Herzig M C, Weigel P H
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
Biochemistry. 1989 Jan 24;28(2):600-10. doi: 10.1021/bi00428a028.
We have developed chemical affinity reagents for the hepatic galactosyl receptor. Asialoorosomucoid (ASOR) was derivatized with five homobifunctional N-hydroxysuccinimide (NHS) ester cross-linkers. NHS/ASOR derivatives were synthesized, purified, and applied within 10 min to isolated rat hepatocytes at 4 degrees C. Specific binding of these 125I-labeled derivatives was approximately 90% in the presence of either EGTA or excess ASOR. Specific cross-linking assessed by the resistance of specifically bound NHS/125I-ASOR to release by EGTA, was 50-75% of the specifically bound ligand. The extent of specific cross-linking correlated with the average number of NHS groups per ASOR and was controlled by varying the molar ratio of cross-linker to ASOR during the synthesis. Cross-linking proceeded rapidly at 4 degrees C as a first-order process (k = 0.25 min-1, t1/2 = 2.8 min). After being cross-linked with any of the NHS/125I-ASOR derivatives, cells were washed with EGTA, solubilized in Triton X-100, and analyzed by SDS-PAGE and autoradiography. Major bands were observed at Mr congruent to 84K, 93K, and 105K corresponding to the expected size of 1:1 adducts between NHS/ASOR (Mr congruent to 41.3K) and the three subunits of the receptor, Mr congruent to 43K, 50K, and 60K. The three subunits, rat hepatic lectin (RHL) 1, 2, and 3, were labeled in the ratio of about 1.0:1.2:1.0, respectively. After cross-linking, a polyclonal goat antibody to the receptor immunoprecipitated up to 100% of the specifically cross-linked NHS/125I-ASOR. Preimmune IgG immunoprecipitated less than 1% of the radiolabeled ligand. Cell surface receptors were cross-linked to NHS-ASOR, extracted with Triton X-100, immunoprecipitated with anti-orosomucoid-Sepharose, and subjected to Western blot analysis. By use of anti-sera specific for RHL 1 or RHL 2/3 (from K. Drickamer), cross-linked complexes of Mr congruent to 85K or approximately 90-115K, respectively, were detected as were un-cross-linked native subunits. The ratio of free to cross-linked subunits was approximately 10:1 for RHL 1 and approximately 0.5:1 for RHL 2/3. We conclude that all three receptor subunits can cross-link to ligand. We propose a model in which the native receptor is a heterohexamer composed of four subunits of RHL 1 and two subunits of RHL 2 and/or RHL 3.
我们已开发出针对肝脏半乳糖基受体的化学亲和试剂。去唾液酸糖蛋白（ASOR）用五种同双功能N-羟基琥珀酰亚胺（NHS）酯交联剂进行衍生化。合成、纯化NHS/ASOR衍生物，并在10分钟内将其应用于4℃下分离的大鼠肝细胞。在存在乙二醇双四乙酸（EGTA）或过量ASOR的情况下，这些125I标记衍生物的特异性结合约为90%。通过特异性结合的NHS/125I-ASOR对EGTA释放的抗性评估的特异性交联为特异性结合配体的50 - 75%。特异性交联程度与每个ASOR上NHS基团的平均数相关，并通过在合成过程中改变交联剂与ASOR的摩尔比来控制。交联在4℃下作为一级过程迅速进行（k = 0.25分钟-1，t1/2 = 2.8分钟）。用任何一种NHS/125I-ASOR衍生物交联后，细胞用EGTA洗涤，在Triton X-100中溶解，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和放射自显影进行分析。在Mr约为84K、93K和105K处观察到主要条带，分别对应于NHS/ASOR（Mr约为41.3K）与受体的三个亚基（Mr约为43K、50K和60K）之间预期大小的1:1加合物。这三个亚基，大鼠肝脏凝集素（RHL）1、2和3，标记比例分别约为1.0:1.2:1.0。交联后，针对该受体的多克隆山羊抗体可免疫沉淀高达100%的特异性交联NHS/125I-ASOR。免疫前IgG免疫沉淀的放射性标记配体不到1%。细胞表面受体与NHS-ASOR交联，用Triton X-100提取，用抗血清类粘蛋白-琼脂糖免疫沉淀，并进行蛋白质免疫印迹分析。使用对RHL 1或RHL 2/3特异的抗血清（来自K. Drickamer），分别检测到Mr约为85K或约90 - 115K的交联复合物以及未交联的天然亚基。游离亚基与交联亚基的比例对于RHL 1约为10:1，对于RHL 2/3约为0.5:1。我们得出结论，所有三个受体亚基都能与配体交联。我们提出一个模型，其中天然受体是由四个RHL 1亚基和两个RHL 2和/或RHL 3亚基组成的异源六聚体。
