Tissue lipoproteins revisited: new proteolipid protein gene family members in elasmobranchs.

The proteolipids (PLPs) are abundant components of mammalian CNS myelin. Recombinant DNA methodologies have enabled us to search for evolutionary antecedents of PLP/DM20. Polymerase chain reactions of Torpedo and Squalus brain cDNA were performed with degenerate primers designed according to the mammalian PLP/DM20 sequence. Three DM20-related products (DM alpha, DM beta, and DM gamma) were amplified; no cDNAs containing the PLP-specific segment were found. Regions of the DM alpha and DM gamma are similar to the pore-forming segments of certain ligand-gated channels. In embryonic Squalus CNS, DM alpha and DM gamma appear to be co-expressed with P0. Antiserum raised against Torpedo DM alpha recognizes a protein in mouse CNS myelin, demonstrating that at least one of the newly recognized fish DMs is also in mammals. Our data, as well as that of other laboratories, supports the existence of a ubiquitously expressed proteolipid gene family.

Proteolipid protein interactions in transfectants: implications for myelin assembly.

The proteolipid proteins (PLP and DM20) are major constituents of CNS myelin, but how they are delivered to and organized within the oligodendrocyte plasma membrane is incompletely understood. We have expressed both PLP and DM20 singly or together in a host cell line, HeLa. In either DM20 or PLP transfectants, at early time points (24 hours), the expressed proteins are found within intracellular compartments. In DM20 transfectants, the protein is delivered to the plasma membrane by 48 hours. In HeLa cells, PLP remains intracellular when expressed in the absence of DM20; only when it is coexpressed with DM20 is it transported to the plasma membrane. In cotransfectants, PLP can also be localized to organelles involved in both the protein biosynthetic and the endocytic pathways. Since, in HeLa cells at least, the delivery of PLP to the plasma membrane is facilitated by the coexpression of DM20, we suggest that the two proteins interact intracellularly to form a complex. In some PLP/DM20 cotransfectants, the proteolipids are concentrated in regions of cell-cell contact. The regional accumulation of these proteins at cell-cell interfaces is highly reminiscent of the behavior in transfected cells of another myelin protein, P0, and certain cadherin polypeptides, both of which have readily demonstrable membrane adhesive properties. Our data suggests that at certain stoichiometric ratios, proteolipids can become stabilized at cell surfaces to form adhesive bonds.

A proteolipid protein gene family: expression in sharks and rays and possible evolution from an ancestral gene encoding a pore-forming polypeptide.

The myelin proteolipid proteins (PLP and DM20) are believed to act as "adhesive struts" in the extracellular apposition of the CNS myelin sheath. These proteins have been considered late evolutionary developments, which arose de novo in the antecedents of early tetrapods. However, PCR primed with degenerate oligonucleotides corresponding to common segments of rat PLP/DM20 revealed three novel mRNAs in the brains of two elasmobranchs. These mRNAs are closely related to each other and to mammalian DM20, but lack the sequence that distinguishes PLP from DM20. We term the novel proteolipid proteins DM alpha, DM beta, and DM gamma. At least DM alpha and DM gamma are highly expressed in white matter in myelinating shark brain. The DMs not only are highly homologous to each other, but also contain regions bearing similarities with segments of channel-forming regions of the nicotinic acetylcholine receptor and the glutamate receptor macromolecular complexes. Significantly, we find that across these segments, DM alpha and DM gamma are more similar to the channel proteins than the two channel proteins are to each other.

The DM20 protein of myelin: intracellular and surface expression patterns in transfectants.

DM20 is an abundant CNS myelin-specific protein whose role in myelinogenesis is unknown. We have cloned the DM20 cDNA from adult mouse brain total RNA using the polymerase chain reaction and expressed it in HeLa cells. DM20, detected by immunofluorescence in stable transfectants, is present in some cells in large, intensely fluorescent intracellular clumps that probably represent elements of the rough endoplasmic reticulum and Golgi apparatus. Frequently, intense DM20 fluorescence could be detected at the plasma membrane. These findings are consistent with previous studies demonstrating that an intracellular "pool" of DM20 and its larger isoform, proteolipid protein, exists and that a substantial lag occurs between synthesis and insertion of these proteins into the expanding myelin membrane. Permanent DM20 expressors in contact with one another do not display any ultrastructural rearrangements at regions of cell-cell contact, in contrast to what we have previously reported for P0, a PNS-specific protein shown to mediate adhesion of the extracellular faces of the Schwann cell during PNS myelinogenesis. We believe that these results indicate that if DM20 is indeed an adhesion molecule, this property is likely to be significantly more subtle than P0-mediated adhesion.

Physiologic properties of myelin proteins revealed by their expression in nonglial cells.

The transfection paradigm described herein can be used to investigate the functional properties of individual nervous system proteins in ways that have not been explored before. In particular, observations on the "structural" proteins of myelin are being made that have already yielded certain unique insights into the physiologic properties of these polypeptides. The ease with which site-directed mutagenesis procedures can be applied to these systems should eventually enable us to define with great precision the "functional domains" within each myelin protein.