Molecular cloning, tissue expression, and partial characterization of the major fish CNS myelin protein 36k.
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 betaalphabeta-barrel structure (Rossman fold), confirming the results of computer-assisted secondary structure predictions.
Moll
Wolfgang
W
Department of Neurobiology, University of Osnabrück, Osnabrück, Germany.
Lanwert
Carsten
C
Stratmann
A
A
Strelau
J
J
Jeserich
Gunnar
G
D000818:Animals; D002490:Central Nervous System / Q000166:cytology / Q000378:metabolism*; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009185:Myelin Proteins / Q000235:genetics* / Q000302:isolation & purification*; D009186:Myelin Sheath / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013057:Spectrum Analysis; D014337:Trout / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D009185:Myelin Proteins