Biomedical subjects

J Bajorath

Publications and source records attributed to J Bajorath.

At least 73 records · Page 4Linked to original sources

PubMed1995

Both extracellular immunoglobin-like domains of CD80 contain residues critical for binding T cell surface receptors CTLA-4 and CD28.

The B7-related molecules CD80 and CD86 are expressed on antigen-presenting cells, bind the homologous T cell receptors CD28 and CTLA-4, and trigger costimulatory signals important for optimal T cell activation. All four molecules are immunoglobulin superfamily members, each comprising an extracellular Ig variable-like (IgV) domain, with CD80 and CD86 containing an additional Ig constant-like (IgC) domain. Despite limited sequence identity, CD80 and CD86 share similar overall receptor binding properties and effector functions. We have identified, by site-directed mutagenesis of soluble forms of CD80 and CD86, residues in both the IgV and IgC domains that are important for CTLA4Ig and CD28Ig binding. Mutagenesis in the IgV domain of CD80 identified 11 amino acids that support receptor binding. Many of these residues are conserved in the B7 family, are hydrophobic, and approximately map to the GFCC'C" beta-sheet face of an IgV fold. Mutagenesis of corresponding residues in CD86 established that some, but not all, of these residues also played a role in CD86 receptor binding. In general, mutations had a similar effect on CTLA4Ig and CD28Ig binding, thereby indicating that both receptors bind to overlapping sites on CD80 and CD86. Further, mutagenesis of several conserved residues in the ABED beta-sheet face of the IgC domain of CD80 completely ablated receptor binding. Point mutagenesis had a more pronounced effect than complete truncation of the IgC domain. Thus, full CTLA4Ig and CD28Ig binding to B7 molecules is dependent upon residues in the GFC'C" face of the IgV domain and the ABED face of the IgC domain.

Abatacept↗

PubMed1995

Affinity maturation of the BR96 anti-carcinoma antibody by codon-based mutagenesis.

We have increased up to 65-fold the avidity of BR96, a mAb recognizing Lewis Y (Le(y))-related Ags expressed on the surface of many human carcinomas. Libraries of mutations in the complementarity-determining regions (CDRs) of BR96 were constructed in an M13 phage Fab expression vector by codon-based mutagenesis, a method that efficiently introduces large numbers and potentially all combinations of amino acid substitutions. Two mutants that improved the affinity of BR96 to tumor Ag were identified by screening the libraries on carcinoma cell lines. One mutant, M1, at position 97 (Asp to Ala) in CDR3 of the heavy chain, resulted in an 8- to 10-fold improvement in Ag binding, as assessed by ELISA. A second mutant, M2, at position 53 (Gly to Asp) in CDR2 of VH increased binding three- to fivefold. When these mutations were combined, the resulting Fab M3 was improved approximately 30-fold. An additional library was constructed in CDR1 of M1. M4, a mutation with three amino acid substitutions in CDR1, was isolated by screening the library with an enzyme conjugate of synthetic Le(y) tetrasaccharide (sLe(y)). This mutant improved BR96 Fab affinity to sLe(y) an estimated 15- to 20-fold by ELISA, and 14-fold as measured by surface plasmon resonance. The M4 IgG had 65-fold improved avidity to sLe(y) relative to the BR96 IgG. The mutants will be useful for comparison of the efficacy of Abs with different affinities for delivery of cytotoxic agents to tumor cells.

Animals↗

PubMed1995

Analysis of gp39/CD40 interactions using molecular models and site-directed mutagenesis.

The interaction between gp39 (CD40L, TRAP, T-BAM) on activated T cells and mast cells and CD40 on antigen-presenting cells modulates immune responses. Gp39 and CD40 are homologous to tumor necrosis factor (TNF) and its receptor (TNFR), respectively. The TNF-beta/TNFR interaction has been analyzed on the basis of mutagenesis experiments and crystal structures. Using the interaction of TNF-beta/TNFR as a guide, we previously reported a site-directed mutagenesis study in which we identified residues in gp39 (K143, Y145) and CD40 (Y82, D84, N86) involved in gp39/CD40 interactions. Here we describe the use of the TNF-beta/TNFR complex crystal structure as a template to prepare molecular models of gp39, CD40, and their approximate interaction. The application of these models has allowed us to extend our mutagenesis analysis of gp39/CD40 interactions. These experiments have led to the identification of additional gp39 (Y146, R203, Q220) and CD40 (E74, E117) residues that contribute to the gp39/CD40 interaction. We also further explored the importance of gp39 residue Y145 and CD40 residue Y82 for the gp39/CD40 interaction by conservatively replacing these residues with Phe. The results of these studies have enabled us to approximately outline the binding sites in gp39 and CD40. It appears that the gp39/CD40 interaction is centered on at least two clusters of residues and involves residues of two adjacent gp39 monomers. The molecular regions involved in the gp39/CD40 interaction essentially correspond to those in the homologous TNF-beta/TNFR system.

Amino Acid Sequence↗

PubMed1995

Binding stoichiometry of the cytotoxic T lymphocyte-associated molecule-4 (CTLA-4). A disulfide-linked homodimer binds two CD86 molecules.

CD28 and CTLA-4 are homologous T cell receptors of the immunoglobulin (Ig) superfamily, which bind B7 molecules (CD80 and CD86) on antigen-presenting cells and transmit important costimulatory signals during T cell activation. Here we have investigated the subunit structure of CTLA-4 and the stoichiometry of its binding to B7 molecules. We demonstrate CTLA-4 is a homodimer interconnected by one disulfide bond in the extracellular domain at cysteine residue 120. Each monomeric polypeptide chain of CTLA-4 contains a high affinity binding site for B7 molecules; soluble CTLA-4 and CD86 form complexes containing equimolar amounts of monomeric CTLA-4 and CD86 (i.e. a 2:2 molecular complex). Thus, CTLA-4 and probably CD28 have a receptor structure consisting of preexisting covalent homodimers with two binding sites. Dimerization of CTLA-4 and CD28 is not required for B7 binding, nor is it sufficient to trigger signaling.

Abatacept↗

PubMed1995

Identification of residues on CD40 and its ligand which are critical for the receptor-ligand interaction.

Interactions between gp39 (CD40L, TRAP, T-BAM) on activated T cells and CD40 on antigen-presenting cells play an important role in regulating antibody production by B cells, cytokine production by monocytes, and other immune responses which require T cell "help". Using structure-based sequence alignments, a molecular model of gp39, site-directed mutagenesis, and receptor-ligand binding assays, we have identified CD40 and gp39 surface residues which are important for receptor-ligand binding. Binding studies with CD40 or gp39 proteins containing single and double amino acid substitutions showed that CD40 residues Y82, D84, and N86 are involved in gp39 binding, while gp39 residues K143 and Y145 are important for CD40 binding. Analysis of the location of amino acid substitutions in the naturally occurring gp39 mutants expressed by the X-linked hyper-IgM (X-HIM) patients studied to date indicated the E129/G substitution found in the S128/R-E129/G double mutant affects a solvent-accessible residue which might participate in CD40/gp39 binding. Binding studies with E129/G and E129/A gp39 point mutants showed that this residue does not contribute directly to CD40/gp39 binding but that its substitution with a glycine disrupts the gp39 structure. Comparison of the gp39 and CD40 residues involved in receptor-ligand contacts with those previously identified as playing an important role in TNF-beta/TNFR binding suggests that some of the identified residues from contacts similar to those found in the TNF-beta/TNFR while others are unique to the CD40-gp39 interaction.

Amino Acid Sequence↗

PubMed1995

Specific binding of Fyn and phosphatidylinositol 3-kinase to the B cell surface glycoprotein CD19 through their src homology 2 domains.

CD19 is a B cell surface protein capable of forming non-covalent molecular complexes with a number of other B cell surface proteins including the CD21/CD81/Leu-13 complex as well as with surface immunoglobulin. CD19 tyrosine phosphorylation increases after B cell activation, and is proposed to play a role in signal transduction through its cytoplasmic domain, which contains nine tyrosine residues. Several second messenger proteins have been shown to immunoprecipitate with CD19, including p59 Fyn (Fyn), p59 Lyn (Lyn) and phosphatidylinositol-3 kinase (PI-3 kinase). These associations are predicted to occur via the src-homology 2 (SH2) domains of the second messenger proteins. Two of the cytoplasmic tyrosines in the CD19 cytoplasmic region contain the consensus binding sequence for the PI-3 kinase SH2 domain (YPO4-X-X-M). However, the reported consensus binding sequence for the Fyn and Lyn SH2 domains (YPO4-X-X-I/L) is not found in CD19. We investigated the capacity of CD19 cytoplasmic tyrosines to bind both Fyn and PI-3 kinase SH2-domain fusion proteins. In activated B cells, both Fyn and PI-3 kinase SH2-domain fusion proteins precipitate CD19. Using synthetic tyrosine-phosphorylated peptides comprising each of the CD19 cytoplasmic tyrosines and surrounding amino acids, we investigated the ability of the Fyn SH2 and PI-3 kinase SH2 fusion proteins to bind to the different CD19 cytoplasmic phosphotyrosine peptides. ELISA revealed that the two CD19 cytoplasmic tyrosine residues contained within the Y-X-X-M sequences (Y484 and Y515) bound preferentially to the PI-3 kinase SH2-domain fusion proteins. Two different tyrosines (Y405 and Y445) bound preferentially to the Fyn SH2-domain fusion protein via a novel sequence, Y-E-N-D/E, different from that previously reported for the Fyn SH2 domain. In precipitation studies, peptide Y484 was able to compete with tyrosine phosphorylated CD19 specifically for binding to the PI-3 kinase SH2 domain fusion proteins, while peptides Y405 and Y445 were able to compete specifically for binding to the Fyn SH2 domain fusion proteins. These results indicate that CD19 may be capable of binding both Fyn and PI-3 kinase concurrently, suggesting a mechanism for CD19 signal transduction, in which binding of PI-3 kinase to the Fyn SH3 domain results in activation of PI-3 kinase.

Amino Acid Sequence↗

PubMed1995

Profiles for the analysis of immunoglobulin sequences: comparison of V gene subgroups.

A format for the structure-oriented analysis of immunoglobulin (Ig) variable region sequences is presented and applied to generate sequence profiles for comparison of heavy- and light-chain subgroups. The profile allows simultaneous evaluation of sequences and structural information and can be used for a number of different applications.

Amino Acid Sequence↗

PubMed1995

Molecular model of the N-terminal receptor-binding domain of the human CD6 ligand ALCAM.

CD6-ligand interactions have been implicated in the regulation of T-cell adhesion and activation. CD6 is a member of the scavenger receptor family, whereas its human ligand (ALCAM) belongs to the immunoglobulin superfamily. The extracellular region of ALCAM includes five immunoglobulin-like domains. As a fusion protein, the N-terminal extracellular domain of ALCAM (ALCAMD1) binds specifically to CD6. We report the construction, assessment, and analysis of a molecular model of ALCAMD1. The model defines the CDR-analogous loops, the location of N-linked glycosylation sites, and residues that form the beta-sheet faces of the immunoglobulin-like domain. Predicted structural characteristics of the A'GFCC'C" face of the model are consistent with the presence of monomeric and dimeric forms of ALCAMD1, which has implications for the receptor-ligand interactions.

Activated-Leukocyte Cell Adhesion Molecule↗

PubMed1995

The membrane-bound and soluble forms of HLA-G bind identical sets of endogenous peptides but differ with respect to TAP association.

The class Ib antigen HLA-G is expressed as a membrane-bound protein like classical class Ia molecules (M.HLA-G) but, unlike typical class I, is also expressed as a soluble protein (S.HLA-G) with a unique C terminus. Our results show that, similar to classical class I proteins, the membrane-bound form of HLA-G associated with TAP, as evidenced by the ability to immunoprecipitate HLA-G class I heavy chain with TAP antisera. In contrast, the soluble G protein did not appear to associate with TAP in the same manner, since similar immunoprecipitation experiments failed to detect soluble G complex. A detailed analysis of peptides bound to the soluble and membrane HLA-G proteins expressed in the B lymphoblastoid cell line 721.221 showed that, like class Ia complexes, both HLA-G proteins consist of heavy and light chains complexed with nonameric peptides in a 1:1:1 ratio. The two proteins bind essentially the same set of peptides, which are derived from a variety of intracellular proteins and define a peptide motif for HLA-G. The peptides contain Leu at the C terminus and Pro or small hydrophobic amino acids in position 3 followed by Pro or Gly in position 4. The complexity of the bound peptides is lower than that found for some class Ia complexes, but is more similar to class Ia than to the limited repertoire of some murine class Ib molecules.

ATP-Binding Cassette Transporters↗

PubMed1995

The x-ray structure of an anti-tumour antibody in complex with antigen.

The crystal structures of the murine BR96 Fab and its human chimera have been determined in complex with the nonoate methyl ester derivative of Lewis Y (nLey) at 2.8 A and 2.5 A resolution, respectively. BR96 binds the carbohydrate in a large pocket which is formed by residues of all CDR loops except L2. The binding of the carbohydrate is mediated predominantly by aromatic residues in BR96. Analysis of the structure suggests that BR96 is capable of recognizing a structure larger than the Le(y) tetrasaccharide, providing a possible explanation for its high tumour selectivity. The structure provides a rationale for mutagenesis experiments that have resulted in BR96 CDR loop mutants with increased affinity for nLey and/or tumour cells.

Animals↗

PubMed1995

Model building of antibody combining sites.

The number of high quality crystal structures of antibody fragments available in the Brookhaven Protein Data Bank is rapidly increasing. These structures provide a substantial knowledge base and support model building of novel antibody combining sites. We review some basic principles of antibody structure, describe structure-based modelling procedures, and indicate the strengths and weaknesses of the modelling approach. Applications of antibody models are discussed.

Animals↗

PubMed1994

Molecular model of the extracellular lectin-like domain in CD69.

CD69 is described as a T cell activation antigen, but the ligand and physiological function of CD69 are currently unknown. The sequence of the extracellular domain of CD69 shows some similarity with that of calcium-dependent (C-type) lectins. Using comparative computer modeling and inverse folding calculations, we have generated and analyzed a detailed three-dimensional model of the extracellular domain of CD69 based on the crystal structure of the mannose binding protein. The sequence of CD69 appears to be highly compatible with the C-type lectin fold, and assessment of the model using inverse folding calculations suggests its overall correctness. Compared with mannose binding protein and the selectins, CD69 displays significant deletions in loop regions. In addition, residues that form conserved calcium binding sites found in the C-type lectin family are not conserved in CD69. This suggests the presence of structural features in CD69 that depart from some of the conserved motifs seen in two crystal structures of C-type lectins. The CD69 model shows cavity-shaped hydrophobic regions surrounded by charged residues. One of these cavities is proximal to a potential low affinity calcium binding site and may be implicated in specific interactions with ligands.

Amino Acid Sequence↗

PubMed1994

Identification of residues in the monoclonal antitumor antibody L6 important for binding to its tumor antigen.

L6 is a monoclonal antitumor antibody which recognizes an epitope located in a 42-residue extracellular domain of a tumor-associated approximately 22 kDa glycoprotein antigen. The L6 mAb localizes to solid tumors in vivo and triggers complement activation and antibody-dependent cellular cytotoxicity. It has been the subject of phase I clinical trials. Previously, we had reported the derivation and analysis of a three-dimensional model of the L6 Fv. The model suggests that L6 displays a generally aromatic CDR surface. We aim at improving the affinity for tumor antigen of L6 by in vitro mutagenesis. As the first step toward this end, we have attempted to identify residues critical for the binding of L6 to tumor antigen. On the basis of the model, seven residues were selected which we thought may be critical for L6 antigen binding. Criteria for the selection of these residues were their accessibility and central position on the CDR surface and the residue character. Large polar or charged residues such as arginine, asparagine, and tyrosine were preferred. Nine site-specific single and double mutants were generated using oligonucleotide-directed mutagenesis in an M13 expression vector encoding the L6 Fab. The binding of these mutant Fabs to the L6 tumor antigen and a set of three anti-idiotypic antibodies was quantified in an ELISA. In eight out of nine mutants, binding to L6 tumor antigen was either abolished or substantially reduced. In contrast, the binding of the mutants to the anti-idiotypic antibodies was largely unaffected, suggesting that no significant structural perturbations were introduced as a consequence of these mutations.

Amino Acid Sequence↗

PubMed1994

Complementarity determining region 1 (CDR1)- and CDR3-analogous regions in CTLA-4 and CD28 determine the binding to B7-1.

T cell surface receptors CD28 and CTLA-4 are homologous members of the immunoglobulin superfamily (IgSF), each comprising a single V-like extracellular domain. CD28 and CTLA-4 bind to the B7-1 and B7-2 counter-receptors on antigen presenting cells (APCs), thereby triggering a costimulatory pathway important for optimal T cell activation in vitro and in vivo. Soluble forms of CD28 and CTLA-4 in which the V-like extracellular domains were fused to Ig constant domains (CD28Ig and CTLA4Ig), have been used to study their interactions with B7-1 and B7-2, with CTLA4Ig binding B7-1 more strongly than CD28Ig (approximately 20-fold higher avidity). We have now, by site-specific and homologue mutagenesis, identified regions in CTLA4Ig important for strong binding to B7-1. A hexapeptide motif (MYPPPY) in the complementarity determining region 3 (CDR3)-like region is fully conserved in all CD28 and CTLA-4 family members. Alanine scanning mutagenesis through the motif in CTLA4Ig and at selected residues in CD28Ig reduced or abolished binding to B7-1. Chimeric molecules HS4, HS4-A, and HS4-B were constructed in which CDR3-like regions of CTLA-4, COOH-terminally extended to include nonconserved residues, were grafted onto CD28Ig. These homologue mutants showed stronger binding to B7-1 than did CD28Ig. Grafting of the CDR1-like region of CTLA-4, which is not conserved in CD28 and is predicted to be spatially adjacent to CDR3, into HS4 and HS4-A, resulted in chimeric molecules (HS7 and HS8) which bound B7-1 even better. Inclusion of the CDR2-like domain of CTLA-4 into HS7 and HS8 did not further increase binding. Thus, the MYPPPY motifs of CTLA4Ig and CD28Ig are important for their binding to B7-1, but the increased strength of this binding by CTLA4Ig is mediated by nonconserved residues in the CDR1- and CDR3-analogous regions.

Abatacept↗

PubMed1994

Membrane topology of the L6 antigen and identification of the protein epitope recognized by the L6 monoclonal antibody.

The murine monoclonal antibody (mAb) L6 recognizes an integral membrane glycoprotein that is highly expressed on lung, breast, colon, and ovarian carcinomas and is referred to as the L6 antigen. This antigen is an attractive target for therapeutic intervention due to its high level expression on malignant cells. We have previously reported the isolation of a cDNA encoding the human L6 antigen (H-L6). Here, we report the isolation of a cDNA clone encoding the murine L6 antigen (M-L6). This cDNA contains one long open reading frame, which encodes a 220-amino acid polypeptide that is 78% homologous to H-L6. This protein contains short NH2- and COOH-terminal hydrophilic domains and four hydrophobic regions, each long enough to span the plasma membrane. Each of these hydrophobic domains is separated by a hydrophilic domain, the longest of which contains one possible N-linked glycosylation site and is located between the third and fourth hydrophobic domains. We have previously demonstrated that the murine L6 mAb recognizes a protein epitope expressed on human tumor-derived cell lines. Now, using chimeric cDNA constructs encoding human-murine L6 antigen hybrids in conjunction with monoclonal antibody binding experiments, we show that the 42-residue hydrophilic domain of the L6 antigen, located between the third and fourth hydrophobic domains, is outside the cell and that residues in the NH2-terminal region of this domain are critical for the binding of the murine L6 mAb to H-L6.

Amino Acid Sequence↗

PubMed1994

CD62/P-selectin binding sites for myeloid cells and sulfatides are overlapping.

P-Selectin (CD62/GMP140/PADGEM) is an inducible cell-surface glycoprotein expressed by endothelial cells and platelets following stimulation by inflammatory mediators such as thrombin, histamine, or peroxides. P-Selectin mediates the binding of leukocytes to activated vascular endothelium at sites of inflammation and plays a role in mediating the binding of activated platelets to leukocytes and the vascular cell wall. The adhesive function of P-selectin is mediated by its calcium-dependent (or C-type) lectin domain, which is known to bind to carbohydrate ligands including fucosyl-N-acetyllactosamine (Lex, CD15), sialyl-Lex, and 3-sulfated galactosylceramides (sulfatides). Sulfatides can efficiently block P-selectin/myeloid cell binding in vitro and are excreted at high levels by activated granulocytes. These observations led to the hypothesis that sulfatide may play a role in facilitating the disengagement of CD62, allowing the efficient exit of granulocytes from the blood stream at sites of inflammation. In this report, we extend our previous mutagenesis analysis of the P-selectin binding site [Hollenbaugh, D., Bajorath, J., Stenkamp, R., & Aruffo, A. (1993) Biochemistry 32, 2960] and show that replacement of Tyr48 with Ser or Lys113 with Arg results in P-selectin mutants that, although correctly folded, do not bind to HL60 cells. These results suggest that the conservation of charged and hydrogen-bonding site chains is not sufficient to maintain the P-selectin function and that the exact stereochemistry provided by the side chains of residues lining the P-selectin binding pocket is critical for P-selectin binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed1994

Crystallization and preliminary X-ray analysis of the monoclonal anti-tumor antibody BR96 and its complex with the Lewis Y determinant.

The monoclonal anti-tumor antibody BR96 binds a tetrasaccharide, Lewis y (Le(y)), in vitro and recognizes a Le(y)-bearing or Le(y)-related tumor-associated antigen in vivo. The Fab of the murine monoclonal antibody, mBR96 (IgG3, kappa), and the Fab' of its human chimera, cBR96 (IgG1, kappa), and their complexes with Le(y) have been screened for crystallization conditions. Crystals suitable for X-ray diffraction have been obtained for uncomplexed cBR96 Fab', cBR96 Fab' in complex with Le(y) and mBR96 Fab in complex with Le(y). The symmetry of the cBR96 Fab' crystals is consistent with space group P2(1)2(1)2, a = 61.1 A; b = 174.3 A; c = 45.6 A; the symmetry of the cBR96 Fab'-Le(y) complex crystals with space group P4(3)2(1)2 (or its enantiomorph), a = b = 82.2 A; c = 167.1 A and the symmetry of the mBR96 Fab-Le(y) complex crystals with space group P2(1)2(1)2(1), a = 69.4 A; b = 84.9 A; c = 86.8 A.

Animals↗

PubMed1994

Extending the B7 (CD80) gene family.

B7-1 and B7-2 are members of the immunoglobulin superfamily (IgSF) and important regulators of T cell-mediated immune responses. Despite sharing only limited sequence identity, B7-1 and B7-2 bind common receptors, CD28 and CTLA-4, on T cells and have similar functional properties. We have found that the extracellular V (ariable)-like domains of B7-1 and B7-2 share significant sequence similarities with 3 major histocompatibility complex (MHC)-encoded members of the IgSF: butyrophilin, myelin/oligodendrocyte glycoprotein, and the chicken MHC molecule, B-G. This raises the question whether there is an evolutionary link between the MHC, which encodes molecules regulating the antigen specificity of T lymphocyte responses, and B7 molecules, which co-stimulate these responses in antigen-nonspecific fashion.

Amino Acid Sequence↗