Construction and Adhesive Properties of a Soluble MAdCAM‐1‐Fc Chimera Expressed in a Baculovirus System: Phylogenetic Conservation of Receptor‐Ligand Interaction

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Abstract
MAdCAM‐1 is a high endothelial venule adhesion molecule composed of immunoglobulin and mucin‐like domains which binds the leucocyte integrin LPAM‐1 (α4β7), and is largely responsible for the selective homing of lymphocytes to mucosal tissues. A novel soluble form of mouse MAdCAM‐1 which is normally membrane bound has been produced by joining the extracellular region of the receptor to the Fc domain of human IgGl. The MAdCAM‐1‐Fc cDNA was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda insect cells infected with the recombinant virus produced MAdCAM‐1‐Fc as a disulfide‐linked homodimer of 82kDa polypeptides, which was secreted into the culture medium at > 1 μg/ml. The product purified by Protein G‐Sepharose was identified as authentic MAdCAM‐1‐Fc by the anti‐MAdCAM‐l monoclonal antibody (MoAb) MECA‐367 using Western blot and ELISA analysis. When immobilized on glass it was fully functional in supporting the binding of mouse α4β1+α4β7+ mesenteric lymph node lymphocytes, and the α4β1−αβ7+ TK1 T cell lymphoma. Binding was enhanced by Mn++ ‐induced integrin activation, and specifically blocked by anti‐integrin α4 subunit and anti‐MAdCAM‐1 MoAbs. Binding was blocked by pretreatment of cells with sodium azide, and EDTA, indicating that binding is an energy‐dependent process which requires divalent cations. Thus the mouse MAdCAM‐1‐Fc chimera produced in insect cells retains certain functional properties that typify the native receptor, and should be valuable in analysing the role of MAdCAM‐1 in lymphocyte recirculation and emigration. However it was not sialylated despite being post‐translational modified with N‐ and O‐linked carbohydrate moieties, suggesting that the ability of MAdCAM‐1 to support cell adhesion under static conditions is sialylation‐independent. A rabbit polyclonal antibody raised against the entire cytopiasmic domain of the human integrin β7 subunit recognized LPAM‐1‐like molecules in human, rat, and mouse cells, suggesting a high degree of conservation of the MAdCAM‐1 receptor across species. In agreement with this notion MAdCAM‐1‐Fc immobilized on glass was fully functional in supporting the cation‐dependent binding of peripheral blood or spleen cells from a range of other species including human, rat, and guinea pig; and for human myeloid HL60 cells, binding was mediated by o4 integrins.