Biomedical subjects

J A López de Castro

Publications and source records attributed to J A López de Castro.

At least 19 recordsLinked to original sources

PubMed2002

HLA-B27 and the pathogenesis of spondyloarthritis.

The association of HLA-B27 with ankylosing spondylitis and other spondyloarthropathies ranks among the strongest between any HLA antigen and a human disease. Yet, in spite of intense research and advanced knowledge of the biochemistry and biology of major histocompatibility complex molecules, the mechanism of this association remains unknown. This review attempts a critical assessment of current pathogenetic hypotheses from evidence concerning the epidemiology of HLA-B27 association with disease, its peptide-binding specificity, and other aspects of the molecular biology and immunology of this molecule.

HLA-B27 Antigen↗

PubMed2001

The Cys-67 residue of HLA-B27 influences cell surface stability, peptide specificity, and T-cell antigen presentation.

Cys-67 of HLA-B27 is located in the B pocket, which determines peptide-binding specificity. We analyzed effects of the Cys-67 --> Ser mutation on cell surface expression, peptide specificity, and T-cell recognition of HLA-B*2705. Surface expression was assessed with antibodies recognizing either native or unfolded HLA proteins. Whereas native B*2705 molecules predominated over unfolded ones, this ratio was reversed in the mutant, suggesting lower stability. Comparison of B*2705- and Cys-67 --> Ser-bound peptides revealed that the mutant failed to bind approximately 15% of the B*2705 ligands, while binding as many novel ones. Two peptides with Gln-2 found in both B*2705 and Cys-67 --> Ser are the first demonstration of natural B*2705 ligands lacking Arg-2. Other effects of the mutation on peptide specificity were: 1) average molecular mass of natural ligands higher than for B*2705, 2) bias against small residues at peptide position (P) 1, and 3) increased P2 permissiveness. The results suggest that the Cys-67 --> Ser mutation weakens B pocket interactions, leading to decreased stability of the mutant-peptide complexes. This may be partially compensated by interactions involving bulky P1 residues. The effect of the mutation on allorecognition was consistent with that on peptide specificity. Our results may aid understanding of the pathogenetic role of HLA-B27 in spondyloarthropathy.

Amino Acid Substitution↗

PubMed2001

Minimal alterations in the HLA-B27-bound peptide repertoire induced upon infection of lymphoid cells with Salmonella typhimurium.

OBJECTIVE: To characterize putative changes in the HLA-B27-bound peptide repertoire following infection of lymphoid cells with Salmonella typhimurium, a bacterium known to trigger reactive arthritis in HLA-B27-positive individuals. METHODS: A protocol was developed for efficient large-scale infection of lymphoblastoid cell transfectants expressing HLA-B*2705. HLA-B27-bound peptide pools were isolated from noninfected and infected B*2705+ cells and comparatively analyzed by high-performance liquid chromatography. Peptide-containing chromatographic fractions from noninfected and infected cells were systematically compared by mass spectrometry (MS) to look for putative differences at the level of individual peptides. RESULTS: The presence of B*2705 did not influence S typhimurium invasion, since this was equally efficient in nontransfected or B27-transfected cells. The chromatographic profiles of B*2705-bound peptides from noninfected and infected cells were virtually identical. A total of 808 molecular species were compared by MS. Of these, 807 were present in both infected and noninfected cells. Only one molecular species from infected cells lacked a detectable counterpart in noninfected cells. CONCLUSION: Intracellular infection of lymphoid cells by S typhimurium induces minimal alterations in the HLA-B27-bound peptide repertoire. Minor changes detectable by cytotoxic T lymphocytes, but not easily amenable to direct biochemical analysis, are not ruled out.

Arthritis, Reactive↗

PubMed2001

Large sharing of T-cell epitopes and natural ligands between HLA-B27 subtypes (B2702 and B2705) associated with spondyloarthritis.

HLA-B*2702 is an ankylosing spondylitis-associated allotype that differs from the more common B*2705 at residues 77, 80, and 81, in the peptide-binding site. The diversity and fine specificity of alloreactive cytolytic T-lymphocyte (CTL) raised against B*2702 were analyzed at the clonal level. Significant crossreaction with B*2705 and B*2709 indicated that the three subtypes share numerous T-cell epitopes. However, some epitopes shared by B*2702 and B*2705 were lost in B*2709, which correlates with weaker association of this subtype to disease. Clonal specificities were donor-dependent, indicating that allo-immunogenicity is variable among individuals. Anti-B*2702 CTL were little affected by single mutations mimicking B*2702/B*2705 polymorphism, but the double mutant at positions 77 and 81 was recognized worse than B*2705, suggesting a compensatory effect of residue 80. Thus, HLA-B27 polymorphism modulated alloreactivity through cooperative and compensatory effects on T-cell epitope structure. Comparison of B*2705- and B*2702-bound peptide repertoires revealed that they overlapped by 73% and 81%, respectively. This was larger than B*2702/B*2705 cross-reaction, indicating that HLA-B27 allospecificity is only partially determined by the nature of peptide repertoires. The large sharing of natural ligands and T-cell epitopes is consistent with a pathogenetic role of B*2702 and B*2705 in spondyloarthritis based on antigen presentation.

Antigen Presentation↗

PubMed2000

A high incidence of Shigella-induced arthritis in a primate species: major histocompatibility complex class I molecules associated with resistance and susceptibility, and their relationship to HLA-B27.

The human major histocompatibility complex (MHC) class I gene, HLA-B27, is a strong risk factor for susceptibility to a group of disorders termed spondyloarthropathies. Rodents that express HLA-B27 develop spondyloarthropathies, implicating HLA-B27 in the etiology of these disorders. To determine whether an HLA-B27-like molecule was associated with spondyloarthropathies in nonhuman primates, we analyzed the MHC class I cDNAs expressed in a cohort of rhesus macaques that developed reactive arthritis after an outbreak of shigellosis. We identified several cDNAs with only limited sequence similarity to HLA-B27. Interestingly, one of these MHC molecules had a B pocket identical to that of HLA-B39. Pool sequencing of radiolabeled peptides bound by this molecule demonstrated that, like HLA-B27 and HLA-B39, it could bind peptides with arginine at the second position. However, extensive analysis of the MHC class I molecules in this cohort revealed no statistically significant association between any particular MHC class I allele and susceptibility to reactive arthritis. Furthermore, none of the rhesus MHC class I molecules bore a strong resemblance to HLA-B27, indicating that reactive arthritis can develop in this animal model in the absence of an HLA-B27-like molecule. Surprisingly, there was a statistically significant association between the rhesus macaque MHC A locus allele, Mamu-A*12, and the absence of reactive arthritis following Shigella infection.

Alleles↗

PubMed2000

Limited plasticity in the recognition of peptide epitope variants by an alloreactive CTL clone correlates directly with conservation of critical residues and inversely with peptide length.

Although self-restricted T cells are peptide-specific and can distinguish among closely related ligands, they have some flexibility in the recognition of sequence variants of their natural peptide epitopes. Alloreactive cytotoxic T lymphocytes (CTL) can recognize specific peptides bound to the allo-major histocompatibility complex (MHC) molecule, but their plasticity in the recognition of related peptide variants has not been properly defined. The anti-B*2705 alloreactive CTL 27S69 specifically recognizes a natural octamer ligand of HLA-B*2705. In this study, we tested the recognition of a nested set of epitope variants by this CTL clone. Although none of these peptides was recognized equally as the natural epitope, two of the peptide variants were recognized with only slightly decreased efficiency. Peptide sensitization assays showed that CTL recognition of epitope variants correlated directly with conservation of two non-anchor residues that were critical for recognition of the natural epitope, and inversely with peptide length. Molecular modeling of the peptide variants complexed with B*2705 provided a rational explanation for their differential recognition. Location of the two critical peptide residues at the right three-dimensional space favored efficient recognition by CTL 27S69. The negative effect of increasing peptide length on recognition was due to the bigger bulging surface between the two critical residues, which precluded for optimal interaction with the specific T-cell receptors (TCR). Our results demonstrate that an alloreactive CTL has a degree of plasticity in the recognition of peptide epitope variants that is comparable to that of peptide-specific self-restricted CTL, and define the structural features determining crossreaction among related peptides.

Cloning, Molecular↗

PubMed2000

Peptide specificity of the Amerindian B*3905 allotype: molecular insight into selection mechanisms driving HLA class I evolution in indigenous populations of the Americas.

HLA-B*3905 is apparently restricted to Amerindian populations and presents a wide geographical distribution, from Mexico to Argentina. It differs from B*3901, one of the founder HLA class I alleles of Central and South Amerindians, by a single nucleotide substitution leading to an Asp74Tyr change in the gene product. The peptide specificity of the B*3905 protein was characterized by pool sequence analysis of B*3905-bound peptides and by sequencing of a set of individual ligands, using electrospray ion trap mass spectrometry. The results indicate a double effect of the B*3905 mutation. First, pocket B specificity was shifted towards an increased preference for His at peptide position 2, which is the main anchor for B39-bound peptides, relative to B*3901. Second, at peptide position 3 acidic residues were favored, and aromatic residues disfavored, relative to B*3901. These features approach the peptide specificity of B*3905 to B*3801 and B*1509, allotypes absent from Central and South Amerindians. Together with B*3909, B*3905 is the second HLA-B39 subtype whose polymorphism results in a shift of peptide specificity towards that of HLA-B allotypes absent from these populations. This suggests that HLA-B39 evolution in Central and South America may be an antigen-driven adaptive response, leading to generate antigen-presenting properties absent from the HLA class I repertoire of the ancestral population.

Amino Acid Motifs↗

PubMed2000

HLA-B27 and immunogenetics of spondyloarthropathies.

The arthritogenic peptide hypothesis has inspired research aimed at defining the peptide-presenting properties of HLA-B27 subtypes and their relation to ankylosing spondylitis. Various studies have shed new light on the influence of HLA-B27 polymorphism in modulating peptide binding and T-cell antigen presentation. Moreover, multiple factors along the antigen processing-loading pathway, including tapasin, contribute to shaping the HLA-B27 repertoire. Other studies have revealed significant peptide-binding similarities between HLA-B27 and subtypes of HLA-B39, supporting a role of this antigen in spondyloarthropathy. A putative pathogenetic role of the HLA-B27 heavy chain, initially suggested from studies in transgenic mice, is claimed on the basis of novel, yet circumstantial, evidence concerning an apparently unusual capacity of the heavy chain to form stable homodimers or misfold after biosynthesis. Finally, it appears that arthritogenic infections might downregulate HLA-B27 expression, favoring bacterial survival. The specificity and mechanism of this phenomenon are yet to be defined.

Alleles↗

PubMed2000

A post-translational modification of nuclear proteins, N(G),N(G)-dimethyl-Arg, found in a natural HLA class I peptide ligand.

Presentation of peptides derived from endogenous proteins by class I major histocompatibility complex molecules is essential both for immunological self-tolerance and induction of cytotoxic T-cell responses against intracellular parasites. Despite frequent and diverse post-translational modification of eukaryotic cell proteins, very few class I-bound peptides with post-translationally modified residues are known. Here we describe a natural dodecamer ligand of HLA-B39 (B*3910) derived from an RNA-binding nucleoprotein that carried N(G),N(G)-dimethyl-Arg. Although common among RNA-binding proteins, this modification was not previously known among natural class I ligands. The sequence of this peptide was determined by Edman degradation and electrospray ion trap mass spectrometry. The fragmentation pattern of the dimethyl-Arg side chain observed with this latter technique allowed us to unambiguously assign the isomeric form of the modified residue. The post-translationally modified ligand was a prominent component (1-2%) of the B*3910-bound peptide repertoire. The dimethyl-Arg residue was located in a central position of the peptide, amenable to interacting with T-cell receptors, and most other residues in the middle region of the peptide were Gly. These structural features strongly suggest that the post-translationally modified residue may have a major influence on the antigenic properties of this natural ligand.

Arginine↗

PubMed1999

High T cell epitope sharing between two HLA-B27 subtypes (B2705 and B2709) differentially associated to ankylosing spondylitis.

HLA-B*2705 is strongly associated with ankylosing spondylitis (AS) and reactive arthritis. In contrast, B*2709 has been reported to be more weakly or not associated to AS. These two molecules differ by a single amino acid change: aspartic acid in B*2705 or histidine in B*2709 at position 116. In this study, we analyzed the degree of T cell epitope sharing between the two subtypes. Ten allospecific T cell clones raised against B*2705, 10 clones raised against B*2703 but cross-reactive with B*2705, and 10 clones raised against B*2709 were examined for their capacity to lyse B*2705 and B*2709 target cells. The anti-B*2705 and anti-B*2703 CTL were peptide dependent as demonstrated by their failure to lyse TAP-deficient B*2705-T2 transfectant cells. Eight of the anti-B*2705 and five of the anti-B*2703 CTL clones lysed B*2709 targets. The degree of cross-reaction between B*2705 and B*2709 was donor dependent. In addition, the effect of the B*2709 mutation (D116H) on allorecognition was smaller than the effect of the other naturally occurring subtype change at this position, D116Y. These results demonstrate that B*2705 and B*2709 are the antigenically closest HLA-B27 subtypes. Because allospecific T cell recognition is peptide dependent, our results imply that the B*2705- and B*2709-bound peptide repertoires are largely overlapping. Thus, to the extent to which linkage of HLA-B27 with AS is related to the peptide-presenting properties of this molecule, our results would imply that peptides within a relatively small fraction of the HLA-B27-bound peptide repertoire influence susceptibility to this disease.

Alleles↗

PubMed1999

Nonapeptide analogues containing (R)-3-hydroxybutanoate and beta-homoalanine oligomers: synthesis and binding affinity to a class I major histocompatibility complex protein.

Crystal structures of antigenic peptides bound to class I MHC proteins suggest that chemical modifications of the central part of the bound peptide should not alter binding affinity to the MHC restriction protein but could perturb the T-cell response to the parent epitope. In our effort in designing nonpeptidic high-affinity ligands for class I MHC proteins, oligomers of (R)-3-hydroxybutanoate and(or) beta-homoalanine have been substituted for the central part of a HLA-B27-restricted T-cell epitope of viral origin. The affinity of six modified peptides to the B2705 allele was determined by an in vitro stabilization assay. Four out of the six designed analogues presented an affinity similar to that of the parent peptide. Two compounds, sharing the same stereochemistry (R,R,S,S) at the four stereogenic centers of the nonpeptidic spacer, bound to B2705 with a 5-6-fold decreased affinity. Although the chiral spacers do not strongly interact with the protein active site, there are configurations which are not accepted by the MHC binding groove, probably because of improper orientation of some lateral substituents in the bound state and different conformational behavior in the free state. However we demonstrate that beta-amino acids can be incorporated in the sequence of viral T-cell epitopes without impairing MHC binding. The presented structure-activity relationships open the door to the rational design of peptide-based vaccines and of nonnatural T-cell receptor antagonists aimed at blocking peptide-specific T-cell responses in MHC-associated autoimmune diseases.

3-Hydroxybutyric Acid↗

PubMed1999

High-sensitivity analysis and sequencing of peptides and proteins by quadrupole ion trap mass spectrometry.

This paper describes experience with the commercially available LCQ quadrupole ion trap mass spectrometer applied to the off-line analysis of peptides and proteins. The standard front end of the electrospray probe was replaced with a micromanipulator which, with the aid of a magnifying device, allowed the use of a variety of miniaturized spraying interfaces. The low sample consumption and extended analysis times of these devices were ideally suitable to obtain improved results in terms of sensitivity and mass accuracy. This needed a careful optimization of the number of ions stored inside the trap (ion target parameter) and required spectrum averaging of many scans. A method is presented for the mathematical fitting of ZoomScan spectra to theoretical isotopic distributions, which allowed the mass determination of large peptides with more accuracy than that achieved by conventional deconvolution algorithms. A very simple on-line desalting configuration is also described which needed no external micro-high-performance liquid chromatographic pumps, and can be easily mounted using the built-in syringe delivery system of the LCQ. This set-up allowed extended analysis times of 'in-gel' protein digests in subpicomole amounts. Finally, the multiple fragmentation capabilities of the ion trap were found to be extremely useful for the analysis of peptide modifications such as phosphorylation and for sequencing individual peptides from highly complex MHC-bound peptide pools.

Animals↗

PubMed1999

Modulation at multiple anchor positions of the peptide specificity of HLA-B27 subtypes differentially associated with ankylosing spondylitis.

OBJECTIVE: To investigate the rules governing peptide binding to HLA-B*2705, and to B*2704 and B*2706, which are 2 subtypes differentially associated with ankylosing spondylitis. METHODS: Poly-Ala analogs carrying the HLA-B27 motif Arg-2, and substitutions at anchor positions P1, P3, or Pomega, were used to determine a binding score for each residue at each position. Binding was assessed in a quantitative epitope stabilization assay, where the cell surface expression of HLA-B27 was measured by flow cytometry as a function of peptide concentration. RESULTS: Peptide anchor residues contributed additively to B*2705 binding. About 15% of the natural B*2705 ligands used a deficient P3 or Pomega anchor, but never both, indicating that detrimental anchoring at one of these positions is always compensated by a good anchor at the other one. About 50% of the B*2705 ligands used suboptimal P1 residues. However, this was compensated with optimal P3 and/or Pomega anchoring. Peptides that were longer than decamers used good anchor residues at the 3 positions, suggesting more stringent binding requirements. B*2704 and B*2706 differed in their residue specificity at P1, P3, and Pomega. The rules derived for B*2705 also applied to the known ligands of these 2 subtypes. CONCLUSION: The B*2705, B*2704, and B*2706 peptide repertoires are limited by the allowed residue combinations described in this study. The differential association of B*2704 and B*2706 with spondylarthropathy correlates with differences in their peptide specificity at multiple anchor positions. However, it is now possible to predict the peptide features that determine this differential binding to both subtypes.

Amino Acid Sequence↗

PubMed1999

A molecular insight on the association of HLA-B27 with spondyloarthropathies.

This article focuses on molecular studies concerning HLA-B27 and their relevance for its pathogenetic role in spondyloarthropathy. The peptide binding and T-cell antigen presenting properties of HLA-B27 are discussed, mainly in connection with differential subtype association to ankylosing spondylitis. Molecular studies in transgenic rodents are also considered, with an emphasis on their relevance to the various pathogenetic mechanisms. Recent studies on the putative role of HLA-B27 in bacteria-host interactions are also discussed, as they suggest another level of implication of HLA-B27 in disease whose molecular basis is obscure.

Animals↗

PubMed1999

The South Amerindian allotype HLA-B*3909 has the largest known similarity in peptide specificity and common natural ligands with HLA-B27.

HLA-B*3909 has only been found among South Amerindians, and presumably arose locally in these populations. It differs from B*3901 by a single Tyr to Ser change at position 99. To analyze the influence of this polymorphism on peptide specificity, pool sequence analysis and sequencing of multiple individual ligands from B*3901 and B*3909 were carried out. Both allotypes bind peptides with Arg2 or His2 and nonpolar C-terminal residues. However, whereas His2 is the predominant B*3901 motif, a majority of the B*3909-bound peptides have Arg2. In addition, B*3909 binds peptides with Pro2, and also shows an increased preference for Pro3. In spite of their differences, both subtypes bind overlapping peptide repertoires, as indicated by the identification of several identical ligands from their respective peptide pools. B*3909 is significantly more similar in its peptide specificity to HLA-B27 than B*3901. This is due to the increased preference of B*3909 for Arg2 and to low suitability of HLA-B27 for His2. The similarity between HLA-B27 and B*3909 was confirmed by identification of a natural ligand common to both allotypes. In addition, multiple HLA-B27 ligands bound efficiently B*3909 in vitro. The results indicate that, among the HLA class I allotypes of known peptide specificity, B*3909 is the most similar in its peptide binding properties to HLA-B27, which is absent in South Amerindians. This may have implications for the susceptibility of individuals in these populations to spondyloarthropathies.

Amino Acid Sequence↗

PubMed1999

Long-range effects in protein--ligand interactions mediate peptide specificity in the human major histocompatibilty antigen HLA-B27 (B*2701).

B*2701 differs from all other HLA-B27 subtypes of known peptide specificity in that, among its natural peptide ligands, arginine is not the only allowed residue at peptide position 2. Indeed, B*2701 is unique in binding many peptides with Gln2 in vivo. However, the mutation (Asp74Tyr) responsible for altered selectivity is far away from the B pocket of the peptide binding site to which Gln/Arg2 binds. Here, we present a model that explains this effect. It is proposed that a new rotameric state of the conserved Lys70 is responsible for the unique B*2701 binding motif. This side chain should be either kept away from pocket B through its interaction with Asp74 in most HLA-B27 subtypes, or switched to this pocket if residue 74 is Tyr as in B*2701. Involvement of Lys70 in pocket B would thus allow binding of peptides with Gln2. Binding of Arg2-containing peptides to B*2701 is also possible because Lys70 could adopt another conformation, H-bonded to Asn97, which preserves the same binding mode of Arg2 as in B*2705. This model was experimentally validated by mutating Lys70 into Ala in B*2701. Edman sequencing of the B*2701(K70A) peptide pool showed only Arg2, characteristic of HLA-B27-bound peptides, and no evidence for Gln2. This supports the computational model and demonstrates that allowance of B*2701 for peptides with Gln2 is due to the long-range effect of the polymorphic residue 74 of HLA-B27, by inducing a conformational switch of the conserved Lys70.

Amino Acid Sequence↗

PubMed1998

The same natural ligand is involved in allorecognition of multiple HLA-B27 subtypes by a single T cell clone: role of peptide and the MHC molecule in alloreactivity.

The human alloreactive CTL clone 27S69, raised against B*2705, cross-reacts with B*2702 and B*2703, but not with B*2701, B*2704, B*2706, or B*2710. Its natural epitope was identified by electrospray/ion trap mass spectrometry, as the proteasome-derived RRFFPYYV octamer. This is the first HLA-B27 ligand shown to be immunogenic in alloreactivity. The RRFFPYYVY nonamer, also found in the B*2705-bound peptide pool, was recognized much less efficiently, demonstrating that an alloreactive CTL distinguishes between very similar natural ligands. Molecular modeling suggested that this was due to the different conformation of each peptide in complex with B*2705. B*2702- and B*2703-RMA-S cells were lysed by CTL 27S69 when sensitized with the octamer, demonstrating that cross-reaction with these subtypes is through recognition of the same peptide as in B*2705. B*2704-, B*2706-, and B*2710-RMA-S cells were not sensitized for lysis, in spite of efficient binding of the octamer, indicating that polymorphism in these subtypes directly impairs allorecognition. B*2701-RMA-S and -C1R cells were sensitized for lysis by the octamer, suggesting lack of the endogenous peptide epitope on this subtype. Absence of the octamer in the B*2701-bound peptide pool further suggested that B*2701 polymorphism impairs the generation of this peptide.

Alleles↗

PubMed1998

The pathogenetic role of HLA-B27 in chronic arthritis.

Population and peptide specificity analyses and studies in transgenic rodents support a role of HLA-B27 as an antigen-presenting molecule in spondyloarthropathy. The interplay between HLA-B27 and arthritogenic bacteria on infected cells suggests that HLA-B27 might also influence disease by other mechanisms. Recent genetic advances promise the identification of additional susceptibility genes.

Animals↗