Main chain hydrogen bond interactions in the binding of proline-rich gluten peptides to the celiac disease-associated HLA-DQ2 molecule

Binding of peptide epitopes to major histocompatibility complex proteins involves multiple hydrogen bond interactions between the peptide main chain and major histocompatibility complex residues. The crystal structure of HLA-DQ2 complexed with the alphaI-gliadin epitope (LQPFPQPELPY) revealed four hydrogen bonds between DQ2 and peptide main chain amides. This is remarkable, given that four of the nine core residues in this peptide are proline residues that cannot engage in amide hydrogen bonding. Preserving main chain hydrogen bond interactions despite the presence of multiple proline residues in gluten peptides is a key element for the HLA-DQ2 association of celiac disease. We have investigated the relative contribution of each main chain hydrogen bond interaction by preparing a series of N-methylated alphaI epitope analogues and measuring their binding affinity and off-rate constants to DQ2. Additionally, we measured the binding of alphaI-gliadin peptide analogues in which norvaline, which contains a backbone amide hydrogen bond donor, was substituted for each proline. Our results demonstrate that hydrogen bonds at P4 and P2 positions are most important for binding, whereas the hydrogen bonds at P9 and P6 make smaller contributions to the overall binding affinity. There is no evidence for a hydrogen bond between DQ2 and the P1 amide nitrogen in peptides without proline at this position. This is a unique feature of DQ2 and is likely a key parameter for preferential binding of proline-rich gluten peptides and development of celiac disease.     

Deamidation and cross-linking of gliadin peptides by transglutaminases and the relation to celiac disease

Activation of small intestinal gluten-reactive CD4+ T cells is a critical event in celiac disease. Such cells predominantly recognise gluten peptides in which specific glutamines are deamidated. Deamidation may be catalysed by intestinal tissue transglutaminase (TG2), a protein which is also the main autoantigen in celiac disease. Our aim was to study how the two main catalytic activities of transglutaminase--deamidation and transamidation (cross-linking) of an immunodominant gliadin epitope--are influenced by the presence of acceptor amines in the intestinal mucosa, and thereby contribute to further elucidation of the pathogenetic mechanisms in celiac disease. We prepared monoclonal antibodies, reacting specifically with the non-deamidated epitope QPFPQPQLPYPQPQ-amide and/or the deamidated epitope QPFPQPELPYPQPQ-amide. A solid phase immunoassay combined with gel filtration chromatography was used to analyse deamidation and cross-linking of these peptides to proteins. Our results show that QPFPQPQLPYPQPQ-amide was deamidated when incubated with purified TG2, with fresh mucosal sheets and with mucosal homogenates. Of other transglutaminases tested, only Streptoverticillium transglutaminase was able to generate the deamidated epitope. A fraction of the non-deamidated epitope was cross-linked to proteins, including TG2. The results suggest that intestinal TG2 is responsible for generation of the active deamidated epitope. As the epitope often occurs in a repeat structure, the result may be cross-linking of a deamidated, i.e., activated cell epitope. Alternatively, the deamidation may occur by reversal of the cross-linking reaction. The results provide a basis for the suggestion that binding of a peptide to a protein, in connection to its modification to a T cell epitope, might be a general explanation for the role of TG2 in celiac disease and a possible mechanism for the generation of autoantigens.     

T cell activation by antibody-like immunoreceptors: the position of the binding epitope within the target molecule determines the efficiency of activation of redirected T cells

Recombinant TCRs confer specificity to T cells and trigger their activation. Receptors with Ab-derived binding domains have the advantages of MHC-independent Ag recognition and of targeting a variety of chemically different molecules. We explored the impact of the position of a defined epitope within the target molecule on the efficacy of receptor-mediated T cell activation. T cells were grafted with recombinant immunoreceptors that recognize either the membrane distal N or the proximal A3 domain of carcinoembryonic Ag (CEA). Upon binding to isolated, solid-phase immobilized CEA, receptor-mediated T cell activation correlates with the binding efficiency, irrespectively, of the epitope position. Upon binding to CEA expressed on the cell membrane, in contrast, the A3 epitope mediates more efficiently T cell activation than the N epitope, although the N epitope is bound with higher affinity. The CEA N epitope when expressed in a more membrane proximal position, however, activated receptor grafted T cells with higher efficiency than in the distal position. The position of the targeted epitope within the molecule obviously has major impact on the efficacy of T cell activation independently of the binding efficiency of the immunoreceptor.     

Molecular mapping of interactions between a Mycobacterium leprae-specific T cell epitope, the restricting HLA-DR2 molecule, and two specific T cell receptors.

A systematic series of 89 single residue substitution analogs of the Mycobacterium leprae 65-kDa protein-derived peptide LQAAPALDKL were tested for stimulation of two HLA-DR2 restricted 65 kDa-reactive T cell clones from a tuberculoid leprosy patient. Some analogs with substitutions outside a "core" region showed enhanced stimulation of the T cell clones. This core region of seven or eight residues was essential for recognition, whereas substitution of amino acids outside this region did not affect T cell recognition although these residues could not be omitted. Thus these core residues interact directly with the presenting HLA-DR2 molecule and/or the TCR. Except for analogs of position 419 for clone 2B6, the majority of the nonstimulatory substitution analogs did not inhibit the presentation of LQAAPALDKL and thus probably failed to bind to the HLA-DR2 molecule. Unless all of the core residues are physically involved in binding to DR2, substitution at a position not directly involved in binding appears to have an influence on other residues that do bind to the DR2 molecule. Active peptide analogs with two or more internal prolines suggest that not all analogs need be helical for activity with clone 2F10.     

Gamma-gliadin specific celiac disease antibodies recognize p31-43 and p57-68 alpha gliadin peptides in deamidation related manner as a result of cross-reaction

Celiac disease (CeD) is a T-cell-dependent enteropathy with autoimmune features where tissue transglutaminase (TG2)-mediated posttranslational modification of gliadin peptides has a decisive role in the pathomechanism. The humoral immune response is reported to target mainly TG2-deamidated γ-gliadin peptides. However, α-gliadin peptides, like p57-68, playing a crucial role in the T-cell response, and p31-43, a major trigger of innate responses, also contain B-cell gliadin epitopes and γ-gliadin like motifs. We aimed to identify if there are anti-gliadin-specific antibodies in CeD patients targeting the p31-43 and p57-68 peptides and to examine whether deamidation of these peptides could increase their antigenicity. We explored TG2-mediated deamidation of the p31-43 and p57-68 peptides, and investigated serum antibody reactivity toward the native and deamidated α and γ-gliadin peptides in children with confirmed CeD and in prospectively followed infants at increased risk for developing CeD. We affinity-purified antibody populations utilizing different single peptide gliadin antigens and tested their binding preferences for cross-reactivity in real-time interaction assays based on bio-layer interferometry. Our results demonstrate that there is serum reactivity toward p31-43 and p57-68 peptides, which is due to cross-reactive γ-gliadin specific antibodies. These γ-gliadin specific antibodies represent the first appearing antibody population in infancy and they dominate the serum reactivity of CeD patients even later on and without preference for deamidation. However, for the homologous epitope sequences in α-gliadins shorter than the core QPEQPFP heptapeptide, deamidation facilitates antibody recognition. These findings reveal the presence of cross-reactive antibodies in CeD patients recognizing the disease-relevant α-gliadins.

     

Dominance of an alternative CLIP sequence in the celiac disease associated HLA-DQ2 molecule

During assembly, HLA class II molecules associate with the invariant chain. As the result, the peptide-binding groove is occupied by an invariant chain peptide termed CLIP (class-II-associated invariant chain peptide; sequence MRMATPLLM). By mass spectrometry, we have now characterized peptides that are naturally present in HLA-DQ2. This analysis revealed that 22 variants of Ii-derived peptides are associated with HLA-DQ2. Strikingly, the large majority of those do not contain the conventional CLIP sequence MRMATPLLM, but instead a peptide that partially overlaps with CLIP, sequence TPLLMQALPM. Peptide binding studies indicate that this alternative CLIP peptide has superior HLA-DQ2 binding properties compared to the conventional CLIP and that the minimal nine-amino-acid binding core consists of the sequence PLLMQALPM, findings that could be corroborated by molecular simulation. The alternative CLIP peptide was also found to be present in HLA-DQ2 molecules isolated from human thymus. Moreover, the alternative CLIP peptide was also found in association with HLA-DQ8. Together, these results indicate that HLA-DQ2 and HLA-DQ8 associate with an alternative CLIP sequence, a property that may relate to the strong association between HLA-DQ molecules and human autoimmune diseases.     

Autoantibodies from patients with celiac disease inhibit transglutaminase 2 binding to heparin/heparan sulfate and interfere with intestinal epithelial cell adhesion

Autoantibodies from patients with celiac disease (CD) can influence transglutaminase 2 (TG2) activity and its cellular functions, but the exact mechanisms have remained unknown. Our objective was to study whether autoantibodies could modulate TG2 binding to heparin/heparan sulfate (HS) and intestinal epithelial cell attachment to fibronectin-TG2 matrix. Anti-TG2 antibodies were purified by TG2 affinity chromatography from sera of patients with active CD. Serum and antibody effects on TG2 binding to heparin/HS, on transamidase activity of TG2, as well as on Caco-2 cell attachment to fibronectin-TG2 matrix were assessed using microplate assays. Both sera and purified anti-TG2 antibodies from CD patients with high anti-TG2 IgA levels reduced TG2 binding to heparin/HS as compared with those with low anti-TG2 IgA or controls. There was a negative correlation between anti-TG2 IgA levels and TG2 binding to heparin/HS. Treatment of fibronectin-TG2 coated wells with CD patients' sera or purified anti-TG2 antibodies reduced attachment of Caco-2 cells onto the plate as compared with the control samples. The effect of CD patients' antibodies on Caco-2 cell attachment to fibronectin-TG2 matrix occurred independently of the inhibition of cell adhesion by Arg-Gly-Asp sequence containing peptides. Anti-TG2 autoantibodies had no effect on transamidase activity of TG2 in vitro. We suggest that modulation of adhesion function of TG2 by autoantibodies from patients with CD could be related to the inhibition of TG2 binding to HS residues of cell surface proteoglycans and could have possible implications for CD pathogenesis.     

Celiac disease association with CD8+ T cell responses: identification of a novel gliadin-derived HLA-A2-restricted epitope

One of the diagnostic hallmarks of the histological lesions associated with celiac disease is the extensive infiltration of the small intestinal epithelium by CD8(+) T cells of unknown Ag specificity. In this study, we report recognition of the gliadin-derived peptide (A-gliadin 123-132) by CD8(+) T lymphocytes from celiac patients. A-gliadin 123-132-specific IFN-gamma production and cytotoxic activity were detected in PBMCs derived from patients on gluten-free diet, but not from either celiac patients on gluten-containing diet or healthy controls. In contrast, A-gliadin 123-132-specific cells were isolated from small intestine biopsies of patients on either gluten-free or gluten-containing diets. Short-term T cell lines derived from the small intestinal mucosa and specific for the 123-132 epitope recognized human APC pulsed with either whole recombinant alpha-gliadin or a partial pepsin-trypsin gliadin digest. Finally, we speculate on a possible mechanism leading to processing and presentation of class I-restricted gliadin-derived epitopes in celiac disease patients.     

The T11 (CD2) molecule is functionally linked to the T3/Ti T cell receptor in the majority of T cells

Most mature human T lymphocytes express both the multichain T3 (CD3)/Ti T cell receptor for antigen (TCR), and the biochemically distinct 55-kDa T11 (CD2) glycoprotein. Stimulating the T11 molecule causes profound T cell proliferation and functional activation in vitro, but the relationship of T11-mediated activation to antigenic stimulation of T lymphocytes in vivo remains unknown. We now present evidence that T11 function is directly linked to TCR components in T3/Ti+ T11+ human T cells. First, we found that stimulating peripheral blood T cells with the mitogenic combination of anti-T11(2) cells with the mitogenic combination of anti-T11(2) plus anti-T11(3) monoclonal antibodies caused the phosphorylation of TCR T3 chains. The predominance of T3-gamma-phosphorylation that occurred in anti-T11(2) plus anti-T11(3)-treated T cells is similar to the pattern previously observed in antigen-stimulated T cell clones. Second, T11 function depended upon concurrent cell-surface expression of the TCR. Thus, when peripheral blood T cells were deprived of cell surface T3/Ti by anti-T3 modulation, anti-T11(2) plus anti-T11(3)-induced mitogenesis and transmembrane signal generation in the form of calcium mobilization were inhibited. The mechanism of TCR-T11 interdependence was investigated in a series of TCR-deficient variants of a T cell lymphoblastoid cell line. T3/Ti negative variants expressed cell surface T11, but anti-T11(2) plus anti-T11(3) failed to cause detectable calcium mobilization. The TCR-deficient variants also failed to express T11(3) activation epitopes after incubation with anti-T11(2) antibodies, suggesting that T11(3) expression is an essential and TCR-dependent intermediate in the T11 activation mechanism in these cells. Taken together, our results suggest that T11 function depends upon cell-surface expression of TCR in many T3/Ti+ T11+ T lymphocytes, and T11-mediated activation is intimately interconnected with TCR activation mechanisms. A model in which stimulating signals delivered via T11 may be a part of antigenic activation of T lymphocytes is presented.     

T cell recognition of bovine ribonuclease. Self/non-self discrimination at the level of binding to the I-Ak molecule

Bovine RNase A specific T-cell hybridomas were generated to study the recognition of foreign Ag by T lymphocytes. One hybrid, TS12, was shown to recognize RNase in association with I-Ak. This hybridoma required bovine RNase to be processed before recognition. The immunogenic determinant on the RNase molecule recognized by TS12 was localized to the tryptic fragment RNase(40-61). All of the stimulatory ability of this determinant was shown to be contained within the synthetic 14mer RNase(43-56). When this segment of bovine RNase was compared with the self murine sequence, only one amino acid difference was found, a substitution of a proline residue at position 50 for a serine residue. This substitution completely abolishes binding to the I-Ak molecule, as shown by both functional and direct binding assays. This finding shows that self/non-self discrimination not only occurs at the level of the T cell, but also can be caused by an inability of the self peptide to associate with a class II molecule.     

Plakophilin-2: a cell-cell adhesion plaque molecule of selective and fundamental importance in cardiac functions and tumor cell growth

Within the characteristic ensemble of desmosomal plaque proteins, the armadillo protein plakophilin-2 (Pkp2) is known as a particularly important regulatory component in the cytoplasmic plaques of various other cell-cell junctions, such as the composite junctions (areae compositae) of the myocardiac intercalated disks and in the variously-sized and -shaped complex junctions of permanent cell culture lines derived therefrom. In addition, Pkp2 has been detected in certain protein complexes in the nucleoplasm of diverse kinds of cells. Using a novel set of highly sensitive and specific antibodies, both kinds of Pkp2, the junctional plaque-bound and the nuclear ones, can also be localized to the cytoplasmic plaques of diverse non-desmosomal cell-cell junction structures. These are not only the puncta adhaerentia and the fasciae adhaerentes connecting various types of highly proliferative non-epithelial cells growing in culture but also some very proliferative states of cardiac interstitial cells and cardiac myxomata, including tumors growing in situ as well as fetal stages of heart development and cultures of valvular interstitial cells. Possible functions and assembly mechanisms of such Pkp2-positive cell-cell junctions as well as medical consequences are discussed.     

Analysis of T cell responses to the major allergens from German cockroach: epitope specificity and relationship to IgE production

Bla g allergens are major targets of IgE responses associated with cockroach allergies. However, little is known about corresponding T cell responses, despite their potential involvement in immunopathology and the clinical efficacy of specific immunotherapy. Bioinformatic predictions of the capacity of Bla g 1, 2, 4, 5, 6, and 7 peptides to bind HLA-DR, -DP, and -DQ molecules, and PBMC responses from 30 allergic donors, identified 25 T cell epitopes. Five immunodominant epitopes accounted for more than half of the response. Bla g 5, the most dominant allergen, accounted for 65% of the response, and Bla g 6 accounted for 20%. Bla g 5 induced both IL-5 and IFN-γ responses, whereas Bla g 6 induced mostly IL-5, and, conversely, Bla g 2 induced only IFN-γ. Thus, responses to allergens within a source are independently regulated, suggesting a critical role for the allergen itself, and not extraneous stimulation from other allergens or copresented immunomodulators. In comparing Ab with T cell responses for several donor/allergen combinations, we detected IgE titers in the absence of detectable T cell responses, suggesting that unlinked T cell-B cell help might support development of IgE responses. Finally, specific immunotherapy resulted in IL-5 down modulation, which was not associated with development of IFN-γ or IL-10 responses to any of the Bla g-derived peptides. In summary, the characteristics of T cell responses to Bla g allergens appear uncorrelated with IgE responses. Monitoring these responses may therefore yield important information relevant to understanding cockroach allergies and their treatment.     

Naive precursor frequencies and MHC binding rather than the degree of epitope diversity shape CD8+ T cell immunodominance

The primary CD8(+) T cell response of C57BL/6J mice against the 28 known epitopes of lymphocytic choriomeningitis virus (LCMV) is associated with a clear immunodominance hierarchy whose mechanism has yet to be defined. To evaluate the role of epitope competition in immunodominance, we manipulated the number of CD8(+) T cell epitopes that could be recognized during LCMV infection. Decreasing epitope numbers, using a viral variant lacking dominant epitopes or C57BL/6J mice lacking H-2K(b), resulted in minor response increases for the remaining epitopes and no new epitopes being recognized. Increasing epitope numbers by using F(1) hybrid mice, delivery by recombinant vaccinia virus, or epitope delivery as a pool in IFA maintained the overall response pattern; however, changes in the hierarchy did become apparent. MHC binding affinity of these epitopes was measured and was found to not strictly predict the hierarchy since in several cases similarly high binding affinities were associated with differences in immunodominance. In these instances the naive CD8(+) T cell precursor frequency, directly measured by tetramer staining, correlated with the response hierarchy seen after LCMV infection. Finally, we investigated an escape mutant of the dominant GP33-41 epitope that elicited a weak response following LCMV variant virus infection. Strikingly, dominance loss likely reflects a substantial reduction in frequencies of naive precursors specific for this epitope. Thus, our results indicate that an intrinsic property of the epitope (MHC binding affinity) and an intrinsic property of the host (naive precursor frequency) jointly dictate the immunodominance hierarchy of CD8(+) T cell responses.     

Kinetic mechanism of uracil phosphoribosyltransferase from Escherichia coli and catalytic importance of the conserved proline in the PRPP binding site

Phosphoribosyltransferases catalyze the formation of nucleotides from a nitrogenous base and 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP). These enzymes and the PRPP synthases resemble each other in a short homologous sequence of 13 amino acid residues which has been termed the PRPP binding site and which interacts with the ribose 5-phosphate moiety in structurally characterized complexes of PRPP and nucleotides. We show that each class of phosphoribosyltransferases has subtle deviations from the general consensus PRPP binding site and that all uracil phosphoribosyltransferases (UPRTases) have a proline residue at a position where other phosphoribosyltransferases and the PRPP synthases have aspartate. To investigate the role of this unusual proline (Pro 131 in the E. coli UPRTase) for enzyme activity, we changed the residue to an aspartate and purified the mutant P131D enzyme to compare its catalytic properties with the properties of the wild-type protein. We found that UPRTase of E. coli obeyed the kinetics of a sequential mechanism with the binding of PRPP preceding the binding of uracil. The basic kinetic constants were derived from initial velocity measurements, product inhibition, and ligand binding assays. The change of Pro 131 to Asp caused a 50-60-fold reduction of the catalytic rate (kcat) in both directions of the reaction and approximately a 100-fold increase in the KM for uracil. The KM for PRPP was strongly diminished by the mutation, but kcat/KM,PRPP and the dissociation constant (KD,PRPP) were nearly unaffected. We conclude that the proline in the PRPP binding site of UPRTase is of only little importance for binding of PRPP to the free enzyme, but is critical for binding of uracil to the enzyme-PRPP complex and for the catalytic rate.     

Immunodeficiency is a tough nut to CRAC: the importance of calcium flux in T cell activation

Severe Combined Immunodeficiency (SCID) is a rare primary immunodeficiency disease often characterized by a block in T cell development, which may also affect the normal development of B cells and NK cells. Several different mutations are known to give rise to SCID, and multiple genes are involved. Consequently, there are several different forms of SCID, which can be classified according to the metabolic and cellular defects that impede normal lymphocyte function. The two most prevalent forms of SCID are X-linked SCID and adenosine deaminase (ADA) deficiency SCID, together accounting for approximately 70-80% of disease cases. Other genetic abnormalities associated with this syndrome range from defective T cell receptor rearrangement to non-functional signaling molecules. Recently, a new genetic defect has been described in which mutations in a key component of Ca(2+) release activated-channels (CRAC) result in T lymphocyte malfunction.     

Heparin prevents the binding of phospholipase A2 to phospholipid micelles: importance of the amino-terminus

The activity of the major isoform of porcine pancreatic phospholipase A2 (PLA2), designated B-PLA2, against micellar substrates is inhibited by heparin. Inhibition is a consequence of binding of the enzyme to heparin, documented by a heparin-induced alteration in the intrinsic fluorescence of B-PLA2 and in the 8-anilino-1-naphthalene sulfonate fluorescence and by the enhanced rate of chemical modification of the active site residue His-48. As a consequence of heparin binding, the conformation of B-PLA2 at the active site and at the amino-terminus is altered, and the enzyme does not bind to phospholipid micelles. In spite of the heparin-induced conformational changes, B-PLA2 retains its ability to catalyze the hydrolysis of monomeric phospholipid. Other glycosaminoglycans can bind to and inhibit the activity of B-PLA2 toward organized phospholipids, but none tested is as effective as heparin. An isoform of the pancreatic enzyme, designated UB-PLA2 and which corresponds to iso-pig PLA2, does not bind to nor is its catalytic activity influenced by heparin. A peptide corresponding to the amino-terminal 26 residues of B-PLA2 can rescue PLA2 from heparin inhibition. A similar peptide corresponding to the amino-terminus of UB-PLA2 has no effect on heparin inhibition. A model for the inhibition of B-PLA2 by heparin is proposed in which the catalytically significant effect of heparin is to interact directly with the amino-terminus of B-PLA2, the interfacial recognition site, to prevent the enzyme from binding to micellar substrates.     

Dual role of the CD44 molecule in T cell adhesion and activation

Studies of T cell adhesion and activation reveal two new functions of the CD44 molecule, a molecule now recognized to be identical to three molecules of functional interest: Pgp-1, Hermes, and extracellular matrix receptor type III (ECMRIII). By screening for mAb which inhibit T cell adhesion to E, we have identified a functionally unique CD44-specific mAb, NIH44-1, which partially inhibits T cell rosetting by binding to CD44 on the E. NIH44-1, which immunoprecipitates a protein of 85 to 110 kDa with broad tissue distribution, was determined to be specific for CD44 based on comparison of its tissue distribution with multiple CD44-specific reference mAb and sequential immunoprecipitation with such mAb. Anticipating a role for many adhesion molecules in signal transduction, we studied the effect of CD44 mAb on T cell activation and observed that CD44 mAb dramatically augments T cell proliferation induced by CD3- and CD2-receptor-mediated activation. The augmentation of the response to immobilized CD3 mAb by exhaustively monocyte-depleted T cells indicates that augmentation can be mediated by binding to the T cell. Thus, our studies demonstrate specific new roles for CD44 in T cell adhesion and activation. Furthermore, we suggest that: 1) CD44 has a role in adhesion of cells of multiple lineages; and 2) CD44 may participate in adhesion not (only) by functioning as an adhesion receptor but rather by serving as an anchorage site for other adhesion molecules.     

Protein dynamics: imidazole and 2-mercaptoethanol binding to the Rhodobacter capsulatus cytochrome c(2) mutant,glycine 95 proline

The Class I c-type cytochromes can bind exogenous ligands in the oxidized state, with the kinetics of ligand binding providing information on naturally occurring intramolecular dynamics. Typically, nitrogenous bases are used as ligands; however, it is less well known that 2-mercaptoethanol (BME), a commonly used cytochrome reducing agent, can form a complex with the heme. To better understand the cytochrome-mercaptan interaction, we have investigated the kinetics of binding of BME to wild type and mutants of Rhodobacter capsulatus cytochrome c(2) and to horse cytochrome c. Complex formation with the G95P mutant is apparent from the formation of a green color and a shift in the Soret peak to 418 nm from 410 nm upon addition of BME. Unlike horse cytochrome c and wild-type R. capsulatus cytochrome c(2), G95P permits the kinetics of formation of the BME-G95P complex to be measured since complex formation and reduction kinetics can be resolved. The affinity constant for the binding of BME to mutant G95P was strong ( approximately 1.5 x 10(5)M(-1)) and the kinetics of formation of the BME-G95P complex were found to undergo a change in rate-limiting step consistent with a concentration-independent protein rearrangement (68s(-1)) followed by second-order binding of BME ( approximately approximately 1.3 x 10(5)M(-1)s(-1)). The most remarkable characteristic of mutant G95P is the relatively large amount of high-spin species in equilibrium with the low- spin form, which can be estimated to be approximately 3% at pH 7. The BME binding kinetics, coupled with the kinetics of imidazole binding to G95P, allow us, for the first time, to specify all four rate constants describing the ligand binding reaction. Moreover, we can use the kinetic results to estimate the rate constants for ligand binding with the wild-type cytochrome c(2). This has also allowed us to quantify and more fully interpret cytochrome dynamics.     

HLA-DR2a is the dominant restriction molecule for the cytotoxic T cell response to myelin basic protein in DR2Dw2 individuals.

The HLA-DR2 restriction of the T cell response to myelin basic protein (MBP) was studied using murine L cells transfected with DRalpha and either DR2a or DR2b beta-chain cDNA. DR2a and DR2b represent the two isotypic DRbeta chains expressed in DR2Dw2 haplotypes. Eleven MBP-specific cytolytic T cell lines derived from patients with multiple sclerosis were isolated. Two of these cell lines recognized MBP-pulsed DR2-expressing L cell transfectants and four of them could only recognize the L cells if the adhesion molecule ICAM-1 was expressed in addition to HLA-DR2. Five of the six lines were restricted by HLA-DR2a; one line recognized Ag in conjunction with DR2b, but only if ICAM-1 was coexpressed. The remaining five lines did not lyse MBP-pulsed L cells. The ability of the DR2b molecules on transfected cells to stimulate T cells was confirmed with DR2b-allospecific T cell clones. Although five MBP-specific lines were restricted by DR2a, they recognized different parts of the MBP molecule, as demonstrated by the presentation of shorter peptides. Thus, our results suggest that DR2a is a dominant restriction molecule in MBP-specific responses by DR2+ MS patients. The results also indicate that the reported heterogeneity in MBP epitopes recognized by DR2-restricted T cells, may not be due to the use of different restriction elements but rather to the binding of different MBP peptides to DR2a molecules.