Mutants of HLA-A2 in the analysis of its structure and function.
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Amino Acid Sequence↗
Biomedical subjects
J L Strominger
Publications and source records attributed to J L Strominger.
Studies of expression of the DQ beta promoter and its 5' deletion derivatives in normal and mutant human B cell lines.
A series of 5' deletion test plasmids harboring promoter sequences of the HLA-DQ beta gene fused to the bacterial chloramphenicol acetyl transferase (CAT) gene were constructed. Transient CAT expression from these constructs in several types of cells was employed to examine the role of the promoter sequence in the regulation of DQ beta gene expression. The DQ beta constructs drove CAT expression in Raji cells (human Burkitt lymphoma cells) to at least 25-fold or 50-fold higher levels than in Hela cells (human cervical carcinoma cells) or Jurkat cells (human T-leukemia cells), respectively. A short promoter sequence of -160 bp containing the conserved X and Y sequences was sufficient for expression in Raji cells, and deletion to -106 bp which interrupted the X sequence abolished the expression. Sequences further upstream to -160 bp as far as -2500 bp which included an Ig-like octamer appeared to have no effect on expression in Raji cells. Thus, the promoter up to -160 bp has all of the sequences required for B cell specific expression of CAT in this assay. CAT expression from the 5' deletion constructs introduced into RJ2.2.5 and 6.1.6 cells (class II-negative mutant B cells lines) was also examined. None of the 5' deletion constructs, including that with -160 bp of promoter, showed CAT expression in these cells, suggesting that a transcriptional factor(s) required for the activity of the DQ beta promoter was missing in these cells. Moreover, the -160 to -66 bp sequence (including the X and Y elements), which functions as a B cell specific enhancer, was inactive in the mutant cells. Thus, the missing factor(s) are required for the enhancer function of this fragment.
B-Lymphocytes↗
Mutational analysis of the structure and function of the CD4 protein.
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Antigens, Differentiation, T-Lymphocyte↗
Novel subsets of human T cells (CD4+ CD8- TCR gamma delta and CD4- CD8- TCR alpha beta) and T-cell development.
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Animals↗
Molecular characterization of serologic recognition sites in the human HLA-A2 molecule.
The localization of the amino acid residues involved in the serologic specificity of the HLA-A2 molecule has been investigated using a combination of site-directed mutagenesis, DNA-mediated gene transfer, indirect immunofluorescence and flow cytometry techniques. Synthetic oligonucleotides were designed to introduce individual and combined amino acid substitutions in both the alpha 1 (positions 9, 43, and the highly polymorphic cluster of residues from aa 62 to 83) and alpha 2 (positions 107, 152, and 156) domains to investigate the effect of the specific mutation on the recognition of the molecule at the surface of transfected human and mouse cell lines by a panel of mAb that recognize monomorphic or polymorphic determinants in MHC class I molecules. At least three non-overlapping serologic epitopes were identified. Mutations in the highly polymorphic region at aa 62 to 66 completely eliminated binding of mAb MA2.1 (A2/B17 cross-reactive). Mutation at position 107 resulted in complete loss of binding of the A2/Aw69-specific mAb PA2.1 and MA2.2 and partial loss of mAb BB7.2 binding. The recognition by other allotypic mAbs was not affected by these mutations and they therefore represent at least a third serologic epitope. Mutations at positions 152 and 156, known to be important for T cell recognition, did not affect serologic recognition. Introduction of residues of HLA-B7 origin in the polymorphic segment spanning aa 70 to 80 created a molecule carrying the -Bw6 supertypic determinant as demonstrated by mAb SFR8-B6 binding.
Amino Acid Sequence↗
Molecular organization of the DQ subregion (DO-DX-DV-DQ) of the human MHC and its evolutionary implications.
An overlapping set of cosmid clones from the homozygous DR7 B lymphoblastoid cell line MANN linking the HLA-DO through -DQ subregions is described. This region encompasses 280 kb of DNA, including DQ alpha, DQ beta, DX alpha, DX beta, DO beta, and recently indentified L chain sequences termed DV beta. The orientation and grouping of the alpha- and beta-chains is comparable with an analogous murine class II subregion and also with the HLA-DR alpha and -DR beta chains, suggesting that the arrangement of the constituent genes of class II subregions predates the mouse/human divergence.
Biological Evolution↗
Characterization of a third form of the human T cell receptor gamma/delta.
A subpopulation of the CD3+ peripheral T lymphocytes express the TCR-gamma/delta complex. Three distinct TCR-gamma forms that differ in size and in the ability to form a disulfide bridge with the TCR-delta subunit have been described. In this study we analyze the structural difference between the non-disulfide-linked 55-kD and 40-kD TCR-gamma chains. The 40-kD TCR-gamma form contains a smaller polypeptide backbone and carries less carbohydrate compared with the 55-kD TCR-gamma form. A cDNA clone corresponding to the 40-kD TCR-gamma subunit lacks one copy of the second exon of the constant region that is present in the other TCR-gamma subunit. This exon copy encodes part of the connector region that is located between the constant domain and the membrane spanning region. We show that the number of potential N-linked glycan attachment sites are the same for the two TCR-gamma forms. Since these attachment sites are located in the connector region we conclude that the connector region influences the amount of N-linked carbohydrates added to the core TCR-gamma polypeptide, probably by affecting the conformation of the protein. In contrast to the TCR-beta constant region usage, the TCR-gamma constant regions are unequally expressed. Virtually exclusive usage of disulfide-linked complexes were found in some individuals, while both the disulfide-linked and the 40-kD, non-disulfide-linked TCR-gamma forms were detected in other subjects. The ability to distinguish these TCR-gamma/delta forms now makes it possible to study the mechanisms that govern their selection and to determine if they correspond to functionally distinct isotypes.
Amino Acid Sequence↗
Functional analysis of a cytoplasmic domain-deleted mutant of the CD4 molecule.
The CD4 molecule is a receptor found on a subset of T lymphocytes. It has been proposed that, upon binding MHC class II molecules expressed on APC, the CD4 molecule enhances the responsiveness of the T cell by increasing intercellular avidity and/or by transducing an intracellular signal. We have analyzed the effect of removing the cytoplasmic domain of the CD4 molecule on the ability of the CD4 molecule to enhance T cell responsiveness. The cytoplasmic domain-deleted mutant of the CD4 molecule (CD4 delta) was found to be as efficient as the CD4 molecule at enhancing responsiveness to cells bearing the appropriate Ag. If subcellular Ag in the form of purified Ag incorporated into liposomes was used, the CD4 molecule was found to be much more efficient than the CD4 delta molecule at enhancing responsiveness. However, the defect in the ability of the CD4 delta molecule to enhance responsiveness could be compensated for by increasing the level of expression of the CD4 delta molecule.
Amino Acid Sequence↗
Extensive junctional diversity of rearranged human T cell receptor delta genes.
The human T cell receptor delta (TCR delta) gene encodes one component of the TCR gamma delta-CD3 complex found on subsets of peripheral blood and thymic T cells. Human TCR delta diversity was estimated by characterizing rearrangements in TCR gamma delta cell lines and determining the structures of complementary DNA clones representing functional and nonfunctional transcripts in these cell lines. One V delta segment and one J delta segment were identified in all functional transcripts, although a distinct J delta segment was identified in a truncated transcript. Further, one D delta element was identified, and evidence for the use of an additional D delta element was obtained. Thus human TCR delta genes appear to use a limited number of germline elements. However, the apparent use of two D delta elements in tandem coupled with imprecise joining and extensive incorporation of N nucleotides generates unprecedented variability in the junctional region.
Amino Acid Sequence↗
Site-directed mutants of a soluble form of penicillin-binding protein 5 from Escherichia coli and their catalytic properties.
Soluble, truncated mutant and wild-type forms of penicillin-binding protein 5 (sPBP 5) from Escherichia coli were produced in large amounts by placing the dacA gene that encodes PBP 5 under the control of the trp-lac fusion promoter. The 3' end of the dacA gene used in this study contains a stop codon that results in the deletion of 15 amino acids from the carboxyl terminus and the production of a soluble protein. Using oligonucleotide-directed mutagenesis, the role of cysteine 115 in the mechanism of sPBP 5 was investigated. Alkylation of cysteine 115 with sulfhydryl reagents has previously been shown to inhibit severely the D-alanine carboxypeptidase activity of PBP 5. Alkylation also inhibits the hydrolysis of bound penicillin G, with only a slight effect on its binding. Cysteine 115 in sPBP 5 was changed to either a serine (sPBP 5C-S) or an alanine (sPBP 5C-A) residue. The wild-type and mutant sPBPs were purified in milligram amounts from induced cultures by ampicillin affinity chromatography. The mutant PBPs showed only a 2-fold increase in the half-life of the penicilloyl-PBP complex, and had a binding affinity for penicillin G identical to wild-type PBP 5. The Km for the release of D-alanine from the peptide L-Ala-D-gamma-Glu-L-Lys-D-Ala-D-Ala was 5.0, 3.5, and 7.8 mM for PBP 5, PBP 5C-S, and PBP 5C-A, respectively, while the values for Vmax were 2.5, 3.3, and 5.1 mumol/min/mg. From these data it was concluded that the cysteine residue does not directly participate in the enzymatic mechanism.
Amino Acid Sequence↗
Expression and function of a CD5 cDNA in human and murine T cells.
In order to define the function of the CD 5 (T1, Leu 1, Tp 67 in the human; Ly-1 in the mouse) molecule, a cDNA clone of human CD 5 was expressed in a CD5-deficient Jurkat cell line and in a murine T cell hybridoma. A Jurkat subclone (Jurkat 9.9) produced interleukin 2 (IL 2) in response to anti-CD 3 monoclonal antibody (mAb) cross-linked to solid support. IL 2 production was enhanced by co-culture with the anti-CD 5 mAb OKT 1. A CD 5-deficient mutant clone Jurkat 1.15 was generated by treatment with ethyl methanesulfonate followed by selection with anti-CD 5 mAb plus complement. Jurkat 1.15 did not demonstrate enhancement of IL 2 production by OKT 1 in the presence of cross-linked anti-CD 3 mAb. A cDNA encoding human CD 5 was introduced into a defective retrovirus which was used to infect Jurkat 1.15. A Jurkat clone stably expressing CD 5 was established. In response to OKT 1, a rise in intracellular calcium was observed in both the parent Jurkat 9.9 and the CD 5+ infectant but not in the CD 5- mutant or a G 418- resistant control. Furthermore, expression of CD 5 restored the augmentation of IL 2 production by OKT 1 in response to cross-linked anti-CD 3 mAb. A murine T cell hybridoma By 155.16 which produces IL 2 in response to HLA-DR antigens was also infected with the CD 5-recombinant retrovirus and three stable CD 5+ infectants were generated. These hybridomas showed enhancement of IL 2 production by stimulation with OKT 1 in the presence of suboptimal concentrations of soluble anti-murine CD 3 mAb. These results provide further evidence that CD 5 provides a co-stimulatory signal for T cell activation.
Animals↗
Two distinct genetic loci regulating class II gene expression are defective in human mutant and patient cell lines.
Heterokaryons were prepared and analyzed shortly after cell fusion using two mutant class-II-negative human B cell lines (RJ 2.2.5 and 6.1.6) and a cell line (TF) from a patient with a class-II-negative Bare Lymphocyte Syndrome. The resulting transient heterokaryons were analyzed by using an anti-HLA-DR monoclonal antibody to assess the cell surface expression of HLA-DR (the major subtype of class II antigens) by immunofluorescence microscopy and by using uniformly 32P-labeled SP6 RNA probes in Northern blots and RNase protection assays to assess mRNA synthesis. We find that class II gene expression in a B cell line from a Bare Lymphocyte Syndrome patient (TF) is rescued by a B cell line which expresses class II antigens indicating that this disease, at least in part, is caused by a defect(s) in a genetic locus encoding a factor(s) necessary for class II gene expression. Secondly, reciprocal genetic complementation was demonstrated in the heterokaryons 6.1.6 x RJ 2.2.5 and TF x RJ 2.2.5 (but not in TF x 6.1.6) by detection of cell surface DR by immunofluorescence microscopy and by a novel class II mRNA typing technique which allows characterization of distinct class II alleles. Thus, the two mutants generated in vitro have defects at two different genetic loci encoding specific regulatory factors necessary for human class II gene expression. One of these mutant cell lines, but not the other, complements the defect in the patient cell line, TF.
B-Lymphocytes↗
A solubilized T-cell receptor from a human leukemia cell line binds to a ligand in the absence of MHC products.
A human T cell alpha beta antigen receptor from the acute lymphoblastoid leukemia line HPB-ALL (also called HPB-MLT) binds and is precipitated in detergent-solubilized form by an antigen present on the surface and secreted by several strains of the gram-positive bacterium Staphylococcus aureus. This binding is completely independent of major histocompatibility complex (MHC) antigens. Receptor/ligand binding is unique to this one cell line (i.e., clonotypic) and furthermore completely blocked by an idiotype-specific monoclonal antibody (mAb) to this receptor, but not by three different nonidiotype-specific mAbs. The nature of this interaction appears more similar to immunoglobulin/antigen binding than to T-cell receptor/antigen/MHC/accessory molecule interactions and would suggest that some T-cell receptors may not require MHC products to interact with antigen.
Antigens, Bacterial↗
T-cell receptor gene rearrangement and expression in human natural killer cells: natural killer activity is not dependent on the rearrangement and expression of T-cell receptor alpha, beta, or gamma genes.
To test the hypothesis that the T-cell receptor (Tcr) gamma gene encodes a natural killer (NK) cell receptor molecule, three human NK clones and fresh peripheral blood lymphocytes with NK activity from two patients with a CD16+ lymphocytosis were analyzed for rearrangements and expression of the human Tcr alpha, beta, and gamma genes. Two of the clones displayed distinct rearrangements of their Tcr beta and gamma genes and expressed mature Tcr alpha, beta, and gamma RNA. However, one of the clones and both patient samples displayed marked NK activity but failed to rearrange or express any of their Tcr genes. These findings demonstrate that human natural killer activity is not dependent on Tcr gamma gene rearrangement and expression. In addition, they confirm previous findings concerning the lack of Tcr alpha and beta gene expression in some natural killer cells. Thus, they suggest the existence of additional NK-specific recognition molecules.
Adult↗
Restriction fragment length polymorphisms and an HLA-DRw52-associated split.
The restriction fragment length polymorphism technique, employing seven restriction endonucleases, and a DR-specific repeated element probe were used to study a large panel of homozygous typing cells in order to delineate haplotypic differences within the HLA-DRw52 supertype. Most of the restriction endonucleases revealed the presence of two allelic restriction fragment length polymorphisms correlating with and splitting the HLA-DRw52 supertypic specificity. One designated A, correlated with the HLA-DRw52 typing in HLA-DR5, HLA-DR3, (non-A1, B8), and some HLA-DRw6 haplotypes. The other, designated a, correlated with the HLA-DRw52 typing in HLA-DR5, HLA-DRw8, HLA-DR3 (A1, B8), and the remaining HLA-DRw6 haplotypes. The general applicability of these findings was validated in 47 HLA-typed laboratory controls.
Alleles↗
Complex transcription of the Epstein-Barr virus BamHI fragment H rightward open reading frame 1 (BHRF1) in latently and lytically infected B lymphocytes.
Several cDNA clones containing the Epstein-Barr virus BamHI fragment H rightward open reading frame 1 (BHRF1) have been recovered from the tightly latent lymphoblastoid cell line IB4. These clones contain the 5' leader exons encoded in the major internal repeat 1 and the viral BamHI fragment Y, identified in the rightwardly transcribed viral mRNAs associated with the latent viral life cycle. In addition, a cDNA clone containing BHRF1 from the Burkitt lymphoma cell line Jijoye was also recovered and exhibits a distinctive splicing pattern. In vitro transcription and translation of BHRF1, followed by immunoprecipitation with Epstein-Barr virus-positive human sera, indicates that this viral antigen is expressed during infection. RNA blot analyses with a wide panel of lymphoblastoid and Burkitt lymphoma cell lines revealed a complex pattern of transcription. Hybridization data obtained with several probes is presented.
Cloning, Molecular↗
B-cell-specific enhancer activity of conserved upstream elements of the class II major histocompatibility complex DQB gene.
A 95-base-pair immediate upstream sequence of the human class II major histocompatibility complex DQB gene containing the conserved X and Y elements showed enhancer activity in a transient expression assay. An "enhancer test plasmid" harboring the bacterial chloramphenicol acetyltransferase gene under the control of a truncated simian virus 40 enhancerless early promoter was employed. The DQB sequence inserted into this plasmid was active as an enhancer in Raji cells (human Burkitt lymphoma cells) but not active in Jurkat cells (human T-cell leukemia cells) or in HeLa cells (human cervical carcinoma cells). This cell-type specificity suggests that this enhancer activity may be involved in the tissue specificity of the DQB gene that is normally expressed only in mature B cells, macrophages, and thymic epithelial cells. Deletion analysis showed that both X and Y box sequences are essential for the full activity of the enhancer sequence and that these two sequences may function in a cooperative manner as cis-acting elements. Further deletions were used to define the 5' border of the X element. These results suggest that previously characterized protein factors that bind to X and Y include transcription factors involved in the cell-type specificity of this enhancer activity.
Acetyltransferases↗
Genomic organization of the human T-cell antigen-receptor alpha/delta locus.
Two clusters of overlapping cosmid clones comprising about 100 kilobases (kb) at the human T-cell antigen-receptor alpha/delta locus were isolated from a genomic library. The structure of the germ-line V delta 1 variable gene segment was determined. V delta 1 is located 8.5 kb downstream of the V alpha 13.1 gene segment, and both V segments are arranged in the same transcriptional orientation. The V alpha 17.1 segment is located between V delta 1 and the D delta, J delta, C delta region (containing the diversity, joining, and constant gene segments). Thus, V delta and V alpha segments are interspersed along the chromosome. The germ-line organization of the D delta 2, J delta 1, and J delta 2 segments was determined. Linkage of C delta to the J alpha region was established by identification of J alpha segments within 20 kb downstream of C delta. The organization of the locus was also analyzed by field-inversion gel electrophoresis. The unrearranged V delta 1 and D delta, J delta, C delta regions are quite distant from each other, apparently separated by a minimum of 175-180 kb.
Base Sequence↗