Ramanathan L, Le H V, Labdon J E, Mays-Ichinco C A, Syto R, Arai N, Nagabhushan T L, Trotta P P
Department of Biotechnology-Biochemistry, Schering Corporation, Bloomfield, NJ 07003.
Biochim Biophys Acta. 1989 Apr 12;1007(3):283-8. doi: 10.1016/0167-4781(89)90149-8.
Recombinant murine interleukin-4 (muIL-4) expressed in COS-7 monkey kidney cells was purified to homogeneity by sequential CM-Sepharose, Sephadex G-100 chromatography and mono-S FPLC to a specific activity of 6.10(7) units per mg of protein based on an in vitro HT-2 cell proliferation assay. Two electrophoretic variants, designated a and b, which migrated on SDS-PAGE as a closely spaced doublet with Mr 19,000, were present in the final product. Gas phase sequencing of the purified protein revealed the presence of an N-terminus corresponding to the mature protein predicted from the cDNA sequence and sequencing of a cyanogen bromide digest confirmed 75 of the 120 predicted amino acids. Elution behavior on gel filtration corresponded to that of a monomer of Mr 19,000. Since there are three potential sites of N-glycosylation predicted by the cDNA sequence, the contribution of glycosylation to the observed heterogeneity was examined by treatment with endoglycosidases. Variant b was digested by either endo-beta-N-acetylglucosaminidase H (endo H) or endo-beta-N-acetylglucosaminidase F (endo F) to protein of Mr 15,000 on SDS-PAGE but was unaffected by treatment with endo-beta-N-acetylglucosaminidase D (endo D), thus indicating the presence of high mannose type of N-glycan. In contrast, variant a was resistant to endo H, F and D. Complete conversion of a mixture of variants a and b to a single protein of Mr 15,000 on SDS-PAGE was obtained only after treatment with N-glycanase. Both variants were resistant to neuraminidase and O-glycanase treatment. These data show that the microheterogeneity observed in purified muIL-4 preparations is due to differences in the nature of the N-linked oligosaccharides. The availability of purified recombinant muIL-4 and a methodology for both total and selective deglycosylation provides a basis for the initiation of structure-function studies of this novel T-cell lymphokine.
在COS-7猴肾细胞中表达的重组鼠白细胞介素-4（muIL-4），通过连续的CM-琼脂糖凝胶、葡聚糖凝胶G-100层析和单-S FPLC纯化至均一，基于体外HT-2细胞增殖试验，其比活性为每毫克蛋白质6.1×10⁷单位。最终产物中存在两种电泳变体，命名为a和b，它们在SDS-PAGE上迁移为紧密间隔的双峰，Mr为19,000。纯化蛋白的气相测序显示存在与从cDNA序列预测的成熟蛋白相对应的N末端，溴化氰消化产物的测序证实了120个预测氨基酸中的75个。凝胶过滤上的洗脱行为与Mr为19,000的单体一致。由于cDNA序列预测有三个潜在的N-糖基化位点，通过用内切糖苷酶处理来研究糖基化对观察到的异质性的贡献。变体b被内切β-N-乙酰氨基葡糖苷酶H（内切H）或内切β-N-乙酰氨基葡糖苷酶F（内切F）消化为SDS-PAGE上Mr为15,000的蛋白质，但不受内切β-N-乙酰氨基葡糖苷酶D（内切D）处理的影响，因此表明存在高甘露糖型N-聚糖。相比之下，变体a对内切H、F和D有抗性。仅在用N-聚糖酶处理后，变体a和b的混合物在SDS-PAGE上才完全转化为单一的Mr为15,000的蛋白质。两种变体对神经氨酸酶和O-聚糖酶处理均有抗性。这些数据表明，纯化的muIL-4制剂中观察到的微观异质性是由于N-连接寡糖性质的差异。纯化的重组muIL-4的可得性以及用于完全和选择性去糖基化的方法为启动这种新型T细胞淋巴因子的结构-功能研究提供了基础。
