Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins

0301 basic medicine Glycosylation QH301-705.5 Immunology EMC OR-01 N-Acetylglucosaminyltransferases Microbiology Madin Darby Canine Kidney Cells Mice 03 medical and health sciences Dogs Virology Chlorocebus aethiops Genetics Animals Humans Biology (General) Molecular Biology Vero Cells Mesocricetus SARS-CoV-2 RC581-607 3. Good health HEK293 Cells A549 Cells Spike Glycoprotein, Coronavirus Parasitology Angiotensin-Converting Enzyme 2 Immunologic diseases. Allergy Protein Multimerization Research Article Protein Binding
DOI: 10.1371/journal.ppat.1009282 Publication Date: 2021-02-09T02:03:27Z
ABSTRACT
Receptor binding studies on sarbecoviruses would benefit from an available toolkit of recombinant spike proteins, or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric Receptor Binding Domain (RBD) proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI-/- mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric, complex glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that complex glycosylated trimeric RBD proteins are attractive to analyze sarbecovirus receptor binding and explore ACE2 expression profiles in tissues.
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