Development of a new tracking tool for the human monomeric laminin-gamma 2 chain in vitro and in vivo.
Laminin-5 (Ln-5), a heterotrimer composed of three different laminin chains [laminin-alpha 3 (Ln-alpha 3), laminin-beta 3 (Ln-beta 3), and laminin-gamma 2 (Ln-gamma 2)], is a major component of the basement membrane in most adult tissues. One of the chains, Ln-gamma 2, is a specific marker of invasive tumors because it is frequently expressed as a monomer in malignant tumors. However, there is no simple and direct method to detect the monomeric form of Ln-gamma 2 selectively in the presence of Ln-5 because all available antibodies recognize both monomeric and heterotrimeric forms of Ln-gamma 2. In this study, we developed a new monoclonal antibody (mAb) termed 1H3 that reacts specifically with human Ln-gamma 2 monomers during immunoprecipitation, ELISA, Western blotting, and immunostaining. Ln-5 was not recognized by mAb 1H3 after denaturation with detergents under nonreducing conditions, but reactivity was recovered when denaturation was done under reducing conditions. The epitope of the antibody was mapped to region on the coiled-coil structure formed between Ln-gamma 2 and its partner chains Ln-alpha 3 and Ln-beta 3 in Ln-5, whose structure is further stabilized by disulfide bonds. In normal tissue samples, the basement membrane was stained with conventional antibody against Ln-gamma 2 but not by mAb 1H3. In contrast, tumor cells in tissue sections could be stained with mAb 1H3 as efficiently as with conventional antibody. Thus, mAb 1H3 holds promise as a powerful tracking tool for the specific detection of monomeric Ln-gamma 2 in vivo and in vitro and is potentially useful as a diagnostic tool for detecting tumors and as a vehicle for drug delivery to cancer tissues.
Koshikawa
Naohiko
N
Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
Minegishi
Tomoko
T
Nabeshima
Kazuki
K
Seiki
Motoharu
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D019281:Dimerization; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007797:Laminin / Q000032:analysis* / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D012107:Research Design; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; C400567:LAMC2 protein, human; D007797:Laminin