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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Research Article Cell biology Genetics

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 6

Direct interaction between normal C1INH and C1INHGly162_Pro206del.

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Direct interaction between normal C1INH and C1INHGly162_Pro206del. (A) C...
(A) Colocalization of normal and mutated C1INH. Widefield microscopy of HeLa cells cultured for 48 hours after cotransfection with SERPING1[WT]-mCherry and SERPING1[WT]-HA or SERPING1[c.551_685del]-HA. C1INH-HA was visualized with HA-specific antibody (green) and nuclei with Hoechst (blue). Scale bars: 10 μm. (B and C) Western blot analysis of total protein in medium as well as soluble and insoluble protein fractions derived from HeLa cells cultured for 72 hours after transfection or cotransfection with 900 ng plasmid DNA in total. Separation of soluble and insoluble C1INH fractions by centrifugation of whole cell lysate at 12,000g for 20 minutes at 4°C. (B) Detection of C1INHGly162_Pro206del in the insoluble fraction. HeLa cells were transfected with 900 ng pSERPING1[WT] or pSERPING1[c.551_685del] or cotransfected with 450 ng pSERPING1[WT] and 450 ng pSERPING1[c.551_685de]. C1INH levels were detected using PAb C1INH antibody. (C) Normal C1INH in the insoluble fraction induced by C1INHGly162_Pro206del. HeLa cells were cotransfected with 450 ng pSERPING1[WT]-HA and either 450 ng pcDNA or pSERPING1[c.551_685del]. Normal C1INH-HA levels were detected with HA-specific antibody. (D) Direct protein-protein interaction between normal and mutated C1INH. Coimmunoprecipitation on whole cell lysate from HeLa cells cultured for 48 hours after transfection or cotransfection with 40 μg plasmid DNA in total. HeLa cells were transfected with pSERPING1[WT]-V5, pSERPING1[WT]-HA, or pSERPING1[c.551_685del]-HA, or cotransfected with pSERPING1[WT]-V5 and pSERPING1[WT]-HA or pSERPING1[c.551_685del]-HA. A small amount of the whole cell lysate was saved (input) and the remaining lysate incubated with anti-V5 coupled beads to capture normal V5-tagged C1INH and interacting proteins (co-IP). Presence of normal C1INH-V5 and C1INH[Variant]-HA was visualized with V5- and HA-specific antibodies as relevant. (A) Data are representative of findings from more than 3 biological replicates. (A–C) Similar results were seen in at least 2 independent experiments. (B–D) Transfections were carried out in triplicate (n = 3).