The Shed Ectodomain of Collagen XVII/BP180 Is Targeted by Autoantibodies in Different Blistering Skin Diseases (1)
Collagen XVII occurs in two forms, as a full-length transmembrane protein and as a distinct, soluble ectodomain that is shed from the cell surface by furin-mediated proteolytic processing. The 120-kd soluble form is a rod-like, flexible molecule that contains 15 collagenous domains flanked by 16 short noncollagenous sequences. The resulting clones, pCEP-ColXVII-Col15 and pCEP-ColXVII-Ecto2, were verified by dideoxynucleotide sequence analysis.
Under physiological conditions the ectodomain was shed into the epidermal basement membrane zone, because it could be extracted with EDTA and a neutral salt buffer from normal human epidermis. Amniotic fluid and, under pathological conditions, pemphigoid blister fluid, also contained this form of collagen XVII.
This authentic ectodomain was recognized by circulating IgG and IgA autoantibodies from patients with blistering skin disorders. The immunoprecipitated by anti recombinant NC16a domain of collagen XVII, 120-kd polypeptide was recognized by IgG and IgA in the sera, indicating that the band targeted by human autoantibodies indeed represented the soluble ectodomain of collagen XVII. The treatment of the antigen preparation with a highly purified collagenase abolished the reactivity of human IgG and IgA autoantibodies and of rabbit anti-collagen XVII antibodies with the 120-kd polypeptide, indicating that the antigen had a collagenous structure. Deglycosylation of the ectodomain with N-glycosidase F accelerated its mobility on SDS-PAGE but did not abolish the reactivity with the autoantibodies.
Of the seven CBDC sera, all were positive with indirect immunofluorescence staining and all recognized the soluble form of collagen XVII, but only three recognized the full-length 180-kd α1(XVII) chain. Most pemphigoid sera contained only IgG, and not IgA, antibodies to collagen XVII, and, in contrast, most LAD sera contained only IgA, and not IgG antibodies, to both forms of collagen XVII.
Twenty-one bullous pemphigoid sera had IgG autoantibodies reactive with the NC16a domain, 11 with the Col15 domain, and 17 with the Ecto2 fragment. Of these, six sera recognized epitopes within all three fragments, 14 in two, and 21 in only one fragment. Of the LAD sera, two recognized NC16a, two Col15, and one Ecto2. One of the sera reacted with two fragments, namely with NC16a and with Col15. Of the seven CBDC sera, only one reacted with the recombinant fragments, specifically with Col15.
Neither full-length collagen XVII nor the recombinant fragments were as sensitive as the authentic shed ectodomain for detection of the autoantibodies, and, therefore, including this form of collagen XVII in the test panels should be considered. Neoepitopes are known to be generated by proteolytic processing of a wide spectrum of biologically active and disease-relevant proteins, such as plasminogen activator inhibitor type 2, IL-1 receptor, or the extracellular matrix proteins collagen II and aggrecan. Matrix metalloproteinase-1, -8, and -13 cleave collagen II in osteoarthritic cartilage, thereby generating neoepitopes not observed in intact collagen. Cleavage by aggrecanase of the peptide bond Asn341-Phe342 in aggrecan generates a neoepitope VDIPEN341, and monoclonal antibodies to this neoepitope can be used to assess the extent of cartilage destruction during inflammatory arthritis.