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Characterization of the antigens recognized by two monoclonal antibodies reactive with basal-layer keratinocytes of human epidermis.

ABSTRACT: Two monoclonal antibodies, GR3 and GR4, reactive with the basal-layer keratinocytes of human epidermis, were derived by immunization of Balb/c mice with glycoproteins isolated from cultured keratinocytes by lectin-affinity chromatography. Immunoprecipitation of Triton X-100 extracts from human keratinocytes metabolically labelled with D-[1-14C]glucosamine revealed that GR3 recognized a major glycoprotein with migration properties identical with those of a glycoprotein (reduced form M(r) 126,000) which was previously shown to be implicated in intercellular adhesion [Roberts and Brunt (1985) Biochem J. 232, 67-70]. In their unreduced forms the antigens recognized by GR3 and GR4 both migrated as two bands with M(r) values of 118,000 and 147,000. Comparison of 125I-labelled glycoproteins immunoprecipitated by GR3, GR4 and integrin antibodies revealed that, under reducing conditions, the major band immunoprecipitated by both GR3 and GR4 co-migrated with the alpha 3 and beta 1 integrin chains. In addition the immunoprecipitate obtained with GR4 contained an additional band co-migrating with the alpha 2 integrin chain. Sequential immunoprecipitation studies with GR3, GR4 and integrin antibodies confirmed that GR3 is directed against the alpha 3 integrin chain, whereas GR4 is directed against the beta 1 chain. These studies also indicate that some of the alpha 2 integrin chains on keratinocytes may be associated with a beta-chain not recognized by the antisera against the beta 1 integrin chain used in this study.

SUBMITTER: Roberts GP

PROVIDER: S-EPMC1138071 | biostudies-other | 1994 May

REPOSITORIES: biostudies-other

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Project description:Pig epidermis separated by 1 M-CaCl2 treatment was homogenized and separated into three fractions by filtration through nylon mesh and high-speed centrifugation. Lectin-binding glycoproteins were isolated from urea/deoxycholate/mercaptoethanol extracts of the residue fraction that resisted filtration, from deoxycholate extracts of the particulate material in the filtrate and from the soluble fraction. Concanavalin A, Ricinus communis (castor bean) agglutinin 1, peanut (Arachis hypogaea) agglutinin and Ulex europaeus (gorse) agglutinin-binding glycoproteins in the three epidermal fractions were analysed by SDS/polyacrylamide-gel electrophoresis. A major neuraminidase-sensitive glycoprotein component of the particulate fraction of Mr 135,000 was strongly bound by concanavalin A and Ricinus communis agglutinin 1, but only weakly by peanut and Ulex europaeus agglutinins. This glycoprotein was not detected in the residue or soluble fractions of the epidermis, indicating that it had only a limited distribution within the tissue. The 135,000-Mr glycoprotein was one of two major glycoprotein antigens in the particulate fraction. Rabbits immunized with total particulate glycoproteins produced antibodies directed mainly against 135,000- and 110,000-Mr components. Monospecific antibodies were obtained from guinea pigs immunized with the 135,000-Mr glycoprotein band excised from polyacrylamide gels. Indirect immunofluorescence with the use of affinity-purified antibodies showed that the 135,000-Mr glycoprotein was present at the surface of cells in the basal layer of the epidermis as well as at that of other stratified epithelia. It was not present on differentiating cells in the suprabasal layers of the epithelium, suggesting an important role in the attachment or proliferative functions of basal cells in stratified epithelia. Metabolic labelling studies with skin explants cultured in the presence of D-[3H]glucosamine showed that this basal-cell glycoprotein was synthesized by cultured tissue. The major D-[3H]glucosamine-labelled glycoprotein component in the residue and particulate fractions of cultured epidermis had an Mr of 135,000, was immunoprecipitated by rabbit antisera raised against particulate epidermal glycoproteins and was bound by concanavalin A. The labelling of this glycoprotein with D-[3H]glucosamine was sensitive to tunicamycin, indicating that the basal-cell glycoprotein contained N-glycosidically linked oligosaccharides.

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Characterization of the antigens recognized by two monoclonal antibodies reactive with basal-layer keratinocytes of human epidermis.

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