Biomedical subjects

J L Strominger

Publications and source records attributed to J L Strominger.

At least 145 records · Page 8Linked to original sources

PubMed1994

Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide.

An influenza virus peptide binds to HLA-DR1 in an extended conformation with a pronounced twist. Thirty-five per cent of the peptide surface is accessible to solvent and potentially available for interaction with the antigen receptor on T cells. Pockets in the peptide-binding site accommodate five of the thirteen side chains of the bound peptide, and explain the peptide specificity of HLA-DR1. Twelve hydrogen bonds between conserved HLA-DR1 residues and the main chain of the peptide provide a universal mode of peptide binding, distinct from the strategy used by class I histocompatibility proteins.

Amino Acid Sequence↗

PubMed1994

A subset of HLA-B27 molecules contains peptides much longer than nonamers.

An unusual monoclonal antibody (MARB4) directed against HLA-B27 that reacts with only approximately 5-20% of the cell surface HLA-B27 was used for large-scale purification of these molecules. Subsequent mass spectrometry of HLA-B27-bound peptides showed that the minor MARB4-reactive population contained peptides primarily from 900 to 4000 Da in size (approximately 8-33 amino acid residues), whereas the major HLA-B27 population contained peptides in the mass range of 900-1400 Da (approximately 8-12 amino acid residues). Thus, a subset of HLA-B27 molecules binds to peptides much longer than nonamers. Typical HLA-B27-binding peptides contain arginine in position 2. Further analysis by Edman sequencing of the pooled bound peptides revealed that the major population contained substantial amounts of arginine at positions 1 and 9 (40-50%) and exclusively arginine at position 2, as expected. The minor population of peptides also contained detectable amounts of arginine at these positions, but at the level of only approximately 10%; no marked enrichment at any position was observed. These long HLA-B27-bound peptides could represent either intermediates in the formation of nonamers or adventitiously bound peptides. Lastly, in the TAP2 mutant cell line BM36.1 transfected with HLA-B*2705, MARB4-reactive HLA-B27 molecules were absent from the cell surface, indicating that the peptide transporter was required for delivery of the long peptides. Thus, during the folding of class I heavy chains, peptides of diverse lengths are available and participating.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Metalloprotease and serine protease are involved in cleavage of CD43, CD44, and CD16 from stimulated human granulocytes. Induction of cleavage of L-selectin via CD16.

CD43, CD44, CD16, and L-selectin have been previously shown to be enzymatically cleaved from stimulated leukocytes. However, little is known about the enzymes involved in these processes. Here, metalloprotease(s) inhibitable by 1,10-phenanthroline together with serine protease(s) inhibitable by N alpha-p-tosyl-L-lysine chloromethyl ketone and 3,4-dichloroisocoumarin are shown to be involved in the cleavage of CD43, CD44, and CD16 but not in the cleavage of L-selectin on granulocytes. In addition, mAbs that recognize these individual receptors and induce their specific cleavage did not initiate cleavage of the others. In one case only, L-selectin, cleavage was also triggered by mAbs interacting with CD16 (the low affinity Fc gamma R). Thus, this mechanism represents a novel pathway of L-selectin cleavage induction.

Antibodies, Monoclonal↗

PubMed1994

Structural requirements for binding of an immunodominant myelin basic protein peptide to DR2 isotypes and for its recognition by human T cell clones.

Immunodominant T cell epitopes of myelin basic protein (MBP) may be target antigens for major histocompatibility complex class II-restricted, autoreactive T cells in multiple sclerosis (MS). Since susceptibility to MS is associated with the DR2 haplotype, the binding and presentation of the immunodominant MBP(84-102) peptide by DR2 antigens were examined. The immunodominant MBP(84-102) peptide was found to bind with high affinity to DRB1*1501 and DRB5*0101 molecules of the disease-associated DR2 haplotype. Overlapping but distinct peptide segments were critical for binding to these molecules; hydrophobic residues (Val189 and Phe92) in the MBP(88-95) segment were critical for peptide binding to DRB1*1501 molecules, whereas hydrophobic and charged residues (Phe92, Lys93) in the MBP(89-101/102) sequence contributed to DRB5*0101 binding. The different registers for peptide binding made different peptide side chains available for interaction with the T cell receptor. Although the peptide was bound with high affinity by both DRB1 and DRB5 molecules, only DRB1 (DRB1*1501 and 1602) but not DRB5 molecules served as restriction elements for a panel of T cell clones generated from two MS patients suggesting that the complex of MBP(84-102) and DRB1 molecules is more immunogenic for MBP reactive T cells. The minimal MBP peptide epitope for several T cell clones and the residues important for binding to DRB1*1501 molecules and for T cell stimulation have been defined.

Amino Acid Sequence↗

PubMed1994

Self-peptides bound to the type I diabetes associated class II MHC molecules HLA-DQ1 and HLA-DQ8.

Genetic susceptibility to several autoimmune disorders is associated with the expression of certain MHC class II alleles. Insight into the etiology of such diseases awaits the identification of the class II restriction elements and the possible pathogenic peptides. Towards these aims, self-peptides bound to HLA-DQ1 and HLA-DQ8, allotypes considered to be neutral and permissive respectively towards the development of insulin-dependent diabetes mellitus, are reported. These naturally processed peptides were isolated from immunoaffinity purified HLA-DQ molecules expressed in cultured B lymphocytes. The chromatographic profiles of the peptide repertoires are unique, whereas the size distributions exhibit general similarity to those reported for naturally processed self-peptides bound to HLA-DR. Twenty-eight individual peptides representing 10 nested sets were identified by combined Edman microsequencing and mass spectrometry. Peptide length varied from 13 to 74 amino acids. Source proteins included MHC molecules and other integral membrane proteins, as well as secretory, cytosolic and mitochondrial proteins. Promiscuous invariant chain peptides were identified among the self-peptides bound to HLA-DQ8. No dominant amino acid markers suggestive of particular enzymatic processing events were detected. Some structural features of DQ1 and DQ8 that may relate to the bound peptides are discussed. Peptide specificity was confirmed in binding assays with purified HLA-DQ and HLA-DR protein.

Amino Acid Sequence↗

PubMed1994

Recombination hotspot associated factors specifically recognize novel target sequences at the site of interchromosomal rearrangements in T-ALL patients with t(8;14)(q24;q11) and t(1;14)(p32;q11).

A DNA binding protein was identified which binds to two novel target-like sequences: (i) at the 5' flanking site of the breakpoint junction of chromosome 8 in a patient with T-acute lymphoblastic leukemia (ALL) carrying the t(8;14)(q24;q11) rearrangement and (ii) on chromosome 1 in three of five T-ALL patients with the t(1;14)(p32;q11) rearrangement. This protein [provisionally called recombination hotspot associated factor (ReHF-1)] was also found to bind to a similar target sequence that is present immediately at the 3' end of the human V delta 3 gene segment. In a small number of lines tested, the ReHF-1 protein was expressed in gamma delta lineage T cells and in a number of B cell precursor ALLs whose TCR delta locus has been rearranged. The molecular weight of ReHF-1 protein was determined to be approximately 30 kDa by UV cross-linking analysis. Gel filtration chromatography and sedimentation velocity centrifugation analyses indicate that the ReHF-1 protein exists as a multimeric protein in its native form. These data might suggest a possible role for this protein in the rearrangement of the TCR delta locus. Furthermore, another protein, ReHF-2, that appears to have strict sequence specificity was found to bind only to the complementary single strand of the target sequence. The interaction of these proteins with a conserved target sequence at the chromosomal breakpoint junction might suggest that they are involved in a novel enzymatic mechanism reminiscent of the general features of DNA recombination or replication events in Escherichia coli or Saccharomyces cerevisiae.

Base Sequence↗

PubMed1994

A factor that regulates the class II major histocompatibility complex gene DPA is a member of a subfamily of zinc finger proteins that includes a Drosophila developmental control protein.

A novel DNA sequence element termed the J element involved in the regulated expression of class II major histocompatibility complex genes was recently described. To study this element and its role in class II gene regulation further, a cDNA library was screened with oligonucleotide probes containing both the S element and the nearby J element of the human DPA gene. Several DNA clones were obtained by this procedure, one of which, clone 18, is reported and characterized here. It encodes a protein predicted to contain 688 amino acid residues, including 11 zinc finger motifs of the C2H2 type in the C-terminal region, that are Krüppel-like in the conservation of the H/C link sequence connecting them. The 160 N-terminal amino acids in the nonfinger region of clone 18 are highly homologous with similar regions of several other human, mouse, and Drosophila sequences, defining a subfamily of Krüppel-like zinc finger proteins termed TAB (tramtrack [ttk]-associated box) here. One of the Drosophila sequences, ttk, is a developmental control gene, while a second does not contain a zinc finger region but encodes a structure important in oocyte development. An acidic activation domain is located between the N-terminal conserved region of clone 18 and its zinc fingers. This protein appears to require both the S and J elements, which are separated by 10 bp for optimal binding. Antisense cDNA to clone 18 inhibited the expression of a reporter construct containing the DPA promoter, indicating its functional importance in the expression of this class II gene.

Amino Acid Sequence↗

PubMed1994

Nuclear proteins binding to a novel target sequence within the recombination hotspot regions of Bcl-2 and the immunoglobulin DH gene family.

The chromosomal breakpoints of follicular lymphomas carrying the t(14;18)(q32;q21) are known to be clustered within a 150-bp region in the major breakpoint region (mbr) of the Bcl-2 oncogene. We have demonstrated that nuclear proteins specifically bind to a novel target sequence within this 150-bp region and a region of Dxp genes, members of the immunoglobulin (Ig) diversity (DH) gene family. One protein, designated BCLF-1, appears to be specifically expressed in lymphoid lineage cells. Two other proteins, BCLF-2 and -3, bind only to the complementary single strand of the target sequence. The manner in which these proteins interact with the target sequence is similar to the interaction of the ReHF-1 and -2 proteins to the signal-like sequence at the chromosomal breakpoint junctions in patients with the t(8;14)(q24;q11) and t(1;14)(p32;q11) translocations. It was further suggested that the BCLF-1 is quite similar to or identical to the ReHF-1. It is therefore hypothesized that these conserved target sequences found in recombination hotspot regions may define novel sequence motifs recognized by two classes of DNA binding proteins. One class of DNA binding proteins is specifically expressed in lymphoid cells while the other class binds to the complementary single strand DNA. These binding activities may play a crucial role in chromosomal translocation in lymphoid neoplasms.

Base Sequence↗

PubMed1993

Human T-cell receptor (TCR) alpha/beta + CD4-CD8- T cells express oligoclonal TCRs, share junctional motifs across TCR V beta-gene families, and phenotypically resemble memory T cells.

Most human T cells express the TCR alpha/beta and either CD4 or CD8 molecules (single positive, SP); however, small numbers lack CD4 and CD8. In inbred mice, alpha/beta CD4-CD8- (double negative, DN) T cells preferentially express certain beta variable region (V beta) families and may arise via unique developmental pathways. Increased percentages of alpha/beta DN T cells have been identified in some human and murine autoimmune and immunodeficiency diseases. However, their contribution to disease pathology or normal immunity is unknown. To study the cell surface phenotype and TCR diversity of human alpha/beta DN T cells, these cells were isolated from the peripheral blood of healthy adults. The proportion of alpha/beta DN T cells expressing molecules associated with activation (HLA-DR), previous exposure to antigen (CD45RO), and cytotoxic function (CD56, CD57, and CD11b) was increased relative to SP T cells. The TCR V beta repertoire of alpha/beta DN T cells was different from that of alpha/beta SP T cells, although most major gene families were present. For example, higher proportions of V beta 11, a minor gene family in peripheral blood leukocytes, were found in most alpha/beta DN T-cell samples. In contrast to mice, no dominant V beta family was used consistently in different human individuals. Within an individual alpha/beta DN T cells possessed an oligoclonal TCR beta repertoire with conservation of several distinct junctional amino acid motifs with one joined to three different V beta genes in two individuals, suggesting that these cells have undergone a selection process driven by a limited set of ligands. The possibility that they may represent, at least in part, originally SP T cells anergized by down-modulation of CD4 or CD8 must also be entertained. Overall, this study demonstrates that human peripheral blood alpha/beta DN T cells possess unique phenotypic and TCR beta repertoire characteristics when compared with the major alpha/beta SP T cell populations and thus may serve specialized immunologic functions and/or have an unusual origin.

Adult↗

PubMed1993

HLA-C is the inhibitory ligand that determines dominant resistance to lysis by NK1- and NK2-specific natural killer cells.

Natural killer (NK) cells recognize alloantigens on normal cells. One of these alloantigens correlates with homozygosity for a dimorphism of HLA-C at positions 77-80, which is shared by a number of HLA-C alleles. A second allelic alloantigen correlates with homozygosity for the alternative HLA-C dimorphism, which is shared by the remaining HLA-C alleles. Moreover, NK1- and NK2-specific NK cell lines can be generated by mixed leukocyte cultures in which donor and stimulator are homozygous for the alternative dimorphisms at positions 77-80 of HLA-C. In the present work, the role of HLA-C in NK cell-mediated allorecognition was directly investigated by analyzing the effects produced by transfection of several HLA-C alleles on NK sensitivity of class I-deleted mutant cell lines. Transfection of cells with HLA-C alleles encoding Asn-77-Lys-80 (including HLA-Cw4, -Cw5, and -Cw6) inhibited the lysis of the targets by NK1-specific NK cells, whereas HLA-C alleles encoding Ser-77-Asn-80 (including HLA-Cw1, -Cw7, and -Cw13) protected the targets from NK2-specific NK cells. Thus, HLA-C alleles are the dominant inhibitory ligands that protect targets from lysis by these allospecific NK cells.

Alleles↗

PubMed1993

Allele-specific peptide ligand motifs of HLA-C molecules.

The consensus motifs of HLA-Cw3, -Cw4, -Cw6, and -Cw7 ligands were determined by pool sequencing. Together with information obtained by sequencing of some prominent individual peptides, the results indicate the following: (i) all four HLA-C molecules are associated with peptides. (ii) These peptides adhere to allele-specific motifs that are similar to those of to HLA-A or -B molecules; they have a preferred length of nine amino acids and an anchor residue at the C terminus. (iii) All four HLA-C molecules analyzed exhibit related peptide motifs, although each allelic product shows individual characteristics in fine specificity. (iv) Processing and origin of peptides appear not to be different from that of other class I molecules. (v) No obvious difference at C-terminal position 9 was present in the peptides isolated from the two dimorphic variants of HLA-C that determine dominant resistance to natural killer NK1-specific cells (HLA-Cw4, -Cw6) or to NK2-specific cells (HLA-Cw3, -Cw7) and that differ in two residues in or near the pocket at position 9.

Amino Acid Sequence↗

PubMed1993

Cell adhesion mediated by CD4 and MHC class II proteins requires active cellular processes.

Human CD4 is an accessory molecule found on the cell surface of a subset of T lymphocytes. We have previously shown by infection of simian fibroblasts with an SV40-CD4 recombinant virus that CD4 acts as an adhesion molecule by binding to human MHC class II Ag expressed on the surface of human B lymphocytes. This report confirms that human B lymphoblastoid cell lines expressing class II molecules at the cell surface can bind to Chinese hamster ovary cells that have been stably transfected with human CD4. This cellular adhesion is a late event, which is first detected after 2 h, but remains stable for up to 16 h. The association between CD4 and class II is energy dependent, as it is detected at 37 degrees C, but not at 4 degrees C or if either cell type is fixed with paraformaldehyde. ATP is required for the establishment and maintenance of stable CD4/class II-mediated cell conjugates. Cytoskeletal interactions also regulate CD4/class II adhesion as treatment with the microtubule and microfilament inhibitors colchicine, cytochalasin-D, and nocodazole rapidly dissociates even preformed cell conjugates. Our observations also indicate that the Ag-independent engagement of class II by CD4 induces homotypic clustering of human B cells. This effect is blocked by reagents directed against lymphocyte function-associated Ag-1 and may result from the induction of a high affinity phenotype of lymphocyte function-associated Ag-1 induced by class II signaling. Finally, we discuss the implications of CD4/class II-mediated adhesion and the role of CD4 in the regulation of T cell adhesion.

Animals↗

PubMed1993

Interactions of CD4 with MHC class II molecules, T cell receptors and p56lck.

CD4 and CD8 are members of the immunoglobulin supergene family of proteins, and function as co-receptors with the T cell receptor (TCR) in binding MHC class II or class I molecules, respectively. Within this multimeric complex, CD4 interacts with three distinct ligands. CD4 interacts through its D1 and D2 domains with MHC class II proteins, through its D3 and D4 domains with T cell receptors, and through its cytoplasmic tail with p56lck, a src-related, protein tyrosine kinase. Each of these interactions is important in the function of CD4 and will be discussed in turn.

Amino Acid Sequence↗

PubMed1993

Identification of a novel cyclosporin-sensitive element in the human tumor necrosis factor alpha gene promoter.

Tumor necrosis factor alpha (TNF-alpha), a cytokine with pleiotropic biological effects, is produced by a variety of cell types in response to induction by diverse stimuli. In this paper, TNF-alpha mRNA is shown to be highly induced in a murine T cell clone by stimulation with T cell receptor (TCR) ligands or by calcium ionophores alone. Induction is rapid, does not require de novo protein synthesis, and is completely blocked by the immunosuppressant cyclosporin A (CsA). We have identified a human TNF-alpha promoter element, kappa 3, which plays a key role in the calcium-mediated inducibility and CsA sensitivity of the gene. In electrophoretic mobility shift assays, an oligonucleotide containing kappa 3 forms two DNA protein complexes with proteins that are present in extracts from unstimulated T cells. These complexes appear in nuclear extracts only after T cell stimulation. Induction of the inducible nuclear complexes is rapid, independent of protein synthesis, and blocked by CsA, and thus, exactly parallels the induction of TNF-alpha mRNA by TCR ligands or by calcium ionophore. Our studies indicate that the kappa 3 binding factor resembles the preexisting component of nuclear factor of activated T cells. Thus, the TNF-alpha gene is an immediate early gene in activated T cells and provides a new model system in which to study CsA-sensitive gene induction in activated T cells.

Base Sequence↗

PubMed1993

Comparison of the P2 specificity pocket in three human histocompatibility antigens: HLA-A6801, HLA-A0201, and HLA-B*2705.

Coordinates from x-ray structures of HLA-A*6801, HLA-A*0201, and HLA-B*2705 were analyzed to examine the basis for their selectivity in peptide binding. The pocket that binds the side chain of the peptide's second amino acid residue (P2 residue) shows a preference for Val, Leu, and Arg in these three HLA subtypes, respectively. The Arg-specific pocket of HLA-B*2705 differs markedly from those of HLA-A*0201 and HLA-A*6801, as a result of numerous differences in the side chains that form the pocket's surface. The cause of the specificity differences between HLA-A*0201 and HLA-A*6801 is more subtle and depends both on a change in conformation of pocket residue Val-67 and on a sequence difference at residue 9. The Val-67 conformational change appears to be caused by a shift in the position of the alpha 1-domain alpha-helix relative to the beta-sheet in the cleft and may, in fact, depend on amino acid differences remote from the P2 pocket. Analysis of the stereochemistry of the P2 side chain interacting with its binding pocket permits an estimate to be made of its contribution to the free-energy change of peptide binding.

Amino Acid Sequence↗

PubMed1993

The regulatory gene, hXBP-1, and its target, HLA-DRA, utilize both common and distinct regulatory elements and protein complexes.

hXBP-1 is a transcription factor of the leucine zipper (b-zip) family important in the expression of the class II major histocompatibility complex gene, DRA. Studies with mouse-human hybrids have mapped hXBP-1 hybridizing fragments to human chromosomes 5 and 22 and the frequent detection of two mRNA transcripts suggested that hXBP-1 may be a member of a small gene family. To analyze the structure and regulation of hXBP-1 further, cosmid clones from both chromosomes were isolated. Mapping and sequence analyses reveal that chromosome 22 contains the functional hXBP-1 gene while chromosome 5 contains a processed pseudogene. hXBP-1 promoter analysis has revealed that cis-active elements within the 5'-untranslated region of hXBP-1 are essential for full promoter activity. One such element, hX2, is identical to the hXBP-1 target sequence in the DRA promoter. Mutagenesis of the hX2 site substantially decreases promoter activity. This element interacts with four distinct protein complexes in mature B cells and cross-competition experiments show that two of these complexes (complex 1 and complex 4) also interact with the hXBP-1 target sequence (X2) from the DRA promoter. The similarities of the hXBP-1 promoter and of the DRA promoter (the gene that the hXBP-1 protein regulates) are further emphasized by the fact that a Y box element is located 3' of both hX2 and X2.

Amino Acid Sequence↗

PubMed1993

Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1.

The three-dimensional structure of the class II histocompatibility glycoprotein HLA-DR1 from human B-cell membranes has been determined by X-ray crystallography and is similar to that of class I HLA. Peptides are bound in an extended conformation that projects from both ends of an 'open-ended' antigen-binding groove. A prominent non-polar pocket into which an 'anchoring' peptide side chain fits is near one end of the binding groove. A dimer of the class II alpha beta heterodimers is seen in the crystal forms of HLA-DR1, suggesting class II HLA dimerization as a mechanism for initiating the cytoplasmic signalling events in T-cell activation.

B-Lymphocytes↗

PubMed1993

Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles.

Naturally processed peptides were acid extracted from immunoaffinity-purified HLA-DR2, DR3, DR4, DR7, and DR8. Using the complementary techniques of mass spectrometry and Edman microsequencing, > 200 unique peptide masses were identified from each allele, ranging from 1,200 to 4,000 daltons (10-34 residues in length), and a total of 201 peptide sequences were obtained. These peptides were derived from 66 different source proteins and represented sets nested at both the amino- and carboxy-terminal ends with an average length of 15-18 amino acids. Strikingly, most of the peptides (> 85%) were derived from endogenous proteins that intersect the endocytic/class II pathway, even though class II molecules are thought to function mainly in the presentation of exogenous foreign peptide antigens. The predominant endogenous peptides were derived from major histocompatibility complex-related molecules. A few peptides derived from exogenous bovine serum proteins were also bound to every allele. Four prominent promiscuous self-peptide sets (capable of binding to multiple HLA-DR alleles) as well as 84 allele-specific peptide sets were identified. Binding experiments confirmed that the promiscuous peptides have high affinity for the binding groove of all HLA-DR alleles examined. A potential physiologic role for these endogenous self-peptides as immunomodulators of the cellular immune response is discussed.

Alleles↗