Abstract
We have recently cloned and characterized the entire human 230-kDa bullous pemphigoid antigen gene, which is expressed at a relatively high level in the basal keratinocytes. A putative AP2 binding sequence (KRE2), identified in the position -1786 to -1778, was cloned in front of a heterologous thymidine kinase chloramphenicol acetyltransferase construct, and transient transfections of normal human keratinocytes indicated a marked enhancement of the promoter activity. Normal human keratinocyte nuclear extracts contained a protein, designated as keratinocyte transcriptional protein-1 (KTP-1), which complexed with the KRE2 oligomer when examined by gel mobility shift assays. This protein was not detected in human skin fibroblast or HeLa cell nuclear extracts that did, however, contain AP2. UV cross-linking studies and Southwestern analyses suggested that KTP-1 binds to DNA as a single polypeptide of approximately 110 kDa. These data suggest that KTP-1 is a DNA-binding protein clearly distinct from AP2, and this protein may be responsible for the basal keratinocyte-specific expression of the bullous pemphigoid antigen gene.
Highlights
Previous studies utilizing immunoelectron microscopy have localized both the 230- and 180-kDa BP antigens to hemidesmosomes, ultrastructurally recognizable attachment comthe KRE2 oligomerwhenexaminedby gel mobility plexes at thebasal keratinocyte-lamina lucida interface of the shift assays
Recent studies have indicated that BPAGl is abundantly expressed by epithelial cells of the stratifying squamous epithelia, such as basal keratinocytes of the epidermis, as determined at themRNA level (Sawamura et al, 1991b;Tamai et al, 1993)
Earlier studies utilizing indirect immunofluorescence with antisera from patients with BP have indicated that the distribution of BP antigens in the skin is restricted to thebasal keratinocytes of the epidermis (Jordon et al, 1967, 1971;Mutasim et al, 1985).the expression of the BPAGl gene, as detected both at the mRNA and protein levels, appears to be highly compartmentalized
Summary
RNA Isolation and Northern Analysis-RNAwas isolated from cultured cells by a single-step extraction method, as described previously (Chomczynski and Sacchi, 1987). RNA was transferred to nitrocellulose filters, and Northern hybridizations were performed with a 2.3-kb human BPAGl cDNA (Sawamura et al, 1991b),labeled radioactive with [cY-~’P]GTPand [cx-~’P]CTPby nick translation (Sambrook et al, 1989). Constructwn of KRE2-containing Plasmids-A double-stranded oligomer KRE2, containingaputative AP2-binding sequence, 5’-. Nuclear proteins were isolated from normal human keratinocytes, skin fibroblasts, and HeLa cells using a small scale preparation method, as reported previously (Schreiber et al, 1989). Southwestern Bbtting Analysis-To demonstrate the binding affinity of KTP-1 protein, nuclear extracts from keratinocytes, fibroblasts, or HeLa cells were fractionated on 8% SDS-polyacrylamide gels after reduction of the disulfide bonds with 2-mercaptoethanol. Nucleotide sequences of oligomers used for gel mobility shift and Southwestern blotting assays
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