Suzu S, Ohtsuki T, Yanai N, Takatsu Z, Kawashima T, Takaku F, Nagata N, Motoyoshi K
Biochemical Research Laboratory, Morinaga Milk Industry Co, Ltd., Kanagawa, Japan.
J Biol Chem. 1992 Mar 5;267(7):4345-8.
Chinese hamster ovary cells transfected with a 4.0-kilobase macrophage colony-stimulating factor (M-CSF) cDNA express two different M-CSF species; one has an apparent molecular weight of 85,000 and is identified as a homodimer of a 43-kDa subunit, and the other has an indeterminate structure greater than 200 kDa. In this study, we investigated the structure of the high molecular weight M-CSF by immunochemical procedures. The high molecular weight M-CSF was easily purified, since it bound tightly to DEAE-Sephacel and eluted at a characteristically high salt concentration. The high molecular weight M-CSF migrated as a diffuse band of over than 200,000 on nonreducing sodium dodecyl sulfate-polyacrylamide gels. Analysis of the same samples under reducing conditions revealed that the larger species consisted of a heteromer of the 43- and 150-200-kDa M-CSF subunits. Digestion of the 150-200-kDa M-CSF subunit with chondroitinase, which degrades the chondroitin sulfate glycosaminoglycan chain, yielded a 100 kDa band. This species was secreted instead of 150-200-kDa species when the cells were cultured in the presence of beta-D-xyloside, which inhibits the elongation of the chondroitin sulfate glycosaminoglycan chain in proteoglycans, providing additional evidence for the existence of a chondroitin sulfate chain in the 150-200-kDa M-CSF subunit. Removal of O- and N-linked carbohydrate from the 150-200-kDa subunit yielded a polypeptide chain with a larger molecular mass (approximately 45 kDa) than that of the 43-kDa subunit (approximately 25 kDa). Collectively, these results indicate that the 150-200-kDa M-CSF subunit is a proteoglycan with a core protein that may be an alternatively processed form of M-CSF.
用一个4.0千碱基的巨噬细胞集落刺激因子（M-CSF）cDNA转染的中国仓鼠卵巢细胞表达两种不同的M-CSF分子；一种的表观分子量为85,000，被鉴定为一个43-kDa亚基的同二聚体，另一种的结构不确定，分子量大于200 kDa。在本研究中，我们通过免疫化学方法研究了高分子量M-CSF的结构。高分子量M-CSF很容易纯化，因为它与DEAE-琼脂糖紧密结合，并在特征性的高盐浓度下洗脱。在非还原十二烷基硫酸钠-聚丙烯酰胺凝胶上，高分子量M-CSF迁移为一条大于200,000的弥散带。在还原条件下对相同样品的分析表明，较大的分子由43-kDa和150 - 200-kDa的M-CSF亚基组成的异聚体。用软骨素酶消化150 - 200-kDa的M-CSF亚基，软骨素酶可降解硫酸软骨素糖胺聚糖链，产生一条100 kDa的条带。当细胞在β-D-木糖苷存在下培养时，该分子被分泌而不是150 - 200-kDa的分子，β-D-木糖苷可抑制蛋白聚糖中硫酸软骨素糖胺聚糖链的延长，这为150 - 200-kDa的M-CSF亚基中存在硫酸软骨素链提供了额外证据。从150 - 200-kDa亚基上去除O-连接和N-连接的碳水化合物后，产生了一条分子量比43-kDa亚基（约25 kDa）更大（约45 kDa）的多肽链。总的来说，这些结果表明150 - 200-kDa的M-CSF亚基是一种蛋白聚糖，其核心蛋白可能是M-CSF的一种可变加工形式。
