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1.
Genes Immun ; 2(1): 48-51, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11294569

ABSTRACT

During the assembly of major histocompatibility complex (MHC) class I molecules transient associations are formed with the endoplasmic reticulum resident chaperones calnexin and calreticulin, ERp57 oxidoreductase, and also with tapasin, the latter mediating binding of the class I molecules to the transporter associated with antigen processing (TAP). We report here the isolation of a cDNA encoding rat tapasin from a DA (RT1av1) library. The cDNA encodes a proline-rich (11.3%) polypeptide of 464 residues with a potential ER-retention KK motif at its COOH-terminus, and a predicted molecular mass of 48 kDa. Matrix-assisted laser-desorption ionisation (MALDI) mass spectrometry of peptides derived from in-gel tryptic digestion of a TAP-associated protein match regions of the predicted translation product. A species of the correct molecular mass and predicted pl was also identified in association with radiolabelled immunoprecipitates of the rat TAP complex analysed by two-dimensional gel electrophoresis. This confirms rat tapasin as a component of the rat MHC class I assembly complex.


Subject(s)
Antiporters/genetics , Histocompatibility Antigens Class I/genetics , Immunoglobulins/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , DNA, Complementary , Electrophoresis, Gel, Two-Dimensional , Humans , Membrane Transport Proteins , Mice , Molecular Sequence Data , Rats , Sequence Homology, Amino Acid
2.
Curr Biol ; 11(4): 242-51, 2001 Feb 20.
Article in English | MEDLINE | ID: mdl-11250152

ABSTRACT

BACKGROUND: The transporter associated with antigen processing (TAP) consists of two polypeptides, TAP1 and TAP2. TAP delivers peptides into the ER and forms a "loading complex" with MHC class I molecules and accessory proteins. Our previous experiments indicated that nucleotide binding to TAP plays a critical role in the uptake of peptide and the release of assembled class I molecules. To investigate whether the conserved nucleotide binding domains (NBDs) of TAP1 and TAP2 are functionally equivalent, we created TAP variants in which only one of the two ATP binding sites was mutated. RESULTS: Mutations in the NBDs had no apparent effect on the formation of the loading complex. However, both NBDs had to be functional for peptide uptake and transport. TAP1 binds ATP much more efficiently than does TAP2, while the binding of ADP by the two chains is essentially equivalent. Peptide-mediated release of MHC class I molecules from TAP was blocked only when the NBD of TAP1 was disrupted. A different NBD mutation that does not affect nucleotide binding has strikingly different effects on peptide transport activity depending on whether it is present in TAP1 or TAP2. CONCLUSIONS: Our findings indicate that ATP binding to TAP1 is the initial step in energizing the transport process and support the view that ATP hydrolysis at one TAP chain induces ATP binding at the other chain; this leads to an alternating and interdependent catalysis of both NBDs. Furthermore, our data suggest that the peptide-mediated undocking of MHC class I is linked to the transport cycle of TAP by conformational signals arising predominantly from TAP1.


Subject(s)
ATP-Binding Cassette Transporters/physiology , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Major Histocompatibility Complex , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP Binding Cassette Transporter, Subfamily B, Member 3 , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Adenosine Monophosphate/metabolism , Binding Sites , Biological Transport , Cell Line , Humans , Mutagenesis , Peptides/metabolism
3.
Curr Biol ; 9(18): 999-1008, 1999 Sep 23.
Article in English | MEDLINE | ID: mdl-10508608

ABSTRACT

BACKGROUND: Newly synthesised peptide-receptive major histocompatibility complex (MHC) class I molecules form a transient loading complex in the endoplasmic reticulum with the transporter associated with antigen processing (TAP) and a set of accessory proteins. Binding of peptide to the MHC class I molecule is necessary for dissociation of the MHC class I molecule from the complex with TAP, but other components of the complex might also be involved. To investigate the role of TAP in this process, mutations that block nucleotide binding were introduced into the ATP-binding site of TAP. RESULTS: Mutant TAP formed apparently normal loading complexes with MHC class I molecules and accessory components, but had no nucleotide-binding or peptide-transport activity. Nevertheless, whereas wild-type loading complexes in detergent lysates could be dissociated by addition of peptides that bind MHC class I molecules, mutant complexes could not be dissociated in this way. Depletion of nucleotide diphosphates or triphosphates from wild-type lysates blocked peptide-mediated dissociation of MHC class I molecules, which could be reversed by readdition of nucleotide diphosphates or triphosphates. Complexes between mutant TAP and MHC class I molecules remained associated in vivo until they were degraded. Disruption of nucleotide binding also eliminated TAP's peptide-binding activity. CONCLUSIONS: Peptide-mediated dissociation of the MHC class I molecule from the loading complex depends on conformational signals arising from TAP. Integrity of the nucleotide-binding site is required not only for transmission of this conformational signal to the loading complex, but also for binding of peptide to TAP. Thus, the dynamic activity of the loading complex is synchronised with the nucleotide-mediated peptide-binding and transport cycle of TAP.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphate/metabolism , Antigen Presentation/physiology , Histocompatibility Antigens Class I/metabolism , Peptide Fragments/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP Binding Cassette Transporter, Subfamily B, Member 3 , ATP-Binding Cassette Transporters/genetics , Allosteric Regulation , Animals , Binding Sites/genetics , HLA-A2 Antigen/metabolism , HLA-B Antigens/metabolism , Humans , Mutagenesis, Site-Directed , Protein Binding , Rats , Recombinant Fusion Proteins/metabolism
4.
J Immunol ; 162(2): 743-52, 1999 Jan 15.
Article in English | MEDLINE | ID: mdl-9916694

ABSTRACT

In common with other mammalian species, the laboratory rat (Rattus norvegicus) expresses MHC class I molecules that have been categorized as either classical (class Ia) or nonclassical (class Ib). This distinction separates the class Ia molecules that play a conventional role in peptide Ag presentation to CD8 T cells from the others, whose function is unconventional or undefined. The class Ia molecules are encoded by the RT1-A region of the rat MHC, while the RT1-C/E/M region encodes up to 60 other class I genes or gene fragments, a number of which are known to be expressed (or to be expressible). Here we report upon novel MHC class Ib genes of the rat that we have expression cloned using new monoclonal alloantibodies and which we term RT1-U. The products detected by these Abs were readily identifiable by two-dimensional analysis of immunoprecipitates and were shown to be distinct from the class Ia products. Cellular studies of these molecules indicate that they function efficiently as targets for cytotoxic killing by appropriately raised polyclonal alloreactive CTL populations. The sequences of these class Ib genes group together in phylogenetic analysis, suggesting a unique locus or family. The combined serological, CTL, and sequence data all indicate that these products are genetically polymorphic.


Subject(s)
Histocompatibility Antigens/genetics , Histocompatibility Antigens/isolation & purification , Amino Acid Sequence , Animals , Antibodies, Monoclonal/metabolism , Antigen Presentation , Antigen-Antibody Reactions , Base Sequence , Cloning, Molecular , DNA, Complementary/isolation & purification , Female , Haplotypes , Histocompatibility Antigens/immunology , Histocompatibility Antigens/metabolism , L Cells , Mice , Molecular Sequence Data , Multigene Family/immunology , Polymorphism, Genetic , Precipitin Tests , Rats , Rats, Inbred Strains , Sequence Homology, Nucleic Acid , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
5.
J Immunol ; 160(6): 2767-79, 1998 Mar 15.
Article in English | MEDLINE | ID: mdl-9510178

ABSTRACT

The transporter associated with Ag processing, TAP, is an endoplasmic reticulum resident heterodimeric member of the ATP-binding cassette transporter family. TAP transports short peptides from cytosol to the endoplasmic reticulum lumen for loading into recently synthesized class I MHC molecules. In the rat, two alleles of the TAP2 chain differ in their permissiveness to the transport of peptides with small hydrophobic, polar, or charged amino acids at the C terminus, and this correlates with differences between the peptide sets loaded into certain class I molecules in vivo. We have used segmental exchanges and site-directed mutagenesis to identify the residues in rat TAP2 responsible for differential transport between the two alleles of peptides terminating above all in the positively charged residue, arginine. Of the 25 residues by which the two functional TAP2 alleles differ, we have localized differential transport of peptides with a C-terminal arginine to two adjacent clusters of exchanges in the membrane domain involving a total of five amino acids. Each cluster, transferred by site-directed mutagenesis from the permissive to the restrictive sequence, can independently confer on TAP a partial ability to transport peptides with arginine at the C terminus. The results suggest that the permissive TAP2-A allele evolved in at least two steps, each partially permissive for peptides with charged C termini.


Subject(s)
ATP-Binding Cassette Transporters/physiology , Antigen Presentation , ATP Binding Cassette Transporter, Subfamily B, Member 3 , ATP-Binding Cassette Transporters/genetics , Alleles , Amino Acid Sequence , Animals , Cell Line , Molecular Sequence Data , Mutagenesis, Site-Directed , Polymorphism, Genetic , Rats , Structure-Activity Relationship , T-Lymphocytes, Cytotoxic/immunology
6.
Immunogenetics ; 40(1): 45-53, 1994.
Article in English | MEDLINE | ID: mdl-8206525

ABSTRACT

We are reporting the cDNA sequences of Tap2 from two cima and two cimb rat strains. Comparison of the cDNA sequences shows that these alleles fall into two groups, which we refer to as Tap2-A and Tap2-B. We found that alleles from the Tap2-B group are more closely related to the mouse homologue than are Tap2-A alleles, and among the 48 nucleotides which differ between the Tap2-A and Tap2-B cDNAs, three affect restriction sites. We defined pairs of oligonucleotides which allow amplification of the regions bearing these restriction sites from genomic DNA or cDNA, and this technique has been successful for the genotyping of all of the 56 laboratory strains of Rattus norvegicus tested and for five cell lines tested so far. All 14 known RT1 standard haplotypes were tested, and 7 found to belong to the Tap2-B group, and 7 to Tap2-A. We also found that intron sizes among the alleles of the Tap2-B group fall into two subgroups, providing further insight into the phylogeny of these various haplotypes.


Subject(s)
ATP-Binding Cassette Transporters , Alleles , Animals, Laboratory/genetics , Carrier Proteins/genetics , Histocompatibility Antigens/genetics , Rats/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 3 , Amino Acid Sequence , Animals , Base Sequence , Biological Transport , Carrier Proteins/classification , Haplotypes , Molecular Sequence Data , Peptides/metabolism , Phylogeny , Restriction Mapping , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
8.
Nature ; 357(6375): 211-5, 1992 May 21.
Article in English | MEDLINE | ID: mdl-1350326

ABSTRACT

Short antigenic peptides bound in the groove of class I major histocompatibility complex molecules enable T cells to detect intracellular pathogens. It has been assumed that structural features of the class I molecule alone select which peptides are bound. It is now demonstrated that a complex polymorphism in one of the major histocompatibility complex-encoded putative peptide-transporter genes is associated with an altered spectrum of bound peptides.


Subject(s)
Carrier Proteins/genetics , Histocompatibility Antigens Class I/genetics , Major Histocompatibility Complex , Membrane Proteins/genetics , Membrane Transport Proteins , Polymorphism, Genetic , Alleles , Amino Acid Sequence , Animals , Carrier Proteins/biosynthesis , Cell Line , Genes, MHC Class I , Histocompatibility Antigens Class I/biosynthesis , Humans , Kinetics , Lymphoma, T-Cell , Membrane Proteins/biosynthesis , Molecular Sequence Data , Poly A/genetics , Polymerase Chain Reaction/methods , Protein Conformation , RNA, Messenger/genetics , Rats , Sequence Homology, Nucleic Acid , Transfection
9.
Anim Genet ; 23(4): 347-59, 1992.
Article in English | MEDLINE | ID: mdl-1503274

ABSTRACT

Previous work made use of nucleic acid probes corresponding to different subtypes of the class II regions of the human and murine major histocompatibility complex (MHC) to isolate seven different alpha and 24 different beta genes of the ovine MHC from two cosmid libraries. In an attempt to identify pairs of alpha and beta genes capable of cell surface expression, all permutations of alpha and beta genes were in turn transfected into mouse L-cells. Two pairs of alpha and beta genes co-expressed and stable ovine MHC class II L-cell lines were developed. The expressed alpha genes had previously been defined as DR-alpha homologues (DRA) by differential Southern hybridization to human subtype specific class II probes. The expressed ovine beta genes were also assigned as ovine DR-beta homologues (DRB) on the basis of their sequence having a higher degree of similarity with human DRB than any other subtype. A total of eight out of 23 anti-sheep class II specific monoclonal antibodies were typed OLA-DR specific by FACScan analysis using the L-cell lines.


Subject(s)
Genes, MHC Class II , Sheep/genetics , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Base Sequence , Blotting, Western , Cells, Cultured , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression , L Cells , Mice , Molecular Sequence Data
10.
Nature ; 354(6354): 528-31, 1991.
Article in English | MEDLINE | ID: mdl-1758495

ABSTRACT

In mammalian cells, short peptides derived from intracellular proteins are displayed on the cell membrane associated with class I molecules of the major histocompatibility complex (MHC). The surface presentation of class I-peptide complexes presumably alerts the immune system to intracellular viral protein synthesis. Peptides derived from the cytosol must reach the cisternae of the endoplasmic reticulum where they are required for the assembly of stable class I molecules, and it has been proposed that the products of the two MHC-encoded ATP-binding cassette (ABC) transporter genes function to deliver the peptides across the membrane of the endoplasmic reticulum. This idea is supported by experiments in which transfection of a human cell line defective in class I expression with a complementary DNA of one of these genes restored cell surface expression levels. Here we show that the complete phenotype of the mouse mutant cell line RMA-S, in which lack of surface expression of stable class I molecules correlates with an inability to present viral peptides originating in the cytosol, is repaired by the cDNA of the other transporter gene. These results are consistent with the possibility that the two transporter polypeptides form a heterodimer.


Subject(s)
ATP-Binding Cassette Transporters , Carrier Proteins/genetics , Major Histocompatibility Complex , Membrane Proteins/genetics , Mutation , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP Binding Cassette Transporter, Subfamily B, Member 3 , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cells, Cultured , Cloning, Molecular , DNA/genetics , DNA/isolation & purification , Flow Cytometry , Gene Library , Genes, MHC Class I , Histocompatibility Antigens Class II , Humans , Kinetics , Molecular Sequence Data , Protein Conformation , Rats , Restriction Mapping , Sequence Homology, Nucleic Acid
11.
Anim Genet ; 22(3): 211-25, 1991.
Article in English | MEDLINE | ID: mdl-1928827

ABSTRACT

The class II genes of the sheep major histocompatibility complex (MHC) have been cloned from two unrelated heterozygous sheep into cosmid vectors. By restriction mapping and hybridization with a number of class II probes of human and mouse origin, the cloned genetic material has been assigned to seven distinct alpha genes, 10 distinct beta genes and 14 beta-related sequences. It was difficult to identify homologues of specific HLA class II genes because of a tendency for the ovine genes to cross-hybridize between HLA probes representing different loci. Such cross-hybridization was especially marked among the beta genes. While DQ and DR homologues have been tentatively identified by several criteria, no genes corresponding to DP have been identified. Cosmids containing class II alpha and beta genes have been transfected into mouse LTK- cells, and surface expression of a sheep class II molecule has been obtained.


Subject(s)
Genes, MHC Class II , Genes , Sheep/genetics , Animals , Cell Line , Cosmids , Fibroblasts/metabolism , Gene Expression , Histocompatibility Antigens Class II/genetics , Humans , Male , Mice , Transfection
12.
Nature ; 348(6303): 738-41, 1990.
Article in English | MEDLINE | ID: mdl-1979660

ABSTRACT

The T-cell immune response is directed against antigenic peptide fragments generated in intracellular compartments, the cytosol or the endocytic system. Peptides derived from cytosolic proteins, usually of biosynthetic origin, are presented efficiently to T-cell receptors by major histocompatibility complex (MHC) class I molecules, with which they assemble, probably in the endoplasmic reticulum (ER). In the absence of recognizable N-terminal signal sequences, such cytosolic peptides must be translocated across the ER membrane by a novel mechanism. Genes apparently involved in the normal assembly and transport of class I molecules may themselves be encoded in the MHC. Here we show that one of these, the rat cim gene, maps to a highly polymorphic part of the MHC class II region encoding two novel members of the family of transmembrane transporters related to multidrug resistance. Other members of this family of transporter proteins are known to be capable of transporting proteins and peptides across membranes independently of the classical secretory pathway. Such molecules are credible candidates for peptide pumps that move fragments of antigenic proteins from the cytosol into the ER.


Subject(s)
Carrier Proteins/genetics , Drug Resistance/genetics , Histocompatibility Antigens Class II/genetics , Amino Acid Sequence , Animals , Base Sequence , Biological Transport , Carrier Proteins/chemistry , Cloning, Molecular , DNA Probes , Endoplasmic Reticulum/metabolism , Histocompatibility Antigens Class I/metabolism , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Nucleic Acid Hybridization , Polymorphism, Restriction Fragment Length , Rats , Restriction Mapping
13.
Nucleic Acids Res ; 12(16): 6523-35, 1984 Aug 24.
Article in English | MEDLINE | ID: mdl-6089118

ABSTRACT

We present the full sequence of an insert of a lambda phage clone which contains a segment of human DNA stretching from the secreted mu(mu s) constant region gene through to the beginning of the constant region gene and including the membrane mu(mu m) segments. The segment of 8.6kb extending from mu s to the first constant domain of delta(C delta 1) has been completely sequenced and reveals little conservation in comparison to the corresponding mouse sequence. The outstanding feature of the mu s-mu m intron is the occurrence of a potential Z-DNA forming region situated at 285bp downstream of the mu s poly A addition signal. A similar DNA stretch exists in mouse and may represent a site for transcriptional control of mu gene expression. The mu m-C delta 1 intron is much longer (6Kb) than the corresponding mouse intron and includes a series of different repeats, which start at 430bp downstream of the mu m poly A addition site and continue for 3.5Kb, ending about 1.5Kb from the beginning of C delta 1. This series of repeats may be a vestigial switch sequence used in the production of the secreting cells which are the progenitors of the rare human IgD myelomas.


Subject(s)
Cloning, Molecular , Genes , Immunoglobulin Heavy Chains/genetics , Immunoglobulin delta-Chains/genetics , Immunoglobulin mu-Chains/genetics , Bacteriophage lambda/genetics , Base Sequence , Computers , DNA/genetics , DNA Restriction Enzymes , Humans , Nucleic Acid Conformation , Transcription, Genetic
14.
Immunology ; 40(4): 657-64, 1980 Aug.
Article in English | MEDLINE | ID: mdl-6776038

ABSTRACT

We report the complete sequence of a cyanogen bromide fragment of a human delta chain (ErI). Its interest lies in the fact that it contains the peptide joining the V delta to the C delta 1 region. The presented sequence is compared with the homologous region of other human heavy chain classes and subclasses. The possibility that human delta chains and other human heavy chains have in their chromosomal DNA a short J sequence, is discussed.


Subject(s)
Immunoglobulin Heavy Chains , Immunoglobulin delta-Chains , Amino Acid Sequence , Amino Acids/analysis , Chemical Phenomena , Chemistry , Chromatography, Gel , Cyanogen Bromide , Humans , Pepsin A , Peptide Fragments/analysis , Trypsin
15.
Biochem J ; 171(1): 73-8, 1978 Apr 01.
Article in English | MEDLINE | ID: mdl-646825

ABSTRACT

1. The electrophoretically fast (F) and slow (S) fragments obtained by tryptic cleavage of bovine iron-saturated transferrin differed in carbohydrate content and peptide 'maps'. 2. A fragment capable of binding one Fe3+ ion per molecule was isolated after brief tryptic digestion of bovine apotransferrin and shown closely to resemble the S fragment obtained from the iron-saturated protein. 3. Fragments F and S are probably derived from the N- and C-terminal halves of the transferrin molecule respectively. 4. Bovine transferrin could donate iron to rabbit reticulocytes, but the monoferric fragments possessed little iron-donating ability.


Subject(s)
Transferrin/analysis , Amino Acids/analysis , Animals , Carbohydrates/analysis , Cattle , Electrophoresis, Polyacrylamide Gel , Iron/metabolism , Mercaptoethanol , Peptide Fragments/analysis , Reticulocytes/metabolism , Transferrin/metabolism , Trypsin
16.
Eur J Immunol ; 5(4): 291-3, 1975 Apr.
Article in English | MEDLINE | ID: mdl-824134

ABSTRACT

Sheep immunoglobulin IgG1 and immunoglobulin IgG2 heavy chains were treated with cyanogen bromide. The fractions from the C-terminal end of the heavy chains were isolated and purified, and the amino acid sequences gamma1 and gamma2 heavy chains had the identical sequence: Met-His-Glx-Ala-Leu-His-Asx-His-Tyr-Thr-Glx-Lys-Ser-Ile-Ser-Lys-Pro-Pro-Gly. Comparison with the C-terminal peptides of other species, reported in the literature, suggests that the subclasses are the results of recent evolutionary processes. Residues at position 4 from the C-terminus may be phylogenetically related.


Subject(s)
Immunoglobulin Allotypes , Immunoglobulin G/analysis , Immunoglobulin Heavy Chains/analysis , Immunoglobulin gamma-Chains/analysis , Sheep/immunology , Amino Acid Sequence , Animals , Cyanogen Bromide
19.
Biochem J ; 123(5): 945-58, 1971 Aug.
Article in English | MEDLINE | ID: mdl-5124396

ABSTRACT

The amino acid sequence of the light chain of the myeloma protein Dee was studied. The light chain is of the kappa type and of subgroup I. The variable part contains some substitutions that are unique and also some that have been observed already in other kappaI chains (repeated variants). Based on these repeated variants a subdivision of the kappaI subgroup is proposed.


Subject(s)
Amino Acid Sequence , Bence Jones Protein/analysis , Alkylation , Chromatography, Paper , Chymotrypsin , Electrophoresis , Formates , Humans , Immunoglobulin G/analysis , Iodoacetates , Oxidation-Reduction , Peptides/analysis , Trypsin
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