Claude Nicot

Limited Proteolysis of Myelin Basic Protein in a System Mimetic of the Myelin Interlamellar Aqueous Space

Abstract: We have investigated the early steps of myelin basic protein (MBP) degradation in a membrane mimetic system (reverse micelles), resembling the interlamellar aqueous spaces where the protein is located in the myelin sheath. MBP, unfolded in buffer, refolds on incorporation into the micelles, resulting in reduced accessibility to three proteolytic enzymes, trypsin, cathespin D, and Staphylococcus aureus V8 protease, in comparison with aqueous solution. Eleven cleavage sites seen in buffer are removed from proteolytic attack in micellar solution. These sites delineate a protected protein domain displaying a potential β‐sheet structure capable of interacting with the myelin membrane. An additional site not seen in buffer is attacked in the micelles. Experiments with a structure inducer, 15% 1‐propanol in buffer, reveal that the refolding pattern of MBP in reverse micelles is specific to the membrane biomimetic system and is not produced by organic solvent per se. Micellar digestions of MBP generate long peptides, two of which, isolated after tryptic digestion, have been found to be immunodominant in multiple sclerosis patients. The findings suggest the structure induced in MBP by the micelles resembles that leading to production of the self‐peptides recognized by T cells during proteolytic breakdown of MBP in autoimmune demyelinating diseases.

Myelin Proteins in Reverse Micelles: Tight Lipid Association Required for Insertion of the Folch‐Pi Proteolipid into a Membrane‐Mimetic System

Abstract: The solubility and reactivity of the Folch‐Pi proteolipid from bovine CNS have been studied in reverse micelles of sodium bis(2‐ethylhexyl)sulfosuccinate, isooctane, and water. Such a membrane‐mimetic system resembles the aqueous spaces of the native myelin sheath in terms of its physicochemical properties. Although the proteolipid is completely insoluble in water, it can be inserted into the water‐containing micellar system. In contrast, the lipid‐depleted protein failed to be incorporated into these organized assemblies. The lipid requirements for insertion of the proteolipid were studied, therefore, after delipidation by several precipitations with isooctane, a nondenaturing solvent. Novel extraction procedures and quantitative analyses by HPLC of the protein‐bound lipids revealed the persistence of a lipidprotein complex (6 ± 1 mol of lipid/mol of protein) displaying optimal micellar solubilization. Competition experiments carried out with brain lipids provide evidence for a preference of the myelin protein for sulfatide, phosphatidylinositol, and phosphatidylserine, in that order. The resulting proteolipid, although differing in relative composition, showed good solubility in the membrane‐mimetic system. In contrast, reconstitution experiments carried out with the lipid‐depleted protein resulted in weak lipid binding and poor micellar incorporation. These results suggest that the tightly bound acidic lipids may stabilize a protein conformation required for insertion into the micellar system.

Study of fluorescent tryptophyl residues and extrinsic probes for the characterization of molecular domains of Folch-Pi apoprotein.

The highly hydrophobic myelin Folch-Pi apoprotein can be solubilized in organic as well as in aqueous media. In order to understand the molecular organization changes consecutive to changes in the solvent medium, the environment of intrinsic probes and extrinsic labels has been studied by fluorescence and accessibility to some reagents. In acqueous solution, only two tryptophan residues per protein molecule of 23,500 molecular weight have been shown to fluoresce, and their fluorescence characterisitics indicate an hydrophobic and/or constrained environment. Two ANS binding sites have also been observed having a high quenching effect on the intrinsic chromophore fluorescence. A large accessibility has been evidenced for the protein sulfhydryl groups in chloroform-methanol 2:1 (v/v), both by kinetic study of the protein reaction with a specific reagent, N-(1-anilino-naphtyl-4) maleimide, and by the fluorescence characteristics of this probe once linked to the protein. The free sulfhydryl groups were still reactive in acqueous solution, but extrinsic fluorescence of the labelled apoprotein transferred from chloroform-methanol 2:1 (v/v) into water gave evidence of constraints on the probe or on its environment. Such constraints may contribute to the solubilization in acqueous solution of this highly hydrophobic protein.

Molecular Dynamics and Structure of Peptide Hormones at Membrane-Water Interfaces

Peptide hormones of low molecular weight are among the most flexible polypeptides in living organisms (Blundell and Wood, 1982). In order to express their specific functions they must acquire a more restricted bioactive conformation conformation (Kaiser and Kezdy, 1983; 1984). As recently pointed out by Braun et al. (1983), it appears rather unlikely that the initial interaction of peptide hormones with the target cells will result in the immediate formation of a specific complex with the membrane-bound receptors at the plasma membrane level. Thermodynamic as well as kinetic considerations indeed suggest that the lipid matrix of the cell plasma membrane may have functional implications in the transmembrane signaling process by playing as an “antenna” for the capture of the peptide (Sargent and Schwyzer, 1986). Moreover, the interaction of the peptide with the membrane lipids may select a bioactive conformational state among the many existing in aqueous solutions (Behnam and Deber, 1984; Deber and Behnam, 1985; Schwyzer, 1986). The interfacial characteristics of the lipid matrix (charge density, head-group packing, availability of hydrogen-bond forming groups, mobility of the interfacial water molecules and local proton activity) are likely to be involved in the selection of a dynamic and conformational state of the peptide.