Additional file 1 of Glycated ACE2 receptor in diabetes: open door for SARS-COV-2 entry in cardiomyocyte
Creators: name: D’Onofrio, Nunzia
name: Scisciola, Lucia
name: Sardu, Celestino
name: Trotta, Maria Consiglia
name: De Feo, Marisa
name: Maiello, Ciro
name: Mascolo, Pasquale
name: De Micco, Francesco
name: Turriziani, Fabrizio
name: Municinò, Emilia
name: Monetti, Pasquale
name: Lombardi, Antonio
name: Napolitano, Maria Gaetana
name: Marino, Federica Zito
name: Ronchi, Andrea
name: Grimaldi, Vincenzo
name: Hermenean, Anca
name: Rizzo, Maria Rosaria
name: Barbieri, Michelangela
name: Franco, Renato
name: Campobasso, Carlo Pietro
name: Napoli, Claudio
name: Municinò, Maurizio
name: Paolisso, Giuseppe
name: Balestrieri, Maria Luisa
name: Marfella, Raffaele
Publisher: name: figshare
Year: 2021
Subjects: subject: Biophysics
subject: Biochemistry
subject: Molecular Biology
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: 69999 Biological Sciences not elsewhere classified
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Cancer
subject: 60506 Virology

description: Additional file 1: Figure SI. Immunofluorescence negative control. Sections from diabetic and non-diabetic patients were performed with blocking solution, supplemented with a non-immune immunoglobulin IgG antibody, following by secondary antibody Alexa Fluor 488 or 633 incubation for 1 hour at RT. All samples were stained with DAPI (5 μg/ml) for 10 min before mounting in Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA). All slides were imaged using a Zeiss LSM 710 confocal microscope (Zeiss, Oberkochen, Germany) with a plan apochromat X63 (NA1.4) oil immersion objective. DM: diabetes mellitus; Non-DM: without diabetes mellitus. Table I: Mass accuracy and retention time (RT) of hACE2 tryptic peptides targeted by glycation. The measurement accuracies were reported in parts per million (ppm) and the chromatographic retention times in minutes (mins). Figure SII: Mass spectra of the triply charged hACE2 tryptic peptide 342-357 in the glycated (panel A) and non-glycated (panel B) form. Figure SIII: Mass spectra of the triply charged hACE2 tryptic peptide 466-475 in the glycated (panel A) and non-glycated (panel B) form. Figure SIV: Mass spectra of the triply charged hACE2 tryptic peptide 560-577 in the glycated (panel A) and non-glycated (panel B) form. Figure SV: Mass spectra of the triply charged hACE2 tryptic peptide 601-621 in the glycated (panel A) and non-glycated (panel B) form. Figure SVI: Mass spectra of the triply charged hACE2 tryptic peptide 626-644 in the glycated (panel A) and non-glycated (panel B) form. Figure SVII: Mass spectra of the triply charged hACE2 tryptic peptide 658-671 in the glycated (panel A) and non-glycated (panel B) form. Figure SVIII: Mass spectra of the triply charged hACE2 tryptic peptide 679-697 in the glycated (panel A) and non-glycated (panel B) form. Figure SIX: hACE2 PNGase F deglycosylation. SDS-PAGE performed on 500 nanograms of hACE2 digested with five units of PNGase F (Promega, catalog n. V4831) overnight at 37 °C and then analyzed on a NuPAGE 4-12 Bis-Tris gel (Thermo, catalog no. NP326) using a NuPAGE MES running buffer (Thermo, catalog no. NP0002). Lane 1=Bio-Rad Precision Standard (catalog no. 161-0373); lane 2=deglycosylation blank; lane 3=PNGase-digested hACE2; lane 4= undigested hACE2. The band at about 35 KDa in lanes 2 and 3 is the PNGase F. Figure SX. Glycated hACE binding to SARS-CoV-2 Spike protein. A, hACE2 control, B, hACE2 at 12 days incubation time with 60 mM and C, 120 mM of glucose. D, Values of kinetic binding parameters of hACE2-SARS-CoV-2 Spike protein obtained from SPR measurement. Binding sensorgrams for SARS-CoV-2 Spike protein and several analyte concentrations of 1 µg of immobilized Spike protein and ACE2. Samples of ACE2 were diluted in HBS-EP+ at different concentration (64 µg/ml, 32µg/ml, 16 µg/ml, 8 µg/ml, and 4 µg/ml) and injected for 120s at a flow rate of 30µl/min on flow cell 3 and 4. Formulation buffer was run as a control. Dissociation was followed for 300sec; regeneration was achieved with a 60-sec pulse of 3M MgCl2. ACE2 concentrations were listed on the right side and were expressed in µg/ml. Ligand-analyte affinity (KD) and kinetic parameters (Association rate, Ka; Dissociation rate, Kd) of Originator and Biosimilar Rituximab was calculated with the Biacore T200 Evaluation Software (version 2.0; GE Healthcare). Figure SXI: RBD amino acid sequence; 319–541 residues and crystal structure of SARS-CoV-2 Spike receptor-binding domain (RBD) bound with ACE2 (PDB ID: 6M0J). Lysine 353 (K353) undergoes mild glycation by high-glucose (12 mM) exposure. Asparagine (N).