Ectodomain Shedding Generates Neoepitopes on Collagen XVII, the Major Autoantigen for Bullous Pemphigoid
Ectodomain Shedding Generates Neoepitopes on Collagen XVII, the Major Autoantigen for Bullous Pemphigoid
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DOI：
10.4049/jimmunol.1001524
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发表时间：
2010-10-15
影响因子：
4.4
通讯作者：
Bruckner-Tuderman, Leena
中科院分区：
文献类型：
--
作者：
Nishie, Wataru;Lamer, Stephanie;Bruckner-Tuderman, Leena
作为基底角质形成细胞中的II型跨膜蛋白，胶原XVII在皮肤中的表皮和真皮之间提供稳定的粘附。它的胞外域可以从细胞表面脱落，并且某些起泡疾病中的自身抗体优先识别脱落形式。胶原蛋白XVII的主要表位聚集在膜外非胶原蛋白第16 A结构域内，并且在该区域内发生胞外域脱落，表明切割产生新表位。然而，候选切割位点一直存在争议，新表位产生的机制尚不清楚。在这项研究中，我们研究了非胶原第16 A结构域的切割位点，以了解新表位的产生及其病理作用。识别Leu（524）-Gly（532）延伸段的多克隆Ab优先与脱落的胞外域反应，但不与全长形式反应，表明新表位位于该位点。新表位特异性抗体固定补体并诱导正常人皮肤冷冻切片中粒细胞依赖性真皮-表皮分离。使用质谱法鉴定生理切割位点。N末端位于Asp（514）、Leu（524）、Glu（525）和Gly（526），其中Asp（514）和Glu（525）被乙酰化和焦氨基化封闭。B细胞表位的计算机模拟预测表明，Leu（524）-Gly（532）区域的抗原性在脱落后显著增加，与切割位点无关。相应地，在大疱性类天疱疮患者的皮肤和水疱液中发现了新表位，并且大疱性类天疱疮血清与肽Leu（524）-Gly（532）反应。总之，这些数据表明胶原XVII的生理性脱落产生新表位，其可作为水疱诱导自身抗体的靶标。免疫学杂志，2010，185：4938-4947。
As a type II transmembrane protein in basal keratinocytes, collagen XVII provides stable adhesion between epidermis and dermis in the skin. Its ectodomain can be shed from the cell surface, and autoantibodies in certain blistering diseases preferentially recognize the shed form. Major epitopes of collagen XVII are clustered within the juxtamembranous noncollagenous 16th A domain, and ectodomain shedding occurs within this region, suggesting that cleavage generates neoepitopes. However, the candidate cleavage sites have been controversial, and the mechanism of neoepitope generation is unclear. In this study, we investigated cleavage sites in the noncollagenous 16th A domain to understand the generation of neoepitopes and their pathological role. Polyclonal Abs recognizing the stretch Leu(524)-Gly(532) preferentially reacted with the shed ectodomain, but not with the full-length form, indicating that a neoepitope was localized at this site. The neoepitope-specific Ab fixed complement and induced granulocyte-dependent dermal-epidermal separation in cryosections of normal human skin. The physiological cleavage sites were identified using mass spectrometry. N termini were found at Asp(514), Leu(524), Glu(525), and Gly(526), among which Asp(514) and Glu(525) were blocked by acetylation and pyroglutaminate. In silico prediction of B cell epitopes indicated that the antigenicity of the Leu(524)-Gly(532) region increased substantially after shedding, regardless of the cleavage sites. Correspondingly, neoepitopes were found in the skin and blister fluids of patients with bullous pemphigoid, and bullous pemphigoid sera reacted with the peptide Leu(524)-Gly(532). Taken together, these data demonstrate that physiological shedding of collagen XVII generates neoepitopes, which may serve as a target of blister-inducing autoantibodies. The Journal of Immunology, 2010, 185: 4938-4947.