Zeng F Y, Kaphalia B S, Ansari G A, Weigel P H
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
J Biol Chem. 1995 Sep 8;270(36):21382-7. doi: 10.1074/jbc.270.36.21382.
Rat hepatic asialoglycoprotein receptors (ASGP-Rs) are hetero-oligomers composed of three homologous glycoprotein subunits, designated rat hepatic lectins (RHL) 1, 2, and 3. ASGP-Rs mediate the endocytosis and degradation of circulating glycoconjugates containing terminal N-acetylgalactosamine or galactose, including desialylated plasma glycoproteins. We have shown in permeable rat hepatocytes that the ligand binding activity of one subpopulation of receptors (designated State 2 ASGP-Rs) can be decreased or increased, respectively, by ATP and palmitoyl-CoA (Weigel, P. H., and Oka, J. A. (1993) J. Biol. Chem. 268, 27186-27190). We proposed that a reversible and cyclic acylation/deacylation process may regulate ASGP-R activity during endocytosis, receptor-ligand dissociation, and receptor recycling. In the accompanying paper (Zeng, F-Y., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21388-21395), we show that the ligand binding activity of affinity-purified State 2 ASGP-Rs is decreased by treatment with hydroxylamine under mild conditions consistent with these ASGP-Rs being fatty acylated in vivo. In this study, we used a chemical method to determine the presence of covalently-bound fatty acids in individual ASGP-R subunits. The affinity-purified ASGP-R preparations were separated by SDS-polyacrylamide gel electrophoresis under nonreducing conditions, and the gel slices containing individual RHL subunits were treated with alkali to release covalently bound fatty acids, which were subsequently analyzed by gas chromatography and confirmed by gas chromatography-mass spectrometry. Both stearic and palmitic acids were detected in all three receptor subunits. Pretreatment of ASGP-Rs with hydroxylamine before SDS-polyacrylamide gel electrophoresis reduced the content of both fatty acids by 66-80%, indicating that most of these fatty acids are attached to cysteine residues via thioester linkages. Furthermore, when freshly isolated hepatocytes were cultured in the presence of [3H]palmitate, all three RHL subunits in affinity-purified ASGP-Rs were metabolically labeled. We conclude that RHL1, RHL2, and RHL3 are modified by fatty acylation in intact cells.
大鼠肝脏去唾液酸糖蛋白受体（ASGP-Rs）是由三个同源糖蛋白亚基组成的异源寡聚体，分别命名为大鼠肝脏凝集素（RHL）1、2和3。ASGP-Rs介导循环中含有末端N-乙酰半乳糖胺或半乳糖的糖缀合物的内吞作用和降解，包括去唾液酸化的血浆糖蛋白。我们已经在可渗透的大鼠肝细胞中表明，一种受体亚群（命名为状态2 ASGP-Rs）的配体结合活性可分别被ATP和棕榈酰辅酶A降低或增加（魏格尔，P.H.，和冈，J.A.（1993）《生物化学杂志》268，27186 - 27190）。我们提出，一个可逆的循环酰化/去酰化过程可能在内吞作用、受体-配体解离和受体再循环期间调节ASGP-R的活性。在随附的论文中（曾，F - Y.，和魏格尔，P.H.（1995）《生物化学杂志》270，21388 - 21395），我们表明，在与这些ASGP-Rs在体内被脂肪酰化相一致的温和条件下，用羟胺处理亲和纯化的状态2 ASGP-Rs会降低其配体结合活性。在本研究中，我们使用化学方法来确定单个ASGP-R亚基中是否存在共价结合的脂肪酸。亲和纯化的ASGP-R制剂在非还原条件下通过SDS-聚丙烯酰胺凝胶电泳进行分离，含有单个RHL亚基的凝胶切片用碱处理以释放共价结合的脂肪酸，随后通过气相色谱进行分析，并通过气相色谱-质谱联用进行确认。在所有三个受体亚基中都检测到了硬脂酸和棕榈酸。在SDS-聚丙烯酰胺凝胶电泳之前用羟胺对ASGP-Rs进行预处理，使两种脂肪酸的含量降低了66 - 80%，这表明这些脂肪酸中的大多数是通过硫酯键连接到半胱氨酸残基上的。此外，当新鲜分离的肝细胞在[3H]棕榈酸存在下培养时，亲和纯化的ASGP-Rs中的所有三个RHL亚基都被代谢标记。我们得出结论，RHL1、RHL2和RHL3在完整细胞中被脂肪酰化修饰。
