Abstract

Heterozygous missense or in-frame insertion/deletion mutations in complement 1 subunits C1r and C1s cause periodontal Ehlers-Danlos Syndrome (pEDS), a specific EDS subtype characterized by early severe periodontal destruction and connective tissue abnormalities like easy bruising, pretibial haemosiderotic plaques, and joint hypermobility. We report extensive functional studies of 16 C1R variants associated with pEDS by in-vitro overexpression studies in HEK293T cells followed by western blot, size exclusion chromatography and surface plasmon resonance analyses. Patient-derived skin fibroblasts were analyzed by western blot and Enzyme-linked Immunosorbent Assay (ELISA). Overexpression of C1R variants in HEK293T cells revealed that none of the pEDS variants was integrated into the C1 complex but cause extracellular presence of catalytic C1r/C1s activities. Variants showed domain-specific abnormalities of intracellular processing and secretion with preservation of serine protease function in the supernatant. In contrast to C1r wild type, and with the exception of a C1R missense variant disabling a C1q binding site, pEDS variants had different impact on the cell: retention of C1r fragments inside the cell, secretion of aggregates, or a new C1r cleavage site. Overexpression of C1R variants in HEK293T as well as western blot analyses of patient fibroblasts showed decreased levels of secreted C1r. Importantly, all available patient fibroblasts exhibited activated C1s and activation of externally added C4 in the supernatant while control cell lines secreted proenzyme C1s and showed no increase in C4 activation. The central elements in the pathogenesis of pEDS seem to be the intracellular activation of C1r and/or C1s, and extracellular presence of activated C1s that independently of microbial triggers can activate the classical complement cascade.

Highlights

  • Periodontitis is a common chronic disease caused by a dysbalance between the oral microbiome and the host immune response leading to inflammatory destruction of the tooth-supporting tissues and loss of teeth

  • Because of auto-activation in the absence of C1 inhibitor (C1Inh; Gene: SERPING1), little full-length C1r (100 kDa) but presence of abnormalities or interactions of the N-terminal domains (A-chain) was detected in all cell lysates

  • Variants resulted in intracellular presence of A-chain, sometimes with an additional smaller C1r fragment that may represent a degradation product or reduced glycosylation

Read more

Summary

Introduction

Periodontitis is a common chronic disease caused by a dysbalance between the oral microbiome and the host immune response leading to inflammatory destruction of the tooth-supporting tissues and loss of teeth. Complement Activation in pEDS (EDS) [1, 2] characterized by early severe and rapidly progressing periodontal destruction and variable connective tissue abnormalities [3]. We recently showed that pEDS is caused by heterozygous missense or in-frame insertion/deletion mutations in C1R or C1S [4]. These genes code for complement 1 subunits C1r and C1s, serine proteases that play a key role in the innate immune response. The penetrance in the individuals with pEDS identified up to now is 100%, and there is no clinical evidence for relevant modifier genes

Methods
Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call