Uptake of severe acute respiratory syndrome coronavirus 2 spike protein mediated by angiotensin converting enzyme 2 and ganglioside in human cerebrovascular cells

Abstract

IntroductionThere is clinical evidence of neurological manifestations in coronavirus disease-19 (COVID-19). However, it is unclear whether differences in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/spike protein (SP) uptake by cells of the cerebrovasculature contribute to significant viral uptake to cause these symptoms.MethodsSince the initial step in viral invasion is binding/uptake, we used fluorescently labeled wild type and mutant SARS-CoV-2/SP to study this process. Three cerebrovascular cell types were used (endothelial cells, pericytes, and vascular smooth muscle cells), in vitro.ResultsThere was differential SARS-CoV-2/SP uptake by these cell types. Endothelial cells had the least uptake, which may limit SARS-CoV-2 uptake into brain from blood. Uptake was time and concentration dependent, and mediated by angiotensin converting enzyme 2 receptor (ACE2), and ganglioside (mono-sialotetrahexasylganglioside, GM1) that is predominantly expressed in the central nervous system and the cerebrovasculature. SARS-CoV-2/SPs with mutation sites, N501Y, E484K, and D614G, as seen in variants of interest, were also differentially taken up by these cell types. There was greater uptake compared to that of the wild type SARS-CoV-2/SP, but neutralization with anti-ACE2 or anti-GM1 antibodies was less effective.ConclusionThe data suggested that in addition to ACE2, gangliosides are also an important entry point of SARS-CoV-2/SP into these cells. Since SARS-CoV-2/SP binding/uptake is the initial step in the viral penetration into cells, a longer exposure and higher titer are required for significant uptake into the normal brain. Gangliosides, including GM1, could be an additional potential SARS-CoV-2 and therapeutic target at the cerebrovasculature.

FAIMS Shotgun Lipidomics for Enhanced HILIC-like Separation and Automated Annotation of Gangliosides

“…To confirm the validity of the suspects, Full MS and DDA scans were analyzed using LDA's shotgun/FAIMS extension described above (version 2.10). The ganglioside specific mass list 7 was limited to the suspects, and the following ion species were searched for: 3and [M-4H] 4-. We applied an m/z tolerance of 5 ppm and 0.2 Da for the analysis of MS1 and MS/MS data, respectively (these settings are activated in LDA version 2.10 by selecting the instrument 'OrbiTrap_Lumos_shotgun' and the fragmentation selection 'Ganglioside_neg_shotgunFAIMS').…”

“…Gangliosides, belonging to the acidic glycosphingolipids, are crucial players in cellular communication and neural function, predominantly present in the brain 1 . Located on the outer leaflet of the plasma membrane, these molecules contribute to neuronal development, cellular signaling, immune modulation, and show a significant impact on several diseases like cancer, Alzheimer's disease, and COVID-19 2,3 . Moreover, gangliosides have emerged as promising marker candidates in stem cell differentiation processes [4][5][6][7] .…”

Risk factors for severe COVID-19 disease increase SARS-CoV-2 infectivity of endothelial cells and pericytes

“…Whether endothelial dysfunction occurs via effects of cytokines borne by the plasma [47] or by infection of endothelial cells themselves remains under debate. Some studies find low ACE2 expression and low infectivity of endothelial cells [48], while other studies find that endothelial cells can bind and internalize SARS-CoV-2 S protein in lung [49] and other tissues such as brain [50] and can cause degeneration of endothelial cells [27]. Pericyte expression of ACE2 has been reported to be much higher than that of endothelial cells, suggesting that vascular infection with SARS-CoV-2 might require breakdown of the blood-brain barrier to enable viral Infectivity of SARS-CoV-2 PVs is higher in mouse pericytes (PC) than endothelial cells (EC) and higher in cultures from APOE4-TR mice.…”

Host-Directed Virus-Mimicking Particles Interacting with the ACE2 Receptor Competitively Block Coronavirus SARS-CoV-2 Entry

“…The Spike protein of SARS-CoV-2 was reported to interact with human cerebrovascular cells, including endothelial cells, pericytes, and smooth muscle cells, mediated by ACE2. 27 We hypothesized that the prepared VMPs functionalized with the Spike S1 RBD bind to ACE2 on the cell surface, which may enhance their uptake into the host cells. To prove that the fabricated VMPs bind to ACE2, we evaluated the cell uptake of VMPs with a human lung carcinoma A549 cell line expressing ACE2 (A549-ACE2), human non-small cell lung cancer Calu-3 cells, and human colorectal adenocarcinoma Caco-2 cells, which are expressed with ACE2 and highly susceptible to SARS-CoV-2 infection.…”

“…The Spike protein of SARS-CoV-2 was reported to interact with human cerebrovascular cells, including endothelial cells, pericytes, and smooth muscle cells, mediated by ACE2 . We hypothesized that the prepared VMPs functionalized with the Spike S1 RBD bind to ACE2 on the cell surface, which may enhance their uptake into the host cells.…”