A reassessment of the MAdCAM-1 structure and its role in integrin recognition.

Mucosal addressin cell-adhesion molecule (MAdCAM-1) is a membrane-bound leukocyte receptor regulating both the passage and retention of leukocytes in mucosal tissues. A crystal structure for the two extracellular amino-terminal domains of human MAdCAM-1 has previously been reported, confirming their expected immunoglobulin superfamily topology. In this study, a second crystal structure of this fragment is described. Although the overall structure is similar to that previously reported, one edge strand in the amino-terminal domain is instead located on the opposite sheet. This alters the arrangement and conformation of amino acids in this region that have previously been shown to be crucial for ligand binding. MAdCAM-1 is also seen to form dimers within the crystal lattice, raising the possibility that oligomerization may influence the biological role of this adhesion molecule.

Intercellular adhesion molecule-4 binds alpha(4)beta(1) and alpha(V)-family integrins through novel integrin-binding mechanisms.

The LW blood group glycoprotein, ICAM-4, is a member of the intercellular adhesion molecule (ICAM) family expressed in erythroid cells. To begin to address the function of this molecule, ligands for ICAM-4 on hemopoietic and nonhemopoietic cell lines were identified. Peptide inhibition studies suggest that adhesion of cell lines to an ICAM-4-Fc construct is mediated by an LDV-inhibitable integrin on hemopoietic cells and an RGD-inhibitable integrin on nonhemopoietic cells. Antibody inhibition studies identified the hemopoietic integrin as alpha(4)beta(1.) Antibody inhibition studies on alpha(4)beta(1)-negative, nonhemopoietic cell lines suggested that adhesion of these cells is mediated by alpha(V) integrins (notably alpha(V)beta(1) and alpha(V)beta(5)). The structure of ICAM-4 modeled on the crystal structure of ICAM-2 was used to identify surface-exposed amino acid residues for site-directed mutagenesis. Neither an unusual LETS nor an LDV motif in the first domain of ICAM-4 was critical for integrin binding. ICAM-4 is the first ICAM family member shown to be a ligand for integrins other than those of the beta(2) family, and the data suggest that ICAM-4 has a novel integrin-binding site(s). These findings suggest a role for ICAM-4 in normal erythropoiesis and may also be relevant to the adhesive interactions of sickle cells.

Expression of a soluble functional form of the integrin alpha4beta1 in mammalian cells.

The integrin alpha4beta1(VLA4) has been expressed as a soluble, active, heterodimeric immunoglobulin fusion protein. cDNAs encoding the extracellular domains of the human alpha4 and beta1 subunits were fused to the genomic DNA encoding the human gamma1 immunoglobulin Fc domain and functional integrin fusion protein was expressed as a secreted, soluble molecule from a range of mammalian cell lines. Specific mutations were introduced into the Fc region of the molecules to promote alpha4beta1 heterodimer formation. The soluble alpha4beta1-Fc fusion protein exhibited divalent cation dependent binding to VCAM-1, which was blocked by the appropriate function blocking antibodies. The apparent Kd for VCAM-1 binding were similar for both the soluble and native forms of alpha4beta1. In addition, the integrin-Fc fusion was shown to stain cells expressing VCAM-1 on their surface by FACs analysis. This approach for expressing soluble alpha4beta1 should be generally applicable to a range of integrins.

RTX toxins recognize a beta2 integrin on the surface of human target cells.

Actinobacillus actinomycetemcomitans leukotoxin and Escherichia coli alpha-hemolysin are RTX toxins that kill human immune cells. We have obtained a monoclonal antibody (295) to a cell surface molecule present on toxin-sensitive HL60 cells that can inhibit cytolysis by both RTX toxins. Utilization of this monoclonal antibody for immunoaffinity purification of detergent-solubilized target cell membranes yielded two polypeptide chains of approximate molecular masses of 100 and 170 kDa. Microsequencing of tryptic peptides from the two proteins showed complete homology with CD11a and CD18, the two subunits of the beta2 integrin, lymphocyte function-associated antigen 1 (LFA-1). Anti-CD11a and CD18 monoclonal antibodies also inhibited RTX toxin-mediated cytolysis. Direct binding experiments demonstrated the ability of an immobilized RTX to bind LFA-1 heterodimers present in a detergent lysate of human HL60 target cells. Transfection of CD11a and CD18 integrin genes into a cell line (K562) that is not sensitive to either RTX toxin resulted in LFA-1 expressing cells, KL/4, that were sensitive to both toxins. These experiments identify LFA-1 as a cell surface receptor that mediates toxicity of members of this family of pore-forming toxins.

7E3 monoclonal antibody directed against the platelet glycoprotein IIb/IIIa cross-reacts with the leukocyte integrin Mac-1 and blocks adhesion to fibrinogen and ICAM-1.

Recent clinical trials suggest that blockade of integrins is a promising strategy for the treatment of acute coronary syndromes. Administration of 7E3 monoclonal antibody (mAb) Fab fragment (c7E3 Fab) directed against platelet integrin IIb/IIIa (alpha IIb beta 3, CD41/CD61) reduces acute ischemic complications of coronary angioplasty and clinical restenosis at 6 months. However, 7E3 mAb is not selective for platelet IIb/IIIa but also cross-reacts with the leukocyte integrin Mac-1 (alpha M beta 2, CD11b/CD18) and the vitronectin receptor (alpha v beta 3, CD51/CD61). Information regarding how this mAb may affect other cells important in vascular repair is scant. Potential interactions of c7E3 Fab with inflammatory (i.e., monocytes and neutrophils), vascular smooth muscle, and endothelial cells may contribute to the in vivo actions of c7E3 Fab. In this study we explored the binding of 7E3 to monocytic cells and the functional effect of 7E3 and c7E3 Fab on Mac-1-mediated adhesion to fibrinogen (FGN) and intercellular adhesion molecule-1 (ICAM-1), ligands abundant in the injured vessel wall. Flow cytometry demonstrated that 7E3 bound to THP-1 monocytic cells and identified a subpopulation (approximately 10%) of Mac-1 that was qualitatively similar to that recognized by CBRM1/5, a mAb directed to an activation-specific neoepitope present on a subset of Mac-1 molecules. mAb 7E3 bound to K562 cells transfected with just the alpha subunit (CD11b) of Mac-1 but not to nontransfected cells, confirming a direct interaction between 7E3 and Mac-1. mAb 7E3 and c7E3 Fab blocked the adhesion of Mac-1-bearing cells to FGN (80 +/- 11% and 78 +/- 9% inhibition, respectively) and ICAM-1 (62 +/- 14% and 62 +/- 17%). Both 7E3 and c7E3 Fab significantly inhibited (70 +/- 6% and 62 +/- 26%) soluble FGN binding to human peripheral blood monocytes. Thus, c7E3 Fab cross-reacts with the CD11b subunit of Mac-1 and interrupts cell-extracellular matrix and cell-cell adhesive interactions and may thereby influence the recruitment of circulating monocytes to sites of vessel injury. Given the recent evidence that adherent and infiltrating monocyte number directly correlates with the extent of neointimal hyperplasia, inhibition of Mac-1-dependent adhesion and IIb/IIIa-dependent function by c7E3 Fab may jointly contribute to the regulation of vascular repair and to the sustained clinical benefits observed with c7E3 Fab after angioplasty.

The beta 2 integrin Mac-1 but not p150,95 associates with Fc gamma RIIA.

In this study we have compared the ligand binding activity of the two closely related beta 2 integrins, Mac-1 and p150,95, which are expressed separately as receptors permanently transfected into K562 cells. Mac-1 has previously been shown to associate with Fc gamma R, particularly Fc gamma RIII, but K562 cells express only endogenous Fc gamma RIIA. We have, therefore, taken advantage of this situation to examine a possible relationship between Fc gamma RIIA with Mac-1 and p150,95 in the absence of other Fc gamma R. The main finding is that anti-Fc gamma RII mAb have a profound inhibitory effect on cell adhesion mediated by Mac-1, but not on the adhesion mediated by p150,95. Thus, in spite of the fact that Mac-1 and p150,95 bind to the same or at least a very similar selection of ligands, their association with other receptors on the cellular membrane, and therefore their mode of regulation may be different.

KIM127, an antibody that promotes adhesion, maps to a region of CD18 that includes cysteine-rich repeats.

A series of fusion proteins have been generated between human and mouse CD18. These proteins have been used to carry out preliminary mapping studies on a number of anti-CD18 antibodies including KIM127 an antibody that promotes CD18-dependent adhesion. This antibody maps to a region of the CD18 molecule between amino acids 406 and 570 in a region containing cysteine-rich repeats.

Temperature dependence of cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1.

We investigated cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1 strain IIIB expressed on the surface of CHO cells. These cells formed syncytia when incubated together with CD4-positive human lymphoblastoid SupT1 cells or HeLa-CD4 cells but not when incubated with CD4-negative cell lines. A new assay for binding and fusion was developed by using fluorescent phospholipid analogs that were produced in SupT1 cells by metabolic incorporation of BODIPY-labeled fatty acids. Fusion occurred as early as 10 min after mixing of labeled SupT1 cells with unlabeled CHO-gp160 cells at 37 degrees C. When both the fluorescence assay and formation of syncytia were used, fusion of SupT1 and HeLa-CD4 cells with CHO-gp160 cells was observed only at temperatures above 25 degrees C, confirming recent observations (Y.-K. Fu, T.K. Hart, Z.L. Jonak, and P.J. Bugelski, J. Virol. 67:3818-3825, 1993). This temperature dependence was not observed with influenza virus-induced cell-cell fusion, which was quantitatively similar at both 20 and 37 degrees C, indicating that cell-cell fusion in general is not temperature dependent in this range. gp120-CD4-specific cell-cell binding was found over the entire 0 to 37 degrees C range but increased markedly above 25 degrees C. The enhanced binding and fusion were reduced by cytochalasins B and D. Binding of soluble gp120 to CD4-expressing cells was equivalent at 37 and 16 degrees C. Together, these data indicate that during gp120-gp41-induced syncytium formation, initial cell-cell binding is followed by a cytoskeleton-dependent increase in the number of gp120-CD4 complexes, leading to an increase in the avidity of cell-cell binding. The increased number of gp120-CD4 complexes is required for fusion, which suggests that the formation of a fusion complex consisting of multiple CD4 and gp120-gp41 molecules is a step in the fusion mechanism.

Antibodies that activate beta 2 integrins can generate different ligand binding states.

A human erythroleukemic cell line (K562) that does not normally express beta 2 integrins has been transfected with the genes encoding these integrins. The resulting cell lines show minimal background adhesion but can be stimulated to bind to appropriate substrates when activated with either of two different antibodies to CD18. The two antibodies appear to generate different ligand binding states in LFA-1 such that different members of the ICAM family are recognized. Antibody-activated complement receptor type 3 and p150,95-transfected cells bind protein-coated surfaces, although they require slightly different activation conditions for optimal binding.

KIM185, a monoclonal antibody to CD18 which induces a change in the conformation of CD18 and promotes both LFA-1- and CR3-dependent adhesion.

Unactivated peripheral blood leukocytes show little tendency to bind to other cells or matrix components, whilst, in the presence of inflammatory mediators, adhesive interactions can rapidly increase. The Leu-CAM (beta 2 integrin) family of adhesion molecules have been shown to mediate a variety of these induced adhesion events. Here we describe a monoclonal antibody against CD18, KIM185, which stimulates JY cell homotypic aggregation by a CD11 a pathway as well as inducing the adherence of neutrophils to protein-coated plastic by a CD11b-dependent mechanism. The antibody recognizes an epitope distinct from the previously described KIM127 antibody and evidence is presented that the binding of KIM185 can cause a change in the conformation of the CD18 molecule.

Effect of mutations in the V3 loop of HIV-1 gp120 on infectivity and susceptibility to proteolytic cleavage.

It has been suggested that the V3 domain of human immunodeficiency virus type 1 (HIV-1) isolates has to interact with a cell-surface-associated or endosomal proteinase during virus entry into susceptible cells. To investigate this hypothesis, we examined the effect of several mutations in the V3 loop on its susceptibility to proteolytic cleavage by thrombin and cathepsin E and compared it with the effect of these mutations on viral infectivity. The data obtained indicate that, if an interaction between the V3 loop and a proteinase is indeed crucial for viral entry, the substrate requirements for such a proteinase(s) would have to be very complex. In particular, it seems unlikely that a single enzyme with a unique specificity would be able to interact with all of the different HIV-1 and HIV-2/SIV strains isolated so far. Therefore, one would have to postulate the involvement of several cellular proteinases, or proteases with multiple specificities, in V3-based viral tropism.

Antibody against the Leu-CAM beta-chain (CD18) promotes both LFA-1- and CR3-dependent adhesion events.

The Leukocytic cell-adhesion molecule (beta 2 integrin) family of adhesion molecules play a key role in the intercellular adhesive interactions necessary for normal immune cell function. In this study, we report an antibody that recognizes an epitope on the Leukocytic cell-adhesion molecule common beta-chain (CD18) and promotes both lymphocyte function-associated Ag-1- and CR3-dependent adhesion events. The antibody recognizes a temperature-sensitive epitope that is not dependent on the presence of divalent cations. It is proposed that antibody binding promotes a conformational change in both lymphocyte function-associated Ag-1 and CR3, which may mimic a natural activation mechanism, resulting in increased cellular adhesion.

Gene expression in a cell-free system on the preparative scale.

A cell-free system for preparative gene expression is described. It is composed of DNA-free Escherichia coli extract and added plasmid DNA; coupled transcription-translation proceeds with a continuous flow of the feeding solution containing nucleoside triphosphates and amino acids. The system works at a high constant rate for tens of hours. The yield of synthesised proteins after 20-50 h is hundreds of micrograms from 1 ml of the reaction mixture. Electrophoretic analysis of translation products confirms synthesis of proteins of the expected molecular mass.

Performing nucleic acid reactions using predispensed lyophilized reaction mixtures.

A system is described in which manipulations of nucleic acids are performed in wells containing predispensed lyophilized reaction mixtures requiring addition of only nucleic acid. This allows increased reproducibility for single-step reactions (e.g., restrictions and ligations), as well as improved productivity for complex reactions (e.g., sequencing). Enzymes, co-factors, nucleotides and buffers can be dried and stored at room temperature without loss of essential function. When used for DNA sequencing, hundreds of templates a day can be sequenced with the potential to determine megabase amounts of sequence per week.

Construction of cloning/sequencing vectors with an alternative polylinker.

This paper describes the construction of a new plasmid and M13 phage cloning vector in which the 54-bp polylinkers of pUC19 and of M13mp8 are replaced with a 45-bp chemically synthesized polylinker with different restriction sites. The new polylinker is inserted in frame at the N-terminal end of the beta galactosidase lac Z fragment. The plasmid was named pLH1, the phage M13LH1.

An improved boiling method for the preparation of bacterial plasmid and phage DNA.

A streamlined, reproducible boiling method for preparing plasmid as well as phage replicative form DNA is described. Both quantity and quality of DNA purified are sufficient for restriction enzyme analysis and sequencing. A small loopful of bacteria will provide enough DNA for several experiments, and multiple samples can be processed and prepared for digestion or sequencing in under 20 minutes. DNA preparations are stable for at least 6 months at -20 degrees C.

Cloning in Bacillus subtilis of an extremely thermostable alpha amylase: comparison with other cloned heatstable alpha amylases.

A heatstable alpha amylase gene was shotgun cloned from Bacillus licheniformis RPO1 into Bacillus subtilis. Restriction endonuclease analysis of the recombinant plasmid revealed a map which was identical to a previously cloned alpha amylase from B. licheniformis FDO2 and very similar to the restriction map of a high temperature amylase from Bacillus coagulans. The thermostability and temperature optimum of the cloned alpha amylase was measureably different from those of the previously reported cloned alpha amylases.

Nucleotide sequence of the 5' region of the Bacillus licheniformis alpha-amylase gene: comparison with the B. amyloliquefaciens gene.

The DNA sequence of the 5' region of the Bacillus licheniformis alpha-amylase gene is reported. Comparison of the inferred amino acid sequence of the B. licheniformis alpha-amylase gene with that of the Bacillus amyloliquefaciens gene shows that whereas the amino acid sequences of the mature proteins have considerable homology, the sequences for the signal peptides are distinct.