Identification and Characterization of Acyl-Protein Thioesterase 1/Lysophospholipase I As a Ghrelin Deacylation/Lysophospholipid Hydrolyzing Enzyme in Fetal Bovine Serum and Conditioned Medium
Identification and Characterization of Acyl-Protein Thioesterase 1/Lysophospholipase I As a Ghrelin Deacylation/Lysophospholipid Hydrolyzing Enzyme in Fetal Bovine Serum and Conditioned Medium
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DOI：
10.1210/en.2010-0412
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发表时间：
2010-10-01
期刊：
影响因子：
4.8
通讯作者：
Sugimoto, Hiroyuki
中科院分区：
文献类型：
--
作者：
Satou, Motoyasu;Nishi, Yoshihiro;Sugimoto, Hiroyuki
Ghrelin在第三个丝氨酸残基处含有辛酸，并且Ghrelin上辛酸的存在对其生理功能至关重要。尽管血清中去酰化生长素释放肽的水平高于酰化生长素释放肽的水平，但循环中生长素释放肽去酰化的确切机制仍有待阐明。在这项研究中，快速鉴定ghrelin脱酰活性的基质辅助激光解吸/电离飞行时间质谱法实现，和ghrelin脱酰酶纯化1515倍，从胎牛血清。色谱分离在SDS-PAGE上显示对应于ghrelin脱酰活性的24-kDa条带，并且蛋白条带被鉴定为酰基蛋白硫酯酶1（APT 1）/溶血磷脂酶I。还纯化了HepG 2细胞培养基中的生长素释放肽脱酰酶，并鉴定为APT 1。尽管缺乏分泌信号序列，但APT 1可由表达APT 1的细胞释放，主要来自体内肝脏。APT 1最初被纯化为胞质溶血磷脂水解酶（溶血磷脂酶I），重组APT 1在体外表现出脱酰活性以及溶血磷脂酶活性。在脂多糖（LPS）刺激后，从RAW264.7巨噬细胞样细胞向培养基中高水平释放APT 1，并且LPS抑制这些细胞中APT 1 mRNA和蛋白的表达。更有效的胃饥饿素脱酰酶活性检测血清中的LPS治疗的大鼠比对照血清。结果提示，血清中APT 1活性在测定循环中去酰基ghrelin浓度中可能起重要作用，尤其是在脓毒性炎症状态下。（内分泌学151：4765-4775，2010）
Ghrelin contains an octanoic acid at the third residue serine, and the presence of octanoic acid on ghrelin is critical to its physiological functions. The precise mechanism for the deacylation of ghrelin in circulation remains to be clarified, although the level of deacylated ghrelin (des-acyl ghrelin) is higher than that of acylated ghrelin in serum. In this study, rapid identification of ghrelin deacylation activity was achieved by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and a ghrelin deacylation enzyme was purified 1515-fold from fetal bovine serum. Chromatographic separation showed a 24-kDa band on SDS-PAGE corresponding to ghrelin deacylation activity, and the protein band was identified as acyl-protein thioesterase 1 (APT1)/lysophospholipase I. A ghrelin deacylation enzyme in medium from HepG2 cells was also purified and identified as APT1. Although it lacks a secretion signal sequence, APT1 may be released by cells expressing APT1, mainly from liver in vivo. APT1 was originally purified as a cytosolic lysophospholipid hydrolyzing enzyme (lysophospholipase I), and recombinant APT1 exhibited deacylation activity as well as lysophospholipase activity in vitro. APT1 is released at high levels from RAW264.7 macrophage-like cells into the culture medium after stimulation with lipopolysaccharide (LPS), and LPS suppresses APT1 mRNA and protein expressions in these cells. More potent ghrelin deacylase activities were detected in sera from LPS-treated rats than in control sera. These results suggested that the serum activity of APT1 may play an important role in determination of the concentration of des-acyl ghrelin in circulation, especially under septic inflammation. (Endocrinology 151: 4765-4775, 2010)