Effect of arginine loss in myelin basic protein, as occurs in its deiminated charge isoform, on mediation of actin polymerization and actin binding to a lipid membrane in vitro

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocyte membranes and is most likely responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also polymerize actin, bundle F-actin filaments, and bind actin filaments to lipid bilayers through electrostatic interactions. MBP consists of a number of posttranslationally modified isoforms of varying charge, including C8, in which six arginines are deiminated to the uncharged residue citrulline. The deiminated form decreases with development, but is increased in patients with the demyelinating disease multiple sclerosis. Here we investigate the effect of decreased net positive charge of MBP on its interaction with actin in vitro by comparing a recombinant murine form, rmC1, of the most highly charged unmodified isoform, C1, and a recombinant analogue of C8 in which six basic residues are converted to glutamine, rmC8. The dissociation constant of the less charged isoform rmC8 for actin was a little greater than that of rmC1, and rmC8 had somewhat reduced ability to polymerize actin and bundle F-actin filaments than rmC1. Moreover, rmC8 was more readily dissociated from actin by Ca(2+)-calmodulin than rmC1, and the ability of the deiminated isoform to bind actin to lipid bilayers was reduced. These results indicate that electrostatic forces are the primary determinant of the interaction of MBP with actin. The spin labeled side chains of a series of rmC1 and rmC8 variants containing single Cys substitutions at seven sites throughout the sequence all became motionally restricted to a similar degree on binding F-actin, indicating that the entire sequence is involved in interacting with actin filaments or is otherwise structurally constrained in actin bundles. Thus, this posttranslational modification of MBP, which occurs early in life and is increased in multiple sclerosis, attenuates the ability of MBP to polymerize and bundle actin, and to bind it to a negatively charged membrane.     

Myelin basic protein undergoes a broader range of modifications in mammals than in lower vertebrates

Myelin basic protein (MBP) is an important component of the myelin sheath surrounding neurons, and it is directly affected in demyelinating diseases. MBP contains a relatively large number of post-translational modifications (PTMs), which have been reported to play a role in multiple sclerosis, while MBPs from lower vertebrates have been reported to be incapable of inducing multiple sclerosis or allergic encephalitis. This study reveals the extent of differences in PTM patterns for mammalian and nonmammalian MBPs. This included intact mass and de novo sequence analysis of approximately 85% of rattlesnake MBP, the first reptile MBP to be characterized, and of bovine MBP. We identified 12 PTMs at 11 sites in the five bovine MBP charge components, which include both previously reported and novel modifications. The most notable modification is an acetylation of lysine 121. Other modifications found in bovine MBP include N-terminal acetylation in components C1, C2, and C3; oxidation of methionine 19 in all five components; all charge isomers having both a mono- and dimethylated (symmetric) arginine at position 106; deimination in arginines 23 and 47 found only in component C8b; deimination of arginine 96 and deamidation in glutamine 102 found in components C2, C3, C8a, and C8b; phosphorylation in threonine 97 restricted to charge components C2 and C3; deimination in arginine 161 only found in component C3; deamidation of glutamine 120 was only observed in C3. All four deiminated arginines and one acetylated lysine were first experimentally revealed in this study for bovine MBP. Mascot database searching combined with de novo sequence analysis of rattlesnake MBP provided more than 85% sequence coverage. A few PTMs were also revealed in rattlesnake MBP: mono- and dimethylated Arg, protein N-terminal acetylation, and deiminated Arg. Overall, snake MBP was found to undergo less modification than bovine MBP on the basis of the mass heterogeneity of the intact protein, the bottom-up structure analysis, and the limited complexity of rattlesnake MBP chromatography. The combined data from this study and information from previous studies extend the known MBP PTMs, and PTMs unique to higher vertebrates are proposed.     

Carboxylmethylation affects the proteolysis of myelin basic protein by Staphylococcus aureus V8 proteinase.

Bovine myelin basic protein (MBP), charge isoform 1 (C1) was carboxylmethylated by the enzyme D-aspartyl/L-isoaspartyl protein methyltransferase (EC. 2.1.1.77) and the carboxylmethylated protein was subjected to proteolysis by sequencing grade staphylococcal V8 proteinase at pH 4.0 to identify its carboxylmethylated modified aspartate and/or asparagine residues which are recognized by this methyltransferase. Native MBP, C1 was treated similarly and the proteolysis products were compared, using electrophoretic, chromatographic and amino acid sequencing techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed differences in the kinetics of proteolysis between the native and the carboxylmethylated MBP, C1 which were confirmed using HPLC. Partial sequencing of the native and carboxylmethylated fragments eluting at about 29 min (P29) revealed cleavage of native MBP, C1 at Gly-127-Gly-128 and of the carboxylmethylated MBP, C1 at Phe-124-Gly-125. Additional evidence including tryptic subdigestion of carboxylmethylated P29 disclosed the following partial sequence for this peptide: Gly-Tyr-Gly-Gly-Arg-Ala-Ser-Asp-Tyr-Lys-Ser-Ala-His-Lys-Gly-Leu-Lys- Gly-His-Asp-Ala-Gln-Gly-Thr-Leu-Ser-Lys-Ileu-Phe-Lys-. This sequence matches MBP residues 125-154. As a result of these findings, Asp-132 and Asp-144 were identified as two of the modified (isomerized or racemized) methyl-accepting L-aspartates in MBP. The results of the proteolysis experiments wherein the sequencing grade staphylococcal V8 proteinase was used at the rarely tested pH of 4.0, rather than at its commonly tested pH of 7.8, also disclose that the proteinase totally failed to recognize and hence cleave the two Glu-X bonds (Glu-82-Asn-83 and Glu-118-Gly-119) of MBP, preferring to cleave the protein at a number of hitherto unreported sites.     

An Antibody Specific for Component 8 of Myelin Basic Protein from Normal Brain Reacts Strongly with Component 8 from Multiple Sclerosis Brain

Myelin basic protein (MBP) consists of several components or charge isomers (C-1 through C-8) generated by one or a combination of posttranslational modifications. One of these, C-8, has been shown to contain citrulline (Cit) at defined sites formed by deimination of six arginyl residues. This unusual modification has allowed us to raise antibodies specific for this charge isomer only. To do this, a synthetic peptide, Gly-Cit-Cit-Cit-Cit, was coupled to keyhole limpet hemocyanin and injected into rabbits. The antibodies so generated reacted only with C-8 and not with any of the other charge isomers. A second antibody fraction was raised against the synthetic peptide ACitHGFLPCitHR naturally occurring between residues 24 and 33 of C-8 (all other charge isomers contain R instead of Cit at positions 25 and 31). These antibodies preferred C-8 but reacted with the other charge isomers, to the extent of approximately 25-30% of the reactivity shown with C-8. In studies with C-8 from multiple sclerosis (MS) MBP, much greater reactivity was obtained with these antibodies when compared with their reactivity with C-8 from normal MBP. Because the total number of Cit residues in C-8 from MS and normal MBP is the same, the difference in reactivity may be related to structural factors. The antibodies raised with the tetra-Cit peptide were reacted with three pairs of synthetic peptides: 24ARHGFLPRHR33 and ACitHGFLPCitHR; 120GQRPGFGYGGRAS132 and GQCitPGFGYGGCitAS; and 157GGRDSRSGSPMARR170 and GGCitDSRSGSPMACitR. They reacted only with the Cit-containing peptides in the order 157-170 greater than 120-130 greater than 24-33.(ABSTRACT TRUNCATED AT 250 WORDS)     

Charge isomers of myelin basic protein: structure and interactions with membranes, nucleotide analogues, and calmodulin

As an essential structural protein required for tight compaction of the central nervous system myelin sheath, myelin basic protein (MBP) is one of the candidate autoantigens of the human inflammatory demyelinating disease multiple sclerosis, which is characterized by the active degradation of the myelin sheath. In this work, recombinant murine analogues of the natural C1 and C8 charge components (rmC1 and rmC8), two isoforms of the classic 18.5-kDa MBP, were used as model proteins to get insights into the structure and function of the charge isomers. Various biochemical and biophysical methods such as size exclusion chromatography, calorimetry, surface plasmon resonance, small angle X-ray and neutron scattering, Raman and fluorescence spectroscopy, and conventional as well as synchrotron radiation circular dichroism were used to investigate differences between these two isoforms, both from the structural point of view, and regarding interactions with ligands, including calmodulin (CaM), various detergents, nucleotide analogues, and lipids. Overall, our results provide further proof that rmC8 is deficient both in structure and especially in function, when compared to rmC1. While the CaM binding properties of the two forms are very similar, their interactions with membrane mimics are different. CaM can be used to remove MBP from immobilized lipid monolayers made of synthetic lipids--a phenomenon, which may be of relevance for MBP function and its regulation. Furthermore, using fluorescently labelled nucleotides, we observed binding of ATP and GTP, but not AMP, by MBP; the binding of nucleoside triphosphates was inhibited by the presence of CaM. Together, our results provide important further data on the interactions between MBP and its ligands, and on the differences in the structure and function between MBP charge isomers.     

Preferential peptide specificity and HLA restriction of myelin basic protein-specific T cell clones derived from MS patients.

A panel of 17 myelin basic protein (MBP)-specific T lymphocyte clones were generated from four multiple sclerosis (MS) patients. All T cell clones expressed CD4 phenotype and 14 clones exhibited substantial cytotoxic activity on MBP-coated target cells. T cell recognition sites of the clones on human MBP were identified by using MBP fragments and synthetic peptides. Despite the fact that at least three epitopes were defined, these T cell clones displayed a striking bias to the C-terminal peptide 149-171 independent of differences in HLA-DR and DQ expression. In addition, the T cell responses of the clones appeared to be restricted by HLA-DR molecules irrespective of peptide specificities. The present study suggests an immunodominant property of the C-terminal peptide for HLA-DR-restricted T cell responses to MBP. However, its association with encephalitogenicity in humans and its potential pathologic importance in MS await further clarification.     

Polymorphism of length of tetranucleotide repeat from the 5'-side from the myelin basic protein gene in multiple sclerosis in Russians

The myelin basic protein gene (MBP) can confer the susceptibility to multiple sclerosis, because its protein product is the main protein component of myelin of the central nervous system and a potential autoimmune antigen in the disease. A possible association of multiple sclerosis with alleles and genotypes of a microsatellite repeat (TGGA)n, located to the 5' side from the first exon of MBP in ethnic Russians (126 patients with reliable multiple sclerosis and 142 healthy controls from Central Russia) was analyzed using the case-control method. Upon separation of the tetranucleotide repeat site amplification products in 1.5% agarose gel, one can see two distinct bands that can be analyzed as two allele groups (A and B). The distribution of allele A and B group frequencies as well as frequency of allele group B and genotype A/A reliably differs in multiple sclerosis patients and healthy controls. Alleles A and the A/A genotype are associated with the development of multiple sclerosis. We also analyzed the association of multiple sclerosis with combined bearing of alleles and genotypes A and B of MBP and groups of alleles of the DRB1 gene of the major histocompatibility complex that correspond to serospecificities DR1-DR18. The comparison of subgroups of multiple sclerosis patients and healthy individuals, formed on the basis of the DRB1 phenotype, has shown a reliable increase in the frequency of allele B in healthy individuals and the genotype A/A frequency in patients, only among DR4- and DR5-positive individuals. No reliable difference was found in the MBP allele and genotype distribution between multiple sclerosis patients and healthy individuals in combined groups of (DR4,DR5)-negative individuals, i.e., no carriers of any phenotype except DR4 and DR5 were revealed. Thus, MBP or some other nearby gene is involved in the multiple sclerosis development in Russians, predominantly (or exclusively) among DR4 and DR5 carriers. In this case, without stratification of analyzed individuals by the MBP alleles, multiple sclerosis is reliably associated only with DR2(15), but not of DR4 and DR5 alleles of DRB1. The results obtained are in favor of the genetic heterogeneity of multiple sclerosis, and suggest the possibility of epistatic interactions between the MBP and DRB1 genes.     

Structural basis for the binding of an immunodominant peptide from myelin basic protein in different registers by two HLA-DR2 proteins

Susceptibility to multiple sclerosis (MS) is associated with certain MHC class II haplotypes, in particular HLA-DR2. Two DR beta chains, DRB1*1501 and DRB5*0101, are co-expressed in the HLA-DR2 haplotype, resulting in the formation of two functional cell surface heterodimers, HLA-DR2a (DRA*0101, DRB5*0101) and HLA-DR2b (DRA*0101, DRB1*1501). Both isotypes can present an immunodominant peptide of myelin basic protein (MBP 84-102) to MBP-specific T cells from MS patients. We have determined the crystal structure of HLA-DR2a complexed with MBP 86-105 to 1.9 A resolution. A comparison of this structure with that of HLA-DR2b complexed with MBP 85-99, reported previously, reveals that the peptide register is shifted by three residues, such that the MBP peptide is bound in strikingly different conformations by the two MHC molecules. This shift in binding register is attributable to a large P1 pocket in DR2a, which accommodates Phe92, in conjunction with a relatively shallow P4 pocket, which is occupied by Ile95. In DR2b, by contrast, the small P1 pocket accommodates Val89, while the deep P4 pocket is filled by Phe92. In both complexes, however, the C-terminal half of the peptide is positioned higher in the binding groove than in other MHC class II/peptide structures. As a result of the register shift, different side-chains of the MBP peptide are displayed for interaction with T cell receptors in the DR2a and DR2b complexes. These results demonstrate that MHC molecules can impose different alignments and conformations on the same bound peptide as a consequence of topological differences in their peptide-binding sites, thereby creating distinct T cell epitopes.     

Adduction of cholesterol 5,6-secosterol aldehyde to membrane-bound myelin basic protein exposes an immunodominant epitope

Myelin degradation in the central nervous system (CNS) is a clinical hallmark of multiple sclerosis (MS). A reduction in the net positive charge of myelin basic protein (MBP) via deimination of arginine to citrulline has been shown to correlate strongly with disease severity and has been linked to myelin instability and a defect that precedes neurodegeneration and leads to autoimmune attack. Recently, we have shown that lipid-derived aldehydes, such as cholesterol 5,6-secosterols atheronal A (1a) and atheronal B (1b), modulate the misfolding of certain proteins such as apolipoprotein B(100), β-amyloid, α-synuclein, and κ- and λ-antibody light chains in a process involving adduction of the hydrophobic aldehyde to lysine side chains, resulting in a decrease in the net positive charge of the protein. In this study, we show that the presence of either atheronal A (1a) or atheronal B (1b) in large unilamellar vesicles (cyt-LUVs) with the lipid composition found in the cytosolic myelin sheath and bovine MBP (bMBP) leads to an atheronal concentration-dependent increase in the surface exposure of the immunodominant epitope (V86-T98) as determined by antibody binding. Other structural changes in bMBP were also observed; specifically, 1a and 1b induce a decrease in the surface exposure of L36-P50 relative to control cyt-LUVs as measured both by antibody binding and by a reduction in the level of cathepsin D proteolysis of F42 and F43. Structure-activity relationship studies with analogues of 1a and 1b point to the aldehyde moiety of both compounds being critical to their effects on bMBP structure. The atheronals also cause a reduction in the size of the bMBP-cyt-LUV aggregates, as determined by fluorescence microscopy and dynamic light scattering. These results suggest that formation of an imine between inflammatory-derived aldehydes, which effectively reduces the cationic nature of MBP, can lead to structural changes in MBP and a decrease in myelin stability akin to deimination and as such may make a hitherto unknown contribution to the onset and progression of MS.     

Variability among the sites by which curaremimetic toxins bind to torpedo acetylcholine receptor, as revealed by identification of the functional residues of alpha-cobratoxin

alpha-Cobratoxin, a long chain curaremimetic toxin from Naja kaouthia venom, was produced recombinantly (ralpha-Cbtx) from Escherichia coli. It was indistinguishable from the snake toxin. Mutations at 8 of the 29 explored toxin positions resulted in affinity decreases for Torpedo receptor with DeltaDeltaG higher than 1.1 kcal/mol. These are R33E > K49E > D27R > K23E > F29A >/= W25A > R36A >/= F65A. These positions cover a homogeneous surface of approximately 880 A(2) and mostly belong to the second toxin loop, except Lys-49 and Phe-65 which are, respectively, on the third loop and C-terminal tail. The mutations K23E and K49E, and perhaps R33E, induced discriminative interactions at the two toxin-binding sites. When compared with the short toxin erabutoxin a (Ea), a number of structurally equivalent residues are commonly implicated in binding to muscular-type nicotinic acetylcholine receptor. These are Lys-23/Lys-27, Asp-27/Asp-31, Arg-33/Arg-33, Lys-49/Lys-47, and to a lesser and variable extent Trp-25/Trp-29 and Phe-29/Phe-32. In addition, however, the short and long toxins display three major differences. First, Asp-38 is important in Ea in contrast to the homologous Glu-38 in alpha-Cbtx. Second, all of the first loop is insensitive to mutation in alpha-Cbtx, whereas its tip is functionally critical in Ea. Third, the C-terminal tail may be specifically critical in alpha-Cbtx. Therefore, the functional sites of long and short curaremimetic toxins are not identical, but they share common features and marked differences that might reflect an evolutionary pressure associated with a great diversity of prey receptors.     

Post-translational Modifications of Chicken Myelin Basic Protein Charge Components

Purified myelin basic protein (MBP) from various species contains several post-translationally modified forms termed charge components or charge isomers. Chicken MBP contains four charge components denoted as C1, C2, C3 and C8. (The C8 isomer is a complex mixture and was not investigated in this study.) These findings are in contrast to those found for human, bovine and other mammalian MBP’s. Mammalian MBP’s, each of which contain seven or eight charge components depending on the analysis of the CM-52 chromatographic curves and the PAGE gels obtained under basic pH conditions. Chicken MBP components C1, C2 and C3 were treated with trypsin and endoproteinase Glu-C. The resulting digests were analyzed by capillary liquid chromatography combined with either an ion trap tandem mass spectrometer or with a Fourier transform ion cyclotron resonance mass spectrometer. This instrumentation permitted establishing the amino acid composition and the determination of the post-translational modifications for each of the three charge components C1-C3. With the exception of N-terminal acetylation, the post-translational modifications were partial. The C1 component lacks any phosphorylated sites, a finding in agreement with the analysis of other MBP species. It also had a single methylation at R105 as did the components C2 and C3. The C2 component contains ten phosphorylated sites (S7, S18, S33, S64, S73, T96, S113, S141, S164, and S168), and modified arginine to citrulline residues at R24, and R165. Component C3 contains eight phosphorylated sites (S7, S33, S64, T96, S113, S141, S164, and S168), and citrulline residues at Arginine 41, R24 and R165. Partial deamidation of glutamine residues Q71, Q101 and Q146 were present in addition to asparagine N90 that was found in all three charge components. The glutamine at residue 3 is partially deamidated in isomers C1 and C2, whereas glutamine 74 and asparagine 83 were found not to be deamidated. Comparison of the PTM’s of MBP’s isolated from several vertebrate species reveals marked differences in their phosphate content. Chicken MBP does not share any phosphorylated sites with dogfish MBP; However, it does contain phosphorylated serine and threonine residues in common with mammalian MBP.     

Interaction of two complementary fragments of the bovine spinal cord myelin basic protein with phospholipid bilayers. An ESR spin-label study

The myelin basic protein (MBP) from bovine spinal cord was cleaved at the single tryptophan residue to produce an N-terminal fragment (F1) of molecular weight 12.6K and a C-terminal fragment (F2) of molecular weight 5.8K. The interactions of the two fragments with bilayers of the acidic lipid dimyristoylphosphatidylglycerol (DMPG) were compared with those of the intact protein, by using both chemical binding assays and spin-label electron spin resonance spectroscopy. The saturation binding stoichiometries of the two fragments were found to sum to that of the MBP, having values of 11, 24, and 36 mol of DMPG/mol of protein for F2, F1, and the MBP, respectively. The strength of binding was found to increase in the order F2 less than F1 less than MBP, which follows that of the net charges on the different fragments. The ionic strength dependence of the protein binding indicated that the interaction is primarily of electrostatic origin. The efficiency of displacement of the proteins by salt was in the order F2 greater than F1 greater than MBP, which correlates with both the strength of binding and the net charge on the different protein fragments. Nitroxide derivatives of phosphatidylglycerol (PG) labeled on the sn-2 chain were used to probe the protein-induced changes in the acyl chain dynamics. Both the fragments and the MBP decreased the lipid chain mobility as recorded by the C-5 atom and C-12 atom position nitroxide-PG spin-labels, in a manner which followed the protein binding curves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine specificity and HLA restriction of myelin basic protein-specific cytotoxic T cell lines from multiple sclerosis patients and healthy individuals.

Myelin basic protein (MBP) is a candidate Ag for the autoimmune process believed to be involved in the pathogenesis of multiple sclerosis (MS). To investigate the fine specificity and HLA restriction of human MBP-specific CTL, long term T cell lines (TCL) were established from 22 MS patients and 16 healthy individuals by repeated antigenic restimulation. By using this approach, MBP-specific cytotoxic TCL were generated from 81% of the lines from MS patients and 69% of those from controls. TCL from both groups expressed the CD3+, CD4+, CD8- phenotype and secreted substantial amounts of IFN-gamma. By using large enzymatic and small synthetic peptides of MBP, TCL were primarily specific for the C-terminal part of the molecule and to a lesser extent for the N-terminal portion. Two regions of the molecule, MBP peptide 87-106 and MBP peptide 154-172, were recognized by the majority of the polyspecific lines and by four and three of 14 monospecific TCL, respectively. These highly immunogenic regions are of interest because they include sequences encephalitogenic in other species. The HLA restriction of each line was determined by using antibody blocking as well as various target cells including EBV-transformed B cells, homozygous typing cells, and fibroblasts transfected with cDNA for DR-alpha and DR-beta genes. All TCL were restricted by HLA-DR Ag. Several HLA-DR molecules restricted multiple cathepsin D-derived and synthetic MBP peptides, including the regions of peptides 87-106 and 154-172 which, respectively, were recognized in conjunction with four and three HLA-DR types. Three of these HLA-DR types are overrepresented in MS patients in different geographic regions. Together, these findings suggest that the MBP-specific cytotoxic T cell response, although not sufficient for disease, may be important for the pathogenesis of MS.     

Structural determinants of ubiquitin-CXC chemokine receptor 4 interaction

Ubiquitin, a post-translational protein modifier inside the cell, functions as a CXC chemokine receptor (CXCR) 4 agonist outside the cell. However, the structural determinants of the interaction between extracellular ubiquitin and CXCR4 remain unknown. Utilizing C-terminal truncated ubiquitin and ubiquitin mutants, in which surface residues that are known to interact with ubiquitin binding domains in interacting proteins are mutated (Phe-4, Leu-8, Ile-44, Asp-58, Val-70), we provide evidence that the ubiquitin-CXCR4 interaction follows a two-site binding mechanism in which the hydrophobic surfaces surrounding Phe-4 and Val-70 are important for receptor binding, whereas the flexible C terminus facilitates receptor activation. Based on these findings and the available crystal structures, we then modeled the ubiquitin-CXCR4 interface with the RosettaDock software followed by small manual adjustments, which were guided by charge complementarity and anticipation of a conformational switch of CXCR4 upon activation. This model suggests three residues of CXCR4 (Phe-29, Phe-189, Lys-271) as potential interaction sites. Binding studies with HEK293 cells overexpressing wild type and CXCR4 after site-directed mutagenesis confirm that these residues are important for ubiquitin binding but that they do not contribute to the binding of stromal cell-derived factor 1α. Our findings suggest that the structural determinants of the CXCR4 agonist activity of ubiquitin mimic the typical structure-function relationship of chemokines. Furthermore, we provide evidence for separate and specific ligand binding sites on CXCR4. As exogenous ubiquitin has been shown to possess therapeutic potential, our findings are expected to facilitate the structure-based design of new compounds with ubiquitin-mimetic actions on CXCR4.     

Different development of myelin basic protein agonist- and antagonist-specific human TCR transgenic T cells in the thymus and periphery

Myelin basic protein (MBP)-specific T cells are thought to play a role in the development of multiple sclerosis. MBP residues 111-129 compose an immunodominant epitope cluster restricted by HLA-DRB1*0401. The sequence of residues 111-129 of MBP (MBP(111-129)) differs in humans (MBP122:Arg) and mice (MBP122:Lys) at aa 122. We previously found that approximately 50% of human MBP(111-129) (MBP122:Arg)-specific T cell clones, including MS2-3C8 can proliferate in response to mouse MBP(111-129) (MBP122:Lys). However, the other half of T cell clones, including HD4-1C2, cannot proliferate in response to MBP(111-129) (MBP122:Lys). We found that MBP(111-129) (MBP122:Lys) is an antagonist for HD4-1C2 TCR, therefore, MS2-3C8 and HD4-1C2 TCRs are agonist- and antagonist-specific TCRs in mice, respectively. Therefore, we examined the development of HD4-1C2 TCR and MS2-3C8 TCR transgenic (Tg) T cells in the thymus and periphery. We found that dual TCR expression exclusively facilitates the development of MBP(111-129) TCR Tg T cells in the periphery of HD4-1C2 TCR/HLA-DRB1*0401 Tg mice although it is not required for their development in the thymus. We also found that MS2-3C8 TCR Tg CD8(+) T cells develop along with MS2-3C8 TCR Tg CD4(+) T cells, and that dual TCR expression was crucial for the development of MS2-3C8 TCR Tg CD4(+) and CD8(+) T cells in the thymus and periphery, respectively. These results suggest that thymic and peripheral development of MBP-specific T cells are different; however, dual TCR expression can facilitate their development.     

Contribution of aromatic-aromatic interactions to the anomalous pK(a) of tyrosine-9 and the C-terminal dynamics of glutathione S-transferase A1-1

Most cytosolic glutathione S-transferases (GSTs) exploit a hydrogen bond between an active site Tyr and the bound glutathione (GSH) cofactor to lower the pK(a) of the GSH and generate the nucleophilic thiolate anion, GS(-). In human (hGSTA1-1) and rat (rGSTA1-1) homologues, the active site Tyr-9 has a low pK(a) of 8.1-8.3, for which the functional significance is unknown. Crystal structures of GSTA1-1 suggest that weakly polar interactions between the electropositive ring edge of Phe-10 and the pi-cloud of Tyr-9, in the apoenzyme, could stabilize the tyrosinate anion and also modulate the pK(a) of GSH. Upon binding a product GSH conjugate, Phe-10 moves away from Tyr-9, allowing the highly dynamic C-terminus to "close" over the active site. To explore the role of Phe-10 in modulating the Tyr-9 pK(a) and in ligand binding, rGSTA1-1 mutants F10Y, F10L, and F10A were characterized. The pK(a)s of Tyr-9 in the apoenzymes were 8.2 +/- 0.2, 8.7 +/- 0.2, and 9.3 +/- 0.1, respectively, for F10Y, F10L, and F10A, compared to 8.3 +/- 0.2 for the "wild type". The experimentally determined pK(a)s qualitatively paralleled the energies required to remove a proton predicted by ab initio calculations using model compounds constrained to the coordinates of rGSTA1-1. The pK(a) of GSH in the binary complex was significantly less affected by these substitutions. In contrast, F220I and F220Y C-terminal mutations caused the pK(a) of Tyr-9 to decrease modestly. For the binary complex with S-hexyl-GSH, which induces the "closed" conformation, Tyr-9 retains a low pK(a) and the Phe-10 substitutions have significant effects. Presumably, Phe-10 plays a critical structural role in stabilizing the closed conformation. The mutations F10L and F10A also slowed the rate of GSH conjugate binding by 10-20-fold, as measured by stopped-flow fluorescence. The effects of Phe-10 substitution were large for both steps of the biphasic binding reaction, suggesting the importance of aromatic interactions throughout the reaction coordinate. A unified view of the C-terminal dynamics of GSTA1-1 is discussed, which emphasizes the coupling between Tyr-9 ionization, active site solvation, and C-terminal dynamics.     

Does the frequency and avidity spectrum of the neuroantigen-specific T cells in the blood mirror the autoimmune process in the central nervous system of mice undergoing experimental allergic encephalomyelitis?

In humans, studies of autoreactive T cells that mediate multiple sclerosis have been largely confined to testing peripheral blood lymphocytes. Little is known how such measurements reflect the disease-mediating autoreactive T cells in the CNS. This information is also not available for murine experimental allergic encephalomyelitis (EAE); the low number of T cells that can be obtained from the blood or the brain of mice prevented such comparisons. We used single-cell resolution IFN-gamma ELISPOT assays to measure the frequencies and functional avidities of myelin basic protein (MBP:87-99)-specific CD4 cells in SJL mice immunized with this peptide. Functional MBP:87-99-specific IFN-gamma-producing cells were present in the CNS during clinical signs of EAE, but not during phases of recovery. In contrast, MBP:87-99-specific T cells persisted in the blood during all stages of the disease, and were also present in mice that did not develop EAE. Therefore, the increased frequency of MBP:87-99-reactive T cells in the blood reliably reflected the primed state, but not the inflammatory activity of these cells in the brain. The functional avidity of the MBP:87-99-reactive T cells was identical in the brain and blood and did not change over 2 mo as the mice progressed from acute to chronic EAE. Therefore, high-affinity T cells did not become selectively enriched in the target organ, and avidity maturation of the MBP:87-99-specific T cell repertoire did not occur in the observation period. The data may help the interpretation of measurements made with peripheral blood lymphocytes of multiple sclerosis patients.     

The role of transmembrane segment TM3 in the xanthine permease XanQ of Escherichia coli

The xanthine permease XanQ of Escherichia coli is used as a study prototype for function-structure analysis of the ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family. Our previous mutagenesis study of polar residues of XanQ has shown that Asn-93 at the middle of putative TM3 is a determinant of substrate affinity and specificity. To study the role of TM3 in detail we employed Cys-scanning mutagenesis. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence 79-107 (YGIVGSGLLSIQSVNFSFVTVMIALGSSM) including TM3 (underlined) and flanking sequences was replaced individually with Cys. Of 29 single-Cys mutants, 20 accumulate xanthine to 40-110% of the steady state observed with C-less, six (S88C, F94C, A102C, G104C, S106C) accumulate to low levels (10-30%) and three (G83C, G85C, N93C) are inactive. Extensive mutagenesis reveals that Gly-83 and, to a lesser extent, Gly-85, are crucial for expression in the membrane. Replacements of Asn-93 disrupt affinity (Thr) or permit recognition of 8-methylxanthine which is not a wild-type ligand (Ala, Ser, Asp) and utilization of uric acid which is not a wild-type substrate (Ala, Ser). Replacements of Phe-94 impair affinity for 2-thio and 6-thioxanthine (Tyr) or 3-methylxanthine (Ile). Single-Cys mutants S84C, L86C, L87C, and S95C are highly sensitive to inactivation by N-ethylmaleimide. Our data reveal that key residues of TM3 cluster in two conserved sequence motifs, (83)GSGLL(87) and (93)NFS(95), and highlight the importance of Asn-93 and Phe-94 in substrate recognition and specificity; these findings are supported by structural modeling on the recently described x-ray structure of the uracil-transporting homolog UraA.     

Increased CD8+ cytotoxic T cell responses to myelin basic protein in multiple sclerosis

Autoreactive T cells of CD4 and CD8 subsets recognizing myelin basic protein (MBP), a candidate myelin autoantigen, are thought to contribute to and play distinct roles in the pathogenesis of multiple sclerosis (MS). In this study we identified four MBP-derived peptides that had high binding affinity to HLA-A2 and HLA-A24 and characterized the CD8(+) T cell responses and their functional properties in patients with MS. There were significantly increased CD8(+) T cell responses to 9-mer MBP peptides, in particular MBP(111-119) and MBP(87-95) peptides that had high binding affinity to HLA-A2, in patients with MS compared with healthy individuals. The resulting CD8(+) T cell lines were of the Th1 phenotype, producing TNF-alpha and IFN-gamma and belonged to a CD45RA(-)/CD45RO(+) memory T cell subset. Further characterization indicated that the CD8(+) T cell lines obtained were stained with MHC class I tetramer (HLA-A2/MBP(111-119)) and exhibited specific cytotoxicity toward autologous target cells pulsed with MBP-derived peptides in the context of MHC class I molecules. These cytotoxic CD8(+) T cell lines derived from MS patients recognized endogenously processed MBP and lysed COS cells transfected with genes encoding MBP and HLA-A2. These findings support the potential role of CD8(+) CTLs recognizing MBP in the injury of oligodendrocytes expressing both MHC class I molecules and MBP.     

The preparation of antibodies reactive against citrulline-containing charge isomers of myelin basic protein but not against the arginine-containing charge isomers.

Human myelin basic protein (MBP) is composed of several charge isomers, the result of various post-translational modifications. One of the charge isomers C-8, has been shown in our laboratory to contain six citrullinyl residues which replace arginyl residues at selected sites in the MBP. In order to determine the disposition of the citrulline-containing charge isomers in the myelin stack, we prepared specific antisera against the citrullinyl group. Since 9-fluorenylmethoxycarbonyl (Fmoc)-citrulline, required for the preparation of the synthetic peptides to be used for antibody production, was not commercially available, synthesis of the Fmoc-citrulline was a necessary prerequisite. The synthesis and purification of the N-9-fluorenylmethyloxycarbonyl derivative of citrulline is described. It was characterized by thin layer chromatography, 1H and 13C NMR spectroscopy, fast-atom bombardment mass spectroscopy, and thermal analyses. It was used in the automated peptide synthesis of a peptide Ala-Cit-His-Gly-Phe-Leu-Pro-Cit-His-Arg corresponding to residues 24-33 and Gly-Cit-Asp-Ser-Arg-Ser-Gly-Ser-Pro-Met-Ala-Cit-Arg, corresponding to residues 158-170 of the C-8 sequence, a naturally occurring charge isomer of human myelin basic protein, and a tetracitrulline peptide, Cit-Cit-Cit-Cit-Gly. The tetracitrulline peptide was used for the production of an antibody shown to react only with synthetic peptides and proteins containing citrulline. This antibody was used to distinguish between a citrulline-containing protein, C-8, a naturally occurring charge isomer of MBP, and a non-citrulline-containing charge isomer of MBP, C-1.