Saito F, Yamada H, Sunada Y, Hori H, Shimizu T, Matsumura K
Department of Neurology and Neuroscience, Teikyo University School of Medicine, Tokyo 173, Japan.
J Biol Chem. 1997 Oct 17;272(42):26708-13. doi: 10.1074/jbc.272.42.26708.
We have shown previously that a bovine peripheral nerve protein with a molecular mass of about 30 kDa binds laminin in blot overlay assay. In this paper, we have characterized this 30-kDa laminin-binding protein (LBP30). LBP30 was extracted from the crude bovine peripheral nerve membranes at pH 12 or by 0.5 M NaCl but not by 2% Triton X-100. LBP30 bound to heparin-Sepharose in the presence of 0.5 M NaCl. The results of lectin staining indicated that LBP30 contained both terminally sialylated and nonsialylated Ser/Thr-linked oligosaccharides. LBP30 bound laminin-2 as well as laminin-1 but not fibronectin or collagen type IV. When immobilized LBP30 was incubated with the crude peripheral nerve membrane extracts, all of the endogenous peripheral nerve laminin chain isoforms, the alpha1, alpha2, beta1, beta2, and gamma1 chains, were detected bound to LBP30. The binding of LBP30 to laminin was inhibited by heparin, heparan sulfate, dextran sulfate, or NaCl but was not affected significantly by chondroitin sulfate, dextran, or EDTA. Although LBP30 bound to laminin-1 denatured with SDS in a nonreducing condition, the binding was reduced drastically when laminin-1 was denatured with SDS in a reducing condition, suggesting that the binding of LBP30 is somewhat dependent on the high order structure of laminin-1. Immunohistochemical analysis demonstrated the broad distribution of LBP30 in the perineurium and endoneurium of bovine peripheral nerve. These results indicate that LBP30 is a laminin- and heparin-binding glycoprotein localized in the perineurium and endoneurium of bovine peripheral nerve.
我们之前已经表明，一种分子量约为30 kDa的牛外周神经蛋白在印迹覆盖试验中能结合层粘连蛋白。在本文中，我们对这种30 kDa的层粘连蛋白结合蛋白（LBP30）进行了表征。LBP30在pH 12条件下或用0.5 M NaCl从粗制牛外周神经膜中提取，但不能用2% Triton X - 100提取。LBP30在0.5 M NaCl存在下与肝素 - 琼脂糖结合。凝集素染色结果表明，LBP30含有末端唾液酸化和非唾液酸化的丝氨酸/苏氨酸连接的寡糖。LBP30能结合层粘连蛋白 - 2以及层粘连蛋白 - 1，但不结合纤连蛋白或IV型胶原。当将固定化的LBP30与粗制外周神经膜提取物一起孵育时，检测到所有内源性外周神经层粘连蛋白链异构体，即α1、α2、β1、β2和γ1链，都与LBP30结合。LBP30与层粘连蛋白的结合受到肝素、硫酸乙酰肝素、硫酸葡聚糖或NaCl的抑制，但不受硫酸软骨素、葡聚糖或EDTA的显著影响。尽管LBP30在非还原条件下能与经SDS变性的层粘连蛋白 - 1结合，但当层粘连蛋白 - 1在还原条件下经SDS变性时，结合显著减少，这表明LBP30的结合在某种程度上依赖于层粘连蛋白 - 1的高级结构。免疫组织化学分析表明，LBP30在牛外周神经的神经束膜和神经内膜中广泛分布。这些结果表明，LBP30是一种定位于牛外周神经神经束膜和神经内膜的层粘连蛋白和肝素结合糖蛋白。
