个Ala和41个Gly之间，44个Ala和45个Gly之间，91个Ala和92个Glu之间，110个Ala和111个Ala之间（氨基酸位置的编号从信号序列开始），表明切割发生在胶原结构域中。这些数据表明，裂解脂联素的白细胞弹性蛋白酶分泌的活化单核细胞和/或中性粒细胞可能是一个候选人的机制的球状片段的脂联素的产生。
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. Aminoterminal sequence analysis revealed that cleavage sites of adiponectin by purified leukocyte elastase were between (38)Thr and (39)Cys, (40)Ala and (41)Gly, (44)Ala and (45)Gly, (91)Ala and (92)Glu, and (110)Ala and (111)Ala (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.