A novel myelin basic protein transcript variant in the murine central nervous system

Abstract

Myelin basic protein is a multifunctional protein whose primary role is to adhere membranes of the myelin sheath. There are various isoforms that have been identified, 6 distinct isoforms in human and 13 distinct isoforms in mice. These distinct isoforms are the product of alternative splicing of a single gene. The present study sought out to identify the different isoforms found in the murine central nervous system. Neuronal tissue (brain) from five different C57BL6/J mice at 2 months of age was harvested and used for mRNA extraction. mRNA was reversed transcribed to cDNA and transcripts were detected through PCR amplification and DNA agarose gel separation. Primers for exon 1, exon 5b and exon 11 of the myelin basic protein gene were used to capture all the possible transcripts that are naturally found in the murine central nervous system. Unknown transcript was sequenced at Genewiz facilities (South Plainfield, NJ) and mass spectrometry protein sequence analysis demonstrated the presence of a novel myelin basic protein transcript variant. We identified a novel transcript variant of myelin basic protein. This novel transcript variant corresponds to a myelin basic protein of 32.5 kDa which has not been previously reported. This novel transcript variant presents relevant clinical significance to various demyelinating diseases due to its contribution to the understanding of the natural state of the murine central nervous system.

Keywords: Central nervous system; Golli myelin basic protein; Myelin basic protein; Myelin sheath; Transcript variant.

Fig. 1

Diagram representation of the different MBP family members and their individual exon composition. The different MBP isoforms are the result of alternative splicing of a single gene

Fig. 2

Representative DNA Agarose gel of transcript amplification. (a) Transcripts obtained from using primers for exon 5B and exon 11. Five different C57BL6/J mouse brains. Band 1 – Unknown transcript (690 bp), Band 2 – MBP-9 (542 bp), Band 3 – MBP-7 (480 bp), Band 4 – MBP-6 (446 bp), and Band 5 – MBP-13 (356 bp). Bands on the rectangle were excised, purified, and sent for sequencing. (b) Unknown transcript obtained from using primers for exon 1 and exon 11. Band 6 – Unknown transcript (806 bp). (c) Coomassie blue stain of brain homogenate and excise band for mass spectrometry protein analysis. Band 7 – excised band

Fig. 3

Novel MBP variant bioinformatics analysis. (a) Exon composition of Golli-MBP isoform 1 and novel MBP variant. (b) Protein sequence alignment of Golli-MBP isoform 1 and novel MBP variant. Score = 485 bits (1248), Expect = 2e-180, Method = Compositional matrix adjust, Identities = 250/302 (83%), Positives = 250/302 (82%), Gaps = 52/302 (17%)