Construction and adhesive properties of a soluble MadCAM-1-Fc chimera expressed in a baculovirus system: phylogenetic conservation of receptor-ligand interaction.
MAdCAM-1 is a high endothelial venule adhesion molecule composed of immunoglobulin 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 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 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 secreted into the culture medium at > 1 microgram/ml. The product purified by Protein G-Sepharose was identified as authentic MAdCAM-1-Fc by the anti-MAdCAM-1 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 alpha 4 beta 1+ alpha 4 beta 7+ mesenteric lymph node lymphocytes, and the alpha 4 beta 1- alpha 4 beta 7+ TK1 T cell lymphoma. Binding was enhanced by Mn(++)-induced integrin activation, and specifically blocked by anti-integrin alpha 4 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 cytoplasmic domain of the human integrin beta 7 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 alpha 4 integrins.
Yang
Y
Y
Department of Molecular Medicine, School of Medicine, University of Auckland, New Zealand.
Sammar
M
M
Harrison
J E
JE
Lehnert
K
K
Print
C G
CG
Leung
E
E
Prestidge
R
R
Krissansen
G W
GW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism*; D007136:Immunoglobulins / Q000235:genetics / Q000378:metabolism*; D007313:Insecta; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009088:Mucoproteins / Q000235:genetics / Q000378:metabolism*; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D013601:T-Lymphocytes / Q000378:metabolism*
D015815:Cell Adhesion Molecules; D007141:Immunoglobulin Fc Fragments; D007136:Immunoglobulins; D016023:Integrins; C504265:MADCAM1 protein, human; C489082:Madcam1 protein, mouse; C504268:Madcam1 protein, rat; D009088:Mucoproteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C071777:integrin alpha4beta7