Characterization of the shark myelin Po protein - PubMed (original) (raw)

Comparative Study

Characterization of the shark myelin Po protein

L Rotenstein et al. Brain Behav Evol. 2008.

Abstract

Myelin, the insulating sheath made by extensive plasma membrane wrapping, is dependent on the presence of highly adhesive molecules that keep the two sides of the membrane in tight contact. The Po glycoprotein (Po) is the major component of the peripheral nervous system (PNS) myelin of mammals. The exact role that Po protein has played in the evolution of myelin is still unclear, but several phylogenetic observations suggest that it is a crucial component in the development of myelin as a multi-lamellar membrane structure. Sharks, which appeared in the fossil record about 400 million years ago, are the first fully myelinated organisms. In this study we investigated the expression pattern of shark myelin Po to suggest a way it might have played a role in the evolution of myelin in the central nervous system. We found that sharks have more than two isoforms (32, 28 and 25 kD), and that some of these might not be fully functional because they lack the domains known for Po homophilic adhesion.

PubMed Disclaimer

Figures

Figure 1. Myelin Po Sequence alignment and analysis

Sequences from Genbank were aligned via the program of ClustalW. Sequences chosen for peptide antibody preparation are underlined. PoEx sequence shares some similarity with rodent Po. PoCy1 sequence has no homology with other Po proteins and PoCy2 corresponds to the cytoplasmic domain of Po which is highly conserved among most of the species we looked. The consensus key for the amino acids sequences is: * (single, fully conserved residue), :(semicolon, conservation of strong groups),.·(black dot, conservation of weak groups) and “blank” (no consensus).

Figure 2. Shark Myelin Po isoforms

Western blot of purified shark myelin (5μg) showed several isoforms for Po. An antibody raised specifically to the shark cytoplasmic region (PoCy1) shows three bands for Po, while antibodies to the extracellular domain (PoEx) or a polyclonal antibody raised against purified Po (RabA) showed two strong bands and a fainter one. The antibody raised against the highly conserved cytoplasmic end of Po showed only one band. A total of 4ug of protein from purified myelin were loaded on each lane.

Figure 3. Endo F treatment of myelin Po

Purified shark myelin was treated with an enzyme (EndoF) to remove all carbohydrates. Western blot showed that enzyme treated Po(+) isoforms run about 6% lower than fully glycosylated Po (−). A total of 4μg of protein from purified myelin were loaded on each lane.

Figure 4. Po isoform distribution in shark brain

Western blots with PoCy1 from different brain regions showed that there was an unequal distribution of shark Po in different areas. 8μg of total brain lysates were prepared from dissected areas and ran on a SDS-PAGE gel. Tel (telencephalon), Di (diencephalon), OT (optic tectum), TN (trigeminal nerve), Cer (cerebellum), Med (medulla) and SSM (spinal cord myelin).

Figure 5. Glycosylation of CNS and PNS shark myelin

Purified shark myelin (5μg) was treated with an enzyme to remove all carbohydrates (Endo F). Western blot showed that enzyme treated Po (+) isoforms run about 6% lower than fully glycosylated Po (−) for both CNS (SSM, shark spinal cord myelin) and PNS (TN, trigeminal nerve myelin). TN myelin showed only two of the three isoforms found in CNS.

Figure 6. Developmental expression of myelin Po

Shark brains from embryos at different developmental stages (4, 4.4 9 and 22cm embryos) were solubilized and ran on an SDS-PAGE gel. Western blot was done with the PoCy1 antibody that recognizes all three shark isoforms. Only the oldest embryo showed significant amounts of Po, and had three isoforms, the 32 and 28kD as well as a novel 34kD (arrowhead). The total protein loaded was: 16μg of protein for the 4, 4.4 and 9cm embryos, 4μg for the 22cm embryo and 2μg for the positive control SSM (shark spinal cord myelin).

Figure 7. Myelin protein expression in CNS of shark embryos

Immuno-histochemistry of 6cm shark embryos showed that although myelination is not widespread in the CNS, there are distinguishable myelinated tract in the shark embryo brain. A and B show tectal region double stained for Po (A) and beta-tubulin III (neuron specific, B), arrows point to myelinated fibers. Hindbrain regions close to the cerebellum (C–D) show fewer Po-positive fibers, D, than MBP in a consecutive section (7C). Arrows point to the fibers positive for MBP and Po. Midbrain section at the level of the eye shows more Po-positive fibers as well myelinated tracts reaching laterally (arrow). Arrowhead points to the trigeminal ganglia.

Figure 8. Myelin protein expression in spinal cord and ganglia embryos

Shark embryo sections (3cm A, C, D) and 7cm (B, E, F) were immunostained for MBP (A,C and E) or Po (B, D and F) proteins. A corresponds to spinal cord at the vagal/medulla level, notice extensive MBP positive tracts (spinal cord ventral area in 8A), but at the tail level there was none (8C for MBP). Cranial ganglia myelination is more extensive and sooner (arrows in B). At the trunk levels (D), we observed Po-positive fibers in the ventral spinal cord but none in sensory ganglia (arrow). In older embryos (E and F) MBP and Po expression respectively was widespread and extensive, including spinal nerve (arrow in E) and ganglia (arrow in F).

References

1. Bakay L, Lee JC. Ultrastructural changes in the edematous central nervous system. 3. Edema in shark brain. Arch Neurol. 1966;14:644–660. -PubMed
1. Ballard WW, Jean Mellinger, Henri Lechenault. A Series of Normal Stages for Development of Scyliorhinus canicula, the Lesser Spotted Dogfish (Chondrichthyes: Scyliorhinidae) J Exp Zool. 1993;267:318–336.
1. Boison D, Bussow H, D’Urso D, Muller HW, Stoffel W. Adhesive properties of proteolipid protein are responsible for the compaction of CNS myelin sheaths. J Neurosci. 1995;15:5502–5513. -PMC -PubMed
1. Bullock TH, Moore JK, Fields RD. Evolution of myelin sheaths: both lamprey and hagfish lack myelin. Neurosci Lett. 1984;48:145–148. -PubMed
1. Butler AB, Hodos W. Comparative vertebrate neuroanatomy : evolution and adaptation. 2. Hoboken, N.J.: Wiley-Interscience; 2005.

Characterization of the shark myelin Po protein - PubMed (original) (raw)