The glycosylation of human angiotensin converting enzyme 2 (hACE2), the receptor for SARS-CoV-2

Understanding the mode of viral attachment, entry, and replication of SARS-CoV-2 is required for the development of new therapeutic strategies to address the COVID-19 pandemic We are interested to understand the glycobiology of SARS-CoV-2 viral infection and how it can help in development of vaccine and therapeutics The coronavirus surface spike (S) protein, which facilitates viral attachment, entry and membrane fusion is heavily glycosylated and also plays a critical role in the host immune response The spike protein is comprised of two protein subunits (S1 and S2) and possess 22 potential N-glycosylation sites and one Oglycosylation site The S protein of SARS-CoV-2 primarily binds to human angiotensin converting enzyme 2 (hACE2) for the host cell entry Even though hACE2 has been known for two decades and has been recognized as the entry point of several coronaviruses, limited studies have been available on glycosylation of human and non-human hACE2 Herein, we describe the comparative glycosylation mapping of hACE2 expressed in HEK293 cells and human plasma by both glycoproteomics and glycomics We have developed methods to easily isolate and purify ACE2 from human plasma which can be used for high throughput screening of ACE2 glycan structures from different individuals In hACE2 expressed in HEK293 cells, we have quantitatively characterized the Nglycosylation on ACE2 and interestingly also have observed O-glycosylation modification We have detected glycosylation on all the seven potential N-glycosylation sites, and are the first to report presence of significant O-glycosylation on Thr 730 The presence of charged sialylated O-glycans on Thr 730, which happens to be in the juxtamembrane stalk region of ACE2 could have implications for "ACE2 shedding"and its dimerization Intriguing questions arise through our data on presence of O-glycans on Thr 730 which could play a role in these processes To understand the detailed structure of glycan epitopes on hACE2, we have characterized the terminal sialic acid linkages, the presence of bisecting GlcNAc, and the pattern of N-glycan fucosylation Elucidation of the sitespecific glycosylation of hACE2 receptor on human plasma can aid in understanding the role of ACE2 glycosylation and the individual susceptibility to SARS-CoV-2 and possibly in the development of novel therapeutics