Identification of β(III) and β(IV)-tubulin isotypes in cold-adapted microtubules from Atlantic cod (Gadus morhua): Antibody mapping and cDNA sequencing

Abstract

Isolated microtubule proteins from the Atlantic cod (Gadus morhua) assemble at temperatures between 8 and 30°C. The cold-adaptation is an intrinsic property of the tubulin molecules, but the reason for it is unknown. To increase our knowledge of tubulin diversity and its role in cold- adaptation we have further characterized cod tubulins using α- and β- tubulin site-directed antibodies and antibodies towards posttranslationally modified tubulin. In addition, one cod brain β-tubulin isotype has been sequenced. In mammals there are five β-tubulins (β(I), β(II), β(III), β(IVa) and β(IVb) expressed in brain. A cod β(III)-tubulin was identified by its electrophoretic mobility after reduction and carboxymethylation. The β(III)-like tubulin accounted for more than 30% of total brain β-tubulins, the highest yield yet observed in any animal. This tubulin corresponds most probably with an additional band, designated β(X), which was found between α- and β-tubulins on SDS-polyacrylamide gels. It was found to be phosphorylated and neurospecific, and constituted about 30% of total cod β- tubulin isoforms. The sequenced cod tubulin was identified as a β(IV)- tubulin, and a β(IV)-isotype was stained by a C-terminal specific antibody. The amount of staining indicates that this isotype, as in mammals, only accounts for a minor part of the total brain β-tubulin. Based on the estimated amounts of β(III)- and β(IV)-tubulins in cod brain, our results indicate that cod has at least one additional β-tubulin isotype and that β- tubulin diversity evolved early during fish evolution. The sequenced cod β(IV)-tubulin had four unique amino acid substitutions when compared to β- tubulin sequences from other animals, while one substitution was in common with Antarctic rockcod β-tubulin. Residues 221, Thr to Ser, and 283, Ala to Ser, correspond in the bovine tubulin dimer structure to loops that most probably interact with other tubulin molecules within the microtubule, and might contribute to cold-adaptation of microtubules.