Molecular cloning, tissue expression, and partial characterization of the major fish CNS myelin protein 36K

Autor(en): Moll, W
Lanwert, C
Jeserich, G
Stichwörter: 2',3'-CYCLIC NUCLEOTIDE 3'-PHOSPHODIESTERASE; CENTRAL-NERVOUS-SYSTEM; CIRCULAR-DICHROISM; CNPase; DEHYDROGENASE; FAMILY; GLYCOPROTEINS; in situ hybridization; LOCALIZATION; Neurosciences; Neurosciences & Neurology; OLIGODENDROCYTES; secondary structure; short-chain dehydrogenase; TROUT; trout brain; VERTEBRATE
Erscheinungsdatum: 2003
Herausgeber: WILEY-LISS
Journal: GLIA
Volumen: 44
Ausgabe: 1
Startseite: 57
Seitenende: 66
A full-length cDNA clone encoding the major structural protein of trout CNS myelin 36K was isolated and sequenced. The deduced amino acid sequence did not reveal a putative transmembrane domain and exhibited no structural homology with any of the known myelin proteins. 36K instead shared characteristic structural elements with enzymes of the short-chain dehydrogenase family. The highest similarity in the database (60%), however, was obtained with a human protein of unknown function. By Northern blotting, a single mRNA species of about 2 kb was identified, which was expressed in brain tissue but not in liver. By in situ hybridization, a selective labeling of myelinating glial cells in the trout CNS but not in the PNS was revealed. The developmental appearance of the 36K transcript closely coincided with a period of active myelin deposition in most regions of the trout brain. As a first step in elucidating the structural and biochemical role of 36K for myelin formation and maintenance, we have overexpressed it in Escherichia coli as a soluble His-tag fusion protein and purified it in high yield by Ni+-chelated affinity chromatography. By SDS-PAGE, a single band of the expected molecular size was revealed, which heavily cross-reacted with polyclonal antibodies generated against the native protein. The results of circular dichroism spectroscopy are compatible with a pup-barrel structure (Rossman fold), confirming the results of computer-assisted secondary structure predictions. (C) 2003 Wiley-Liss, Inc.
ISSN: 08941491
DOI: 10.1002/glia.10269

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