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J. Biol. Chem., Vol. 283, Issue 30, 20830-20840, July 25, 2008

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Opalin, a Transmembrane Sialylglycoprotein Located in the Central Nervous System Myelin Paranodal Loop Membrane^*

Fumio Yoshikawa, Yumi Sato, Koujiro Tohyama, Takumi Akagi^¶, Tsutomu Hashikawa^¶, Yuko Nagakura-Takagi, Yukiko Sekine, Noriyuki Morita¹, Hiroko Baba^||, Yutaka Suzuki^**, Sumio Sugano^**, Akira Sato², and Teiichi Furuichi³

From the Laboratory for Molecular Neurogenesis and ^¶Laboratory for Neural Architecture, RIKEN Brain Science Institute, Wako 351-0198, The Center for Electron Microscopy and Bio-Imaging Research, Iwate Medical University, Morioka 020-8505, the ^||Tokyo University of Pharmacy and Life Sciences, Hachioji 192-0392, and the ^**Department of Frontier Science, University of Tokyo, Tokyo 108-8639, Japan

In contrast to compact myelin, the series of paranodal loops located in the outermost lateral region of myelin is non-compact; the intracellular space is filled by a continuous channel of cytoplasm, the extracellular surfaces between neighboring loops keep a definite distance, but the loop membranes have junctional specializations. Although the proteins that form compact myelin have been well studied, the protein components of paranodal loop membranes are not fully understood. This report describes the biochemical characterization and expression of Opalin as a novel membrane protein in paranodal loops. Mouse Opalin is composed of a short N-terminal extracellular domain (amino acid residues 1–30), a transmembrane domain (residues 31–53), and a long C-terminal intracellular domain (residues 54–143). Opalin is enriched in myelin of the central nervous system, but not that of the peripheral nervous system of mice. Enzymatic deglycosylation showed that myelin Opalin contained N- and O-glycans, and that the O-glycans, at least, had negatively charged sialic acids. We identified two N-glycan sites at Asn-6 and Asn-12 and an O-glycan site at Thr-14 in the extracellular domain. Site-directed mutations at the glycan sites impaired the cell surface localization of Opalin. In addition to the somata and processes of oligodendrocytes, Opalin immunoreactivity was observed in myelinated axons in a spiral fashion, and was concentrated in the paranodal loop region. Immunogold electron microscopy demonstrated that Opalin was localized at particular sites in the paranodal loop membrane. These results suggest a role for highly sialylglycosylated Opalin in an intermembranous function of the myelin paranodal loops in the central nervous system.

Received for publication, February 19, 2008 , and in revised form, April 22, 2008.

^* This work was supported by Grants-in-Aid for Scientific Research and for Advanced Medical Science Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Japan Society for the Promotion of Science (JSPS), and by the Institute of Physical and Chemical Research (RIKEN). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5.

¹ Current address: Mimasaka Junior College, Tsuyama 708-8511, Japan.

² Current address: RIKEN Advanced Science Institute, Yokohama 230-0045, Japan.

³ To whom correspondence should be addressed. Tel.: 81-48-467-5906; Fax: 81-48-467-6079; E-mail: tfuruichi{at}brain.riken.jp.

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