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180‐kD bullous pemphigoid antigen/type XVII collagen: Tissue‐specific expression and molecular interactions with keratin 18
Author(s) -
Aho Sirpa,
Uitto Jouni
Publication year - 1999
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/(sici)1097-4644(19990301)72:3<356::aid-jcb5>3.0.co;2-m
Subject(s) - biology , complementary dna , microbiology and biotechnology , keratin , keratinocyte , in vitro , biochemistry , genetics , gene
The 180‐kD bullous pemphigoid antigen (BPAG2) is a hemidesmosomal transmembrane protein, also known as type XVII collagen. In this study, potential interactions of BPAG2 with other proteins expressed in epidermal keratinocytes were explored by yeast two‐hybrid system using the amino‐terminal intracellular domain of BPAG2 as a bait. Several independent interacting clones encoding keratin 18 (K18) were identified when the keratinocyte cDNA library, cloned into the yeast two‐hybrid activation domain vector, was screened. The peptide sequence responsible for the interaction of BPAG2 was restricted to amino acids 15–25, and substitution of a valine residue in the middle of this sequence by a proline (V23P) by site‐directed mutagenesis abolished the interaction. Further examination of the K18 sequences by restricted cDNA constructs in yeast two‐hybrid system identified a carboxyl‐terminal segment corresponding to helix 2B domain as critical for BPAG2 binding. The interaction of BPAG2/K18 was confirmed by an in vitro protein‐protein interaction assay, which also confirmed that normal human keratinocytes express K18 in culture. The tissue specific expression of BPAG2 was first examined using a multi‐tissue RNA blot. Human multiple tissue cDNA panels representing a variety of adult and fetal tissues as well as tumor cells were used as PCR‐templates to study the expression patterns of both BPAG2 and K18. The results demonstrated significant level of expression of BPAG2, besides in epidermal keratinocytes, also in a variety of tissues with predominant epithelial component, such as mammary, salivary and thyroid glands, colon, prostate, testis, placenta, and adult and fetal thymus, as well as in colon, pancreatic and prostatic adenocarcinoma cell lines, and an ovarian carcinoma. As expected, K18 transcript is present in liver, pancreas, colon, placenta, and in fetal kidney. Collectively, the results suggest that BPAG2 has a relatively broad tissue distribution including specialized and simple epithelia, and that within the tissues such as colon and placenta, BPAG2 may have direct interactions with K18, a keratin characteristically expressed in a simple epithelia. J. Cell. Biochem. 72:356–367, 1999. © 1999 Wiley‐Liss, Inc.