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Biomedical subjects

J Bajorath

Publications and source records attributed to J Bajorath.

At least 19 recordsLinked to original sources
PubMed1999

Escherichia coli and Porphyromonas gingivalis lipopolysaccharide interactions with CD14: implications for myeloid and nonmyeloid cell activation.

Porphyromonas gingivalis, a gram-negative bacterium, is an etiologic agent for adult periodontitis. Lipopolysaccharide (LPS) released from this bacterium can react with numerous host cell types. P. gingivalis LPS stimulates tumor necrosis factor alpha and interleukin-1beta secretion from monocytes (myeloid) but does not elicit E-selectin expression from human endothelial cells (nonmyeloid). In contrast, Escherichia coli LPS facilitates expression of these inflammatory mediators through CD14-dependent pathways on both myeloid and nonmyeloid cells. LPS binding studies have revealed that although P. gingivalis and E. coli LPSs bind to CD14 differently, this fact does not adequately explain the lack of endothelial cell activation by P. gingivalis LPS. Rather, LPS binding site and blocking monoclonal antibody epitope mapping studies have suggested that CD14 presents a charged surface that captures different microbial ligands by electrostatic interactions. We propose that human endothelial cells do not respond to P. gingivalis LPS because of their inability to "recognize" CD14-P. gingivalis LPS complexes.

Amino Acid Sequence↗
PubMed1998

Detailed comparison of two molecular models of the human CD40 ligand with an x-ray structure and critical assessment of model-based mutagenesis and residue mapping studies.

The interactions between the B cell receptor CD40 and its ligand on T cells are critical for the integrity of immune responses. The human CD40 ligand gp39, a tumor necrosis factor-like protein, has been the subject of intense efforts to identify the receptor-binding site and to analyze naturally occurring mutations that compromise gp39 function in vivo. These investigations relied heavily on molecular models of gp39, built in the presence of only approximately 25% sequence identity to tumor necrosis factor. The x-ray structure of gp39 has made it possible to assess modeling accuracy and to evaluate the results of model-based mutagenesis analyses. Although the models display local errors, their accuracy was sufficient to predict the CD40-binding site, to map natural mutations, and to rationalize their effects. One of five gp39 residues critical for CD40 binding was displaced in the models, and 1 of 21 point mutants was incorrectly classified. Factors most important for the reliability of the molecular models and their successful applications were valid sequence alignments and the focus of experimental studies on regions of high prediction confidence. Analysis of mutagenesis experiments correlated with anti-gp39 monoclonal antibody binding studies to assess the conformational integrity of mutant proteins.

Amino Acid Sequence↗
PubMed1998

Analysis of the ligand binding site in Fas (CD95) by site-directed mutagenesis and comparison with TNFR and CD40.

Fas and its ligand (FasL) are members of the tumor necrosis factor receptor (TNFR) and tumor necrosis factor (TNF) superfamilies, respectively. Fas-FasL interactions trigger controlled cell death (apoptosis) in the immune system and thus play a key role in the regulation of immune responses. Structural details of the Fas-Fas ligand interaction are currently unknown. Previously, six Fas residues were identified by mutagenesis as important for ligand binding. We have now extended our mutagenesis analysis and identified additional residues which contribute to the Fas-FasL interaction. Candidate and control residues were selected based on a molecular model of the Fas extracellular region. Although residues in all three extracellular domains were identified to contribute to binding, the Fas-FasL interaction is centered on the second TNFR-like domain. Important residues were compared to critical positions in TNFR and CD40, another member of the TNFR family.

Amino Acid Sequence↗
PubMed1998

Analysis of BR96 binding sites for antigen and anti-idiotype by codon-based scanning mutagenesis.

We performed a scanning mutagenesis study of heavy chain complementarity-determining region (CDR) residues to identify how mutations affected binding of the anti-carcinoma mAb BR96 to Ag, Lewis Y, and to an anti-Id Ab (anti-Id). By ELISA, we demonstrated that the anti-Id bound close to the Ag binding site of BR96, but the anti-Id and Ag sites were not identical. Immunoblot analysis and screening of light and heavy chain CDR libraries with multiple mutations in each CDR suggested that the heavy chain had greater involvement in anti-Id binding. We then analyzed contributions of individual residues in the heavy chain CDRs to binding of Ag and anti-Id. In a filamentous phage vector containing BR96 V region sequences, mutations were introduced by codon-based mutagenesis at single positions within the three heavy chain CDRs. The resulting libraries of Fab fragments had all amino acids represented at a CDR position. We evaluated the expressed Fabs for binding to Ag and anti-Id by plaque lift assay. We identified the positions with mutations that had the greatest negative effect on binding to the anti-Id and to Ag and analyzed them on the basis of the BR96 x-ray structure. The residues most important for binding to the anti-Id were located in heavy chain CDR1 and CDR2 and were peripheral to the residues within the Lewis Y binding pocket.

Animals↗
PubMed1998

Identification of CD44 residues important for hyaluronan binding and delineation of the binding site.

CD44 is a widely distributed cell surface protein that plays a role in cell adhesion and migration. As a proteoglycan, CD44 is also implicated in growth factor and chemokine binding and presentation. The extracellular region of CD44 is variably spliced, giving rise to multiple CD44 isoforms. All isoforms contain an amino-terminal domain, which is homologous to cartilage link proteins. The cartilage link protein-like domain of CD44 is important for hyaluronan binding. The structure of the link protein domain of TSG-6 has been determined by NMR. Based on this structure, a molecular model of the link-homologous region of CD44 was constructed. This model was used to select residues for site-specific mutagenesis in an effort to identify residues important for ligand binding and to outline the hyaluronan binding site. Twenty-four point mutants were generated and characterized, and eight residues were identified as critical for binding or to support the interaction. In the model, these residues form a coherent surface the location of which approximately corresponds to the carbohydrate binding sites in two functionally unrelated calcium-dependent lectins, mannose-binding protein and E-selectin (CD62E).

Amino Acid Sequence↗
PubMed1997

Sulfated galactocerebrosides as potential antiinflammatory agents.

Native sulfatides, as well as many sulfated glycolipids, have been shown to avidly bind to the selectin receptors. In vivo, native sulfatides significantly block activity in selectin-dependent inflammatory responses. The fact that nonsulfated galactocerebrosides did not inhibit selectin-mediated adhesion identified a critical role for the anionic sulfate residue. We therefore initiated a program to evaluate the activity of position isomers. This study showed a binding selectivity for the positions 2 and 3 of the sulfate group on the carbohydrate ring as well as enhanced activity for the disulfated analogs. Furthermore, it was discovered that the attachment of lipophilic substituents on the carbohydrate ring was tolerated, consistent with the presence of a lipophilic pocket in the binding activity. This resulted in compounds with a 6-fold increased potency.

Animals↗
PubMed1997

Identification of amino acid residues important for ligand binding to Fas.

The interaction of Fas (CD95), a member of the tumor necrosis factor receptor (TNFR) family, and its ligand (FasL) triggers programmed cell death (apoptosis) and is involved in the regulation of immune responses. Although the Fas-FasL interaction is conserved across species barriers, little is currently known about the molecular details of this interaction. Our aim was to identify residues in Fas that are important for ligand binding. With the aid of a Fas molecular model, candidate amino acid residues were selected in the Fas extracellular domain 2 (D2) and D3 and subjected to serine-scanning mutagenesis to produce mutant Fas molecules in the form of Ig fusion proteins. The effects of these mutations on FasL binding was examined by measuring the ability of these proteins to inhibit FasL-mediated apoptosis of Jurkat cells and bind FasL in ELISA and BIAcore assays. Mutation of two amino acids, R86 and R87 (D2), to serine totally abolished the ability of Fas to interact with its ligand, whereas mutants K84S, L90S, E93S (D2), or H126S (D3) showed reduced binding compared with wild-type Fas. Two mutants (K78S and H95S) bound FasL comparably to wild type. Therefore, the binding of FasL involves residues in two domains that correspond to positions critical for ligand binding in other family members (TNFR and CD40) but are conserved between murine and human Fas.

Binding Sites↗
PubMed1997

Analysis of 4-1BBL and laminin binding to murine 4-1BB, a member of the tumor necrosis factor receptor superfamily, and comparison with human 4-1BB.

The T cell activation antigen 4-1BB (CDw137) is a distantly related member of the tumor necrosis factor receptor family of cell surface receptors. We previously reported that murine 4-1BB (m4-1BB) bound to extracellular matrix (ECM) proteins. Recently, a tumor necrosis factor-like ligand of m4-1BB, m4-1BBL, as well as the human counterparts of 4-1BB (ILA) and 4-1BBL (h4-1BB and h4-1BBL, respectively) have been cloned. No information is currently available on how binding of m4-1BB to ECM proteins affects its binding to m4-1BBL and vice versa and if the ability of m4-1BB to bind ECM proteins is conserved across species. We report that binding of m4-1BBL to m4-1BB blocked its ability to bind laminin (LN), while binding of m4-1BB to LN did not block its ability to bind m4-1BBL. Furthermore, binding of m4-1BBL to the m4-1BB.LN complex did not displace LN. These findings suggest the two ligands bind to proximal but distinct sites on m4-1BB. This is supported by the observation that six of eight anti-m4-1BB monoclonal antibodies blocked the interaction between 4-1BB and 4-1BBL, while seven blocked LN binding. Ligand and monoclonal antibody binding studies with a truncated protein lacking the amino-terminal LN-homologous domain of m4-1BB demonstrated that regions downstream of the LN-homologous domain participate in LN binding and that the intact protein is required for m4-1BBL binding. Studies with h4-1BB showed that h4-1BB only bound h4-1BBL, indicating that the ECM binding activity of 4-1BB is not conserved across species. This finding allowed the construction of murine/human 4-1BB chimeras, which permitted further dissection of the regions of 4-1BB involved in LN and 4-1BBL binding and suggests that sequence differences in the LN-homologous domain of h4-1BB in part account for the inability of h4-1BB to bind ECM proteins.

4-1BB Ligand↗
PubMed1997

Identification of residues in CD6 which are critical for ligand binding.

CD6 is a member of the scavenger receptor cysteine rich protein superfamily (SRCRSF). This family includes many cell surface proteins whose three-dimensional structures and functions are presently not well understood. The extracellular region of CD6 includes 3 SRCR domains. The membrane proximal SRCR domain specifically binds the activated leukocyte cell adhesion molecule (ALCAM), a CD6 ligand belonging to the immunoglobulin superfamily. CD6-ALCAM interactions mediate immune cell adhesion and are implicated in T cell maturation and the regulation of T cell function. On the basis of SRCRSF sequence comparison, a mutagenesis analysis of the membrane proximal SRCR domain of CD6 (CD6D3) has been carried out. Fifteen mutants were characterized. Three CD6 residues were identified in a region of low sequence conservation which, when mutated, abolish ligand binding but not the binding to a panel of conformationally sensitive anti-CD6 mAbs. This study provides the first analysis of residues critical for ligand binding to a member of the SRCRSF.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1997

Characterization of mouse ALCAM (CD166): the CD6-binding domain is conserved in different homologs and mediates cross-species binding.

Activated leukocyte cell adhesion molecule (ALCAM; CD166) is a member of the immunoglobulin gene superfamily (IgSF) which is expressed by activated leukocytes and thymic epithelial cells and is a ligand for the lymphocyte antigen CD6. Herein, we report on the isolation and characterization of cDNA clones encoding mouse ALCAM (mALCAM). Comparison of the predicted amino acid sequence of mALCAM and human ALCAM (hALCAM) showed an overall identity of 93%. Binding studies with truncated forms of the extracellular region of mALCAM showed that the CD6 binding site is located in the N-terminal Ig-like domain and that mALCAM is capable of binding both human and mouse CD6. Mutagenesis studies on hALCAM suggested that residues critical for CD6 binding map to the predicted A'GFCC'C" beta-sheet of ALCAM's N-terminal binding domain. Residue differences in the N-terminal domains of mALCAM and hALCAM were analyzed with the aid of a molecular model of ALCAM. All residues critical for CD6 binding are conserved in both mALCAM and hALCAM, whereas residue differences map to the predicted BED face which is opposite the CD6 binding site on hALCAM. These findings provide a molecular rationale for the observed cross-species CD6/ALCAM interaction and the apparent inability to generate monoclonal antibodies (mAb) against the CD6 binding site. RNA blot analysis showed that mRNA transcripts encoding mALCAM are expressed in the brain, lung, liver, and the kidney, as well as by activated leukocytes and a number of cell lines. A rat mAb specific for mALCAM was produced and by two-color immunofluorescence studies was shown to bind to both activated CD4+ and CD8+ T cells.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1997

CD6 recognizes the neural adhesion molecule BEN.

CD6 and its ligand activated leukocyte cell adhesion molecule (ALCAM, CD166) have been detected on various immune cells and in the brain. CD6-ligand interactions have been implicated in the regulation of T cell function. ALCAM shares the same extracellular domain organization and significant sequence homology with the chicken neural adhesion molecule BEN. Although ALCAM's CD6 binding site is only partially conserved in BEN, CD6 specifically binds BEN, albeit with approximately 10-fold lower avidity than ALCAM. Differences in binding avidity are not detected when ALCAM and BEN fusion proteins containing the full-length extracellular regions are tested. Homotypic interactions between full-length forms are likely to account for these observations. The identified cross-species interaction between CD6 and BEN suggests that CD6-ligand interactions are highly conserved.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1997

CD6-ligand interactions: a paradigm for SRCR domain function?

The scavenger receptor cysteine-rich (SRCR) superfamily, which includes proteins expressed by leukocytes, can be subdivided into groups A and B. Group B contains the lymphocyte cell-surface receptor CD6. This article reviews recent progress in understanding the interaction between CD6 and its ligand, activated leukocyte cell adhesion molecule (ALCAM). Analysis of the CD6-ALCAM interaction may help to understand how other SRCR domains bind to their ligands.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1997

Molecular modeling of CD28 and three-dimensional analysis of residue conservation in the CD28/CD152 family.

CD28/CD152-CD80/CD86 receptor-ligand interactions result in costimulatory signals critical for optimal T cell activation. CD28/CD152 and CD80/CD86 are members of the immunoglobulin superfamily (IgSF). Despite common receptor-ligand interactions, both receptor and ligand pairs share only limited sequence identity. A detailed molecular model of the extracellular Ig-like domain of human CD28 was constructed using a combination of different modeling methods. The model was based on the solution structure of CD152 and sequence comparison of the CD28/CD152 family. Assessment of the model revealed good stereochemical quality and sequence-structure compatibility. The CD28 model was used to map surface residues, N-linked glycosylation sites, and to compare residue conservation in CD28 and CD152. The location of N-linked glycosylation sites in CD28/CD152 restricts the surface area available for binding. Rigorous sequence conservation in CD28 and CD152 is limited to core IgSF consensus positions and surface residues implicated in ligand binding. Other surface residues vary greatly in CD28/CD152. Residues critical for ligand binding are surrounded by surface patches conserved only in either CD28 or CD152.

Abatacept↗
PubMed1997

Prediction of the three-dimensional structure of the human Fas receptor by comparative molecular modeling.

The Fas antigen, a cell surface receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily, triggers programmed cell death (apoptosis) in the immune system. The three-dimensional structure of Fas and molecular details of the interaction between Fas and its ligand are currently unknown. A three-dimensional model of the Fas extracellular region was generated by comparative modeling. Inverse folding analysis suggested good sequence-structure compatibility of the model and thus reasonable accuracy. The model was analyzed in the light of information provided by studies on TNFR and CD40, another member of the TNFR family, and the Fas ligand binding site was predicted.

Amino Acid Sequence↗
PubMed1997

Mutational analysis of the CD6 ligand binding domain.

CD6 belongs to the scavenger receptor cysteine-rich protein superfamily (SRCRSF), which includes a large number of cell surface proteins. The extracellular region of CD6 is composed of three SRCR domains. The membrane proximal SRCR domain of CD6 (CD6D3) specifically binds activated leukocyte cell adhesion molecule (ALCAM), a cell surface protein which is a member of the immunoglobulin superfamily (IgSF). CD6-ligand interactions have been implicated in immune cell adhesion, T cell maturation and the regulation of T cell activation. We tested 13 CD6D3 mutant proteins for binding to ALCAM and a panel of conformationally sensitive anti-CD6D3 monoclonal antibodies (mAbs). CD6D3 residues were classified according to their importance for structural integrity and ligand binding. The results were analyzed in the light of SRCR domain sequence comparison. A number of residues critical for ligand binding or important for structural integrity cluster in the C-terminal region of CD6D3 which is not conserved in other SRCR proteins.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1997

Identification of CD14 residues involved in specific lipopolysaccharide recognition.

CD14 is a key molecule responsible for the innate host inflammatory response to microbial infection. It is able to bind a wide variety of microbial ligands and facilitate the activation of both myeloid and nonmyeloid cells. However, its specific contribution to the innate recognition of bacteria is not known. Presently there is no information on the contribution of individual CD14 residues to Escherichia coli lipopolysaccharide (LPS) binding or on the molecular basis of the interaction between CD14 and LPS from other bacteria. LPS obtained from Porphyromonas gingivalis, a bacterium associated with chronic inflammatory disease, binds CD14 and activates myeloid cells but does not facilitate the activation of nonmyeloid cells. The transfer and binding of these two LPS species to soluble CD14 recombinant globulin proteins with single point mutations was examined. Functional activity of the mutant proteins was monitored by E-selectin expression on human umbilical cord endothelial cells. The analysis identified a charge reversal mutation in a single residue, E47, that demonstrated selective binding to E. coli LPS but not to P. gingivalis LPS. E-selectin activation assays indicated that proteins with mutations at position E47 maintained their structural integrity. Other mutations, including a charge reversal mutation of residue E58, did not significantly reduce the binding of either LPS ligand or the ability of the molecule to facilitate E-selectin activation. These data demonstrate that CD14 can selectively recognize different LPS ligands.

Amino Acid Sequence↗
PubMed1997

Construction and analysis of a detailed three-dimensional model of the ligand binding domain of the human B cell receptor CD40.

The interaction between the human B cell receptor CD40 and its ligand on T cells is critical for B cell proliferation and the regulation of humoral immune responses. CD40 is a member of the tumor necrosis factor receptor (TNFR) family. We report here the construction and analysis of a detailed three-dimensional model of the TNFR-homologous extracellular region of CD40. This study provides an example for structure-based model building in the presence of low sequence similarity. The assessment of model quality and sequence-structure compatibility is emphasized, and limitations of the model are discussed. The current CD40 model predicts structural details beyond the backbone level. Features of the CD40 ligand binding site are discussed in conjunction with the results of a previous mutagenesis study.

Amino Acid Sequence↗