Rudd C E, Bodmer J G, Bodmer W F, Crumpton M J
J Biol Chem. 1985 Feb 10;260(3):1927-36.
Two-dimensional polyacrylamide gel analyses of immunoprecipitates of HLA-D region antigens prepared from [35S]methionine-labeled B lymphoblastoid cells revealed a number of invariant polypeptides (Ii and theta) that co-precipitate with the alpha and beta polypeptides of the class II (Ia) antigens. The invariant polypeptides comprised at least three Ii spots of Mr = 31,000 (Ii1-Ii3) and a series of six theta spots of Mr = 34,000 (theta 1-theta 6). The structural inter-relationships of these polypeptides have been investigated. Tryptic peptide fingerprints showed that Ii and theta have closely related amino acid sequences. In contrast, the fingerprints of the HLA-DR alpha and beta polypeptides clearly differed from those of theta and Ii as well as from each other. Analyses of immunoprecipitates prepared from cells cultured in the presence of tunicamycin revealed the presence of two N-linked oligosaccharides on each invariant polypeptide and suggested that the more acidic theta polypeptides (theta 1 and theta 2) differed from the other invariant polypeptides by the presence of sialic acid on one or both N-linked oligosaccharides. Removal of sialic acid by neuraminidase simplified the pattern of theta spots into three distinct Ii-related polypeptides. Endo-beta-N-acetylglycosaminidase H digestion indicated that the individual theta polypeptides represent stages in carbohydrate processing whereby Ii with two N-linked immature oligosaccharides are converted initially to theta 6-theta 3 with one immature and one complex, but nonsialylated, oligosaccharide and finally to theta 2-theta 1 with two complex oligosaccharides. Digestion of the theta polypeptides with N-acetylgalactosamine oligosaccharidase indicated that the theta spots are also derived by O-glycosylation from the Ii polypeptides. This assignment is supported by results obtained using monensin to block glycosylation within the Golgi. At least three spots persisted after complete removal of the N- and O-linked oligosaccharides, suggesting the presence of a family of invariant polypeptides differing in amino acid sequence.
对从[35S]甲硫氨酸标记的B淋巴母细胞制备的HLA - D区抗原免疫沉淀物进行的二维聚丙烯酰胺凝胶分析显示，有许多恒定多肽（Ii和theta）与II类（Ia）抗原的α和β多肽共沉淀。恒定多肽包括至少三个分子量为31,000的Ii斑点（Ii1 - Ii3）和一系列六个分子量为34,000的theta斑点（theta 1 - theta 6）。已经研究了这些多肽的结构相互关系。胰蛋白酶肽指纹图谱表明Ii和theta具有密切相关的氨基酸序列。相比之下，HLA - DRα和β多肽的指纹图谱明显不同于theta和Ii的指纹图谱，彼此之间也不同。对在衣霉素存在下培养的细胞制备的免疫沉淀物的分析表明，每个恒定多肽上存在两个N - 连接的寡糖，并表明酸性更强的theta多肽（theta 1和theta 2）与其他恒定多肽的不同之处在于一个或两个N - 连接的寡糖上存在唾液酸。用神经氨酸酶去除唾液酸后，theta斑点的模式简化为三种不同的与Ii相关的多肽。内切β - N - 乙酰氨基葡糖苷酶H消化表明，各个theta多肽代表碳水化合物加工的阶段，其中具有两个N - 连接的未成熟寡糖的Ii最初转化为具有一个未成熟和一个复杂但非唾液酸化寡糖的theta 6 - theta 3，最后转化为具有两个复杂寡糖的theta 2 - theta 1。用N - 乙酰半乳糖胺寡糖酶消化theta多肽表明，theta斑点也通过O - 糖基化从Ii多肽衍生而来。使用莫能菌素阻断高尔基体中的糖基化获得的结果支持了这一归属。在完全去除N - 和O - 连接的寡糖后，至少有三个斑点持续存在，表明存在一个氨基酸序列不同的恒定多肽家族。
