Characterization of a 30-kDa Peripheral Nerve Glycoprotein That Binds Laminin and Heparin*
- Fumiaki Saito‡,
- Hiroki Yamada‡,
- Yoshihide Sunada‡,
- Hisae Hori§,
- Teruo Shimizu‡ and
- Kiichiro Matsumura‡¶
- From the ‡Department of Neurology and Neuroscience, Teikyo University School of Medicine, Tokyo 173 and the§Department of Tissue Physiology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 101, Japan
Abstract
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 0.5m 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 α1, α2, β1, β2, and γ1 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.
Footnotes
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↵* This work was supported by grants from the Kato Memorial Bioscience Foundation, the Cell Science Research Foundation, the Science Research Promotion Fund of the Japan Private School Promotion Foundation, Research Grants 8A-1 and 8A-2 for Nervous and Mental Disorders from the Ministry of Health and Welfare, and Research Grants 08457195, 09470156, 09770460, and 09877121 from the Ministry of Education, Science, Sports and Culture.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed: Department of Neurology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173, Japan. Tel.: 81-3-3964-1211; Fax: 81-3-3964-6394; E-mail: k-matsu{at}med.teikyo-u.ac.jp.
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↵1 The abbreviations used are: HSPG, heparan sulfate proteoglycan; PVDF, polyvinylidene difluoride; EHS, Engelbreth-Holm-Swarm; PAGE, polyacrylamide gel electrophoresis; LBP, laminin-binding protein; LBB, 10 mm triethanolamine, pH 7.6, 140 mm NaCl, 1 mm CaCl2, and 1 mm MgCl2; MLBB, LBB + nonfat dry milk.
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↵2 F. Saito and K. Matsumura, unpublished data.
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- Received July 2, 1997.
- Revision received August 7, 1997.
