Identification of putative ligand-binding sites of the integrin α4β1 (VLA-4, CD49d/CD29)

Integrin alpha 4 beta 1 recognizes both fibronectin (CS-1 sequence) and vascular cell adhesion molecule-1 (VCAM-1). To localize the ligand-binding sites of alpha 4, we located the epitopes for function-blocking anti-alpha 4 monoclonal antibodies (mAbs), including those that recognize previously described (but not yet physically localized) functional epitopes (A, B1, B2 and C) using interspecies alpha 4 chimeras expressed in mammalian cells. Epitopes B1 and B2 were associated with ligand binding, and epitopes A and B2 with homotypic cellular aggregation. mAbs P4C2 (epitope B2), 20E4 and PS/2 were mapped within residues 108-182; mAbs HP2/1 (epitope B1), SG/73 and R1-2 within residues 195-268; mAbs HP1/3 (epitope A) and P4G9 within residues 1-52; and B5G10 (epitope C) within residues 269-548. The data suggest that residues 108-268, which do not include bivalent-cation-binding motifs, are related to VCAM-1 and CS-1 binding, and more N-terminal portions of alpha 4 (residues 1 and 52 and 108-182) to homotypic aggregation. Since mAbs PS/2 and HP2/1 block alpha 4 beta 7 binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the MAdCAM-1-binding site is close to, or overlapping with, VCAM-1- and CS-1-binding sites. The role of Asp-130 of beta 1 in the binding to VCAM-1 and CS-1 peptide was examined. Chinese hamster ovary (CHO) cells expressing beta 1 (D130A) (Asp-130 to Ala mutant of beta 1) and alpha 4 showed much less binding to both ligands than CHO cells expressing wild-type beta 1 and alpha 4 [a dominant negative effects of beta 1 (D130A)], suggesting that Asp-130 of beta 1 is critical for binding to both ligands and that the two ligand share common binding mechanisms [corrected].

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Development of a novel mammalian display system for selection of antibodies against membrane proteins

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015053 ◽

2020 ◽

Vol 295 (52) ◽

pp. 18436-18448

Author(s):

Nathan Robertson ◽

Nancy Lopez-Anton ◽

Shalom A. Gurjar ◽

Hena Khalique ◽

Zainab Khalaf ◽

...

Keyword(s):

Cell Adhesion ◽

Epithelial Cell ◽

Membrane Proteins ◽

Membrane Protein ◽

Adhesion Molecule ◽

Chinese Hamster Ovary ◽

Mammalian Cells ◽

Cell Adhesion Molecule ◽

Chinese Hamster ◽

Epithelial Cell Adhesion Molecule

Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 109 variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (KD) as low as 0.8 nm. We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule–positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies.

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Activation Dependent and Independent VLA-4 Binding Sites on Vascular Cell Adhesion Molecule-1

Cell Adhesion and Communication ◽

10.3109/15419069409004429 ◽

1994 ◽

Vol 2 (2) ◽

pp. 87-99 ◽

Cited By ~ 32

Author(s):

Lindsey A. Needham ◽

Sonja Van Dijk ◽

Rod Pigott ◽

R. Mark Edwards ◽

Maura Shepherd ◽

...

Keyword(s):

Cell Adhesion ◽

Adhesion Molecule ◽

Binding Sites ◽

Cell Adhesion Molecule ◽

Vascular Cell Adhesion ◽

Vascular Cell ◽

Vascular Cell Adhesion Molecule ◽

Cell Adhesion Molecule 1

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A novel region of the α4 integrin subunit with a modulatory role in VLA-4-mediated cell adhesion to fibronectin

Biochemical Journal ◽

10.1042/bj3270727 ◽

1997 ◽

Vol 327 (3) ◽

pp. 727-733 ◽

Cited By ~ 15

Author(s):

Marisa MUÑOZ ◽

Juan SERRADOR ◽

Marta NIETO ◽

Alfonso LUQUE ◽

Francisco SÁNCHEZ-MADRID ◽

...

Keyword(s):

Cell Adhesion ◽

Ligand Binding ◽

Binding Sites ◽

Close Association ◽

Cell Aggregation ◽

Integrin Subunit ◽

Vascular Cell Adhesion ◽

Ligand Binding Sites ◽

Potential Ligand ◽

Cell Adhesion Molecule 1

The integrin VLA-4 (α4β1) is a receptor for fibronectin and vascular cell-adhesion molecule 1 (VCAM-1). Four functionally different epitopes, designated A, B1, B2 and C, have previously been defined on the α4 subunit. Using K562 α4 mutant transfectants we found that α4 amino acids Tyr151, Gln152, Asp153, Tyr154 and Val155 are important for the structure of the epitope B2. Mutations at α4 Gln152 substantially impaired the transfectant adhesion to a CS-1-containing fragment of fibronectin (FN-H89), whereas this adhesion was not affected on the other α4 mutant transfectants. None of the α4 mutations significantly altered the adhesion of the different α4 transfectants to VCAM-1. In addition, we have identified residues Gln152, Asp153 and Tyr154 as part of the α4 epitope B2 involved in homotypic cell aggregation. The decrease in adhesion to FN-H89 shown by Gln152 α4 mutant transfectants was the result of an inefficient binding of FN-H89 by VLA-4 mutated at this residue. Also, mutant VLA-4 displayed an altered reactivity with HUTS-21, an anti-β1 monoclonal antibody that reacts with functionally active VLA integrins. Adhesion to FN-H89 was not restored unless stimuli that increase the ligand-binding affinity of VLA heterodimers were added, suggesting that cell adhesion was affected in the initial phases. These results indicate that α4 Gln152 modulates cell adhesion to FN-H89 by playing important roles in the maintenance and/or the acquisition of an active state of VLA-4, an integrin that is normally expressed on the cell surface in a range of multiple activation states. The location of the α4 Gln152 residue on a loop of the upper surface of the proposed β-propeller structure suggests a close association with potential ligand-binding sites.

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Deletions in the cytoplasmic domain of platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) result in changes in ligand binding properties

The Journal of Cell Biology ◽

10.1083/jcb.124.1.195 ◽

1994 ◽

Vol 124 (1) ◽

pp. 195-203 ◽

Cited By ~ 71

Author(s):

HM DeLisser ◽

J Chilkotowsky ◽

HC Yan ◽

ML Daise ◽

CA Buck ◽

...

Keyword(s):

Cell Adhesion ◽

Ligand Binding ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Cytoplasmic Domain ◽

Wild Type ◽

Platelet Endothelial Cell Adhesion ◽

Cell Adhesion Molecule 1 ◽

Endothelial Cell Adhesion ◽

Cell Cell

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a member of the immunoglobulin superfamily present on platelets, endothelial cells, and leukocytes that may function as a vascular cell adhesion molecule. The purpose of this study was to examine the role of the cytoplasmic domain in PECAM-1 function. To accomplish this, wild-type and mutated forms of PECAM-1 cDNA were transfected into murine fibroblasts and the functional characteristics of the cells analyzed. Wild-type PECAM-1 localized to the cell-cell borders of adjacently transfected cells and mediated heterophilic, calcium-dependent L-cell aggregation that was inhibitable by a polyclonal and two monoclonal anti-PECAM-1 antibodies. A mutant protein lacking the entire cytoplasmic domain did not support aggregation or move to cell-cell borders. In contrast, both forms of PECAM-1 with partially truncated cytoplasmic domains (missing either the COOH-terminal third or two thirds of the cytoplasmic domain) localized to cell-cell borders in 3T3 cells in a manner analogous to the distribution seen in cultured endothelial cells. L-cells expressing these mutants demonstrated homophilic, calcium-independent aggregation that was blocked by the polyclonal anti-PECAM-1 antibody, but not by the two bioactive monoclonal antibodies. Although changes in the cytoplasmic domain of other receptors have been shown to alter ligand-binding affinity, to our knowledge, PECAM-1 is the first example of a cell adhesion molecule where changes in the cytoplasmic domain result in a switch in the basic mechanism of adhesion leading to different ligand-binding specificity. Variations in the cytoplasmic domain could thus be a potential mechanism for regulating PECAM-1 activity in vivo.

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Activated endothelium binds lymphocytes through a novel binding site in the alternately spliced domain of vascular cell adhesion molecule-1.

Journal of Experimental Medicine ◽

10.1084/jem.176.1.99 ◽

1992 ◽

Vol 176 (1) ◽

pp. 99-107 ◽

Cited By ~ 92

Author(s):

L Osborn ◽

C Vassallo ◽

C D Benjamin

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Adhesion Molecule ◽

Binding Sites ◽

Cell Adhesion Molecule ◽

Vascular Cell Adhesion ◽

Vascular Cell ◽

Vascular Cell Adhesion Molecule ◽

Cell Adhesion Molecule 1

Vascular cell adhesion molecule-1 (VCAM-1) is induced on endothelial cells by inflammatory cytokines, and binds mononuclear leukocytes through the integrin very late antigen-4 (VLA-4) (alpha 4 beta 1). This adhesion pathway has been implicated in a diverse group of physiological and pathological processes, including B cell development, leukocyte activation and recruitment to sites of inflammation, atherosclerosis, and tumor cell metastasis. The major form of VCAM-1 (VCAM-7D) has seven extracellular immunoglobulin (Ig)-like domains, of which the three NH2-terminal domains (domains 1-3) are similar in amino acid sequence to domains 4-6. By functional analysis of VCAM-7D relative to VCAM-6D (a minor 6-domain form of VCAM-1 in which domain 4 is deleted because of alternative splicing), and chimeric constructs between VCAM-1 and its structural relative intercellular adhesion molecule-1 (ICAM-1), we show that either the first or the homologous fourth domain of VCAM-1 is required for VLA-4-dependent adhesion. Either of these binding sites can function in the absence of the other. When both are present, cell binding activity is increased relative to monovalent forms of the molecule. The homologous binding regions appear to have originated by internal duplication of a portion of a monovalent ancestral gene, before the mammalian radiation. Thus VCAM-1 exemplifies evolution of a functionally bivalent cell-cell adhesion molecule by intergenic duplication. We have also produced a new class of anti-VCAM-1 monoclonal antibodies that block domain 4-dependent adhesion, and demonstrate that both binding sites participate in the adhesion function of VCAM-1 on endothelial cells in vitro. Therefore both sites must be blocked in clinical, animal, or in vitro studies depending on the use of anti-VCAM-1 antibodies to inactivate the VCAM-1/VLA-4 adhesion pathway.

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