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1.
A panel of unique HLA-A2 mutant molecules define epitopes recognized by HLA-A2-specific antibodies and cytotoxic T lymphocytes 总被引:7,自引:0,他引:7
K T Hogan C Clayberger E J Bernhard S F Walk J P Ridge P Parham A M Krensky V H Engelhard 《Journal of immunology (Baltimore, Md. : 1950)》1989,142(6):2097-2104
HLA-A2.1 and HLA-A2.3, which differ from one another at residues 149, 152, and 156, can be distinguished by the mAb CR11-351 and many allogeneic and xenogeneic CTL. Site-directed mutagenesis was used to incorporate several different amino acid substitutions at each of these positions in HLA-A2.1 to evaluate their relative importance to serologic and CTL-defined epitopes. Recognition by mAb CR11-351 was completely lost when Thr but not Pro was substituted for Ala149. A model to explain this result based on the 3-dimensional structure of HLA-A2.1 is presented. In screening eight other mAb, only the substitutions of Pro for Val152 or Gly for Leu156 led to the loss of mAb binding. Because other non-conservative substitutions at these same positions had no effect, these results suggest that the loss of serologic epitopes is in many cases due to a more indirect effect on molecular conformation. Specificity analysis using 28 HLA-A2.1-specific alloreactive and xenoreactive CTL clones showed 19 distinct patterns of recognition. The epitopes recognized by alloreactive CTL clones demonstrated a pronounced effect by all substitutions at residue 152, including the very conservation substitution of Ala for Val. Overall, the most disruptive substitution at amino acid residue 152 was Pro, followed by Glu, Gln, and then Ala. In contrast, substitutions at 156 had little or no effect on allogeneic CTL recognition, and most clones tolerated either Gly, Ser, or Trp at this position. Similar results were seen using a panel of murine HLA-A2.1-specific CTL clones, except that substitutions at position 156 had a greater effect. The most disruptive substitution was Trp, followed by Ser and then Gly. In addition, when assessed on the entire panel of CTL, the effects of Glu and Gln substitutions at position 152 demonstrated that the introduction of a charge difference is no more disruptive than a comparable change in side chain structure that does not alter charge. Taken together, these results indicate that the effect of amino acid replacements at positions 152 and 156 on CTL-defined epitopes depends strongly on the nature of the substitution. Thus, considerable caution must be exercised in evaluating the significance of particular positions on the basis of single mutations. Nonetheless, the more extensive analysis conducted here indicates that there are differences among residues in the class I Ag "binding pocket," with residue 152 playing a relatively more important role in formation of allogeneic CTL-defined epitopes than residue 156. 相似文献
2.
M B Brenner J McLean S Y Yang J J van der Poel D Pious J L Strominger 《Journal of immunology (Baltimore, Md. : 1950)》1985,135(1):384-390
A human alloimmune cytotoxic T lymphocyte (CTL) clone (4E4) was generated against the HLA-A2 molecule. Lysis of 51Cr-labeled HLA-A2 target cells was blocked by monoclonal antibodies (mAb), including mAb PA2.1 (anti-HLA-A2), mAb BB7.2 (anti-HLA-A2), mAb 4B (anti-HLA-A2-plus-A28), mAb MA2.1 (anti-HLA-A2-plus-B17), and mAb W6/32 (anti-HLA-A,B,C), which are directed against different serologic epitopes on the HLA-A2 molecule. However, HLA-A2 mutant lines lacking the serologic epitope recognized by mAb BB7.2 (anti-HLA-A2) were efficiently lysed by CTL 4E4. Thus, although mAb may block cytolysis, the HLA-A2 epitope recognized the 4E4 CTL clone is distinct from the HLA-A2-specific epitope recognized by serologic reagents. Moreover, analysis of HLA-A2 population variants revealed that only the predominant HLA-A2.1 subtype molecule was recognized by CTL 4E4. No cross-reactivity on other, biochemically related HLA-A2 population subtypes was observed, including HLA-A2.2 cells (Hill, CVE, ZYL, M7), HLA-A2.3 cells (TENJ, DK1), or HLA-A2.4 cells (CLA, KNE). This CTL clone appears to recognize a single epitope and, like monoclonal antibody counterparts, can be used to discriminate among immunogenic cellular and serologic epitopes on closely related HLA-A2 molecules. On the basis of the known sequence changes in mutant and subtype HLA-A2 molecules, it appears that the sequence spanning residues 147 to 157 may be critical for cellular recognition of this Class I MHC molecule. 相似文献
3.
K T Hogan C Clayberger E J Bernhard S F Walk J P Ridge P Parham A M Krensky V H Engelhard 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(7):2519-2525
Site-directed mutagenesis of HLA-A2.1 has been used to identify the amino acid substitutions in HLA-A2.3 that are responsible for the lack of recognition of the latter molecule by the HLA-A2/A28 specific antibody, CR11-351, and by HLA-A2.1 specific CTL. Three genes were constructed that encoded HLA-A2 derivatives containing one of the amino acids known to occur in HLA-A2.3: Thr for Ala149, Glu for Val152, and Trp for Leu156. Three additional genes were constructed that encoded the different possible combinations of two amino acid substitutions at these residues. Finally, a gene encoding all three substitutions and equivalent to HLA-A2.3 was constructed. These genes were transfected into the class I negative, human cell line Hmy2.C1R. Analysis of this panel of cells revealed that recognition by the antibody CR11-351 was completely lost when Thr was substituted for Ala149, whereas substitutions at amino acids 152 and 156, either singly or in combination, had no effect on the binding of this antibody. The epitopes recognized by the allogeneic and xenogeneic HLA-A2.1 specific CTL clones used in this study were all affected by either one or two amino acid substitutions. Of those epitopes sensitive to single amino acid changes, none were affected by the substitution of Thr for Ala149, whereas all of them were affected by at least one of the substitutions of Glu for Val 152 or Trp for Leu156. Overall, amino acid residue 152 exerted a stronger effect on the epitopes recognized by HLA-A2.1 specific CTL than did residue 156. Of those epitopes affected only by multiple amino acid substitutions, double substitutions at residues 149 and 152 or at 152 and 156 resulted in a loss of recognition, whereas a mutant with substitutions at residues 149 and 156 was recognized normally. This reemphasizes the importance of residue 152 and indicates that residue 149 can affect epitope formation in conjunction with another amino acid substitution. These results are discussed in the context of current models for the recognition of alloantigens and in light of the recently published three-dimensional structure of the HLA-A2.1 molecule. 相似文献
4.
We used site-directed mutagenesis of HLA-DR beta-chains to localize the binding sites for two polymorphic DR-binding mAb to residues in the first and second external domains, respectively. Transfer of three first domain alpha-helical residues, G73, R74 and N77, normally present in DR3a and DRw52a, to a DR4 beta-chain was sufficient for recognition of this mutant DR molecule by a DR3-specific mAb, NDS 9. A polymorphism controlling the binding of a DR4-specific mAb, GS 359-13F10, was mapped to a tyrosine at position 96 of the DR4 beta-chain second domain by the construction fo a chimeric DR molecule containing a DR2-first domain and DR4-second domain. The mapping of these two polymorphic epitopes to specific positions on the DR beta-chain will allow further structural and functional analysis of the DR molecule. 相似文献
5.
N Shimojo E P Cowan V H Engelhard W L Maloy J E Coligan W E Biddison 《Journal of immunology (Baltimore, Md. : 1950)》1989,143(2):558-564
Previous studies have demonstrated that certain amino acid substitutions in the alpha two domain at positions 152 and 156 in the alpha two helix of the HLA-A2 molecule can affect presentation of the influenza virus matrix peptide M1 55-73 without abolishing binding of the M1 peptide. HLA-A2.1-restricted M1 55-73 peptide-specific CTL lines obtained from almost all HLA-A2.1+ individuals fail to recognize the M1 peptide presented by site-directed mutants of HLA-A2 that have either a Val----Ala or Val----Gln substitution at position 152 or a Leu----Trp substitution at position 156. Only one HLA-A2+ individual (donor Q66, HLA-A2,-B53,-B63) has been found who is able to generate a unique repertoire of HLA-A2-restricted M1 peptide-specific CTL that can recognize peptide presented by HLA-A2 mutants with either an Ala or Gln substitution at position 152 or a Trp substitution at position 156. These Q66 M1 peptide-specific CTL could be selected by stimulation with M1 peptide-pulsed transfectants that express the mutant HLA-A2 gene with the Trp substitution at 156. To determine if the presence of the unique CTL repertoire could be attributed to a variant HLA-A2 molecule in Q66, sequences were determined from polymerase chain reaction-amplified segments of the HLA-A2 RNA. Two different HLA-A2 genes were found expressed in Q66 cells: one is identical to HLA-A2.1 and the other is identical to HLA-A2.2F (Gln----Arg at position 43, Val----Leu at position 95, and Leu----Trp at position 156). These results demonstrate that a different CTL repertoire specific for HLA-A2 plus the M1 55-73 peptide is generated in an individual that expresses both HLA-A2.1 and HLA-A2.2F compared to individuals who express HLA-A2.1 alone, and that the unique repertoire can be selected by the presence of an HLA-A2 molecule with a single amino acid substitution at position 156. 相似文献
6.
D H Mattson N Shimojo E P Cowan J J Baskin R V Turner B D Shvetsky J E Coligan W L Maloy W E Biddison 《Journal of immunology (Baltimore, Md. : 1950)》1989,143(4):1101-1107
Crystallographic studies of the HLA-A2 molecule have led to the assignment of a putative peptide binding site that consists of a groove with a beta-pleated sheet floor bordered by two alpha-helices. A CTL-defined variant of HLA-A2, termed HLA-A2.2F, differs from the common A2.1 molecule by three amino acids: a Leu to Trp substitution at position 156 in the alpha-2 helix, a Val to Leu substitution at position 95 in the beta-sheet floor of the groove, and a Gln to Arg substitution at position 43 in a loop outside of the groove. Another HLA-A2 variant, termed CLA, has a single Phe to Tyr substitution at position 9 that is sterically located adjacent to position 95 in the beta-sheet floor of the groove. We have determined which of the amino acid substitutions at positions 9, 43, 95, or 156 could individually affect recognition by panels of A2.1 allospecific and A2.1-restricted influenza viral matrix peptide-specific CTL lines, using a panel of site-directed mutants and CLA. Recognition by allospecific CTL lines was generally unaffected by any one of the amino acid substitutions, but was eliminated by the double substitution at positions 95 and 156. Allorecognition by some CTL lines was eliminated by a single substitution at position 9 or 95. In contrast, recognition by A2.1-restricted matrix peptide specific CTL was totally eliminated by a single substitution at position 9 or 156. The substitution at position 43 in a loop away from the peptide binding groove had no effect on allorecognition or matrix peptide recognition. These results indicate that amino acid residues in the floor or alpha-2 helical wall of the peptide binding groove of the HLA-A2 molecule can differentially affect allorecognition and viral peptide recognition. 相似文献
7.
M H Newberg J P Ridge D R Vining R D Salter V H Engelhard 《Journal of immunology (Baltimore, Md. : 1950)》1992,149(1):136-142
The alpha 1 and alpha 2 domains of the class I MHC molecule constitute the putative binding site for processed peptides and the TCR, although the alpha 3 domain has been implicated as a binding site for the CD8 molecule. Species specificity in the binding of CD8 to the alpha 3 domain has been suggested as an explanation for the low xenogeneic T cell response to class I molecules, but results on this point have been conflicting and controversial. We have addressed this issue using CTL lines from HLA-A2.1 transgenic mice that specifically recognize and lyse A2.1-expressing cells infected with influenza A/PR/8 or pulsed with influenza matrix peptide M1(57-68). Species specificity was examined using transfectants that expressed hybrid molecules containing the alpha 1 and alpha 2 domains from HLA-A2.1 and the alpha 3 domain from a murine class I molecule. Lower levels of M1(57-68) peptide were required to sensitize L cell transfectants expressing a chimera that contained an H-2Dd alpha 3 domain than targets expressing the intact A2.1 molecule. However, at high doses of peptide, lysis of these two targets was similar. However, no reproducible difference in sensitization was observed using EL4 or Jurkat transfectants expressing A2.1 or A2.1 chimeric molecules that contained an H-2Kb alpha 3 domain. In all cases, however, lysis of peptide-pulsed A2.1 expressing targets was more sensitive to inhibition with anti-CD8 mAb than lysis of cells expressing these chimeric molecules. Thus, under suboptimal conditions such as low Ag density or in the presence of anti-CD8 mAb, these CTL preferentially recognize class I molecules with a murine alpha 3 domain. This suggests that there is some species specificity in the interaction of CD8 with the alpha 3 domain of the class I molecule. However, CTL recognition was inhibited by point mutations in the alpha 3 domain of HLA-A2.1 that have been shown to inhibit binding of human CD8 and recognition by human CTL, suggesting that murine CD8 interacts to some degree with human alpha 3 domains, and that similar alpha 3 domain residues may be important for murine and human CD8 binding. The relevance of these results to an understanding of low xenogeneic responses is discussed. 相似文献
8.
Cytotoxic T lymphocyte-defined epitope differences between HLA-A2.1 and HLA-A2.2 map to two distinct regions of the molecule 总被引:3,自引:0,他引:3
K T Hogan C Clayberger A X Le S F Walk J P Ridge P Parham A M Krensky V H Engelhard 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(11):4005-4011
Hemi-exon shuffling and site-directed mutagenesis have been used to determine which amino acid differences between HLA-A2.1 and HLA-A2.2 alter the CTL-defined epitopes on these two molecules. Two genes were constructed that encode novel molecules in which the effect of amino acid differences at residues 9, 43, and 95, or at residue 156 could be separately evaluated. Using both human and murine CTL that were specific for either HLA-A2.1 or HLA-A2.2, four types of epitopes were identified: 1) epitopes that were insensitive to substitutions at either residues 9, 43, and 95, or residue 156 but were lost when all four positions were changed; 2) epitopes that were dependent on the residues 9, 43, 95, but not residue 156; 3) epitopes that were dependent on residue 156, but not amino acid residues 9, 43, and 95; and 4) epitopes that were dependent on residues 9, 43, and 95, as well as amino acid residue 156. Overall, there was a roughly equal distribution of clones recognizing each of these types of epitopes. Additional molecules were constructed by hemi-exon shuffling between the HLA-A2.2 and HLA-A2.3 genes, and by site-directed mutagenesis, to analyze the epitopes recognized by two HLA-A2.2/A2.1 cross-reactive murine CTL that do not recognize HLA-A2.3. Although the epitopes recognized by these CTL were unaffected by changes occurring at residues 9, 43, and 95, or at residues 149, 152, and 156 alone, simultaneous changes in both of these regions acted in concert to destroy the epitopes. Both of the CTL recognized epitopes that were lost when substitutions were made at residues 9, 43, 95, 149, and 152. The epitope recognized by one of the CTL was also destroyed by the substitution of residues 9, 43, 95, 152, and 156. Overall, these results indicate that residues 9, 43, and 95, as well as residues in the alpha-helical region of the molecule, are all capable of contributing to the definition of the epitopes recognized by HLA-A2.1- and HLA-A2.2-specific CTL. They further indicate that some epitopes can be mapped to a particular region of the molecule, whereas other epitopes are formed through a complex interaction of residues in distant regions of the molecule. 相似文献
9.
Giovanna Lombardi Masanori Matsui Robert Moots Gerald Aichinger Sid Sidhu Richard Batchelor Jeff Frelinger Robert Lechler 《Immunogenetics》1991,34(3):149-156
The regions of the HLA-A2 molecule controlling anti-A2 alloreactivity were explored using naturally occurring allelic variants of HLA-A, and a panel of transfectants expressing the products of A2.1 genes that had been mutated at multiple positions encoding residues in the 2 domain -helix. As a means of detecting distant conformational effects, these altered A2.1 molecules were also examined serologically. Amino acid substitutions at the carboxy-terminal end of the 2 domain -helix led to diminished staining with the monoclonal antibody (mAb) MA2.1. The epitope for this antibody has previously been mapped to the 1 domain -helix (residues 62–65). This suggests that interdomain contacts may cause conformational alteration, and that mutants can have distant, as well as local effects. Of the 24 positions where substitutions were made, only six led to loss of the anti-A2 alloresponse by the three clones and three lines that were tested. In addition, the mutations that altered the MA2.1 epitope, located on the 1 domain -helix, did not inhibit allorecognition. This suggests that a limited number of regions on the A2.1 molecule are responsible for allodeterminant expression. The most influential substitutions were those at positions 152, 154, 162, and 166. It is notable that three of these are predicted to be T-cell receptor (Tcr)-contacting residues, and one (152) to contribute to peptide binding. These results suggest that the specificity of alloreactive T cells is determined by exposed polymorphisms, directly contacted by the Tcr, and by concealed polymorphisms which influence peptide binding. 相似文献
10.
The complete primary structure of HLA-Bw58 总被引:12,自引:0,他引:12
Serological studies indicate that HLA-B17 molecules are unusually cross-reactive with products of the HLA-A locus. In particular, a mouse monoclonal antibody MA2.1 defines an epitope that is shared by HLA-A2 and the two subtypes (Bw57 and Bw58) of B17. To investigate these relationships at the structural level, we have isolated a gene coding for Bw58 from the WT49 B cell line. The gene was transfected into mouse L cells and its protein product was characterized with a panel of monoclonal anti-HLA antibodies. The nucleotide sequence of 3520 base pairs of DNA encompassing the seven exons coding for Bw58 and associated introns was determined. The deduced protein sequences for Bw58 and eight other HLA-A,B,C molecules were compared. In the first polymorphic domain (alpha 1), Bw58 is unusual in that it is as homologous to HLA-A locus products as to HLA-B locus products. In the second polymorphic domain (alpha 2), Bw58 has greater homology to B locus products. In the alpha 1 domain of Bw58, small segments of amino acid and nucleotide sequence homology with A2 (residues 62-65) and with Aw24 (residues 75-83) are found in the major region of polymorphic diversity (residues 62-83). These similarities provide structural correlates for the serological relationships between Bw58 and A locus molecules, with residues 62-65 possibly being involved in the MA2.1 epitope. From comparisons of four HLA-A and four HLA-B sequences, there is a difference in the patterns of variation for A and B locus molecules. For B locus molecules there is greater variation in the alpha 1 domain than in the alpha 2 domain. For A locus molecules, variation in the two domains is similar and like that for B locus alpha 2 domains. In comparison to other HLA-A,B,C genes, novel inverted repeat sequences were found in the nucleotide sequence of HLA-Bw58. These sequences flank the putative RNA splicing sites at the 3' end of the exons encoding the alpha 2 and alpha 3 protein domains. 相似文献
11.
Humphries JD Askari JA Zhang XP Takada Y Humphries MJ Mould AP 《The Journal of biological chemistry》2000,275(27):20337-20345
Different beta(1) integrins bind Arg-Gly-Asp (RGD) peptides with differing specificities, suggesting a role for residues in the alpha subunit in determining ligand specificity. Integrin alpha(5)beta(1) has been shown to bind with high affinity to peptides containing an Arg-Gly-Asp-Gly-Trp (RGDGW) sequence but with relatively low affinity to other RGD peptides. The residues within the ligand-binding pocket that determine this specificity are currently unknown. A cyclic peptide containing the RGDGW sequence was found to strongly perturb the binding of the anti-alpha(5) monoclonal antibody (mAb) 16 to alpha(5)beta(1). In contrast, RGD peptides lacking the tryptophan residue acted as weak inhibitors of mAb 16 binding. The epitope of mAb 16 has previously been localized to a region of the alpha(5) subunit that contains Ser(156)-Trp(157). Mutation of Trp(157) (but not of Ser(156) or surrounding residues) to alanine blocked recognition of mAb 16 and perturbed the high affinity binding of RGDGW-containing peptides to alpha(5)beta(1). The same mutation also abrogated recognition of the alpha(5)beta(1)-specific ligand peptide Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Based on these findings, we propose that Trp(157) of alpha(5) participates in a hydrophobic interaction with the tryptophan residue in RGDGW, and that this interaction determines the specificity of alpha(5)beta(1) for RGDGW-containing peptides. Since the RGD sequence is recognized predominantly by amino acid residues on the beta(1) subunit, our results suggest that Trp(157) of alpha(5) must lie very close to these residues. Our findings therefore provide new insights into the structure of the ligand-binding pocket of alpha(5)beta(1). 相似文献
12.
Comparison between two peptide epitopes presented to cytotoxic T lymphocytes by HLA-A2. Evidence for discrete locations within HLA-A2 总被引:7,自引:0,他引:7
P A Robbins L A Lettice P Rota J Santos-Aguado J Rothbard A J McMichael J L Strominger 《Journal of immunology (Baltimore, Md. : 1950)》1989,143(12):4098-4103
An influenza B virus nucleoprotein (BNP) peptide, residues 82-94, defined by limited sequence homology with an HLA-A2-restricted peptide from influenza A matrix protein, was recognized by HLA-A2-restricted CTL. Reciprocal inhibition of T cell recognition by the two peptides suggest that the BNP peptide may have lower avidity for HLA-A2 molecules than the matrix peptide. The interaction between this peptide and HLA-A2 was explored by studying the CTL recognition of BNP 82-94 presented by mutant HLA-A2 molecules. Mutations at residues 9, 99, 70, 74, 152 and 156 were found to abolish T cell recognition of the BNP peptide. These results were compared with results previously obtained with the influenza A matrix peptide and suggest that the two peptides bind differently in the peptide binding site. 相似文献
13.
Creation of an HLA-A2/HLA-Aw69 alloantigenic determinant on an HLA-A3 molecule by site-directed mutagenesis 总被引:1,自引:0,他引:1
C Layet B Kahn-Perles P Pontarotti P Ferrier J Sire F A Lemonnier 《Journal of immunology (Baltimore, Md. : 1950)》1987,138(7):2197-2201
The HLA-A2 and HLA-Aw69 molecules share an antigenic determinant not expressed by HLA-Aw68 and HLA-A3. Comparison of the amino acid (aa) sequences of these molecules and previous studies of the antigenic determinant expressed by different HLA-A2 X HLA-A3 hybrid molecules had established that three aa at positions 95, 97, and 107 were possibly involved in the formation of this determinant. The HLA-A3 gene was therefore mutagenized to replace successively at these positions the HLA-A3-specific aa by the HLA-A2 residues. A single substitution at position 107 of a glycine by a tryptophan residue is sufficient for full expression by HLA-A3 molecules of the HLA-A2/Aw69 shared antigenic determinant without modification of the other serological reactivities characteristic of the HLA-A3 molecules. Previous studies of ethyl methanesulfonate mutants having shown the involvement of aa 161 in this determinant, we assume that the two aa residues 107 and 161 are close to each other. 相似文献
14.
von der Mark H Pöschl E Lanig H Sasaki T Deutzman R von der Mark K 《Journal of molecular biology》2007,371(5):1188-1203
The binding specificity of alpha7beta1 integrins for different laminin isoforms is defined by the X1 and X2 splice domains located in the beta-propeller domain of the alpha7 subunit. In order to gain insight into the mechanism of specific laminin-integrin interactions, we defined laminin-binding epitopes of the alpha7X1 and -X2 domains by single amino acid substitutions and domain swapping between X1 and X2. The interaction of mutated, recombinantly prepared alpha7X1beta1 and alpha7X2beta1 heterodimers with various laminin isoforms was studied by surface plasmon resonance and solid phase binding assays. The data show that distinct clusters of surface-exposed acidic residues located in different positions of the X1 and the X2 loops are responsible for the specific recognition of laminins. These residues are conserved between the respective X1 or X2 splice domains of the alpha7 chains of different species, some also in the corresponding X1/X2 splice domains of alpha6 integrin. Interestingly, ligand binding was also modulated by mutating surface-exposed hydrophobic residues (alpha7X1L205, alpha7X2Y208) at positions corresponding to the fibronectin binding synergy site in alpha5beta1 integrin. Mutations in X1 that affected binding to laminin-1 also affected binding to laminin-8 and -10, but not to the same extent, thus allowing conclusions on the specific role of individual surface epitopes in the selective recognition of laminin-1 versus laminins -8 and -10. The role of the identified epitopes was confirmed by molecular dynamics simulations of wild-type integrins and several inactivating mutations. The analysis of laminin isoform interactions with various X1/X2 chimaera lend further support to the key role of negative surface charges and pointed to an essential contribution of the N-terminal TARVEL sequence of the X1 domain for recognition of laminin-8 and -10. In conclusion, specific surface epitopes containing charged and hydrophobic residues are essential for ligand binding and define specific interactions with laminin isoforms. 相似文献
15.
Clonal analysis of the BALB/c secondary B cell repertoire specific for a self-antigen, cytochrome c 总被引:3,自引:0,他引:3
mAb to rat cytochrome c (cyt c), totaling 556, were produced by individual clones of secondary B lymphocytes from nine groups of five BALB/c mice each in vitro using the splenic focus culture system. Inasmuch as rat and mouse cyt c are identical, these B cells can be considered specific for a self-antigen. The mAb were categorized into specificity groups based on their reactivities with a panel of seven cyts c that differ at two to six amino acid residues. The number of distinct specificities for the native protein was restricted to fewer than 20. Different groups of mice expressed the same specificities at comparable frequencies, including a single dominant one, and the total number of secondary cyt c-specific B cells was constant among groups of mice. This suggests that the acquisition of the secondary B cell specificity repertoire for this self-antigen is regulated. However, it is indeed possible that each specificity group may comprise a number of distinct mAb molecules that have arisen stochastically. Specificities expressed by as few as 1% of the total mAb were observed. Thus, it is likely that the identified specificities reflect the secondary B cell specificity repertoire for rat cyt c. The dominant specificity expressed by 50% of the mAb was characterized by elimination of antigen recognition as a result of replacement of aspartic acid by glutamic acid at position 62. Minor specificities expressed by 19% of the mAb were characterized by more subtle affects of an amino acid change at position 62 and/or an amino acid substitution from rat cyt c at position 60. Antibodies in other specificity groups reacted with epitopes in the region of residues 44 and 47. Whereas substitutions at positions 44, 47, 60, and 62 eliminated recognition by most of the mAb, changes at position 92 and at 103 also appeared to affect the binding of some mAb in the region around residues 60 and 62. The amino acid residues implicated in the recognition by murine mAb of murine cyt c have been shown previously to be involved in the epitopes of foreign mammalian cyt c. Therefore, self-tolerance cannot fully explain the restriction of the epitopes to these regions on foreign mammalian cyt c. 相似文献
16.
Ramelot TA Cort JR Yee AA Liu F Goshe MB Edwards AM Smith RD Arrowsmith CH Dever TE Kennedy MA 《Journal of molecular biology》2002,322(5):943-954
Phosphorylation of the translation initiation factor eIF2 on Ser51 of its alpha subunit is a key event for regulation of protein synthesis in all eukaryotes. M156R, the product of the myxoma virus M156R open reading frame, has sequence similarity to eIF2alpha as well as to a family of viral proteins that bind to the interferon-induced protein kinase PKR and inhibit phosphorylation of eIF2alpha. In this study, we demonstrate that, like eIF2alpha. M156R is an efficient substrate for phosphorylation by PKR and can compete with eIF2alpha. To gain insights into the substrate specificity of the eIF2alpha kinases, we have determined the nuclear magnetic resonance (NMR) structure of M156R, the first structure of a myxoma virus protein. The fold consists of a five-stranded antiparallel beta-barrel with two of the strands connected by a loop and an alpha-helix. The similarity between M156R and the beta-barrel structure in the N terminus of eIF2alpha suggests that the viral homologs mimic eIF2alpha structure in order to compete for binding to PKR. A homology-modeled structure of the well-studied vaccinia virus K3L was generated on the basis of alignment with M156R. Comparison of the structures of the K3L model, M156R, and human eIF2alpha indicated that residues important for binding to PKR are located at conserved positions on the surface of the beta-barrel and in the mobile loop, identifying the putative PKR recognition motif. 相似文献
17.
Sriyans Jain Rahul Shubhra Mandal Swadha Anand Souvik Maiti Srinivasan Ramachandran 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(12):2736-2749
We investigated the interacting amino acids critical for the stability and ATP binding of Mycobacterium tuberculosis PII protein through a series of site specific mutagenesis experiments. We assessed the effect of mutants using glutaraldehyde crosslinking and size exclusion chromatography and isothermal titration calorimetry. Mutations in the amino acid pair R60–E62 affecting central electrostatic interaction resulted in insoluble proteins. Multiple sequence alignment of PII orthologs displayed a conserved pattern of charged residues at these positions. Mutation of amino acid D97 to a neutral residue was tolerated whereas positive charge was not acceptable. Mutation of R107 alone had no effect on trimer formation. However, the combination of neutral residues both at positions 97 and 107 was not acceptable even with the pair at 60–62 intact. Reversal of charge polarity could partially restore the interaction. The residues including K90, R101 and R103 with potential to form H-bonds to ATP are conserved throughout across numerous orthologs of PII but when mutated to Alanine, they did not show significant differences in the total free energy change of the interaction as examined through isothermal titration calorimetry. The ATP binding pattern showed anti-cooperativity using three-site binding model. We observed compensatory effect in enthalpy and entropy changes and these may represent structural adjustments to accommodate ATP in the cavity even in absence of some interactions to perform the requisite function. In this respect these small differences between the PII orthologs may have evolved to suite species specific physiological niches. 相似文献
18.
R M Horton W H Hildebrand J M Martinko L R Pease 《Journal of immunology (Baltimore, Md. : 1950)》1990,145(6):1782-1787
The H-2Kf allele and the spontaneous mutant Kfm1 have been cloned using locus-specific sequences. The mutation consists of a cluster of four nucleotide changes, resulting in amino acid substitutions at positions 95 (Leu----Ile) and 97 (Val----Arg). This finding has structural, genetic, and technical implications. The amino acid substitutions are located on the beta-strands of the antigen recognition site. Their influence on the allogeneic properties of the Kf glycoprotein is consistent with the hypothesis that alloreactivity results from alterations in the spectrum of peptides presented to T cells. These substitutions would not, however, be predicted to be directly accessible for binding to antibodies. Nonetheless, the fm1 mutant binds anti Kf alloantisera and mAb much less strongly than the parent molecule, suggesting some indirect effect of these residues on serologic phenotype. The mutant is also interesting genetically because the sequence of the mutated region is identical to the sequence of the Df gene. This implies that there is a gene conversion-like mutational mechanism operating in the H-2f haplotype. Finally, the strategy used to obtain these K-locus cDNA should prove generally useful for isolating other MHC alleles. 相似文献
19.
Variability and conformation of HLA class I antigens: a predictive approach to the spatial arrangement of polymorphic regions 总被引:2,自引:0,他引:2
Comparison of available sequences of HLA-A and HLA-B antigens shows that variable positions are predominantly localized in four segments spanning residues 63-85, 105-116, 138-156, and 177-194. The fourth segment is unique in that it contains no differences between antigens of the same locus. Secondary folding of HLA heavy chain was estimated by three independent predictive methods and areas of defined structure were correlated with the distribution of local hydrophobicity to outline putative internal and external portions. The three analyses each independently predict a high probability for beta structure in the alpha 1, alpha 2, and alpha 3 domains. A single alpha-helix is predicted within residues 146-160, a segment of likely importance in cytotoxic T cell recognition and graft rejection. Substitutions within this segment are spatially related by the helical turn. Variable residues usually lie in areas of high local hydrophilicity, and therefore they are probably on the surface of the molecule. The model predicts that they are frequently located in beta strands, beta-turns, or the above-mentioned alpha-helix, so that most substitutions would be accommodated within rigid frameworks that may impose structural constraints to variability. The secondary structure of alpha 1, alpha 2, and alpha 3 domains presents some analogies that suggest that they might share common features in their tertiary folding. The predicted structure of alpha 3 is strongly reminiscent of that of immunoglobulin constant domains. Possible arrangements of elements of secondary structure are discussed, as an attempt to situating the polymorphic regions of HLA class I antigens in a spatial context. 相似文献
20.
Russell D. Salter 《Immunogenetics》1994,39(4):266-271
Intracellular transport of class I MHC complexes is dependent on assembly of class I heavy chains with 2-microglobulin (2m) and peptides. This suggests that amino acid residues of individual class I molecules which are important for their stability and transport are likely to include those which contribute to binding of a majority of the cleft-associated peptides. To identify such critical residues, substitutions at polymorphic positions within the peptide binding cleft were introduced into a mutant HLA-A*0201 molecule bearing an additional gly>lys substitution at position 242 (242K). The 242K mutation weakens association of the HLA-A*201 heavy chain with 2m and was used to enhance potential effects of substitutions in the peptide binding groove on class I stability. Critical in choosing which binding cleft positions to mutate was the observation that HLA-A*6801 was less sensitive to the effects of 242K mutation than HLA-A*0201 and A*6901. This suggested that one or more of the six residues in the 2 domain differing between HLA-A*6901 and A*6801 were likely to affect class I complex stability. Positions 95, 97, 107, 114, 116, and 156 in either 242K or wild-type HLA-A*0201 molecules were therefore each converted to those residues found in HLA-A*6801. One of the second-site substitutions, arg>met at position 97, increased stability and restored surface expression of the 242K molecule. Five other substitutions either had no additional effect or further impaired 242K stability. Substitution of his>arg at position 114 blocked surface expression of both 242K and wild-type HLA-A*0201 molecules. These results demonstrate that polymorphic residues in the binding cleft influence the stability of class I complexes, and suggest that position 97 plays a critical role in stabilizing class I molecules for transport. 相似文献