Accelerated Publications Proteolipid Protein (PLP) of CNS Myelin: Positions of Free, Disulfide-Bonded, and Fatty Acid Thioester-Linked Cysteine Residues and Implications for the Membrane Topology of PLPT

Proteolipid protein (PLP), the major integral membrane protein of central nervous system myelin, contains 14 cysteine residues within its 276-residue polypeptide chain. We determined the state of all cysteine residues and localized four of them as free thiols a t positions 24, 32, 34, and 168. Four cysteines are connected by disulfide bonds: Cys200-Cys219 and Cys183-Cys227. The remaining six cysteine residues at positions 5,6,9, 108, 138, and 140 are modified by long-chain fatty acids, mainly palmitic acid, in thioester linkage. The extreme hydrophobicity of PLP can therefore be explained by two structural features: a composition of approximately 50% apolar amino acid residues and a high degree of fatty acid acylation. A differential fluorescent-labeling technique was developed for the structural studies: the cysteine residues belonging to one of the three states were derivatized by N-(iodoacetylaminoethy1)-5-naphthylamine1 -sulfonic acid (I-AEDANS) either directly (a), after thioester cleavage with hydroxylamine (b), or after disulfide cleavage with dithiothreitol (c). The protein was then proteolytically digested with thermolysin, and the labeled peptides were isolated by reversed-phase HPLC followed by sequence analysis. The results were further confirmed by determination of the fatty acid to protein stoichiometry. The structural data not only demand the revision of our concept of the membrane topology of PLP but will also promote more sophisticated studies on the mechanism of myelination and new functions of PLP. Myelin proteolipid protein (PLP)' is the most abundant protein component and the major integral membrane protein of the myelin of central nervous system. It is one of the most hydrophobic proteins in nature, insoluble in aqueous solution but soluble in organic solvents like mixtures of chloroform and methanol (Folch-Pi & Lees, 1951). PLP awaited its structural elucidation for 30 years due to these unusual hydrophobic properties (Stoffel et al., 1982). The 276-amino acid polypeptide contains approximately 50% hydrophobic amino acids and is distinctly structured into four hydrophobic and five hydrophilic domains. The hydrophobicity is further enhanced by the presence of covalently linked long-chain fatty acids (Stoffyn & Folch-Pi, 1971). So far, only structural functions have been assigned to PLP which are believed to be essential for the wrapping and close and compact apposition of oligodendrocyte plasma membrane processes around the axon. Besides the newly synthesized PLP, MBP, other minor protein constituents, and vast amounts of sphingoand phospholipids are also targeted during myelination with the formation of the highly ordered myelin membrane system of CNS. The contribution of the hydrophobic domains of PLP to the high order of the membrane becomes strikingly apparent when point mutations cause minute structural changes which in Supported by the DFG, SFB 243, the Fritz Thyssen Foundation, and the Hertie Foundation. Abbreviations: PLP, proteolipid protein; DM-20, isoform of PLP, originating from alternative splicing; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; HPLC, high-pressure liquid chromatography; I-AEDANS, N-(iodoacetylaminoethyl)-5-naphthylamine-lsulfonic acid; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate. 0006-2960/92/043 112289$03.00/0 most cases have lethal effects. Point mutations of the PLP locus have been analyzed in several animal species, e.g., the md rat (Boison & Stoffel, 1989), msd mouse (Gencic & Hudson, 1990), human dysmyelinoses of the type PelizaeusMerzbacher (Hudson et al., 1989; Weimbs et al., 1990), and a point mutation (A-G transition) at the splicing acceptor site of intron IV of the jimpy mouse (Nave et al., 1987) which leads to an alternative splicing associated with a frame shift. The amino acid sequence of PLP shows a most remarkable homology between mammalian species (Stoffel, 1990) and phylogenetically distant species like frog, chicken, and rodents (Schliess & Stoffel, 1991). Several models of the topology of PLP in the lipid bilayer of the myelin membrane have been suggested as working hypotheses. They were either hypothetical models (Laursen et al., 1984; Popot et al., 1991) or derived from results obtained by different techniques such as proteolytic attack of hypoosmotically shocked myelin with subsequent characterization of the main proteolytic fragments (Stoffel et al., 1984), immunochemical analysis with antipeptide antibodies (Hudson et al., 1989; Stoffel et al., 1989), photoaffinity labeling (Kahan & Moscarello, 1985), and X-ray diffraction (Inouye & Kirschner, 1989). These models altogether lack the essential information about the correct state of the 14 cysteine residues within the complete amino acid sequence of PLP. Cysteine residues of PLP have been claimed to be present partly as free thiols and partly as disulfide bonds (Lees et al., 1969). So far, one disulfide bond between Cys2O0 and Cys219 has been reported (Shaw et al., 1989), and Cys108 was found to be acylated by a long-chain fatty acid (Bizzozero et al., 1990b).