A 70,000-mol-wt
protein was isolated from A431
carcinoma cell extracellular matrix that promotes cell substratum adhesion of these epidermoid
tumor cells. Extracellular matrix was isolated by a modification of a procedure described by Hedman et al. (Hedman, K., M. Kurkinen, K. Alitalo, A. Vaheri, S. Johansson, and M. Höök, 1979 J. Cell Biol., 81:83-91) and Yamada and Weston (Yamada, K., and J. A. Weston, 1974, Proc. Natl. Acad. Sci. USA, 71:3492-3496). Cells were solubilized with 0.5%
deoxycholate, 10 mM Tris,
0.9% NaCl, and 1 mM
phenylmethylsulfonyl fluoride, pH 8.0. The residual matrix was then removed from the plates with 6 M
urea and 1 mM
phenylmethylsulfonyl fluoride and
phosphate-buffered saline. SDS PAGE
gels of the 6 M
urea extract showed one major band at 70,000-mol-wt by
Coomassie Blue staining. A 70,000-mol-wt isotopically-labeled band could also be extracted from the matrix of cells incubated with [35S]
methionine. Because of the presence of this
protein on squamous-derived epithelial cells we have called the 70,000-mol-wt molecule
epinectin. Indirect immunofluorescence with polyclonal rabbit
antibodies against
epinectin stained A431 cells pericellularly in dense punctate accumulations and along the plasma membrane.
Enzyme-linked immunoassays and gel-transfer immunolocalization studies showed that the extract did not cross-react with
antibodies to
fibronectin,
laminin,
serum-spreading factor, epibolin, or
keratin. Additionally,
antibodies to
epinectin did not cross-react with these
proteins. Further studies showed that
epinectin does not bind to
gelatin. Cell-adhesion assay, using radiolabeled A431
carcinoma cells on various adhesion-promoting substrates, showed that
epinectin has similar adhesion-promoting capacity as
serum-spreading factor, was somewhat less active than
fibronectin, but more effective than
laminin or epibolin.
Epinectin appears to be a unique
protein isolated from epidermoid
tumor cells that is distinct from other known adhesion
proteins.