Plasmin Plays a Role in the In Vitro Generation of the Linear IgA Dermatosis Antigen LADB97

Abstract

Collagen XVII (BP180) and its shed ectodomain represent major autoantigens in dermatoses of the pemphigoid group. The 120 kDa ectodomain is constitutively shed from the cell surface by disintegrin-metalloproteinases (ADAMs). Part of it is further processed to a 97 kDa fragment (LABD97), an autoantigen in linear IgA dermatosis (LAD), but the responsible proteinases remain elusive. In this study, we identified the 120 and the 97 kDa ectodomain in blister fluids of bullous pemphigoid patients using new mAbs. As blister fluids contain significant plasmin-like serine protease activity, HaCaT keratinocytes or purified 120 kDa ectodomain were incubated with several human serine proteases. In vitro, only plasmin generated a stable 97 kDa fragment that was also targeted by LAD sera. Characterization of the plasmin-derived 97 kDa fragment with domain-specific collagen XVII antibodies, heparin binding and N-glycosylation studies indicates that the N-terminus is located approximately at AA 515 and the C-terminus N-terminally from AA 1,421. Interestingly, plasmin-derived LABD97 was also generated in the presence of ADAM inhibitors and remained stable over more than 12 hours incubation at 37 degrees C, indicating that this disease relevant collagen XVII fragment can also arise in an ADAM-independent manner through direct action by plasmin.

Context-Dependent Regulation of Collagen XVII Ectodomain Shedding in Skin

“…In addition, it is known that autoAbs from LAD 16 and certain BP patients 17 preferentially react with the shed ectodomain but not with the full-length form of COL17. This unique characteristic was also observed 3 in several monoclonal Abs (mAbs) directing the ectodomain of COL17 11,[18][19][20] , suggesting that neoepitopes appeared on the shed ectodomain of COL17.…”

“…However, epitopes can be significantly changed in regions far from the cleavage sites. For example, the mAb NC16A-3, whose epitope is at least 19 aa C-terminal from the physiological cleavage site within the NC16A domain of COL17, preferentially reacts with the shed ectodomain but not with the full-length form 20 . In addition, the 15 th collagenous domain is known to be a candidate epitope for autoAbs from LAD patients 35 , 20 which suggests that the conformation of the COL17 ectodomain may change drastically after cleavage within the NC16A domain.…”

Dynamic Interactions of Epidermal Collagen XVII with the Extracellular Matrix

“…Abs, including mouse monoclonal Abs NC16A-1 and NC16A-3, directed to distinct epitopes in the NC16A domain of collagen XVII, 24 were used in a 1:10 dilution for IIF (NC16A-1) and in a 1:2000 dilution for immunoblotting (NC16A-3). The cleavage-site-specific rabbit polyclonal Ab HK139 was prepared as previously described 17 and was used at a concentration of 10 g/mL for IIF and 1 g/mL for immunoblotting.…”

Chronic Bullous Disease or Linear IgA Dermatosis of Childhood -Revisited

“…Plasmin is formed by the activation of plasminogen and is able to cleave type XVII collagen. Thus, fragments may be recognized as immunogenic epitopes (including a 97-kDa fragment) by the sera of patients with CBDC [19,20].…”

“…This enzyme is essential for the inactivation of α1-proteinase inhibitor leading to chemoattraction of neutrophils and allowing neutrophil elastase to induce epithelial -dermal detachment [20,21].…”