Neutral Cysteine Protease Bleomycin Hydrolase Is Essential for the Breakdown of Deiminated Filaggrin into Amino Acids
Neutral Cysteine Protease Bleomycin Hydrolase Is Essential for the Breakdown of Deiminated Filaggrin into Amino Acids
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DOI：
10.1074/jbc.m807908200
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发表时间：
2009-05-08
影响因子：
4.8
通讯作者：
Takeda, Atsushi
中科院分区：
文献类型：
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作者：
Kamata, Yayoi;Taniguchi, Aya;Takeda, Atsushi
丝状蛋白是角质化细胞膜的一种成分，也是角质层中作为天然保湿因子的游离氨基酸的前体。脱亚胺是丝聚蛋白降解成游离氨基酸的关键。在这项研究中，我们试图确定在体外切割脱酰亚胺微丝蛋白的酶(S)。首先，我们用双层荧光酶谱技术研究了新生大鼠表皮提取液中瓜氨酸氨基肽酶的活性，并在中性pH下检测到了较强的活性。通过对瓜氨酸释放活性的监测，我们纯化了一个280 kDa的酶，它由6个相同的48 kDa亚基组成。其NH2末端与大鼠博莱霉素水解酶(BH)的序列完全一致。该酶能从β-萘胺类化合物中释放除Pro以外的多种氨基酸，其中对瓜氨酸-β-萘胺的水解率最高。因此，要分解脱亚胺的微丝蛋白，需要另一种蛋白酶。基质辅助激光解吸/电离飞行时间质谱图显示，在所测试的各种酶中，calain I能有效地将脱脂后的微丝蛋白降解成许多不同质量的多肽。我们证实了BH从这些多肽中释放了包括瓜氨酸在内的各种氨基酸。另一方面，半胱氨酸天冬氨酸氨基转移酶14将脱酰亚胺的微丝蛋白降解成一些有限质量的多肽。对正常人皮肤的免疫组织化学分析显示，BH和微丝蛋白在颗粒层中共存。总而言之，我们的结果表明，BH对于合成天然保湿因子是必不可少的，而calain I将作为上游蛋白酶在微丝蛋白的降解中发挥作用。
Filaggrin is a component of the cornified cell envelope and the precursor of free amino acids acting as a natural moisturizing factor in the stratum corneum. Deimination is critical for the degradation of filaggrin into free amino acids. In this study, we tried to identify the enzyme(s) responsible for the cleavage of deiminated filaggrin in vitro. First, we investigated citrulline aminopeptidase activity in the extract of newborn rat epidermis by double layer fluorescent zymography and detected strong activity at neutral pH. Monitoring the citrulline-releasing activity, we purified an enzyme of 280 kDa, comprised of six identical subunits of 48 kDa. The NH2 terminus of representative tryptic peptides perfectly matched the sequence of rat bleomycin hydrolase (BH). The enzyme released various amino acids except Pro from beta-naphthylamide derivatives and hydrolyzed citrulline-beta-naphthylamide most effectively. Thus, to break down deiminated filaggrin, another protease would be required. Among proteases tested, calpain I degraded the deiminated filaggrin effectively into many peptides of different mass on the matrix-assisted laser desorption/ionization-time of flight mass spectrum. We confirmed that various amino acids including citrulline were released by BH from those peptides. On the other hand, caspase 14 degraded deiminated filaggrin into a few peptides of limited mass. Immunohistochemical analysis of normal human skin revealed co-localization of BH and filaggrin in the granular layer. Collectively, our results suggest that BH is essential for the synthesis of natural moisturizing factors and that calpain I would play a role as an upstream protease in the degradation of filaggrin.