[PMC free content] [PubMed] [Google Scholar] 3. selection of O-glycans and N- seeing that receptor ligands. These outcomes indicate multiple routes for SARS-CoV-2 to connect to human cells and suggest alternative strategies for therapeutic intervention. Introduction: SARS-CoV-2 is a positive-sense RNA enveloped virus characterized by a surface Spike (S) glycoprotein1. During host cell invasion, S binds to receptors on cell membranes, such as angiotensin-converting enzyme 2 (ACE2)1, 2, 3. However, the nature and function of the S glycosylation is not fully understood. Densely glycosylated with multiple Asn-linked (N-glycans) and a few Ser/Thr-linked (O-glycans)4, 5, the S protein of SARS-CoV-2 potentially presents ligands for a variety of innate immune receptors, including C-type lectin receptors (CLRs), that are known to bind specific glycans mostly in a Ca2+-dependent manner6, 7. CLRs such as DC-SIGN/CD209, L-SIGN/CD209L/CLEC4M, mannose receptor/MR/MRC1/CD206, MGL/CLEC10A/CD301, and Dectin-2/CLEC6A, are highly expressed within the human immune system including monocytes, dendritic cells, and macrophages, functioning as the first-line of defense against invading viruses and pathogens8, 9. As known pattern-recognition receptors, BAPTA CLRs, especially DC-SIGN, can direct host immune responses against numerous pathogens in a glycan-specific manner by modulating Toll-like receptor-induced activation10. Evidence implicates innate immune cells in the pathogenesis of SARS-CoV-2. Over 80% of patients with SARS-CoV-2 infection present with lymphopenia and an increased neutrophil-lymphocyte ratio11. Patients with severe COVID-19 exhibit hyperactive macrophages in the bronchoalveolar lavage fluid (BALF) and oropharyngeal swab12. Likewise, increased infiltration and activation of macrophages is observed in biopsy or autopsy specimens from COVID-19 patients13. Previous studies of the closely-related SARS-CoV demonstrated that primary human monocytes and dendritic cells can be infected14, 15, and SARS-CoV S can bind DC-SIGN and L-SIGN16, 17, 18, 19. Eight glycosylation sites of SARS-CoV S were identified to be involved in their interactions20, 21, among which six are conserved in SARS-CoV-2 (Supplementary Fig. 1). However, it is not known whether SARS-CoV-2 interacts with a variety of CLRs. Here, we demonstrate that many different CLRs directly bind in a glycan-dependent manner to the S glycoprotein of SARS-CoV-2 with picomolar affinities. Binding of DC-SIGN can trigger the internalization of S in 3T3-DC-SIGN+ cells, which implies the potential involvement in viral entry. Furthermore, we dissect the Ppia N- and O-glycan sequences on the recombinant SARS-CoV-2 S, and identify glycan features that are crucial for interactions with these CLRs. The analyses of open accessible single-cell RNA sequencing data confirm that various human tissues and their resident immune cells differentially express CLRs, including MR, MGL and DC-SIGN in bronchoalveolar macrophages in patients BAPTA with SARS-CoV-2. This is in direct contrast to the absence of ACE2 expression within the same cell types across the tissues. Our study identifies new SARS-CoV-2 binding receptors expressed on innate immune cells, particularly on macrophages and dendritic cells, which could accentuate severe pathological inflammation along with cytokine release syndrome. The results suggest potential additional routes for viral infection and new anti-viral strategies. Results: Multiple CLRs bind SARS-CoV-2 S in a glycan-dependent manner Multiple CLRs including DC-SIGN, L-SIGN, MR (C-type lectin domains 4C7) and MGL exhibited strong binding to the recombinant full-length S produced in human embryonic BAPTA kidney HEK293 cells (Fig. 1aCc & e). HEK293 cells are known to present a spectrum of human glycosylation reflective of the kidney and other epithelial tissues22. DC-SIGN, L-SIGN and MGL also bound to recombinant S1, the subunit involved in ACE2 recognition. By contrast, another CLR, Dectin-2, did not bind S or S1, but bound the positive control, a yeast extract (EBY-100) containing mannan-type ligands (Fig. 1d). Binding of DC-SIGN, L-SIGN, MR and MGL was glycan-dependent, as it was sensitive to treatment with sequence-specific glycosidases (Fig. 1f). Binding of DC-SIGN and L-SIGN was attenuated by Endoglycosidase H (Endo H, oligomannose and hybrid type N-glycan-targeting) and eliminated by PNGase F (N-glycan-targeting), suggesting that the two CLRs bind to S via both oligomannose and complex N-glycans. BAPTA MR binding was abolished by both Endo H and PNGase F (Fig. 1c), suggesting that oligomannose N-glycans are the ligands. A profound reduction in BAPTA MGL binding was observed by exposure to PNGase F, indicating that MGL ligands reside primarily on N-glycans. Sialic acid appeared to mask some of the MGL ligands as neuraminidase (Neu) treatment slightly increased the binding (Fig. 1e), which was similar to the effect on bovine submaxillary mucin (BSM) with abundant O-glycans comprised of sialylated N-acetylgalactosamine (STn antigen). The effects of glycosidase treatment were confirmed by loss or gain of binding by.