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A corin variant identified in hypertensive patients that alters cytoplasmic tail and reduces cell surface expression and activity.

Zhang Y, Li H, Zhou J, Wang A, Yang J, Wang C, Liu M, Zhou T, Zhu L, Zhang Y, Dong N, Wu Q - Sci Rep (2014)

Bottom Line: Corin is a membrane-bound protease that regulates blood pressure by activating the natriuretic peptides.Analysis of two independent cohorts showed that the variant was preferentially present in hypertensive patients (38/795 or 4.78% vs. 4/632 or 0.63% in normal individuals, p = 4.14E-6).In cell-based studies, the corin variant exhibited poor trafficking in the Golgi, reduced cell surface expression and zymogen activation, and low natriuretic peptide processing activity.

View Article: PubMed Central - PubMed

Affiliation: Cyrus Tang Hematology Center, MOE Engineering Center of Hematological Disease, MOH Key Lab of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital, and Collaborative Innovation Center of Hematology, Soochow University, Suzhou, China.

ABSTRACT
Corin is a membrane-bound protease that regulates blood pressure by activating the natriuretic peptides. CORIN variants have been associated with hypertension and heart disease in African Americans. In this study, we conducted targeted exome sequencing and identified an insertion variant, c.102_103insA, in exon 1 of the CORIN gene. Analysis of two independent cohorts showed that the variant was preferentially present in hypertensive patients (38/795 or 4.78% vs. 4/632 or 0.63% in normal individuals, p = 4.14E-6). The insertion shifted the reading frame, resulting in a corin variant with a truncated cytoplasmic tail. In cell-based studies, the corin variant exhibited poor trafficking in the Golgi, reduced cell surface expression and zymogen activation, and low natriuretic peptide processing activity. Compared with normal individuals with the wild-type allele, individuals with the variant allele had lower levels of plasma corin [0.59 ± 0.07 ng/mL (n = 25) vs. 0.91 ± 0.02 ng/mL (n = 215), p<0.001] and higher levels of plasma N-terminal pro-atrial natriuretic peptide (NT-pro-ANP) [2.39 ± 3.6 nmol/L (n = 21) vs. 0.87 ± 0.6 nmol/L (n = 48), p = 0.005]. These results indicate that the variant altered corin structure and impaired the natriuretic peptide processing activity in vivo. The results highlight corin defects as an important underlying mechanism in hypertension.

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Zymogen activation of the insA variant in HEK293 cells.(A) Western analysis of corin proteins in cell lysate. The ~40-kDa band represented the corin protease domain (corin-p) from activation cleavage. This band was absent in mutant R801A, in which the cleavage site was abolished. Data are representative of five independent experiments. (B) Percentage of the activated protease domain fragment (corin-p) vs. corin zymogen protein (corin) was estimated by densitometry. Data are mean ± S.D. from five independent experiments.
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f3: Zymogen activation of the insA variant in HEK293 cells.(A) Western analysis of corin proteins in cell lysate. The ~40-kDa band represented the corin protease domain (corin-p) from activation cleavage. This band was absent in mutant R801A, in which the cleavage site was abolished. Data are representative of five independent experiments. (B) Percentage of the activated protease domain fragment (corin-p) vs. corin zymogen protein (corin) was estimated by densitometry. Data are mean ± S.D. from five independent experiments.

Mentions: One possible explanation for the insA variant expression is that a down-stream methionine was used as the translation initiation site. Indeed, there is a methionine at position 30 that may be used as an alternative initiation site (Fig. 1B). To test this hypothesis, we expressed the variant in HEK293 cells together with a corin isoform that contains methionine 30 (Met-30) but lacks methionine 1 (Met-1)15. By Western analysis, similar expression levels of the insA variant, the Met-30 isoform (Met-30), WT, and mutants R801A and S985A were observed (Fig. 3A). On the Western blots, an ~40-kDa band (corin-p), representing the corin protease domain cleaved at the activation site Arg-801 (Fig. 1B), was detected in WT (Fig. 3A, lane 2). This band also was detected in the catalytic site mutant S985A (Fig. 3A, lane 5), but not in mutant R801A, in which the cleavage site is abolished (Fig. 3A, lane 4). The intensity of this band in the insA variant was markedly reduced (Fig. 3A, lane 3). Similar results were observed in the Met-30 isoform (Fig. 3A, lane 6). By densitometric analysis, the ~40-kDa band in WT represented 12.73 ± 1.97% of the total corin protein in lysate, whereas the band in the insA variant was 4.04 ± 1.02%, significantly lower than that in WT (n = 5, p<0.01) (Fig. 3B). In the Met-30 isoform, this band was 3.99 ± 2.67% of the total corin protein, which was similar to that in the insA variant (Fig. 3B). The data support that the insertion resulted in a variant corin with a shorter cytoplasmic tail and reduced zymogen activation and activity.


A corin variant identified in hypertensive patients that alters cytoplasmic tail and reduces cell surface expression and activity.

Zhang Y, Li H, Zhou J, Wang A, Yang J, Wang C, Liu M, Zhou T, Zhu L, Zhang Y, Dong N, Wu Q - Sci Rep (2014)

Zymogen activation of the insA variant in HEK293 cells.(A) Western analysis of corin proteins in cell lysate. The ~40-kDa band represented the corin protease domain (corin-p) from activation cleavage. This band was absent in mutant R801A, in which the cleavage site was abolished. Data are representative of five independent experiments. (B) Percentage of the activated protease domain fragment (corin-p) vs. corin zymogen protein (corin) was estimated by densitometry. Data are mean ± S.D. from five independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260221&req=5

f3: Zymogen activation of the insA variant in HEK293 cells.(A) Western analysis of corin proteins in cell lysate. The ~40-kDa band represented the corin protease domain (corin-p) from activation cleavage. This band was absent in mutant R801A, in which the cleavage site was abolished. Data are representative of five independent experiments. (B) Percentage of the activated protease domain fragment (corin-p) vs. corin zymogen protein (corin) was estimated by densitometry. Data are mean ± S.D. from five independent experiments.
Mentions: One possible explanation for the insA variant expression is that a down-stream methionine was used as the translation initiation site. Indeed, there is a methionine at position 30 that may be used as an alternative initiation site (Fig. 1B). To test this hypothesis, we expressed the variant in HEK293 cells together with a corin isoform that contains methionine 30 (Met-30) but lacks methionine 1 (Met-1)15. By Western analysis, similar expression levels of the insA variant, the Met-30 isoform (Met-30), WT, and mutants R801A and S985A were observed (Fig. 3A). On the Western blots, an ~40-kDa band (corin-p), representing the corin protease domain cleaved at the activation site Arg-801 (Fig. 1B), was detected in WT (Fig. 3A, lane 2). This band also was detected in the catalytic site mutant S985A (Fig. 3A, lane 5), but not in mutant R801A, in which the cleavage site is abolished (Fig. 3A, lane 4). The intensity of this band in the insA variant was markedly reduced (Fig. 3A, lane 3). Similar results were observed in the Met-30 isoform (Fig. 3A, lane 6). By densitometric analysis, the ~40-kDa band in WT represented 12.73 ± 1.97% of the total corin protein in lysate, whereas the band in the insA variant was 4.04 ± 1.02%, significantly lower than that in WT (n = 5, p<0.01) (Fig. 3B). In the Met-30 isoform, this band was 3.99 ± 2.67% of the total corin protein, which was similar to that in the insA variant (Fig. 3B). The data support that the insertion resulted in a variant corin with a shorter cytoplasmic tail and reduced zymogen activation and activity.

Bottom Line: Corin is a membrane-bound protease that regulates blood pressure by activating the natriuretic peptides.Analysis of two independent cohorts showed that the variant was preferentially present in hypertensive patients (38/795 or 4.78% vs. 4/632 or 0.63% in normal individuals, p = 4.14E-6).In cell-based studies, the corin variant exhibited poor trafficking in the Golgi, reduced cell surface expression and zymogen activation, and low natriuretic peptide processing activity.

View Article: PubMed Central - PubMed

Affiliation: Cyrus Tang Hematology Center, MOE Engineering Center of Hematological Disease, MOH Key Lab of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital, and Collaborative Innovation Center of Hematology, Soochow University, Suzhou, China.

ABSTRACT
Corin is a membrane-bound protease that regulates blood pressure by activating the natriuretic peptides. CORIN variants have been associated with hypertension and heart disease in African Americans. In this study, we conducted targeted exome sequencing and identified an insertion variant, c.102_103insA, in exon 1 of the CORIN gene. Analysis of two independent cohorts showed that the variant was preferentially present in hypertensive patients (38/795 or 4.78% vs. 4/632 or 0.63% in normal individuals, p = 4.14E-6). The insertion shifted the reading frame, resulting in a corin variant with a truncated cytoplasmic tail. In cell-based studies, the corin variant exhibited poor trafficking in the Golgi, reduced cell surface expression and zymogen activation, and low natriuretic peptide processing activity. Compared with normal individuals with the wild-type allele, individuals with the variant allele had lower levels of plasma corin [0.59 ± 0.07 ng/mL (n = 25) vs. 0.91 ± 0.02 ng/mL (n = 215), p<0.001] and higher levels of plasma N-terminal pro-atrial natriuretic peptide (NT-pro-ANP) [2.39 ± 3.6 nmol/L (n = 21) vs. 0.87 ± 0.6 nmol/L (n = 48), p = 0.005]. These results indicate that the variant altered corin structure and impaired the natriuretic peptide processing activity in vivo. The results highlight corin defects as an important underlying mechanism in hypertension.

Show MeSH
Related in: MedlinePlus