CONSTRUCTION AND ADHESIVE PROPERTIES OF A SOLUBLE MADCAM-1-FC CHIMERAEXPRESSED IN A BACULOVIRUS SYSTEM - PHYLOGENETIC CONSERVATION OF RECEPTOR-LIGAND INTERACTION

MAdCAM-1 is a high endothelial venule adhesion molecule composed of im munoglobulin and mucin-like domains which binds the leucocyte integrin LPAM-1 (alpha 4 beta 7), and is largely responsible for the selective homing of lymphocytes to mucosal tissues. A novel soluble form of mou se MAdCAM-1 which is normally membrane bound has been produced by join ing the extracellular region of the receptor to the Fc domain of human IgG1. 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 82 kDa polypeptides, which was sec reted into the culture medium at > 1 mu g/ml. The product purified by Protein G-Sepharose was identified as authentic MAdCAM-1-Fc by the ant i-MAdCAM-1 monoclonal antibody (MoAb) MECA-367 using Western blot and ELISA analysis. When immobilized on glass it was fully functional in s upporting the binding of mouse alpha 4 beta 1(+) alpha 4 beta 7(+) mes enteric lymph node lymphocytes, and the alpha 4 beta 1(-) alpha 4 beta 7(+) TK1 T cell lymphoma, Binding was enhanced by Mn++-induced integr in activation, and specifically blocked by anti-integrin alpha 4 subun it and anti-MadCAM-1 MoAbs. Binding was blocked by pretreatment of cel ls with sodium azide, and EDTA, indicating that binding is an energy-d ependent process which requires divalent cations. Thus the mouse MAdCA M-1-Fc chimera produced in insect cells retains certain functional pro perties that typify the native receptor, and should be valuable in ana lysing the role of MAdCAM-1 in lymphocyte recirculation and emigration . However it was not sialylated despite being post-translational modif ied with N- and O-linked carbohydrate moieties, suggesting that the ab ility of MAdCAM-1 to support cell adhesion under static conditions is sialylation-independent. A rabbit polyclonal antibody raised against t he entire cytoplasmic domain of the human integrin beta 7 subunit reco gnized LPAM-1-like molecules in human, rat, and mouse cells, suggestin g a high degree of conservation of the MAdCAM-1 receptor across specie s. In agreement with this notion MAdCAM-1-Fc immobilized on glass was fully functional in supporting the cation-dependent binding of periphe ral blood or spleen cells from a range of other species including huma n, rat, and guinea pig; and for human myeloid HL60 cells, binding was mediated by alpha 4 integrins.