Dominant-negative SERPING1 variants cause intracellular retention of C1 inhibitor in hereditary angioedema
Didde Haslund, … , Lene N. Nejsum, Jacob Giehm Mikkelsen
Didde Haslund, … , Lene N. Nejsum, Jacob Giehm Mikkelsen
Published November 6, 2018
Citation Information: J Clin Invest. 2019;129(1):388-405. https://doi.org/10.1172/JCI98869.
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Dominant-negative SERPING1 variants cause intracellular retention of C1 inhibitor in hereditary angioedema

Abstract

Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent edema attacks associated with morbidity and mortality. HAE results from variations in the SERPING1 gene that encodes the C1 inhibitor (C1INH), a serine protease inhibitor (serpin). Reduced plasma levels of C1INH lead to enhanced activation of the contact system, triggering high levels of bradykinin and increased vascular permeability, but the cellular mechanisms leading to low C1INH levels (20%–30% of normal) in heterozygous HAE type I patients remain obscure. Here, we showed that C1INH encoded by a subset of HAE-causing SERPING1 alleles affected secretion of normal C1INH protein in a dominant-negative fashion by triggering formation of protein-protein interactions between normal and mutant C1INH, leading to the creation of larger intracellular C1INH aggregates that were trapped in the endoplasmic reticulum (ER). Notably, intracellular aggregation of C1INH and ER abnormality were observed in fibroblasts from a heterozygous carrier of a dominant-negative SERPING1 gene variant, but the condition was ameliorated by viral delivery of the SERPING1 gene. Collectively, our data link abnormal accumulation of serpins, a hallmark of serpinopathies, with dominant-negative disease mechanisms affecting C1INH plasma levels in HAE type I patients, and may pave the way for new treatments of HAE.

Authors

Didde Haslund, Laura Barrett Ryø, Sara Seidelin Majidi, Iben Rose, Kristian Alsbjerg Skipper, Tue Fryland, Anja Bille Bohn, Claus Koch, Martin K. Thomsen, Yaseelan Palarasah, Thomas J. Corydon, Anette Bygum, Lene N. Nejsum, Jacob Giehm Mikkelsen

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Figure 3

Negative impact of C1INH encoded by HAE-causing SERPING1 gene variants on secretion of normal C1INH-mCherry correlates with increased intracellular protein levels.

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Negative impact of C1INH encoded by HAE-causing SERPING1 gene variants o...
(A) Schematic representation of the cellular assay system. Cells were cotransfected with pSERPING1[Variant] and pSERPING1[WT]-mCherry. Expression of mCherry-fused normal C1INH allowed evaluation of the effect of different SERPING1 gene variants on (i) secretion, (ii) intracellular retention, and (iii) intracellular localization of normal C1INH-mCherry. (B and C) Levels of secreted and intracellular C1INH-mCherry in HepG2 cells cotransfected with 450 ng of both pSERPING1[Variant] and pSERPING1[WT]-mCherry. (D and E) Levels of secreted and intracellular C1INH-mCherry in HeLa cells transfected as in B and C. Secretion of normal C1INH-mCherry was measured by fluorescence scanning (B and D) and the intracellular level by flow cytometry (C and E). (F) Increasingly restricted C1INH secretion with higher levels of mutant C1INH. Dose-response experiment was carried out in HeLa cells. Cells were cotransfected with 450 ng pSERPING1[WT]-mCherry and 50, 200, 450, 700, or 900 ng pSERPING1[WT] or pSERPING1[c.551_685del]. pcDNA plasmid was included in all transfections to ensure a total amount of 1,350 ng plasmid DNA in each transfection reaction. (G) Intracellular levels of C1INH-mCherry in cells described in F. (B–G) Transfections were carried out in triplicate (n = 3) and similar results were seen in at least 3 independent experiments. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, compared with pcDNA. Statistical analyses were performed by 1-way ANOVA with Dunnett’s multiple comparison test. MFI, median fluorescence intensity.