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

Introduction

The myelin sheath provides an electrical insulation through the multilayers of nonconductive plasma membrane. Insulating myelin sheath allows rapid transduction of nerve impulses [1]. There are two major components of the myelin sheath, a protein component that is geared towards maintaining structural integrity and a lipid component that is asymmetrically distributed across the membrane [2]. Myelin basic protein (MBP) is one of the major structural proteins, which is also the second most abundant protein in the central nervous system (CNS) myelin. MBP has been associated with various neurological diseases, underscoring its importance in health and disease [3-6].

MBP is a multifunctional protein critically involved in the maintenance of the myelin sheath compact structure [7,8]. There are 6 distinct MBP isoforms reported in humans and 13 distinct MBP isoforms reported in mice, all of which arise from alternative splicing of a single gene [8]. These transcript variants are categorized into two main families: 1) the Golli (Genes of Oligodendrocyte Lineage) family ubiquitously expressed throughout the immune and nervous system and 2) the classic family that is primarily expressed in oligodendrocytes [9,10]. There are 11 exons that comprise the murine MBP gene, which give rise to MBP isoforms 1 to 3 in the Golli family, and MBP isoforms 4 to 13 in the classic family [9,7].

The versatility of MBP gives rise to a broad functional spectrum which encompasses many biological processes, all of which remain to be fully understood [7,8,11]. The Golli family appears to be involved in calcium regulation in T-cells and oligodendrocytes, suggesting their involvement in T-cell activation, oligodendrocyte development and calcium dependent biological processes [7]. On the other hand, the classic MBP family plays a major role within oligodendrocytes, which is primarily associated with stability of the myelin sheath [8,9,5]. However, certain members of the classic MBP family have been associated with other functions including cell signaling, nuclear translocation, lipid interactions, interaction with cytoskeletal elements, and regulation of gene expression [12-21]. Given the wide range of functions that MBP seems to be involved in and the numerous isoforms it has, the present report explored the various transcripts that are naturally found in the murine CNS.

Materials and Methods

Neuronal tissue acquisition, RNA extraction, and cDNA synthesis

All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Miami (IACUC protocol number: 16-235). Neuronal tissue (brain) from five C57BL6/J mice at 2 months of age was dissected and 30 mg of neuronal tissue was utilized for RNA extraction. The Qiagen RNeasy Mini Kit (74104) was used for RNA extraction and all surfaces and tools for dissection were cleaned utilizing an RNase decontamination solution (Thermo, AM9780). Extracted RNA was purified from contaminant DNA using the TURBO DNA-free Kit (Ambion, AM1907). cDNA was made from DNA-free RNA utilizing oligo (dT) to specifically target the reverse transcription of mRNA transcripts (Invitrogen Superscript III, 18080-051).

Primer design

Primers were designed specifically for exon 5B (forward), exon 11 (reverse) and exon 1 (forward) of the MBP gene. Exon 5B forward primer: 5’-TGGCATCACAGAAGAGACCC-3’, exon 11 reverse primer: 5’-AGCGGCTGTCTCTTCCTCC-3’, and exon 1 forward primer 5’-ATGGGAAACCACTCTGGAAAGA-3’.

PCR and product purification

The Amresco PCR Kit (N555) was utilized for amplification of transcript and optimization of primer annealing temperature (65.5°C). PCR products were separated in a 3% agarose gel and bands of interest were excised under UV lamp visualization. The PCR product was extracted from the excised band utilizing the Qiagen QIAquick Gel Extraction Kit (28704) and the eluted product was send for sequencing (Genewiz, South Plainfield, NJ).

Enzymatic digestion and mass spectrometry analysis

Neuronal tissue was separated using the XCell SureLock Mini-Cell system (Thermo, EI0001). Gel was stained using SimplyBlue (Invitrogen, LC6060). Band excised at 32.5 kDa was denature using 6 M urea, reduced in 10 mM Dithiothreitol, alkylated in 15 mM Iodoacetamide, and quenched in 20 mM Dithiothreitol. Samples were digested with either Trypsin (Promega, V511A), Chymotrypsin (Promega, V106A), or Proteinase K (Thermo, 17916). Mass spectrometry was performed on a Q-Exactive instrument after fractionation on a coupled Easy nLC 1000 nano-liquid chromatography system (Thermo Fisher Scientific, Waltham, MA) as described in our other published reports [22].

Results and Discussion

Primers for MBP transcripts were designed to encompass the first and last exons that are common in all the classic MBP transcripts (exon 5B and exon 11, respectively) in order to capture the transcripts present in the CNS (Fig. 1). Agarose DNA gel analysis demonstrated the presence of five major bands, which could be accounted for by the molecular weight of known MBP transcripts (Fig. 2a). These encompassed classic-MBP isoform 9 (band 2, 542 bp), classic-MBP isoform 7 (band 3, 480 bp), classic-MBP isoform 6 (band 4, 446 bp), and classic-MBP isoform 13 (band 5, 356 bp). However, band 1 corresponded to a transcript of about 690 bp, which could not be accounted for by any of the known MBP transcripts. The highest transcript molecular weight corresponds to Golli-MBP isoform 1 (MBP-1, 750 bp) followed by the classic-MBP isoform 4 (MBP-4, 588 bp), demonstrating the lack of an intermediate transcript that could account for the 690 bp unknown transcript. The sequence for MBP-1 (NM_010777.3) is the canonical sequence that is used as a reference for the comparison of other MBP transcript variants. MBP-1 is composed of exons 1, 2, 3, 5A, 5B, 7, 8, and 11, which gives rise to the possibility that the 690 bp unknown transcript is part of the larger MBP-1 transcript. However, MBP-1 transcript analysis demonstrated that the primer designed for exon 5B is localized 400 bp downstream of the start site. This indicates that the unknown transcript corresponding to 690 bp could not be part of the region detected by primers against exon 5B and exon 11 of the MBP-1 transcript, since it would result in a transcript of 350 bp.

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

To verify the nature of this unknown transcript variant, band 1 was excised, purified, and sent for sequencing (Table 1). Sequence analysis demonstrated that the unknown transcript could be part of a larger transcript. This larger transcript corresponds to a 32.5 kDa MBP isoform (XM_006526456.2) which has not been reported in the literature. Subsequent transcript analysis utilizing a primer for exon 1, revealed the presence of an 806 bp unknown transcript (band 6, Fig. 2b). Given that the highest molecular weight MBP isoform reported is the Golli-MBP isoform 1 (750 bp), this finding further supported the possibility that the 806 bp unknown transcript corresponds to the transcript for the 32.5 kDa MBP isoform.

Table 1.

DNA sequence and composite protein sequence for excised bands. The composite sequence is composed of underlined sequence corresponding to band 6 and non-underlined sequence corresponds to band 1. Lowercase protein sequence correspond to the partial sequence of exon 11 not detected by DNA sequence analysis

Band 1	TGGCATCACAGAAGAGACCCTCACAGCGATCCAAGTACCTGGCCACAGCAAGTACCATGGACCATGCC AGGCATGGCTTCCTCCCAAGGCACAGAGACACGGGCATCCTTGACTCCATCGGGCGCTTCTTTAGCGG TGACAGGGGTGCGCCCAAGCGGGGCTCTGGCAAGGACTCACACACGAGAACTACCCATTATGGCTCC CTGCCCCAGAAGTCGCAGCACGGCCGGACCCAAGATGAAAACCCAGTAGTCCATTTCTTCAAGAACAT TGTGACACCTCGAACACCACCTCCATCCCAAGGGAAGGGGAGAGGCCTGTCCCTCAGCAGATTTAGCT GGGGGGCCGAGGGGCAGAAGCCAGGATTTGGCTACGGAGGCAGAGCTTCCGACTATAAATCGGCTCA CAAGGGATTCAAGGGGGCCTACGACGCCCAGGGCACGCTTTCCAAAATCTTTAAGCTGGGAGGAAGAG ACAGCCGCT
Band 6	ATGGGAAACCACTCTGGAAAGAGAGAATTATCTGCTGAGAAGGCCAGTAAGGATGGAGAGATTCACCG AGGAGAGGCTGGAAAGAAGAGAAGCGTGGGCAAGCTTTCTCAGACGGCCTCAGAGGACAGTGATGTG TTTGGGGAGGCAGATGCGATCCAGAACAATGGGACCTCGGCTGAGGACACGGCGGTGACAGACTCCA AGCACACAGCAGACCCAAAGAATAACTGGCAAGGCGCCCACCCAGCTGACCCAGGGAACCGCCCCCA CTTGATCCGCCTCTTTTCCCGAGATGCCCCGGGAAGGGAGGACAACACCTTCAAAGACAGGCCCTCAG AGTCCGACGAGCTTCAGACCATCCAAGAAGACCCCACAGCAGCTTCCGGAGGCCTGGATGTGATGGCA TCACAGAAGAGACCCTCACAGCGATCCAAGTACCTGGCCACAGCAAGTACCATGGACCATGCCAGGCA TGGCTTCCTCCCAAGGCACAGAGACACGGGCATCCTTGACTCCATCGGGCGCTTCTTTAGCGGTGACA GGGGTGCGCCCAAGCGGGGCTCTGGCAAGGACTCACACACGAGAACTACCCATTATGGCTCCCTGCC CCAGAAGTCGCAGCACGGCCGGACCCAAGATGAAAACCCAGTAGTCCATTTCTTCAAGAACATTGTGA CACCTCGAACACCACCTCCATCCCAAGGGAAGGG
Composite sequence	ATGGGAAACCACTCTGGAAAGAGAGAATTATCTGCTGAGAAGGCCAGTAAGGATGGAGAGATTCACCG AGGAGAGGCTGGAAAGAAGAGAAGCGTGGGCAAGCTTTCTCAGACGGCCTCAGAGGACAGTGATGTG TTTGGGGAGGCAGATGCGATCCAGAACAATGGGACCTCGGCTGAGGACACGGCGGTGACAGACTCCA AGCACACAGCAGACCCAAAGAATAACTGGCAAGGCGCCCACCCAGCTGACCCAGGGAACCGCCCCCA CTTGATCCGCCTCTTTTCCCGAGATGCCCCGGGAAGGGAGGACAACACCTTCAAAGACAGGCCCTCAG AGTCCGACGAGCTTCAGACCATCCAAGAAGACCCCACAGCAGCTTCCGGAGGCCTGGATGTGATGGCA TCACAGAAGAGACCCTCACAGCGATCCAAGTACCTGGCCACAGCAAGTACCATGGACCATGCCAGGCA TGGCTTCCTCCCAAGGCACAGAGACACGGGCATCCTTGACTCCATCGGGCGCTTCTTTAGCGGTGACA GGGGTGCGCCCAAGCGGGGCTCTGGCAAGGACTCACACACGAGAACTACCCATTATGGCTCCCTGCC CCAGAAGTCGCAGCACGGCCGGACCCAAGATGAAAACCCAGTAGTCCATTTCTTCAAGAACATTGTGA CACCTCGAACACCACCTCCATCCCAAGGGAAGGGGAGAGGCCTGTCCCTCAGCAGATTTAGCTGGGG GGCCGAGGGGCAGAAGCCAGGATTTGGCTACGGAGGCAGAGCTTCCGACTATAAATCGGCTCACAAG GGATTCAAGGGGGCCTACGACGCCCAGGGCACGCTTTCCAAAATCTTTAAGCTGGGAGGAAGAGACAG CCGCT
Predicted composite protein sequence	MGNHSGKRELSAEKASKDGEIHRGEAGKKRSVGKLSQTASEDSDVFGEADAIQNNGTSAEDTAVTDSKHT ADPKNNWQGAHPADPGNRPHLIRLFSRDAPGREDNTFKDRPSESDELQTIQEDPTAASGGLDVMASQKRP SQRSKYLATASTMDHARHGFLPRHRDTGILDSIGRFFSGDRGAPKRGSGKDSHTRTTHYGSLPQKSQHGR TQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQKPGFGYGGRASDYKSAHKGFKGAYDAQ GTLSKIFKLGGRDSRsgspmarr

Composite sequence GeneBank Accession Number: MH926013

The band corresponding to this 806 bp unknown transcript was excised, purified and sent for sequencing, which matched with the predicted sequence for the 32.5 kDa MBP isoform (Table 1). The sequence was submitted to the GeneBank database (accession number MH926013). Bioinformatics analysis revealed the protein sequence for this 806 bp unknown transcript and comparison with the sequence from MBP-1 demonstrated that the 806 bp unknown transcript shares the same exon composition with the addition of exon 9 and 10 (Table 1, Fig. 3). To determine the presence of this novel MBP protein, CNS tissue from the same animal was separated via SDS-PAGE gel electrophoresis and the band corresponding to the molecular weight of 32.5 kDa was excised and analyzed by mass spectrometry (band 7, Fig. 2c). Mass spectrometry analysis of three enzymatic digestions (Trypsin, Chymotrypsin, and Proteinase K) demonstrated a 99% coverage of the predicted protein sequence (Table 2).

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%)

Table 2.

Peptides identified from mass spectrometric analysis using enzymatic digestions. Identification of peptide sequence from digestions with Trypsin, Chymotrypsin, and Proteinase K

Enzyme	Exp. MH+ *	Peptide
Trypsin	1046.5	DTGILDSIGRF
	728.3	FFSGDRG
	1800.8	FSWGAEGQKPGFGYGGRA
	726.4	HGFLPRH
	1339.7	HRDTGILDSIGRF
	1460.7	TQDENPVVHFFKN
	4015.9	DRPSESDELQTIQEDPTAASGGLDVMASQKRPSQRSK
	3473.5	LSQTASEDSDVFGEADAIQN NGTSAEDTAVTDSK
	2653.4	NNWQGAHPADPGNRPHLIRL FSR
	2044.0	YLATASTMDHARHGFLPR
	1727.9	DSHTRTTHYGSLPQK
	1006.5	ASDYKSAHK
	832.4	RELSAEK
	774.4	SAHKGFK
	753.3	EDNTFK
Chymotrypsin	841.4	DSIGRFF
	1801.9	FSGDRGAPKRGSGKVPW
	2508.2	GSLPQKSQHGRTQDENPVVHFF
	1498.7	KGAYDAQGTLSKIF
	1733.8	KLGGRDSRSGSPMARR
	2003.1	KNIVTPRTPPPSQGKGRGL
	2596.4	KNIVTPRTPPPSQGKGRGLSLSRF
	1654.8	SGDRGAPKRGSGKVPW
	882.4	SLSRFSW
	1383.6	SWGAEGQKPGFGY
Proteinase K	1128.5	MGNHSGKREL
	648.3	SKDGEI
	976.4	EKASKDGEI
	1513.7	TDSKHTADPKNNW
	1224.6	HRGEAGKKRSV
	1107.4	QNNGTSAEDTA
	1364.6	SRDAPGREDNTF
	651.2	SEDSDV
Experimentally determined composite protein sequence	MGNHSGKRELSAEKASKDGEIHRGEAGKKRSVGKLSQTASEDSDVFGEADAIQNN GTSAEDTAVTDSKHTADPKNNWQGAHPADPGNRPHLIRLFSRDAPGREDNTFKDR PSESDELQTIQEDPTAASGGLDVMASQKRPSQRSKYLATASTMDHARHGFLPRHRD TGILDSIGRFFSGDRGAPKRGSGKDSHTRTTHYGSLPQKSQHGRTQDENPVVHFFK NIVTPRTPPPSQGKGRGLSLSRFSWGAEGQKPGFGYGGRASDYKSAHKGFKGAYD AQGTLSKIFKLGGRDSRSGSPMARR

Mass spectrometric peptide sequence identification showed 99% coverage of predicted protein sequence. Trypsin (red), Chymotrypsin (blue) and Proteinase K (purple) digestion identified peptide sequences are shown in the predicted protein sequence.

^*Experimental MH+ determined by Q-Exactive was found to be in complete agreement with Theoretical MH+.

These findings expand the number of murine myelin basic protein transcript variants that are found in nature, which is crucial for understanding the different factors contributing to myelin associated diseases. This is crucial for clinical significance given that it provides an understanding of the natural state of the central nervous system myelin, which is often perturbed during disease. Given that this novel MBP variant contains exons from the Golli family (exons 1 to 3), this classifies the novel MBP variant as a member of the MBP Golli family and we propose for this novel MBP variant to be named Golli-MBP isoform 14. As it is the case with other members of the MBP Golli family their functions are not completely understood and the scope of this study limits the conclusions that can be made regarding this novel MBP’s biological function. However, the similarity it holds with the Golli family members suggests that this novel MBP variant could share similar functions in oligodendrocyte development and calcium dependent biological processes. Future experiments will seek to understand the biological role it may potentially have in the murine CNS.