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The Journal of Cell Biology, Vol 100, 216-225, Copyright © 1985 by The Rockefeller University Press
ARTICLES |
DF Sas, MJ Sas, KR Johnson, AS Menko and RG Johnson
A monoclonal antibody (mcAb) that recognizes an intracellular domain of the major lens membrane protein in both chicken and bovine lenses is described. Mice were immunized with chicken lens fiber cell membranes that had been washed with 7 M urea. Hybridomas were screened by means of enzyme-linked immunosorbent assays and the molecular specificities of the mcAbs were determined using electrophoretic transfer procedures, "Westerns." One of these mcAbs, an IgG designated B2, reacted with a single band of 28,000 Mr from the chicken embryo lens (MP28) and the analogous 26,000 Mr protein in the bovine lens (MP26). Monoclonal B2 was shown to be specific for these proteins, since (a) heating in SDS caused MP26 to aggregate and reduced B2 binding to the protein band at an Mr of 26,000 in Western transfer analysis; (b) apparent dimers were bound by B2 in Western transfers; (c) soluble protein fractions from the lens contained no detectable B2 antigens; and (d) a cyanogen bromide fragment of MP26 was bound by B2. Studies with several proteases indicated that the antigenic site for B2 resides on a 2-kd, protease-sensitive region at the C-terminal end of MP26 and MP28. Evidence for B2 binding on the cytoplasmic side of the membrane comes from labeling studies done at the ultrastructural level. These studies, utilizing indirect methods with peroxidase and colloidal gold markers, clearly demonstrated that B2 labels two types of junctional profiles. In our calf lens membrane preparations after tannic acid staining, the predominant type (80%) measured 16-18 nn thick, with the second type measuring only 12-14 nm. Chick embryo lens cells that had differentiated in vitro and formed groups of lens fiber-like cells (termed lentoids), fluoresced brightly only when they had been permeabilized before labeling with B2 and a fluorochrome-conjugated antibody. This binding was concentrated at the plasma membranes of cells within the lentoids, even outside areas of cell-cell contact. Surrounding epithelioid cells were not stained. Solubilized lens cultures, examined by Westerns, displayed a single immunoreactive band, which co-migrated with MP28.
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