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Two different missense C1S mutations, associated to periodontal Ehlers-Danlos syndrome, lead to identical molecular outcomes

Abstract : Ehlers-Danlos syndromes (EDS) are clinically and genetically heterogeneous disorderscharacterized by soft connective tissue alteration like joint hypermobility and skinhyper-extensibility. We previously identified heterozygous missense mutations in theC1RandC1Sgenes, coding for the complement C1 proteases, in patients affected byperiodontal EDS, a specific EDS subtype hallmarked by early severe periodontitis leadingto premature loss of teeth and connective tissue alterations. Up to now, there is no clearmolecular link relating the nominal role of the C1r and C1s proteases, which is to activatethe classical complement pathway, to these heterogeneous symptoms of periodontalEDS syndrome. We aim therefore to elucidate the functional effect of these mutations,at the molecular and enzymatic levels. To explore the molecular consequences, a setof cell transfection experiments, recombinant protein purification, mass spectroscopyand N-terminal analyses have been performed. Focusing on the results obtained on twodifferentC1Svariants, namely p.Val316del and p.Cys294Arg, we show thatHEK293-Fcells stably transfected with the corresponding C1s variant plasmids, unexpectedly, donot 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 twoC1s variants show that they are identical, which was also unexpected. This suggeststhat local misfolding of the CCP1 module containing the patient mutation exposes anovel cleavage site, between Lys353 and Cys354, which is notnormally accessible. Themutation-induced Fg40 fragment contains the intact C-terminal serine protease domainbut 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 ofC1s activity within C1, potentially providing a loss-of-control. Comparative enzymaticanalyses show that Fg40 retains the native esterolytic activity of C1s, as well as itscleavage efficiency toward the ancillary alarmin HMGB1 substrate, for example, whereasthe nominal complement C4 activation cleavage is impaired.These new results open theway to further molecular explorations possibly involving subsidiary C1s targets
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Isabelle Bally, Fabien Dalonneau, Anne Chouquet, Rebekka Gröbner, Albert Amberger, et al.. Two different missense C1S mutations, associated to periodontal Ehlers-Danlos syndrome, lead to identical molecular outcomes. Frontiers in Immunology, Frontiers, 2019, 10, ⟨10.3389/fimmu.2019.02962⟩. ⟨hal-02417468⟩

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