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1.
Structure ; 9(12): 1213-24, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11738047

RESUMO

BACKGROUND: Qa-2 is a nonclassical MHC Ib antigen, which has been implicated in both innate and adaptive immune responses, as well as embryonic development. Qa-2 has an unusual peptide binding specificity in that it requires two dominant C-terminal anchor residues and is capable of associating with a substantially more diverse array of peptide sequences than other nonclassical MHC. RESULTS: We have determined the crystal structure, to 2.3 A, of the Q9 gene of murine Qa-2 complexed with a self-peptide derived from the L19 ribosomal protein, which is abundant in the pool of peptides eluted from the Q9 groove. The 9 amino acid peptide is bound high in a shallow, hydrophobic binding groove of Q9, which is missing a C pocket. The peptide makes few specific contacts and exhibits extremely poor shape complementarity to the MHC groove, which facilitates the presentation of a degenerate array of sequences. The L19 peptide is in a centrally bulged conformation that is stabilized by intramolecular interactions from the invariant P7 histidine anchor residue and by a hydrophobic core of preferred secondary anchor residues that have minimal interaction with the MHC. CONCLUSIONS: Unexpectedly, the preferred secondary peptide residues that exhibit tenuous contact with Q9 contribute significantly to the overall stability of the peptide-MHC complex. The structure of this complex implies a "conformational" selection by Q9 for peptide residues that optimally stabilize the large bulge rather than making intimate contact with the MHC pockets.


Assuntos
Apresentação de Antígeno , Genes MHC Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/química , Animais , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Ligação de Hidrogênio , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/metabolismo
2.
Immunogenetics ; 53(6): 455-67, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11685456

RESUMO

We reexamined expression patterns of one of the best characterized mouse class Ib MHC molecules, Qa-2. Transcripts encoding glycosylphosphatidylinositol-linked and soluble forms of Qa-2 are expressed in all organs except brain. The membrane-bound Qa-2 proteins are detectable, to varying degrees, in many cell types of immunological interest: on professional antigen-presenting cells capable of inducing anti-Qa-2 allogeneic responses, on thymic epithelial cells essential for T-cell positive selection, on mature as well as immature thymocytes, in immunologically privileged sites (testis/spermatazoa), and on cells implicated in mucosal immunity (lymphoid-derived and epithelial gut cells and hepatocytes). Although Qa-2 has a nearly ubiquitous tissue distribution similar to H2-Kb and Db molecules, the relative levels of Qa-2 and class Ia displayed on cell surfaces vary in a cell-specific fashion. Analyses of primary cell lines derived from normal mouse tissues also support the conclusion that Qa-2 is present in all cells that can express class Ia antigens. In contrast, tumor lines from Qa-2-positive mice are frequently Qa-2 deficient, suggesting that the Qa-2-negative phenotype of malignant cells is selected in vivo.


Assuntos
Células da Medula Óssea/imunologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Animais , Células Apresentadoras de Antígenos/imunologia , Linhagem Celular , Hepatócitos/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Mucosa Intestinal/imunologia , Masculino , Camundongos , Neoplasias/metabolismo , RNA Mensageiro/biossíntese , Testículo/imunologia , Timo/imunologia , Distribuição Tecidual , Transcrição Gênica , Células Tumorais Cultivadas
3.
J Immunol ; 164(4): 1906-15, 2000 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-10657640

RESUMO

The biological properties of the nonclassical class I MHC molecules secreted into blood and tissue fluids are not currently understood. To address this issue, we studied the murine Q10 molecule, one of the most abundant, soluble class Ib molecules. Mass spectrometry analyses of hybrid Q10 polypeptides revealed that alpha1alpha2 domains of Q10 associate with 8-9 long peptides similar to the classical class I MHC ligands. Several of the sequenced peptides matched intracellularly synthesized murine proteins. This finding and the observation that the Q10 hybrid assembly is TAP2-dependent supports the notion that Q10 groove is loaded by the classical class I Ag presentation pathway. Peptides eluted from Q10 displayed a binding motif typical of H-2K, D, and L ligands. They carried conserved residues at P2 (Gly), P6 (Leu), and Pomega (Phe/Leu). The role of these residues as anchors/auxiliary anchors was confirmed by Ala substitution experiments. The Q10 peptide repertoire was heterogeneous, with 75% of the groove occupied by a multitude of diverse peptides; however, 25% of the molecules bound a single peptide identical to a region of a TCR V beta-chain. Since this peptide did not display enhanced binding affinity for Q10 nor does its origin and sequence suggest that it is functionally significant, we propose that the nonclassical class I groove of Q10 resembles H-2K, D, and L grooves more than the highly specialized clefts of nonclassical class I Ags such as Qa-1, HLA-E, and M3.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Fígado/imunologia , Oligopeptídeos/metabolismo , Transportadores de Cassetes de Ligação de ATP/fisiologia , Animais , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Oligopeptídeos/isolamento & purificação , Ligação Proteica/imunologia , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Solubilidade
4.
J Immunol ; 159(6): 2771-81, 1997 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-9300698

RESUMO

Studies of naturally processed peptides eluted from membrane-bound and soluble isoforms of murine class Ib Qa-2 molecules determined several features of these ligands, such as the conserved nonameric length and the preferred usage of specific residues at four to six of nine peptide positions. The structural information derived from these studies proved insufficient to distinguish between two interpretations: 1) that Qa-2 are peptide receptors of higher stringency than ordinary class I molecules, and 2) that Qa-2 molecules, like classical class I Ags, bind diverse arrays of peptides. We have addressed this issue by a systematic analysis of peptide residues involved in the binding of membrane-bound Qa-2 molecule, MQ9b. The optimal binding of synthetic peptides in vitro occurs at neutral pH. Two dominant anchors are required for peptide binding to MQ9b: His at position 7 and a hydrophobic residue, Leu, Ile, or Phe, at position 9. In addition, one or two auxiliary anchors participate in binding. The identity and the position of the auxiliary anchors differ from peptide to peptide, suggesting that the binding motifs defined from pool sequencing are composed of many superimposed alternative motifs present in individual peptides. The number of anchors used by Qa-2 peptides is similar to that found in ligands of classical class I Ags. Consequently, the Qa-2 are predicted to bind large repertoires of self and nonself peptides. In support of this interpretation we demonstrate that MQ9b binds strongly 5 of 17 motif-positive, pathogen-derived synthetic peptides.


Assuntos
Antígenos de Histocompatibilidade Classe I/química , Animais , Sítios de Ligação , Linhagem Celular , Antígenos de Histocompatibilidade Classe I/metabolismo , Camundongos , Peptídeos/química , Ligação Proteica , Conformação Proteica
6.
J Immunol ; 152(11): 5268-74, 1994 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-8189046

RESUMO

The assembly of class Ia MHC Ags is thought to occur in the endoplasmic reticulum (ER) where H chains, beta 2m, and peptides come together to form trimers. Several types of proteins are implicated in the regulation of class Ia MHC assembly, including: 1) TAP1/TAP2 transporters, which translocate peptides derived from naturally processed endogenous proteins from the cytosol into the ER and which are necessary for expression of "peptide-filled" class Ia Ags, and 2) calnexin, a chaperone protein, which was proposed to retain unassembled class Ia chains in the ER. In our study, we examined if the expression of class Ib Qa-2 molecules depends on the TAP1/TAP2 peptide delivery system. The glycosylphosphatidylinositol-linked GPIQa-2 and soluble SQa-2 molecules lack transmembrane regions and consensus calnexin binding sites. Because of these structural features, they were thought to differ from class Ia Ags in cellular trafficking pathways and peptide-binding mechanisms. We find that in TAP2 negative RMA-S cells, the great majority of GPIQa-2 and SQa-2 behave as "empty" heterodimers: They cannot maintain stable conformations at 37 degrees C, but their half-lives can be significantly extended by reducing the temperature to 26 degrees C. These results suggest that the Qa-2 binding peptides are delivered to Qa-2 molecules in a manner similar to the class Ia MHC Ag system and, therefore, that both GPIQa-2 and SQa-2 may be assembled in the ER. Detection of a small population of heat-resistant Qa-2 molecules in RMA-S is indicative of an alternative, but minor, peptide delivery pathway, or "leakiness" of the RMA-S mutation.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Transporte/fisiologia , Glicosilfosfatidilinositóis/fisiologia , Antígenos de Histocompatibilidade Classe I/análise , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transporte Biológico , Linhagem Celular , Retículo Endoplasmático/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Temperatura , Transfecção
7.
Proc Natl Acad Sci U S A ; 91(5): 1883-7, 1994 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-8127900

RESUMO

Class Ib Qa-2 molecules are expressed in tissue culture cells as approximately 40-kDa membrane-bound, glycophosphatidylinositol-linked antigens and as approximately 39-kDa soluble polypeptides. Recently, alternative splicing events which delete exon 5 from a portion of Qa-2 transcripts were demonstrated to give rise to truncated secreted Qa-2 molecules in transfected cell lines. To determine whether this mechanism operates in vivo and to find out whether Qa-2 can be detected in soluble form in circulation, murine blood samples were analyzed. Critical to these experiments was preparation of an anti-peptide antiserum against an epitope encoded by a junction of exon 4 and exon 6. We find that supernatants of splenocytes cultured in vitro as well as serum or plasma contain two forms of soluble Qa-2 molecules. One form corresponds to a secreted molecule translated from transcripts from which exon 5 has been deleted; the other is derived from membrane-bound antigens or their precursors. The levels of both soluble forms of Qa-2 are inducible upon stimulation of the immune system, suggesting an immunoregulatory role for these molecules or for the mechanism leading to the reduction of cell-associated Qa-2 antigens in vivo.


Assuntos
Processamento Alternativo , Antígenos de Histocompatibilidade Classe I/sangue , Antígenos de Histocompatibilidade Classe I/genética , Complexo Principal de Histocompatibilidade , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Sequência de Bases , Concanavalina A/farmacologia , DNA Complementar/genética , Éxons , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Poli I-C/farmacologia , Coelhos , Solubilidade , Baço/imunologia , Transcrição Gênica
8.
J Exp Med ; 179(2): 579-88, 1994 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-8294869

RESUMO

Unlike the highly polymorphic major histocompatibility complex (MHC) class Ia molecules, which present a wide variety of peptides to T cells, it is generally assumed that the nonpolymorphic MHC class Ib molecules may have evolved to function as highly specialized receptors for the presentation of structurally unique peptides. However, a thorough biochemical analysis of one class Ib molecule, the soluble isoform of Qa-2 antigen (H-2SQ7b), has revealed that it binds a diverse array of structurally similar peptides derived from intracellular proteins in much the same manner as the classical antigen-presenting molecules. Specifically, we find that SQ7b molecules are heterodimers of heavy and light chains complexed with nonameric peptides in a 1:1:1 ratio. These peptides contain a conserved hydrophobic residue at the COOH terminus and a combination of one or more conserved residue(s) at P7 (histidine), P2 (glutamine/leucine), and/or P3 (leucine/asparagine) as anchors for binding SQ7b. 2 of 18 sequenced peptides matched cytosolic proteins (cofilin and L19 ribosomal protein), suggesting an intracellular source of the SQ7b ligands. Minimal estimates of the peptide repertoire revealed that at least 200 different naturally processed self-peptides can bind SQ7b molecules. Since Qa-2 molecules associate with a diverse array of peptides, we suggest that they function as effective presenting molecules of endogenously synthesized proteins like the class Ia molecules.


Assuntos
Antígenos H-2/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Antígenos H-2/química , Antígenos de Histocompatibilidade Classe I/química , Tolerância Imunológica , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Mapeamento por Restrição , Solubilidade , Células Tumorais Cultivadas , Água/química
9.
Int J Cancer ; 43(5): 768-73, 1989 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-2654024

RESUMO

The 7F7-antigen is a widespread 85-kDa membrane adherence molecule involved in heterotypic adhesion between PHA-blasts and fibroblasts. Immunofluorescence analysis of COS cells transfected with clone pICAM-I indicated that 7F7-antigen is identical with ICAM-I, the ligand for the contact molecule LFA-I. We have investigated the expression of ICAM-I on several human carcinomas. Tumor cells in most carcinomas, with the exception of squamous-cell carcinomas, expressed very little ICAM-I or none at all. In contrast, marked expression of this molecule was observed on fibrous tissue in the vicinity of carcinoma cells, its intensity correlating with lymphatic infiltration in these tumors. We also examined the expression of ICAM-I on carcinoma cell lines and its induction by treatment with interferon-gamma (IFN-gamma). The inducibility of ICAM-I expression on cultured fibroblasts by several lymphokines suggests that the expression of ICAM-I in the vicinity of carcinoma cells is effected by lymphokines produced by activated lymphocytes/macrophages within the tumor.


Assuntos
Antígenos de Superfície/análise , Glicoproteínas de Membrana/análise , Proteínas de Neoplasias/análise , Neoplasias/patologia , Antígenos de Superfície/genética , Adesão Celular , Moléculas de Adesão Celular , Linhagem Celular , Feminino , Fibroblastos/citologia , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Transfecção
10.
Immunogenetics ; 29(5): 297-307, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2714852

RESUMO

Several new HLA-B (B8, B51, Bw62)- and HLA-C (Cw6, Cw7)-specific genes were isolated either as genomic cosmid or cDNA clones to study the diversity of HLA antigens. The allele specificities were identified by sequence analysis in comparison with published HLA-B and -C sequences, by transfection experiments, and Southern and northern blot analysis using oligonucleotide probes. Comparison of the classical HLA-A, -B, and -C sequences reveals that allele-specific substitutions seem to be rare events. HLA-B51 codes only for one allele-specific residue: arginine at position 81 located on the alpha 1 helix, pointing toward the antigen binding site. HLA-B8 contains an acidic substitution in amino acid position 9 on the first central beta sheet which might affect antigen binding capacity, perhaps in combination with the rare replacement at position 67 (F) on the alpha 1 helix. HLA-B8 shows greatest homology to HLA-Bw42, -Bw41, -B7, and -Bw60 antigens, all of which lack the conserved restriction sites Pst I at position 180 and Sac I at position 131. Both sites associated with amino acid replacements seem to be genetic markers of an evolutionary split of the HLA-B alleles, which is also observed in the leader sequences. HLA-Cw7 shows 98% sequence identity to the JY328 gene. In general, the HLA-C alleles display lower levels of variability in the highly polymorphic regions of the alpha 1 and alpha 2 domains, and have more distinct patterns of locus-specific residues in the transmembrane and cytoplasmic domains. Thus we propose a more recent origin for the HLA-C locus.


Assuntos
Evolução Biológica , Genes MHC Classe I , Variação Genética , Antígenos HLA-B/genética , Antígenos HLA-C/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Humanos , Dados de Sequência Molecular
11.
Immunol Lett ; 6(6): 327-9, 1983 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-6629427

RESUMO

In a population of E-rosette forming human peripheral blood cells, about 20% of the rosettes were formed by monocytes. About 60% of the peripheral blood monocytes bound SRBC in a standard E-rosette test.


Assuntos
Monócitos/imunologia , Formação de Roseta , Animais , Eritrócitos/metabolismo , Humanos , Monócitos/metabolismo , Ovinos/sangue
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