Characterization of ACE2 naturally occurring missense variants: impact on subcellular localization and trafficking

Figures

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  Fig. 1. ACE2 protein domains, coding missense variants, and full-length three-dimensional structure. A A schematic diagram displaying the different domains of human ACE2 including the signal peptide domain (SP), peptidase domain and collectrin homology domain. Dark blue regions correspond to the residues interacting with SARS-CoV-2 S protein. Residues labeled in red correspond to the 7N-glycosylation sites in ACE2. The studied coding variants are labeled in black and distributed based on their domain. B 3D structure of ACE2 protein along with its functional domains. Twenty-seven positions are only shown where 2 variants are available in Leu731 position. Signal peptide (pink), peptidase domain (gray), collectrin domain (orange), transmembrane domain (cyan), and cytoplasmic domain (purple) are shown in cartoon representation and amino acids are shown in stick representation

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  Fig. 2. The enlarged view of the residues surrounding missense variants shown in Fig. 1B. A1 wild type Ser19; A2 mutant Pro19; B1wild type Ile21; B2 mutant Thr21; C1 wild type Lys26; C2 mutant Arg26; D1 wild type Asn33; D2 mutant Asp33; E1 wild type Asp38; E2 mutant Glu38; F1 wild type Asn149; F2 mutant Ser149; G1 wild type Asn159; G2 mutant Ser159; H1 wild type Gly173; H2 mutant Ser173; I1 wild type Met383; I2 mutant Ile383; J1 wild type Ile468; J2 mutant Val468; K1 wild type Ala501; K2 mutant Thr501; L1 wild type Gly575; L2 mutant Val575; M1 wild type Phe592; M2 mutant Leu592; N1 wild type Ala627; N2 mutant Val627; O1 wild type Asn638; O2 mutant Ser638; P1 wild type Val658; P2 mutant Ile658; Q1 wild type Glu668; Q2 mutant Lys668; R1 wild type Ser692; R2 mutant Pro692; S1 wild type Arg708; S2 mutant Trp708; T1 wild type Arg710; T2 mutant His710; U1 wild type Asn720; U2 mutant Asp720; V1 wild type Ile727; V2 mutant Val727; W1 wild type Leu731; W2 mutant Ile731; X1 wild type Leu731; X2 mutant Phe731; Y1 wild type Ile753; Y2 mutant Thr753; Z1 wild type Arg768; Z2 mutant Trp768; Z3 wild type Asp785; Z4 mutant Asn785; Z5 wild type Val801; and Z6 mutant Gly801. Signal peptide (pink),…

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  Fig. 3. Overexpressed WT ACE2 subcellular localization and stability. A Immunofluorescence confocal imaging of permeabilized HeLa cells transfected with Flag-tagged ACE2 (red), GFP-tagged HRas (plasma membrane marker in green) and Calnexin (ER marker in blue). Images were acquired using 100X magnification and manipulated by ImageJ. Scale bar = 50 μm. B HEK293 cells transiently transfected with GFP and Flag-tagged WT ACE2 plasmids for 48 Hrs. Lysates were then digested in presence or absence of Endo H and PNGase F enzymes for 3 Hrs and 1 Hrs, respectively. Anti-Flag primary antibody was used to stain WT ACE2 and anti-GFP antibody was used to satin GFP that was used as a transfection control. C Flag-tagged WT ACE2 transfected cells were treated with DMSO and 100 μg/ml cycloheximide for a period of 18 Hrs. Cells were lysed at the indicated time points (0, 4, 8, 12, and 18 Hrs) and lysates were analyzed by western blotting. Anti-Flag antibody was used to stain WT ACE2. Mock sample represent un-transfected cells. In all the experiments actin was used as a loading control. D Graph representing the relative expression of WT ACE2 compared to DMSO treated cells at the indicated time points. Error…

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  Fig. 4. ACE2 variants subcellular localization. HeLa cells were grown on coverslips and transiently co-transfected with Flag-tagged ACE2 WT or missense variants and GFP-tagged HRas (plasma membrane marker). 24 Hrs post transfection, cells were fixed and stained with anti-Flag and anti-Calnexin (ER marker) antibodies. Images were acquired using 100X magnification and manipulated by ImageJ. Scale bar = 50 μm

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  Fig. 5. ACE2 variants glycosylation profile. HEK293 cells transiently co-transfected transfected with Flag-tagged ACE2 WT or missense variants and GFP plasmids for 48 Hrs. Lysates were then digested in presence or absence of Endo H enzymes for 3 Hrs. Anti-Flag primary antibody was used to stain WT ACE2 and anti-GFP antibody was used to satin GFP that was used as a transfection control. Actin was used as a loading control. Images were manipulated using ImageJ