Abstract
Collagen XVII is a hemidesmosomal transmembrane molecule important for epithelial adhesion in the skin. It exists in two forms, as a full-length protein and as a soluble ectodomain that is shed from the keratinocyte surface by furin-mediated proteolysis. To obtain information on the conformation and the functions of this unusual collagen, its largest collagenous domain, Col15, was expressed in a eukaryotic episomal expression system and purified by DEAE and fast protein liquid- Mono S chromatography. The protein was triple-helical (T(m) of 26.5 degrees C) when produced in cultures containing ascorbic acid. When the vitamin supply was limited, the 4-hydroxyproline content was reduced from 74 to 9%, which, in turn, resulted in a drastic reduction of the stability of the triple helix. The glycine substitution mutation G627V associated with junctional epidermolysis bullosa, a human blistering skin disease, also had a striking effect on thermal stability of rCol15 causing partial unfolding already at 4 degrees C. Col15 promoted cell adhesion of epithelial and fibroblastic cell lines with a beta1 integrin-mediated mechanism. In concert with this, in acquired autoimmune blistering skin diseases, circulating IgG and IgA autoantibodies were found to target rCol15r.
Highlights
Collagen XVII is a hemidesmosomal transmembrane molecule important for epithelial adhesion in the skin
Expression and Purification of the rCol15 Domain of Collagen XVII—The Col15 domain of collagen XVII consists of 242 amino acids and is
Its calculated molecular mass deduced from the cDNA sequence is 2245 kDa, and its predicted amino acid sequence is characterized by 80 perfect Gly-Xaa-Yaa triplets with one GlyXaa-Gly (Gly-Ser-Gly) interruption in positions 639 – 641
Summary
Collagen XVII is a hemidesmosomal transmembrane molecule important for epithelial adhesion in the skin. It exists in two forms, as a full-length protein and as a soluble ectodomain that is shed from the keratinocyte surface by furin-mediated proteolysis. It was established that collagen XVII exists as two molecular forms, i.e. as a full-length transmembrane homotrimer of three 180kDa ␣1(XVII) chains and as a 120-kDa soluble form that corresponds to the extracellular domain and is presumably released from the cell surface through furin-mediated proteolytic processing [6, 7]. Mutations in the collagen XVII gene, COL17A1, lead to junctional epidermolysis bullosa, a hereditary blistering skin disease with epidermal detachment from the basement membrane [15,16,17,18,19,20,21]. The conformation, the functions, and most ligands of the collagenous ectodomain have remained elusive
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