Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular Outcomes
Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular Outcomes
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DOI：
10.3389/fimmu.2019.02962
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发表时间：
2019-12-18
影响因子：
7.3
通讯作者：
Gaboriaud, Christine
中科院分区：
文献类型：
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作者：
Bally, Isabelle;Dalonneau, Fabien;Gaboriaud, Christine
ehers - danlos综合征（EDS）是一种临床和遗传异质性疾病，其特征是关节过度活动和皮肤过度伸展等软结缔组织改变。我们之前在患有牙周EDS的患者中发现了编码补体C1蛋白酶的C1R和C1S基因的杂合错义突变，这是一种特殊的EDS亚型，其特征是早期严重牙周炎导致牙齿过早脱落和结缔组织改变。C1r和C1s蛋白酶的名义作用是激活经典补体途径，但目前还没有明确的分子联系表明C1r和C1s蛋白酶在牙周EDS综合征这些异质性症状中的作用。因此，我们的目标是阐明这些突变的功能效应，在分子和酶的水平。为了探索分子效应，进行了一系列细胞转染实验、重组蛋白纯化、质谱分析和n端分析。关注两种不同的C1S变体，即p.Val316del和p.Cys294Arg，我们发现HEK293-F细胞稳定地转染了相应的C1S变体质粒，出乎意料的是，不分泌全长突变的C1S，而只分泌40 kDa的截断的Fg40片段，产生非常低的水平。对两个C1s变体纯化的Fg40片段的详细分析表明它们是相同的，这也是出乎意料的。这表明含有患者突变的CCP1模块的局部错误折叠暴露了一个新的切割位点，位于Lys353和Cys354之间，这是通常无法进入的。突变诱导的Fg40片段包含完整的c端丝氨酸蛋白酶结构域，但不包含介导C1s与其他C1亚基（C1r和C1q）相互作用的n端结构域。因此，Fg40酶活性逃避C1内C1s活性的正常生理控制，可能导致失控。比较酶分析表明，Fg40保留了C1s的天然酯分解活性，以及其对辅助警告蛋白HMGB1底物的裂解效率，而名义补体C4激活的裂解受到损害。这些新结果为可能涉及辅助C1s靶点的进一步分子探索开辟了道路。
Ehlers-Danlos syndromes (EDS) are clinically and genetically heterogeneous disorders characterized by soft connective tissue alteration like joint hypermobility and skin hyper-extensibility. We previously identified heterozygous missense mutations in the C1R and C1S genes, coding for the complement C1 proteases, in patients affected by periodontal EDS, a specific EDS subtype hallmarked by early severe periodontitis leading to premature loss of teeth and connective tissue alterations. Up to now, there is no clear molecular link relating the nominal role of the C1r and C1s proteases, which is to activate the classical complement pathway, to these heterogeneous symptoms of periodontal EDS syndrome. We aim therefore to elucidate the functional effect of these mutations, at the molecular and enzymatic levels. To explore the molecular consequences, a set of cell transfection experiments, recombinant protein purification, mass spectroscopy and N-terminal analyses have been performed. Focusing on the results obtained on two different C1S variants, namely p.Val316del and p.Cys294Arg, we show that HEK293-F cells stably transfected with the corresponding C1s variant plasmids, unexpectedly, do not secrete the full-length mutated C1s, but only a truncated Fg40 fragment of 40 kDa, produced at very low levels. Detailed analyses of the Fg40 fragments purified for the two C1s variants show that they are identical, which was also unexpected. This suggests that local misfolding of the CCP1 module containing the patient mutation exposes a novel cleavage site, between Lys353 and Cys354, which is not normally accessible. The mutation-induced Fg40 fragment contains the intact C-terminal serine protease domain but not the N-terminal domain mediating C1s interaction with the other C1 subunits, C1r, and C1q. Thus, Fg40 enzymatic activity escapes the normal physiological control of C1s activity within C1, potentially providing a loss-of-control. Comparative enzymatic analyses show that Fg40 retains the native esterolytic activity of C1s, as well as its cleavage efficiency toward the ancillary alarmin HMGB1 substrate, for example, whereas the nominal complement C4 activation cleavage is impaired. These new results open the way to further molecular explorations possibly involving subsidiary C1s targets.