Katz H R, Benson A C, Austen K F
Department of Medicine, Harvard Medical School, Boston, MA 02115.
J Immunol. 1989 Feb 1;142(3):919-26.
As assessed by immunoprecipitation analyses, expression of the epitope recognized by the rat mAb B23.1 is approximately sevenfold greater on the surface of mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) than on serosal mast cells (SMC) obtained directly from the peritoneal cavity. Immunoprecipitation of B23.1 antibody-binding molecules from Na[125I] surface-labeled BMMC and SMC followed by sizing on SDS-polyacrylamide gels under reducing conditions demonstrated that the epitope is located on molecules of 49,000 and 47,500 Mr, respectively. An additional immunoprecipitated molecule of 42,000 Mr was detected from BMMC intrinsically radiolabeled with [35S]methionine, and pulse-chase analyses revealed that this species was a biosynthetic precursor of the 49,000 Mr cell surface form of the Ag. Treatment of the immunoprecipitated 42,000 and 49,000 Mr forms with endoglycosidase F reduced the Mr of both to 37,000, as did intrinsic radiolabeling of BMMC in the presence of tunicamycin, indicating that both the 42,000 Mr precursor form and the 49,000 Mr cell surface molecule (gp49) contained N-linked carbohydrate. Activation of [32P]orthophosphate-labeled BMMC by sensitization with mouse monoclonal IgE anti-TNP and challenge with TNP-BSA or by exposure to the calcium ionophore A23187 elicited the rapid phosphorylation of gp49 but not of its precursor forms, as did treatment of the cells with PMA. Elution of phosphorylated and immunoprecipitated gp49 from SDS-polyacrylamide gels followed by partial acid hydrolysis of the protein and phosphoamino acid analysis by high voltage thin-layer electrophoresis on cellulose plates indicated that serine, but not threonine or tyrosine, was phosphorylated upon stimulation of BMMC with IgE/Ag, calcium ionophore, or PMA. Cholera toxin did not elicit phosphorylation of gp49. These data suggest that gp49, a plasma membrane glycoprotein preferentially expressed by mouse BMMC, may be either directly or indirectly phosphorylated via protein kinase C during mast cell activation-secretion.
通过免疫沉淀分析评估，大鼠单克隆抗体B23.1识别的表位在小鼠白细胞介素-3依赖的骨髓培养来源肥大细胞（BMMC）表面的表达比直接从腹腔获得的浆膜肥大细胞（SMC）高约7倍。从用Na[125I]进行表面标记的BMMC和SMC中免疫沉淀B23.1抗体结合分子，然后在还原条件下在SDS-聚丙烯酰胺凝胶上进行大小分离，结果表明该表位分别位于49,000和47,500 Mr的分子上。从用[35S]甲硫氨酸进行内在放射性标记的BMMC中检测到另一种42,000 Mr的免疫沉淀分子，脉冲追踪分析表明该物种是该抗原49,000 Mr细胞表面形式的生物合成前体。用内切糖苷酶F处理免疫沉淀的42,000和49,000 Mr形式，两者的Mr均降至37,000，在衣霉素存在下对BMMC进行内在放射性标记时也是如此，这表明42,000 Mr前体形式和49,000 Mr细胞表面分子（gp49）都含有N-连接的碳水化合物。用小鼠抗TNP单克隆IgE致敏并用TNP-BSA攻击或通过暴露于钙离子载体A23187激活[32P]正磷酸盐标记的BMMC，会引起gp49的快速磷酸化，但其前体形式不会，用PMA处理细胞时也是如此。从SDS-聚丙烯酰胺凝胶上洗脱磷酸化并免疫沉淀的gp49，然后对蛋白质进行部分酸水解，并通过在纤维素板上进行高压薄层电泳进行磷酸氨基酸分析，结果表明在用IgE/抗原、钙离子载体或PMA刺激BMMC时，丝氨酸被磷酸化，而苏氨酸或酪氨酸没有。霍乱毒素不会引起gp49的磷酸化。这些数据表明，gp49是一种优先由小鼠BMMC表达的质膜糖蛋白，在肥大细胞激活-分泌过程中可能通过蛋白激酶C直接或间接被磷酸化。
