Zeng F Y, Weigel P H
Department of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
J Biol Chem. 1996 Dec 13;271(50):32454-60. doi: 10.1074/jbc.271.50.32454.
Functional rat or human asialoglycoprotein receptors (ASGP-Rs) are hetero-oligomeric integral membrane glycoproteins. Rat ASGP-R contains three subunits, designated rat hepatic lectins (RHL) 1, 2, and 3; human ASGP-R contains two subunits, HHL1 and HHL2. Both receptors are covalently modified by fatty acylation (Zeng, F.-Y., Kaphalia, B. S., Ansari, G. A. S., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21382-21387; Zeng, F.-Y., Oka, J. A., and Weigel, P. H. (1996) Biochem. Biophys. Res. Commun. 218, 325-330). We report here that the single Cys residue in the cytoplasmic domain of each RHL or HHL subunit is fatty acylated. The degree of acylation is >/=90% per subunit. Deacylation of affinity-purified ASGP-Rs with hydroxylamine results in the spontaneous formation of dimers through reversible disulfide bonds, indicating that deacylation concomitantly generates free thiol groups. Reaction of hydroxylamine-treated ASGP-R with [14C]iodoacetamide resulted in the specific incorporation of radioactivity into all RHL and HHL subunits, verifying that fatty acids are attached via thioester linkages. To identify the Cys residue involved in the thioester linkages, 14C-carboxyamidomethylated RHL subunits were separated by SDS-polyacrylamide gel electrophoresis and digested in-gel with trypsin, and the resulting peptides were separated by reverse-phase high performance liquid chromatography. Amino acid sequence of radioactive peptides revealed that Cys35 in RHL1 and Cys54 in RHL2 and RHL3 were radiolabeled and, therefore, are fatty acylation sites. Fatty acylation of HHL subunits was analyzed by site-directed mutagenesis. Metabolic labeling of Cos7 cells transfected with wild type HHL1 cDNA resulted in substantial incorporation of [3H]palmitate into purified HHL1. Incorporation of [3H]palmitate into a C36S mutant of HHL1 was negligible ( approximately 1%) compared with wild type. This result also shows that Cys57 within the transmembrane domain of HHL1 is not normally palmitoylated. We conclude that Cys35 in RHL1, Cys54 in RHL2 and RHL3, and Cys36 in HHL1 are fatty acylated. Cys57 in HHL1 and probably Cys56 in RHL1 are not palmitoylated.
功能性大鼠或人类去唾液酸糖蛋白受体（ASGP-Rs）是异源寡聚体整合膜糖蛋白。大鼠ASGP-R包含三个亚基，分别命名为大鼠肝凝集素（RHL）1、2和3；人类ASGP-R包含两个亚基，HHL1和HHL2。两种受体都通过脂肪酰化进行共价修饰（曾，F.-Y.，卡法利亚，B.S.，安萨里，G.A.S.，以及韦格尔，P.H.（1995年）《生物化学杂志》270，21382 - 21387；曾，F.-Y.，冈田，J.A.，以及韦格尔，P.H.（1996年）《生物化学与生物物理研究通讯》218，325 - 330）。我们在此报告，每个RHL或HHL亚基细胞质结构域中的单个半胱氨酸残基被脂肪酰化。每个亚基的酰化程度≥90%。用羟胺对亲和纯化的ASGP-Rs进行脱酰化会通过可逆的二硫键自发形成二聚体，这表明脱酰化同时产生了游离的巯基。用[¹⁴C]碘乙酰胺处理羟胺处理过的ASGP-R，导致放射性特异性掺入所有RHL和HHL亚基，证实脂肪酸是通过硫酯键连接的。为了鉴定参与硫酯键连接的半胱氨酸残基，¹⁴C - 羧酰胺甲基化的RHL亚基通过SDS - 聚丙烯酰胺凝胶电泳分离，然后在凝胶中用胰蛋白酶消化，所得肽段通过反相高效液相色谱分离。放射性肽段的氨基酸序列显示，RHL1中的Cys35、RHL2和RHL3中的Cys54被放射性标记，因此是脂肪酰化位点。通过定点诱变分析HHL亚基的脂肪酰化。用野生型HHL1 cDNA转染的Cos7细胞的代谢标记导致[³H]棕榈酸大量掺入纯化的HHL1中。与野生型相比，[³H]棕榈酸掺入HHL1的C36S突变体中的量可忽略不计（约1%）。该结果还表明，HHL1跨膜结构域内的Cys57通常不被棕榈酰化。我们得出结论，RHL1中的Cys35、RHL2和RHL3中的Cys54以及HHL1中的Cys36被脂肪酰化。HHL1中的Cys57以及可能RHL1中的Cys56不被棕榈酰化。
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