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 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.

Complement C1s and C4d as Prognostic Biomarkers in Renal Cancer: Emergence of Noncanonical Functions of C1s

“…The subcellular localization of C1s in the cytoplasm hints at potential mechanisms by which it can affect fundamental cellular functions. C1s interacts and cleaves HMGB-1 [52], [53], which shuttles between the cytoplasm and the nucleus [54]. In the nucleus, HMGB1 interacts with nucleosomes, transcription factors, and histones, regulating transcription.…”

“…We are grateful to Veronique Rossi and Isabelle Bally (IBS, Grenoble) for providing the pcDNA3.1 C1sexpressing vector, described in [52].…”

Complement System and Alarmin HMGB1 Crosstalk: For Better or Worse

“…This observation therefore suggests that alterations in C1r or C1s functions might trigger sterile inflammation in this specific pEDS context. Two studies aimed to explore the molecular consequences of these specific mutations in C1r and C1s ( 118 , 119 ). Defective secretion of these proteases is always observed in the presence of all pEDS mutations, with the only exception of a particular mutation in C1r which affects a C1q-binding residue ( 118 ).…”

Functional recombinant human complement C1q with different affinity tags

“…The latter was generated by site-directed mutagenesis using the pcDNA3.1/Neo(+) plasmid encoding human C1s with a C-terminal FLAG tag (Bally et al, 2019a) as a template and the mutagenic primers described in Table S1. Generation of stably co-transfected 293-F cells and purification of the recombinant proenzyme tetramer from the culture supernatant by anti-FLAG affinity chromatography were performed as described in (Bally et al, 2013).…”