During the course of the study of brain sulfatides with Lees MB, Folch J described for the first time in 1951 the presence of special proteins in rat brain myelin which could be solubilized in organic solvents (chloroform / methanol / water mixtures) (Folch J et al., J Biol Chem 1951, 191, 807). These substances were named “proteolipides” and were considered as a novel lipoprotein but quite different from the other known lipoproteins.
The discovery of these special proteins was made after drying and extracting a brain lipid extract with organic solvents. All the lipids re-dissolved except for an insoluble residue which accounted for about 15% of the initial lipid extract. The residue was completely insoluble in any organic solvents and also in aqueous solutions. As it contained about 13% amino acid nitrogen and some phosphorus, the residue was considered an insoluble protein with some contaminant lipids. For an exhaustive history of proteolipids, see the review of Marjorie B Lees (Neurochem Res 1998, 23, 261).
These proteolipids were shown to be present mainly in neural tissues but also in heart, kidney, liver, and muscles but absent from blood plasma.
During thirty years the definition of proteolipids was exclusively used to refer to a family of various proteins which are related by their solubility in mixtures of chloroform and methanol (Lees MB et al., Biochim Biophys Acta 1979, 559, 209). Thus, the archetypal proteolipid found initially in myelin is now known as “proteolipid protein” or PLP.
The presence of fatty acids covalently associated with hydrophobic proteins was first described in Gram-negative bacteria (Braun V et al., Eur J Biochem 1969, 10, 426 and 1972, 28, 51) but rapidly extended to myelin PLP (Sherman G et al., Biochem Biophys Res Comm 1971, 44, 157) and to the Ca++-dependent ATPase complex of sarcoplasmic reticulum (McLennan DH, Can J Biochem 1975, 53, 251). These discoveries led to the new definition for proteolipid : a protein that contains a lipid moiety as part of its primary structure (Schlesinger MJ, Ann Rev Biochem 1981, 50, 193).
Thus, fatty acylated proteins should now be characterized by more strict criteria than hydrophobicity, i.e. presence of lipids even after exhaustive extraction with organic solvents and boiling SDS and identification of covalent lipids after chemical cleavage of the protein-lipid linkage.
Since that restrictive definition, a wide variety of fatty acylated proteins were reported to be present in virus, bacteria and eukaryotic cells leading to the acceptance that acylation may be one of the most widespread modifications of proteins (Schultz AM et al., Ann Rev Cell Biol, 1988, 4, 611; Schmidt MFG, Biochim Biophys Acta 1989, 988, 411).
Curiously, only two types of acylated proteins have been identified :
Myristic acid (C14:0) is bound to the amino-terminal glycine residue (stable amide linkage)
Palmitic acid (C16:0) is bound to side chains of cystein residues (labile thioester linkage). Other fatty acids can also be present (C16:1, C18:2, C20:0 ..)
The first proteins to be demonstrated to contain myristic acid were calcineurin B (Aitken A et al., FEBS Lett 1982, 150, 314) and the catalytic subunit of the cyclic AMP-dependent protein kinase (Carr SA et al., Proc Natl Acad Sci USA 1982, 79, 6128).
It was shown that myristic acid (R2) was attached through an amide linkage to the a-amino group of glycine (R1) at the N-terminus of both proteins :
Later, a wide range of proteins of viral and cellular origin have been shown to be modified by acylation with myristic acid (Olson EN, Prog Lipid Res 1988, 27, 177).
Myristoylated proteins are localized to the cytosol or to cellular membranes and sometimes to both. Membrane-bound myristoylated proteins interact tightly with the bilayer so that drastic conditions may be used to release them from membranes (Olson EN et al., J Biol Chem 1986, 261, 2458). It is now well established that myristoylation is able to direct soluble proteins to membranes but the specificity of targeting remains unclear.
The function for myristoylation is also not well known. It was speculated that these proteins may represent enzymes involved in lipid metabolism or carrier proteins.
These proteins are the most extensively studied among proteolipids and the first member among them to be identified was the myelin PLP which represents the major component of the myelin proteins (at least 40%). The long-chain fatty acids (R2, mainly C16:0, C18:0 and C18:1) constitute about 2-4% of the PLP dry weight and are covalently bound by thioester linkages to cystein residues (R1).
The presence of thioester bonds was demonstrated by in vitro and in vivo acylation (Ross NW et al., J Neurosci Res 1988, 21, 35; Bizzozero OA et al., J Neurochem 1990, 55, 1986).
PLP was shown to be palmitoylated with acyl-CoA by a non-enzymatic mechanism and depalmitoylated by a specific myelin-associated acyltransferase.
The extreme hydrophobicity of PLP is easily explained by a composition of about 50% apolar amino acid residues and a high degree of fatty acid acylation (Weimbs T et al., Biochemistry 1992, 31, 12289).
Besides myelin PLP, several other membrane proteins were shown to be S-palmitoylated. The best known examples are the followings :
– myelin P0 glycoprotein in peripheral nervous system (Bizzozero OA et al., Anal Biochem 1989, 180, 59).
– ligatin in neonatal enterocytes (Jakoi ER et al., J Biol Chem 1987, 262, 1300).
– lung surfactant proteolipid (Stults JT et al., Am J Physiol 1991, 261, L118).
– rhodopsin in retina cells (O’Brien P et al., J Biol Chem 1987, 262, 5210).
– sodium channel polypeptide (Levinson SR et al., Biophys J 1986, 49, 378A).
– P-selectin in vascular endothelium (Fujimoto T et al., J Biol Chem 1993, 268, 11394).
– band 3 protein in erythrocytes (Okudo K et al. J Biol Chem 1991, 266, 16420).
– hepatic asialoglycoprotein receptor (Zeng FY et al., J Biol Chem 1995, 270, 21382).
– glycoprotein proteolipids from Sindbis virus (Schmidt MFG et al., Proc Natl Acad Sci USA 1979, 76, 1687).
More than 20 proteins modified by covalent palmitic acid were reviewed in 1988 (Olson EN, Prog Lipid Res 1988, 27, 177) and 14 were added in 1994 (Bizzozero OA et al., Neurochem Res 1994, 19, 923).
A phylogenetic conservation of fatty acid acylation was demonstrated in studying brain myelin from amphibians, reptiles, birds and mammals, suggesting a critical role of this post-translational modification for PLP function (Bizzozero OA et al., Neurochem Res 1999, 24, 269). In all species, PLP contains about 3% (w/w) of bound fatty acids, 78% of them being C16:0, C16:1, C18:0 and C18:1. Curiously, hydroxy and branched-chain fatty acids are absent. While discrepancies are found concerning the fatty acid to protein stoichiometry, it is now accepted that no more than 3 moles of fatty acids are bound to one mole of PLP (MW = 25000). Interestingly, PLP appears to be strongly associated in situ with acidic phospholipids, mostly phosphatidylserine. It is estimated that about 15 molecules of phospholipids form a boundary lipid matrix around a molecule of PLP.