A small deletion in SERPINC1 causes type I antithrombin deficiency by promoting endoplasmic reticulum stress

Abstract

Antithrombin (AT) deficiency is an autosomal dominant disorder, and identification of mutation AT variants would improve our understanding of the anticoagulant function of this serine protease inhibitor (SERPIN) and the molecular pathways underlying this disorder. In the present study, we performed whole-exome sequencing of a Chinese family with deep vein thrombosis, and identified a new small deletion that eliminates four amino acids (INEL) from exon 4 of SERPINC1 gene. This causes type I AT deficiency by enhancing the intracellular retention of this protein. AT retention leads to endoplasmic reticulum (ER) stress, which further inhibits AT release. In addition, ER stress activates ER-associated degradation, which promotes AT degradation. Suppression of ER stress enhanced the secretion of AT, while inhibition of ER-associated degradation suppressed AT release. Thus, our study identified a new mutation (INEL deletion) causing type I AT deficiency, and uncovered a novel mechanism for AT retention through enhanced ER stress, which may provide an innovative approach for treating AT deficiency.

Keywords: SERPINC1; antithrombin deficiency; deletion mutation; endoplasmic reticulum stress.

Figure 1. Identification of AT deficiency due to a small deletion

A. Pedigree of proband. The proband is indicated by the arrow. Family members affected with thrombosis are indicated in black, and III-2, IV-2/12/15, and V-6 are the small deletion carriers. B. Representation of the residues deleted (ΔINEL) in the mutant AT aligned with the wild-type (WT) sequence. C. The sequencing graph for mutant AT.

Figure 2. The accumulation of mutant AT activates ER stress

HEK293 or LO2 cells were transiently transfected with ATWT, ATΔINEL or ATK241E plasmids for 48 hours. A. HEK293 cells were transiently transfected with the indicated plasmids. Total RNA was extracted and reverse-transcribed, and PCR performed with AT-specific primer pairs. B. LO2 cells were transiently transfected with the indicated plasmids, and culture medium was collected for ELISA detection of AT levels. C. LO2 cells were transiently transfected with the indicated plasmids, and cell lysates were analyzed by immunoblotting with AT antibodies. The band density was quantified with ImageJ software. The quantitative data were obtained from three independent experiments, and are shown as the mean ± SEM. D. LO2 cells were transiently transfected with the indicated plasmids. Total RNA was extracted and reverse-transcribed, and PCR was performed with the indicated specific primer pairs. E. Cell lysates were analyzed by immunoblotting with antibodies for the expression of the indicated molecules. The image is representative of three experiments. F. LO2 cells were plated in 96-well plates at a density of 5×10² cells per well and transfected with a plasmid containing WT or mutant AT. The cell viability was assessed at various time-periods (0, 1, 2, 3, 4 d) after transfection. The absorbance of each well was measured at 570 nm with an ELISA reader. The cell viability was calculated as the percentage of the absorbance with respect to the control (WT). All the results are the mean ± SEM of three independent experiments (A-F). (*p<0.05)

Figure 3. Enhanced ER stress contributes to AT deficiency by promoting AT degradation

A, B. LO2 cells were transiently transfected with AT^WT, AT^ΔINEL and AT^K241E plasmids for 48 hours, and then treated with tunicamycin (TM) (2 μM), thapsigargin (TG) (1 μM) or 4-phenyl butyric acid (PBA) (500 μM) for 4 hours. (A) Cell lysates from LO2 cells were analyzed by immunoblotting with specific antibodies for the expression of the indicated molecules. The band density was quantified with ImageJ software. The quantitative data were obtained from three independent experiments, and are shown as the mean ± SEM. (B) Cell culture medium was collected and subjected to ELISA. Data are the mean ± SEM of three independent experiments. C, D. LO2 cells were transiently transfected with AT^WT, AT^ΔINEL and AT^K241E plasmids for 48 hours and then treated with Eeyarestatin I (Eerl) (2 μM), MG-132 (1.2 μM), or E64d (10 μM) for 12 hours. (C) Cell lysates from LO2 cells were analyzed by immunoblotting with specific antibodies for the expression of the indicated molecules. The band density was quantified with ImageJ software. The quantitative data were obtained from three independent experiments, and are shown as the mean ± SEM. (D) Cell culture medium was collected and subjected to ELISA. A representative image of three experiments is shown, and the quantitative data are presented as the mean ± SEM of three experiments. Ctl, control. (*p < 0.05)