Elisabeth Trifilieff

Arrest of proteolipid transport through the Golgi apparatus in Jimpy brain

SummaryImmunocytochemical investigations were performed on Jimpy and control mouse brains using three specific anti-myelin proteolipids antisera: immunoaffinity purified multivalent anti-(PLP + DM-20) proteolipid antibodies, anti-C-terminal hexapeptide 271–276 and anti-tridecapeptide 117–129 antisera. The results show that oligodendrocytes and myelin sheaths in normal mouse brain are labelled to the same extent by the three specific antisera; in contrast, in Jimpy brain these cellular structures are only stained by the multivalent antibodies and the site-specific, anti-tridecapeptide antiserum. The absence of labelling with C-terminal hexapeptide antiserum in mutant brain is interpreted as the result of either a large deletion or a point mutation producing a frameshift in the C-terminal part of the sequences of the proteolipids PLP and DM-20. Furthermore, we show that this mutation prevents the normal transport of proteolipid molecules through the Golgi apparatus. The existence of a minor, extra-Golgi apparatus metabolic route for proteolipids to myelin structures is also discussed.

Monoclonal antipeptide antibodies: Affinity and kinetic rate constants measured for the peptide and the cognate protein using a biosensor technology

The interaction of antipeptide antibodies with the corresponding peptide and the cognate protein has been compared using a novel biosensor technology (BIAcore, Pharmacia). The peptide corresponds to residues 110-135 of the coat protein of tobacco mosaic virus, known to encompass an alpha-helical region reactive with antiprotein antibodies. A panel of 33 monoclonal antibodies raised against the peptide was studied and the epitope recognized by these antibodies was determined by the pepscan method. Further discrimination between the antibodies was performed by measurements of association and dissociation kinetic constants. Several antibodies showed an heterogeneous binding profile when reacting with the 25 residue long peptide but not with a shorter 10 residue peptide suggesting that they recognized different conformational states in the longer peptide. Equilibrium affinity constants were calculated for five of the antibodies and were found to be 10-50 times higher for the peptide than for the protein, the difference being caused mainly by a lower association rate constant.

Antigenic cross-reactivity potential of synthetic peptides immobilized on polyethylene rods.

A number of continuous epitopes of tobacco mosaic virus protein (TMVP) have been defined by the pepscan technique using polyclonal and monoclonal antibodies to TMVP as well as antisera raised against synthetic peptides. In general, the location of continuous epitopes agreed with the results of earlier studies with peptides synthesized by classical methods although there were some notable exceptions. Results obtained with the different types of antibodies used in this study indicated that a homology of three residues was sufficient to give rise to antigenic cross-reactions. In the case of antibodies raised against a peptide conjugated to ovalbumin, some unexpected cross-reactivities could be explained by assuming that antibodies to the carrier molecule recognized homologous tripeptide sequences in TMVP and ovalbumin.

Developmental Study of Proteolipids in Bovine Brain: A Novel Proteolipid and DM-20 Appear Before Proteolipid Protein (PLP) During Myelination

Abstract: In a developmental study, we have shown that DM‐20 is present before proteolipid protein (PLP) in the fetal bovine cerebral hemispheres. When the white matter appears (27–30 weeks of gestation), the amount of DM‐20 drastically increases. DM‐20 remains the major proteolipid until birth. PLP is detected only 2–4 weeks after the appearance of white matter, that is, more than 4 weeks after the appearance of DM‐20. The early appearance of DM‐20 at the beginning of myelination raises the question of its particular function. In the adult bovine cerebral hemispheres, PLP is the major proteolipid but DM‐20 remains quantitatively important because the PLP/DM‐20 ratio ranges from 1.5 to 1.7. In the same developmental study we have, in the fetal cerebral hemispheres, isolated and characterized a novel proteolipid (apparent Mr 20,000), which appears even before DM‐20 and is not detected in the adult brain. It is structurally related to PLP and DM‐20 because the first 31 N‐terminal amino acid residues are the same. However, in immunoblot. it did not react either with the antitridecapeptide 117–129 antiserum of PLP or with the anti‐C‐terminal hexapeptide antiserum of PLP.

Thiopalmitoylation of Myelin Proteolipid Protein Epitopes Enhances Immunogenicity and Encephalitogenicity

Proteolipid protein (PLP) is the most abundant protein of CNS myelin, and is posttranslationally acylated by covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage. Two of the acylation sites are within epitopes of PLP that are encephalitogenic in SJL/J mice (PLP104–117 and PLP139–151) and against which increased immune responses have been detected in some multiple sclerosis patients. It is known that attachment of certain types of lipid side chains to peptides can result in their enhanced immunogenicity. The aim of this study was to determine whether thioacylated PLP peptides, as occur in the native protein, are more immunogenic than their nonacylated counterparts, and whether thioacylation influences the development of autoreactivity and experimental autoimmune encephalomyelitis. The results show that in comparison with nonacylated peptides, thioacylated PLP lipopeptides can induce greater T cell and Ab responses to both the acylated and nonacylated peptides. They also enhanced the development and chronicity of experimental autoimmune encephalomyelitis. Synthetic peptides in which the fatty acid was attached via an amide linkage at the N terminus were not encephalitogenic, and they induced greater proportions of CD8+ cells in initial in vitro stimulation. Therefore, the lability and the site of the linkage between the peptide and fatty acid may be important for induction of encephalitogenic CD4+ T cells. These results suggest that immune responses induced by endogenous thioacylated lipopeptides may contribute to the immunopathogenesis of chronic experimental demyelinating diseases and multiple sclerosis.

Evidence for a phosphorylation‐induced conformational change in phospholamban cytoplasmic domain by CD analysis

Phospholamban (PLB), an integral membrane protein of cardiac sarcoplasmic reticulum (SR), is described as the regulator of the Ca2+‐ATPase pump, via its phosphorylation‐dephosphorylation of Ser‐16. Recently it has been shown that a direct interaction between the N‐terminal hydrophilic domain of PLB and Ca2+‐ATPase may be one of the mechanisms of regulation. In order to show that this interaction could be modulated by a phosphorylation‐induced conformational change in PLB, we ran CD studies on the synthetic peptide PLB(2‐33) in its phosphorilated and non‐phosphorylated forms, at various pHs, concentrations and in the absence or presence of trifluoroethanol. The results show a clear difference in structure of the phosphorylated and non‐phosphorylated peptide.

Antagonistic action of cholesterol towards the toxicity of hydroxysterols on cultured hepatoma cells

The cytostatic and cytolytic action of 22R - hydroxydesmosterol on hepatoma cells cultured in a medium containing 10% newborn-calf serum can be reversed within certain concentration limits by adding cholesterol to the culture medium. In contrast, under the same conditions, the cytotoxicity of 7 beta -hydroxycholesterol could not be reversed, whatever the concentrations of cholesterol added. However, in a lipoprotein-poor and in a chemically defined medium, the cytolytic action of both hydroxysterols can be reversed by adding cholesterol, but growth inhibition cannot be suppressed. This demonstrates the importance of serum lipids and lipoproteins for the toxicity of the hydroxysterols and for the antagonistic effect of cholesterol. Our results suggest that the action mechanisms of 7 beta-hydroxycholesterol and 22R - hydroxydesmosterol on HTC hepatoma cells are not fully identical.

Synthesis of palmitoyl‐thioester T‐cell epitopes of myelin proteolipid protein (PLP). Comparison of two thiol protecting groups (StBu and Mmt) for on‐resin acylation

In order to test the effect of thiopalmitoylation on the encephalitogenic properties of two proteolipid protein (PLP) T‐cell epitopes, we have studied the on‐resin S‐palmitoylation of peptides, synthesized using the Fmoc/tBu strategy. The use of two Cys protecting groups was investigated: the tert‐butylsulfenyl (StBu) and the methoxytrityl (Mmt). Our studies show that the ease of deprotection of the thiol protected with StBu was sequence dependent. The deprotection of Cys(StBu) was difficult in the case of the two peptides PLP(104–117) and PLP(139–151). Neither of the two Cys(StBu) (Cys108 and Cys140, respectively) could be deprotected with tributylphosphine. β‐mercaptoethanol was only efficient for the deprotection of Cys(StBu)140 at 85°C and at 135°C for Cys108. The two palmitoylated peptides could be obtained in good yield starting from Cys protected with Mmt. Our conclusion is that the Mmt group is the more versatile protecting group of the thiol for use in the on‐resin synthesis of thiopalmitoylated peptides. Copyright

Solid phase synthesis of a self complementary (antiparallel) chiral peptidic nucleic acid strand

Abstract The solid phase synthesis (Boc protocol) of a new chiral peptidic nucleic acid formed exclusively by α-amino acids is described. The product showed a defined T m suggesting the possibility of a DNA-like self-aggregation in solution

Route of uptake of palmitoylated encephalitogenic peptides of myelin proteolipid protein by antigen-presenting cells: Importance of the type of bond between lipid chain and peptide and relevance to autoimmunity

Previously, we have shown that thiopalmitoylation of peptides of myelin proteolipid protein, as occurs naturally in vivo, increases their ability to induce experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis, and skews the autoimmune response toward a CD4+-mediated response. In contrast, the same peptide, when synthesized with a stable amide bond between peptide and lipid, inhibits experimental autoimmune encephalomyelitis and skews the response toward a CD8+ response. The aim of the current study was to determine the mechanisms responsible for these observations. We show that proteolipid protein lipopeptides, when synthesized with a thioester bond between the lipid and the peptide, are taken up into APCs via an actin-independent endocytic route, the thioester bond is cleaved in the endosome, and the peptide is subsequently displayed on the surface of the APC in the context of MHC class II. The same peptide, when synthesized with the lipid attached via a stable amide bond, rapidly enters into the cytoplasm of the APC and forms micelles; however, the bond between peptide and lipid is not cleaved, and the micelles travel via the endoplasmic reticulum to complex with MHC class I. These findings have implications for vaccine development and for the development of MHC class II-restricted autoimmune diseases, as many human autoantigens thus far identified are thioacylated.