Substrate adherence of newborn and adult human keratinocytes

A method to detect and analyze the proteins transferred to a collagen substrate following interaction of normal human keratinocytes with a collagen substrate is described. Keratinocytes are allowed to attach to a collagen substrate for 1 hour, and the attached cells are lysed with 0.1 N NaOH. When the keratinocytes are prelabeled with 3H-amino acids, cell surface proteins are transferred from the keratinocyte to the collagen substrate. The transferred proteins cannot be removed from the substrate by sulfhydryl reducing reagents, high concentrations of urea, or metal chelators. Treatment of the attached proteins with proteolytic enzymes (trypsin chymotrypsin) or with 2 M NaBr partially releases the proteins. More specific labeling of the attachment proteins can be obtained using 3H-tyrosine instead of a complete amino acid mixture. 3H-fucose is also incorporated and transferred, suggesting that one of the components of the attachment protein(s) is a glycoprotein. Antibodies prepared in rabbits to the attachment proteins inhibit the adherence of both primary and first-passaged keratinocytes, suggesting that these proteins may be a part of the protein glycoprotein complexes on the surface of keratinocytes involved in the adherence of keratinocytes to basement membranes.