Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular Outcomes
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...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2019-12-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fimmu.2019.02962/full |
| _version_ | 1819057272153702400 |
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| author | Isabelle Bally Fabien Dalonneau Anne Chouquet Rebekka Gröbner Albert Amberger Ines Kapferer-Seebacher Heribert Stoiber Johannes Zschocke Nicole M. Thielens Véronique Rossi Christine Gaboriaud |
| author_facet | Isabelle Bally Fabien Dalonneau Anne Chouquet Rebekka Gröbner Albert Amberger Ines Kapferer-Seebacher Heribert Stoiber Johannes Zschocke Nicole M. Thielens Véronique Rossi Christine Gaboriaud |
| author_sort | Isabelle Bally |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-21T13:36:40Z |
| format | Article |
| id | doaj.art-a3474fe3975b4cf8a32afe8ea80ca13c |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-12-21T13:36:40Z |
| publishDate | 2019-12-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-a3474fe3975b4cf8a32afe8ea80ca13c2022-12-21T19:02:09ZengFrontiers Media S.A.Frontiers in Immunology1664-32242019-12-011010.3389/fimmu.2019.02962495372Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular OutcomesIsabelle Bally0Fabien Dalonneau1Anne Chouquet2Rebekka Gröbner3Albert Amberger4Ines Kapferer-Seebacher5Heribert Stoiber6Johannes Zschocke7Nicole M. Thielens8Véronique Rossi9Christine Gaboriaud10University Grenoble Alpes, CEA, CNRS, IBS, Grenoble, FranceUniversity Grenoble Alpes, CEA, CNRS, IBS, Grenoble, FranceUniversity Grenoble Alpes, CEA, CNRS, IBS, Grenoble, FranceDivision of Human Genetics, Institute of Human Genetics, Medical University Innsbruck, Innsbruck, AustriaDivision of Human Genetics, Institute of Human Genetics, Medical University Innsbruck, Innsbruck, AustriaDepartment for Operative and Restorative Dentistry, Medical University Innsbruck, Innsbruck, AustriaInstitute of Virology, Medical University Innsbruck, Innsbruck, AustriaDivision of Human Genetics, Institute of Human Genetics, Medical University Innsbruck, Innsbruck, AustriaUniversity Grenoble Alpes, CEA, CNRS, IBS, Grenoble, FranceUniversity Grenoble Alpes, CEA, CNRS, IBS, Grenoble, FranceUniversity Grenoble Alpes, CEA, CNRS, IBS, Grenoble, FranceEhlers-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.https://www.frontiersin.org/article/10.3389/fimmu.2019.02962/fullC1s proteasecomplement systemperiodontalEhlers-DanlosHMGB1 |
| spellingShingle | Isabelle Bally Fabien Dalonneau Anne Chouquet Rebekka Gröbner Albert Amberger Ines Kapferer-Seebacher Heribert Stoiber Johannes Zschocke Nicole M. Thielens Véronique Rossi Christine Gaboriaud Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular Outcomes Frontiers in Immunology C1s protease complement system periodontal Ehlers-Danlos HMGB1 |
| title | Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular Outcomes |
| title_full | Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular Outcomes |
| title_fullStr | Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular Outcomes |
| title_full_unstemmed | Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular Outcomes |
| title_short | Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular Outcomes |
| title_sort | two different missense c1s mutations associated to periodontal ehlers danlos syndrome lead to identical molecular outcomes |
| topic | C1s protease complement system periodontal Ehlers-Danlos HMGB1 |
| url | https://www.frontiersin.org/article/10.3389/fimmu.2019.02962/full |
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