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1.
Mouse anti-HLA-A, B monoclonal antibodies have been used to study the homologues of HLA-A, B antigens in other primate species. Immunoprecipitates of primate histocompatibility antigens from extracts of radioactively labeled lymphocytes were analyzed by SDS-polyacrylamide electrophoresis. Primate histocompatibility antigens appear to have similar molecular structure to human HLA antigens. Owl monkeys, which react polymorphically with some monomorphic anti-HLA antibodies, showed biochemical differences which correlated with the serological polymorphism. An antibody (W6/32) which only reacts with the HLA/β 2-microglobulin complex in humans and not with the free HLA heavy chain has the reverse specificity in some owl monkeys. 相似文献
2.
Dog peripheral blood lymphocytes, when cultured with 35S-methionine in the presence of tunicamycin, synthesize DLA molecules consisting of
2-microglobulin and a heavy chain approximately 3000 daltons lower in apparent mol. wt. than observed in control cases. This difference in mol. wt. is consistent with the fact that a single N-linked carbohydrate side chain is present on the heavy chain of DLA class I antigens. There is no evidence of polymorphism in the DLA light chain (
2m). Both glycosylated and nonglycosylated forms of the heavy chain, however, show microheterogeneity, which can be related to tissue-type. Analysis by two-dimensional electrophoresis shows that the biochemical heterogeneity in the DLA heavy chain is less than expected from DLA serology, and less than found in HLA class I antigens. The data are consistent with the fact that the products of only a single DLA class I locus are detected.Abbreviations used in this paper
2m
beta-2-microglobulin
- 2D
two dimensional
- DLA
dog MHC
- HLA
human MHC
- Ia
I-region associated
- MHC
major histocompatibility complex
- PBL
peripheral blood lymphocytes
- PHA-M
phytohaemagglutinin-muco
- pI
isoelectric point
- RLA
rabbit MHC
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis 相似文献
3.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1990,1037(2):209-215
The major histocompatibility complex (MHC) class I antigens contain a light chain β2-microglobulin, non-covalently associated to the transmembrane heavy α-chain carrying the allotypic determinants. Since the C1q complement component is known to associate with β2-microglobulin, and we recently found that activated C1s complement was capable of cleaving β2-microglobulin, we decided to investigate the proteolytic activity of C1 complement towards the heavy chain of class I antigens. Our results demonstrate that human C1s complement cleaves the heavy chain of human class I antigens into at least two fragments, with apparent molecular weights of 22 000 and 24 000 g/ mol on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), under both reducing and non-reducing conditions. The cleavage of the heavy chain is inhibited by the presence of C1 esterase inhibitor. The molecular weights of the fragments are in agreement with the cleavage located in the area between the disulphide loops of the α2-andα3-domains of the heavy chain. In addition human C1s complement is able to cleave H-2 antigens from mouse in a similar fashion but not rat MHC class I antigen or mouse MHC class II antigen (I-Ad). Mouse MHC class I antigen-specific determinants could also be detected in supernatant from mouse spleen cells incubated with C1r and C1s. These results indicate the presence in the body fluids of a non-membrane-bound soluble form of the α1andα2-domains which represent the binding site for atnigenic peptide. 相似文献
4.
《生物化学与生物物理学报:疾病的分子基础》2007,1772(11-12):1258-1269
Refolding of the heavy chain of the Class I HLA molecule, HLA-B27, in the absence of β2m, yields soluble high molecular weight (HMW) oligomers reminiscent of the oligomeric forms of β2m-free heavy chains (FHCs) of class I HLA antigens observed on cell surfaces. Here we examine the structural characteristics of HMW B27 in respect of features potentially relevant to autoimmunity, such as: (a) retention of native-like structure, since this could facilitate non-canonical interactions with T-cell receptors even in the absence of bound β2m and peptide, or (b) presence of non-native structure, since this could yield novel (non-self) antigenic conformational epitopes that could elicit immune attack. We report that HMW B27 is characterized by high secondary structural content, structural stability, stability to proteolysis by trypsin, and structural features that are both partly native-like, and partly non-native-like, as assessed through the binding of conformationally-distinguishing and cross-reacting scFv antibodies specifically selected against HMW B27. We also present cell ELISA data with conformation-specific scFv antibodies that distinguish between lymphocytes from individuals who are healthy and B27 positive, and those who are B27 positive but suffering from ankylosing spondylitis. 相似文献
5.
We have produced a series of hybrid IgG1-IgG2a mouse immunoglobulins with identical light chains (L) and variable regions to facilitate the identification of structural features associated with functional differnces between immunoglobulin isotypes. Hybrid heavy chain (H) constant region gene segments were generated by genetic recombination in Escherichia coli between plasmids carrying mouse γ1 and γ2a gene segments. Crossovers occured through out these segments although the frequency was highest in regions of high nucleotide sequence homology. Eleven variant immunoglobulins produced by transfected hybridoma cell lines are assembled into H2L2 tetramers and properly glycosylated. In addition, all 11 immunoglbulins have identical antigen combining sites specific for the fluorescent hapten ε-dansyll-L-lysine. Protein A binding was used as probe of the structural integrity of the Fc portion of the variant antibodies. Differeneces in protein A binding between IgG1 and IgG2a appear to be due to amino acid differances at postions 252 (Thr→Met) and 254 (Thr→Ser) of the heavy chain (EU numbering). 相似文献
6.
Raif G. Vasilov Alfred Hahn Horst Mölders Jon J. van Rood Martijn Breuning Hidde L. Ploegh 《Immunogenetics》1983,17(4):333-356
Class I antigens were isolated by immunoprecipitation from cell extracts prepared from mitogenically stimulated and internally radiolabeled peripheral blood lymphocytes (PLBs). The precipitating antibodies used are monomorphic and recognize a determinant on the heavy chain of HLA-A, B, C antigens regardless of their allelic specificities when complexed with
2m, or determinants on
2m itself. Comparison of class I molecules isolated from 25 different homozygous typing cels (HTC) and analyzed by two-dimensional (2-D) gel electrophoresis allowed the identification of those HLA-A,13 locus specificities most common in the European Caucasoid population. Class I antigens isolated from HTC that are HLA identical are biochemically indistinguishable also. Evidence was obtained for the expression of additional class I antigens besides the HLA-A, B, C locus products: for some haplotypes, up to six class I genes may be active in mitogenically activated PBLs. No differences in molecular weight and isoelectric point of the class I heavy chains were observed between the antigens recognized by W6/32, the anti-heavy chain reagent, and anti-
2m reagents. The nature of the mitogenic stimulus, i. e., pokeweed mitogen or phytohemagglutinin, was irrelevant with respect to the class I antigens isolated by this method. Using the HTCs as reference, a panel of HLA-B27 positive heterozygous cells was analyzed. Two types of HLA-B27 antigens, distinct by CML typing were represented. These two forms differed also in their biochemical properties. In addition, we obtained evidence for the existence of an A2 variant. This finding was likewise confirmed by CML typing. 相似文献
7.
Highly purified, papain-solubilized HLA-A, -B, and -C antigens comprising a mixture of a great number of allelic forms from at least three loci have been fragmented by limited proteolysis, acid cleavage, and cyanogen bromide treatment. Limited proteolysis of 125I-labeled HLA-A, -B, and -C antigens with trypsin, chymotrypsin, thermolysin, and pepsin resulted in the production of two large fragments. One fragment was associated with beta 2-microglobulin and contained all of the carbohydrate. The other fragment, which had a molecular weight of about 13,000, is most probably derived from the COOH-terminal part of the heavy chain. Acid cleavage of the HLA antigen heavy chain gave rise to two main fragments with molecular weights of 22,000 and 11,000. Both fragments contained disulfide bonds. Two minor components, representing further cleavage products of the 22,000-dalton fragment, were also observed. Cleavage of the HLA antigen heavy chain at methionyl residues gave rise to one carbohydrate-containing, cysteine-free 14,000-dalton fragment and one 20,000-dalton fragment that contained all cysteines but no carbohydrate. NH2-terminal amino acid sequence analyses demonstrated that the 22,000-dalton acid cleavage fragment and the 14,000-dalton cyanogen bromide fragment were derived from the NH2-terminal part of the HLA antigen heavy chain. 相似文献
8.
Beta2-microglobulin-bound T-cell membrane components containing both human TL-like antigens and HLA(A, B, C) antigens were partially purified from Renex 30-solubilized membrane material of cells of a human T-cell-type leukemia cell line, HPB-ALL. The radioiodinated preparation was subjected to limited papain digestion; the HLA(A, B, C) antigens split, whereas a large portion of the human TL-like antigens remained intact. The antigen molecules were recovered by lentil-lectin affinity chromatography and separated by gel filtration on the basis of the induced difference in molecular size. The human TL-like-antigen preparation thus obtained was essentially free of HLA(A, B, C) antigens. The human TL-like antigens were immunospecifically precipitated and the component polypeptide, heavy and light, chains were separated by acid dissociation followed by gel filtration. The component chains were compared with the corresponding chains of HLA(A, B, C) antigens obtained similarly from the same HPB-ALL cells with respect to their fragmentation patterns on chemical or enzymatic cleavage. The results provided convincing evidence for the identity of the light chains of human TL-like antigens and HLA(A, B, C) antigens, and also evidence suggesting the presence of substantial differences in the fundamental structure of the heavy chains of human TL-like antigens and HLA(A, B, C) antigens.A unit of the New York State Department of Health. 相似文献
9.
William P. Lafuse Shumpei Yokota Chella S. David 《Journal of cellular biochemistry》1980,14(1):97-106
Ia specificities 22 and 23 were found to be determinants on hybrid Ia molecules, formed by the noncovalent binding of a 26,000–28,000 dalton beta polypeptide chain (Ae) coded by the I-A subregion and a 32,000–35,000 dalton alpha chain (Eα) coded by the I-E subregion. For expression of Ia. 23 the Ae chain, coded by the I-A subregion, must be derived from the H-2d haplotype, while Ab, As, or Ak can provide the complementing beta chain for the expression of Ia. 22. For expression of Ia. 22 and Ia. 23, most Ia. 7 positive strains can provide the complementing alpha chain (Eα), with the one exception of B 10. PL (Eu), which is Ia. 7 positive but will not complement with Ad to express Ia. 23. Antisera were also produced against hybrid Ia antigens by immunizing with F1 cells expressing Ia. 22 or Ia. 23 generated by transcomplementation. These antisera detect the same specificities as conventional anti-Ia. 22 and anti-Ia. 23 sera produced against cis-complementing Ia antigens. It is postulated that hybrid Ia determinants are involved in recognition and generation of immune response to antigens under dual Ir gene control. It is also suggested that there are 2 types of Ia specificities: (1) allotypic Ia specificities expressed on the alpha or beta chains (for example, Ia. 7 on the Eα chain) and (2) hybrid Ia specificities, which are unique interaction determinants formed by the association of alpha and beta chains (for example, Ia. 22 and Ia. 23). These interaction gene products may be involved in antigen recognition and presentation. 相似文献
10.
Human and murine class I MHC antigens share conserved serine 335, the site of HLA phosphorylation in vivo 总被引:8,自引:0,他引:8
The site of phosphorylation of the HLA-B7 antigen in vivo is serine 335, which is located in the intracellular region. Pseudomonas fragi protease was used in limited proteolysis experiments of HLA antigens to identify the position of the phosphoserine residue. The intracellular region is composed of 30 amino acids at the carboxyl terminus of the heavy chain; up to nine serine residues are located in this region. Four of the serines are found at the distal end, and are encoded by exon 7 in both human and murine class I MHC antigens. Alignment of the protein and DNA sequences of the intracellular regions of human and murine class I antigens demonstrates conservation of the serine positions located within this exon. Realignment of exon 7, by introducing a gap in the murine sequences, increases homology across species, and reveals two conserved serines at 332 and 335 within the conserved sequence Ser-Asp/Glu-X-Ser(P)-Leu. The preservation of this sequence and the site of phosphorylation suggests that this modification of the intracellular region of histocompatibility antigens is functionally significant. 相似文献
11.
Papain-solubilized HLA-A, -B, and -C antigens have been isolated from cadaveric spleens. The isolated material was homogeneous and comprised subunits with the apparent molecular weights 33,000 and 12,000. Amino acid analyses of a mixture of HLA antigen heavy chains obtained from a great number of spleens with different HLA antigen phenotypes revealed a composition that is very similar to that of individual HLA-A and -B antigens. Likewise, the NH2-terminal 30 residues of the HLA-antigen heavy chain mixture were virtually identical with that recorded for individual specificities. The circular dichroism spectra for the isolated HLA antigens and for free beta2-microglobulin revealed similarities with spectra recorded for immunoglobulin chains and domains. The HLA-antigen heavy chain may contain an appreciable amount of beta structure. Antibodies raised against free beta2-microglobulin react better with beta2-microglobulin in free form than when bound to the HLA-A, -B, and -C antigen heavy chains. This is due to the fact that free beta2-microglobulin can bind a maximum of four Fab fragments simultaneously, whereas the HLA-antigen-associated beta2-microglobulin can bind only two Fab fragments without dissociating from the heavy HLA-antigen subunit. 相似文献
12.
Lia M. E. Dobbe Nico J. Stam Jacques J. Neefjes Marius J. Giphart 《Immunogenetics》1988,27(3):203-210
Human serum was found to contain a variety of class I-like molecules by Western blotting with anti-class I heavy chain reagents: major bands usually are observed around M
r 44 000, 40 000, and 35 000–37 000. HLA-A24-positive individuals are distinguished by higher serum levels of M
r 44 000 and 40 000 class I-like molecules than those found in HLA-A24-negative individuals. The M
r 44 000 serum molecules are probably intact class I molecules that have been shed from the cell membrane, because they contain both a transmembrane segment (TM), as deduced from detergent-binding experiments, and a cytoplasmic tail (CT), as inferred from reactivity with an antipeptide serum specific for the cytoplasmic domain of class I antigens (RaCT). The M
r 35 000 and 37 000 molecules contain neither a TM nor a CT region and therefore are probably proteolytic breakdown products of cellular and/or serum M
r 44 000 molecules, although the existence of Q10-like molecules in man cannot be ruled out. The M
r 40 000 molecules do not contain a TM region. M
r 40 000 molecules reactive with the RaCT serum were found in the minority (2/13) of sera tested. We conclude that alternative splicing resulting in a precise excision of the TM exon plays a minor role in the generation of serum HLA class I antigens.Abbreviations used in this paper B2m
beta-2 microglobulin
- BSA
bovine serum albumin
- EBV-BLCL
Epstein-Barr virus-transformed B lymphoblastoid cell line
- mAb
monoclonal antibody
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- TCA
trichloroacetic acid 相似文献
13.
Desai SA Wang X Noronha EJ Zhou Q Rebmann V Grosse-Wilde H Moy FJ Powers R Ferrone S 《Journal of immunology (Baltimore, Md. : 1950)》2000,165(6):3275-3283
The association of HLA class I heavy chains with beta2-microglobulin (beta2m) changes their antigenic profile. As a result, Abs react with either beta2m-free or beta2m-associated HLA class I heavy chains. An exception to this rule is the mAb TP25.99, which reacts with both beta2m-associated and beta2m-free HLA class I heavy chains. The reactivity with beta2m-associated HLA class I heavy chains is mediated by a conformational determinant expressed on all HLA-A, -B, and -C Ags. This determinant has been mapped to amino acid residues 194-198 in the alpha3 domain. The reactivity with beta2m-free HLA class I heavy chains is mediated by a linear determinant expressed on all HLA-B Ags except the HLA-B73 allospecificity and on <50% of HLA-A allospecificities. The latter determinant has been mapped to amino acid residues 239-242, 245, and 246 in the alpha3 domain. The conformational and the linear determinants share several structural features, but have no homology in their amino acid sequence. mAb TP25.99 represents the first example of a mAb recognizing two distinct and spatially distant determinants on a protein. The structural homology of a linear and a conformational determinant on an antigenic entity provides a molecular mechanism for the sharing of specificity by B and TCRs. 相似文献
14.
鼠抗HFRSV衣壳蛋白McAb F3株可变区基因的获取及特性分析 总被引:1,自引:0,他引:1
培养鼠抗肾综合征出血热病毒衣壳蛋白F3杂交瘤细胞株,提取总RNA,根据鼠源IgG抗体基因家族可变区基因碱基序列的特点,设计简并引物,通过逆转录聚合酶链反应,获得抗体轻链可变区和重链可变区基因。分别将其克隆入载体PT7BlueT Vector,选取阳性重组克隆各两个,分别测定了所载重链可变区和轻链可变区基因的碱基序列,比较了不同克隆轻链可变区基因之间和重链可变区基因之间碱基序列的差异;分析了各自的氨基酸框架及其对应蛋白的亲水性。结果显示,两个重链可变区基因碱基序列有4处不同,同源性为979%;其中重组克隆ZG364 5F所载重链可变区基因有完整的开放阅读框架,对应的蛋白含有丰富的亲水基因,第112氨基酸处亲水性最高;另一重组克隆ZG364 4F所载重链可变区基因不能通读。两个轻链基因碱基序列有4处不同,同源性为991%,重组克隆ZG365 5F和ZG365 7F所载轻链可变区基因均有完整的开放阅读框架,对应的蛋白均含有丰富的亲水基因,ZG365 5F所载基因对应蛋白第67氨基酸亲水性最高,ZG365 7F所载基因对应蛋白第34氨基酸亲水性最高。 相似文献
15.
A self-derived-peptide with the same amino acid sequence (N-RRYLENGKETLQR-C) as residues 169–181 of the human leukocyte antigen (HLA) B27 heavy chain is known to bind to MHC Class I complexes containing the HLA-B27 heavy chain. This observation has been invoked previously in at least two different (but related) molecular explanations for the disease-association of the HLA-B27 allele. Here, we use a combination of fluorescence polarization, competitive inhibition and gel filtration chromatographic studies to show that a fluorescently-labeled peptide of the above sequence binds to two disease-associated subtypes of HLA-B27 (namely HLA-B*27:04 and HLA-B*27:05) but not to non-disease-associated subtypes (HLA-B*27:06 or HLA-B*27:09). This differential binding behavior is seen both in (a) peptide binding to complexes of heavy chain (HLA-B27) and light chain (β2 microglobulin), and in (b) peptide binding to β2 microglobulin-free heavy chains in the aggregated state. Such subtype-specific differences are not seen with two other control peptides known to bind to HLA-B27. Our results support the likelihood of differential peptide binding holding at least one of the keys to HLA-B27’s disease association. 相似文献
16.
J P Allison M A Pellegrino S Ferrone G N Callahan R A Reisfeld 《Journal of immunology (Baltimore, Md. : 1950)》1977,118(3):1004-1009
HLA antigens of both the A and B loci were shown to be associated with the high density lipoprotein fraction of serum prepared by ultracentrifugal flotation. HLA-A9 antigens were purified 100-fold with essentially complete recovery by a simple procedure of high density lipoprotein preparation involving precipitation with polyanions and ultracentrifugal flotation. The purified lipid-associated antigen was immunogenic since it elicited the formation of cytotoxic xenoantibodies in rabbits. Serum HLA-A9 antigens were found by immunoprecipitation and gel electrophoresis to consist of a 45,000 m.w. heavy chain associated with beta2-microglobulin. The size of the HLA-lipid complex (less than 190,000 m.w.) and of the HLA-deoxycholate complex (less than 102,000 m.w.) suggests that HLA antigens are shed into plasma as a complex of a single HLA molecule and a single beta2-microglobulin chain, associated with boundary lipid. 相似文献
17.
18.
Mouse and human β2-microglobulin (β2m), which differ by 30% in their primary sequence, give rise to disparate levels of HLA class I heavy chain expression in
transfectants of the β2m-null FO-1 human melanoma cell line, i.e., mouse β2m directs expression of HLA class I heavy chains that is only ∼20%–30% of that observed for heavy chains assembled with human
β2m. In this report we describe our efforts to better understand the structural basis of this regulatory phenomenon. Initial
insight into the importance of the N-terminus of β2m on HLA expression came from studies with FO-1 cells transfected with chimeric (human X mouse) B2m genes. Chimeric β2m molecules containing residues 1–69 from human β2m and residues 70–99 from mouse β2m (designated HM- β2m) induced expression of HLA heavy chains to a significantly greater extent (∼80% of level observed with cognate β2m) than the reverse chimeric construct (designated MH- β2m) (10%–15% of level observed with cognate β2m). These data are consistent with the view that the major determinants of HLA class I heavy chain expression map to the portion
of the β2m molecule which forms the four-stranded β-pleated sheet, comprised of S1, S2, S4, and S5, and one strand of the three-stranded
sheet (S3). The mapping of class I regulatory sites to the portion of β2m containing the four-stranded β-pleated sheet supports the interpretation that the heavy chain contact residues on β2m play the major role in regulating major histocompatibility (MHC) class I expression. To further dissect β2m-mediated regulation of HLA class I expression, site-directed mutants of β2m were prepared by conversion of human β2m to the mouse sequence at individual amino acid positions within the four-stranded and three-stranded β-pleated sheets. Human
to mouse amino acid substitutions were made in each divergent residue between positions 1–66, and as controls for COOH-terminal
modification, several residues between positions 75 and 94. Cytofluorometry with HLA class I-specific antibodies indicated
that cell surface expression of HLA class I heavy chains was largely insensitive to each of the individual substitutions.
It is concluded that a combination of divergent residues mapping to positions of heavy chain contact are responsible for the
differences observed in MHC class I expression by heterologous forms of β2m.
Received: 18 March 1997 / Revision: 21 April 1997 相似文献
19.
F Lottspeich J Kellermann A Henschen B Foertsch W Müller-Esterl 《European journal of biochemistry》1985,152(2):307-314
The complete amino acid sequence of the light chain of human high-molecular-mass kininogen has been determined. The peptide chain contains 255 amino acid residues. The half-cystine, which forms the disulfide bridge to the heavy chain, was identified in position 225. Nine carbohydrate attachment sites were found. All carbohydrate side chains are O-glycosidically linked. Alignment of the present sequence with the bovine kininogen light chain sequence shows a high degree of homology, except for an extension of 22 amino acids within the histidine-rich part of the sequence. The histidine-rich region may have arisen by gene multiplication during evolution. 相似文献
20.
Archbold JK Macdonald WA Miles JJ Brennan RM Kjer-Nielsen L McCluskey J Burrows SR Rossjohn J 《The Journal of biological chemistry》2006,281(45):34324-34332
Our understanding of the molecular mechanisms of T cell alloreactivity remains limited by the lack of systems for which both the T cell receptor allo- and cognate ligand are known. Here we provide evidence that a single alloreactive T cell receptor interacts with analogous structural regions of its cognate ligand, HLA-B*0801(FLRGRAYGL), as its allogeneic ligand, HLA-B*3501(KPIVVLHGY). The crystal structures of the binary peptide-major histocompatibility complexes show marked differences in the conformation of the heavy chains as well as the bound peptides. Nevertheless, both epitopes possess a prominent solvent-exposed aromatic residue at position 7 flanked by a small glycine at position 8 of the peptide determinant. Moreover, regions of close structural homology between the heavy chains of HLA B8 and HLA B35 coincided with regions that have previously been implicated in "hot spots" of T cell receptor recognition. The avidity of this human T cell receptor was also comparable for the allo- and cognate ligand, consistent with the modes of T cell receptor binding being broadly similar for these complexes. Collectively, it appears that highly focused structural mimicry against a diverse structural background provides a basis for the observed alloreactivity in this system. This cross-reactivity underpins the T cell degeneracy inherent in the limited mature T cell repertoire that must respond to a vast diversity of microbial antigens. 相似文献