Myelin sheaths are formed with proteins that originated in vertebrate lineages

Abstract

All vertebrate nervous systems, except those of agnathans, make extensive use of the myelinated fiber, a structure formed by coordinated interplay between neuronal axons and glial cells. Myelinated fibers, by enhancing the speed and efficiency of nerve cell communication allowed gnathostomes to evolve extensively, forming a broad range of diverse lifestyles in most habitable environments. The axon-covering myelin sheaths are structurally and biochemically novel as they contain high portions of lipid and a few prominent low molecular weight proteins often considered unique to myelin. Here we searched genome and EST databases to identify orthologs and paralogs of the following myelin-related proteins: (1) myelin basic protein (MBP), (2) myelin protein zero (MPZ, formerly P0), (3) proteolipid protein (PLP1, formerly PLP), (4) peripheral myelin protein-2 (PMP2, formerly P2), (5) peripheral myelin protein-22 (PMP22) and (6) stathmin-1 (STMN1). Although widely distributed in gnathostome/vertebrate genomes, neither MBP nor MPZ are present in any of nine invertebrate genomes examined. PLP1, which replaced MPZ in tetrapod CNS myelin sheaths, includes a novel 'tetrapod-specific' exon (see also Möbius et al., 2009). Like PLP1, PMP2 first appears in tetrapods and like PLP1 its origins can be traced to invertebrate paralogs. PMP22, with origins in agnathans, and STMN1 with origins in protostomes, existed well before the evolution of gnathostomes. The coordinated appearance of MBP and MPZ with myelin sheaths and of PLP1 with tetrapod CNS myelin suggests interdependence - new proteins giving rise to novel vertebrate structures.

Fig. 1. Electron micrographs of rodent (A) PNS and (B) CNS myelin sheaths (originals kindly provided by Dr. Jack Rosenbluth, New York University School of Medicine)

Compact myelin (bordered by red bands in PNS and by blue bands in CNS) is surrounded by glial cell cytoplasm (*), containing organelles where myelin-destined lipids and proteins are synthesized. For peripheral nerves, a supportive basement membrane (arrowhead) is also present and functions to stabilize the fibers and signal myelination (Jessen and Mirsky, 2005). Regions of compact myelin, where MBP, MPZ and PLP1 reside, are separated from regions of non-compacted myelin where MBP, MPZ and PLP1 are made, by tight junctions that form inner and outer mesaxons (arrows). Of many proteins synthesized in glial cell cytoplasm, few accumulate in and help structure myelin. A Schmidt–Lanterman incisure, with cytoplasm included between layers is common in MPZ-based though not PLP1-based myelin ( ).

Fig. 2

Phylogenetic tree constructed with the UniTree program (Methods) with murine MBP (NP_001020426.1) as ‘BAIT’ (red) and with a search specified for 35×UR90 and 7×UR50 genes (3507) and re-rooted to teleost fish clade of MBP using Figtree 2.1 software (http://beast.bio.ed.ac.uk/FigTree). Each Uniprot hit is annotated with common species name, protein name and number of amino acids. In cases in which Uniprot accessions are not annotated, top hits obtained from BLASTP queries of the mouse NR database with FASTA sequences are listed (*). Some accession numbers were inactive (i). The branches, for mammals (black), birds/reptiles (pink/brown), amphibians (blue), teleosts (dark green), elasmobranchs (light green) and interspersed poorly related bacterial sequences (gold) are color coded. Crossbars indicate where branches are shortened to allow the tree to fit the page.

Fig. 3

Pair-wise amino acid identity comparisons of all full-length sequences (see supplementary Tables 1A and 1B online) from each taxon with its murine counterpart are plotted. Mean and standard deviations are included. Statistical analyses (Prism 4, Graphpad program) were limited to comparisons among mammalian and teleost sequences and significance differences between MBP versus other myelin proteins are displayed (* – P < 0.05, ** – P < 0.0001, all others). Except for mammalian PLP1 and STMN1, comparisons show that conservations of each protein were different from other myelin proteins. Detailed information on comparative studies is presented in supplementary Table 2 online.

Fig. 4. Alignments of MBP sequences (isoform 3, contains all but exon II) from species that represent each gnathostome taxon

Accession numbers are listed in supplementary Tables 1A and 1B online. Alignments are made, exon-by-exon with Clustal W (Methods), merged into a single alignment, and finally modified by eye. Comparisons among all listed species, excepting human use white letters on a black background ( ) for complete identity, white letters on gray background for blocks of identity (>2) and conserved substitutions ( ); the background shading is set to the number of sequences with the same amino acid – darker background are for greater number of sequences. For simplicity, conserved substitutions are considered the same as identical amino acids. Exon boundaries are marked (θ) with exon numbers above the human sequence. Positions of all basic (K, R) amino acids are shown with coloring indicating complete identity ( ) and ( ) conservation and presence in sequences of one or two species (*). Sixty-nine HMM logo (PFAM) amino acids were selected based on individual or conserved substitution contributions >1.0 and shown with white lettering on orange backgrounds. As above, darker background shading indicates greater sequence identity. Letters and basic residue designations are placed between human and murine sequences. A single arginine methylation site is shown below the human sequence (M). A portion of exon III, absent in the common zebrafish 88 amino acid isoform is indicated [ ] and assigned IIIA (see supplementary Table 3 online).

Fig. 5. Phylogenetic tree constructed with the Unitree program, with murine MPZ (NP_032649) as ‘BAIT’ and with the (40 × 15 stringency, Methods) used in most instances; this tree is re-rooted to horn shark MPZ using Figtree 2.1

Each hit is annotated, and clusters that include: MPZ, MPZL1, MPZL2/EVA and MPZL3, are separated from one another with brackets. Shading of branches is as described in Fig. 2.

Fig. 6. Alignment of MPZ sequences from species representing different vertebrate taxa

Alignments are made on complete sequences with Clustal W program and positions of exon boundaries, based on mammalian sequences, are shown (▼). Accession numbers for all sequences are listed in supplementary Tables 1A and 1B online. A horizontal line separates agnathan/lamprey sequence from the gnathostome sequences and only the first 118 amino acids from this model are included as the remainder shows insignificant match. The signal peptide, transmembrane domain and the exon VI missing from many teleost fish MPZ sequences are similarly shaded with the first two regions marked between the human and murine sequences and the third region placed below the zebrafish sequence ([ ]). Included are the single glycosylation site (N), two cysteine residues (C) that form the disulfide bond, a third (C) that is acylated in mammals. Two tryptophan residues (W) suggested from the X-ray diffraction data (Shapiro et al., 1996) to intercalate the apposing lipid bilayers, a glycine zipper (G) in the transmembrane domain, a tyrosine (Y) and two serine (S) represent phosphorylation sites. The (L), (Y) and (R) are cholesterol recognition motifs are highlighted. Amino acids that form the myelin-PO_C PFAM HMM logo are depicted as in Fig. 4, i.e. they are shaded in orange. A region of high conservation in exon 3 is also marked ([ ]).