Yoshikawa W, Hara H, Takehara T, Shimonishi M, Sakai H, Shimizu N, Shimizu S, Wang M H, Hagiya M, Skeel A, Leonard E J
Toyobo Co., Ltd., 2-1-1 Katata, Ohtsu, 520-02, Japan.
Arch Biochem Biophys. 1999 Mar 15;363(2):356-60. doi: 10.1006/abbi.1998.1090.
Human serum macrophage-stimulating protein (MSP) induces motile activity of murine resident peritoneal macrophages and is a growth and motility factor for epithelial cells. It belongs to the plasminogen-related family of kringle proteins, and is secreted as a single-chain, 78-kDa, biologically inactive pro-MSP. Proteolytic cleavage of pro-MSP at a single site yields active MSP, a disulfide-linked alphabeta-chain heterodimer. However cleavage of recombinant pro-MSP yielded not only the disulfide-linked heterodimer, but also free alpha- and beta-chains, indicating that some of the recombinant molecules lacked an alphabeta-chain disulfide. We purified the free chains for characterization. The beta-chain of MSP has three extra cysteines, Cys527, Cys562, and Cys672, which are not found in the plasminogen beta-chain. Disulfide bond analysis showed a Cys527-Cys562, but also a Cys588-Cys672. Coopting Cys588 by Cys672 prevented the expected formation of a disulfide between alpha-chain Cys468 and beta-chain Cys588. Concomitant studies determined structures of oligosaccharides at the three Asn-linked glycosylation sites of MSP. The oligosaccharides at the three Asn loci are heterogeneous; 11 different sugars were identified, all being sialylated fucosyl biantennary structures. We also located the pro-MSP signal peptide cleavage site at Gly18-Gln19 and the scissile bond for formation of mature MSP at Arg483-Val484.
人血清巨噬细胞刺激蛋白（MSP）可诱导小鼠驻留腹膜巨噬细胞的运动活性，是上皮细胞的生长和运动因子。它属于 kringle 蛋白的纤溶酶原相关家族，以单链、78 kDa、无生物学活性的前体 MSP 形式分泌。前体 MSP 在单个位点的蛋白水解切割产生活性 MSP，即一种二硫键连接的αβ链异二聚体。然而，重组前体 MSP 的切割不仅产生了二硫键连接的异二聚体，还产生了游离的α链和β链，这表明一些重组分子缺乏αβ链二硫键。我们纯化了游离链以进行表征。MSP 的β链有三个额外的半胱氨酸，即 Cys527、Cys562 和 Cys672，在纤溶酶原β链中未发现。二硫键分析显示存在 Cys527-Cys562，也存在 Cys588-Cys672。用 Cys672 取代 Cys588 可阻止α链 Cys468 和β链 Cys588 之间预期的二硫键形成。同时进行的研究确定了 MSP 三个天冬酰胺连接糖基化位点的寡糖结构。三个天冬酰胺位点的寡糖是异质的；鉴定出 11 种不同的糖，均为唾液酸化岩藻糖基双天线结构。我们还确定了前体 MSP 信号肽切割位点在 Gly18-Gln19 处，以及成熟 MSP 形成的可裂解键在 Arg483-Val484 处。
