A Single Asparagine-Linked Glycosylation Site of the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Facilitates Inhibition by Mannose-Binding Lectin through Multiple Mechanisms

Abstract

Mannose-binding lectin (MBL) is a serum protein that plays an important role in host defensesA novel coronavirus (CoV), severe acute respiratory syndrome-CoV (SARS-CoV), is the causal agent of severe acute respiratory syndrome, which afflicted thousands of people worldwide in 2002 and 2003 (10, 39). SARS-CoV is an enveloped, single-and positive-strand RNA virus that encodes four major structural proteins: S, spike glycoprotein (GP); E, envelope protein; M, membrane glycoprotein; and N, nucleocapsid protein (46, 55). Similar to other coronaviruses, the S glycoprotein of the virus mediates the initial attachment of the virus to host cell receptors, angiotensinconverting enzyme 2 (ACE2) (44) and/or DC-SIGNR (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-related molecule; also CD209L or L-SIGN[liver/lymph node-SIGN]) (32) and subsequent fusion of the viral and cellular membranes to allow viral entry into susceptible target cells. The S glycoprotein of SARSCoV (SARS-S) is a 1,255-amino-acid (aa) type I membrane glycoprotein (46) with 23 potential N-linked glycosylation sites (55). The S glycoproteins of some coronaviruses are translated as a large polypeptide that is subsequently proteolytically cleaved into two functional subunits, S1 (harboring the receptorbinding domain [RBD]) and S2 (containing the membrane fusion domains) (1, 31, 51), during biogenesis, but others are not. The S glycoprotein on mature SARS-CoV virions does not appear to be cleaved (50, 61), but sequence alignments with other coronavirus S glycoproteins allow definition of S1 and S2 regions (46, 55). More recently, studies showed the proteolysis of the S glycoprotein of SARS-CoV on mature virions by cathepsin L (CTSL) (28, 59), as well as trypsin (43, 61) and factor Xa (11), suggesting that a critical cleavage event may occur during cell entry rather than during virion biogenesis.Mannose-binding lectin (MBL; also known as mannosebinding or mannan-binding protein [MBP]) is a Ca 2ϩ -dependent (C-type) serum lectin that plays an important role in innate immunity by binding to carbohydrates on the surface of a wide range of pathogens (including bacteria, viruses, fungi, and protozoa) (8,14,18), where it activates the complement system or acts directly as an opsonin (30,40,52). In order to activate the complement system, MBL must be in complex with a group of MBL-associated serine proteases (MASPs), MASP-1, -2, and -3. Currently, only the role of MASP-2 in complement activation has been clearly defined (65). The MBL-MASP-2 complex cleaves C4 and C2 to form C3 convertase (C4bC2a), which, in turn, activates the downstream complement cascade. MBL is a pattern recognition molecule (9), and surface recognition is mediated through its C-terminal carbohydrate recognition domains (CRDs), which are linked to collagenous stems by a short coiled-coil of alpha-helices. MBL is a mixture of oligomers assembled from subunits that are formed from three identical polypeptide chains (9) and usually has two to six clusters of CRDs. Withi...

Chicken mannose binding lectin has antiviral activity towards infectious bronchitis virus

“…rcMBL acts by direct binding the viral glycoprotein spike through its CRD, thereby aggregating virus particles. For mammalian MBL, direct virus neutralization through interaction with viral glycoproteins, thereby blocking virus binding to receptors on host cells, has been described before (Brown et al, 2010;Ip et al, 2005;Ji et al, 2005;Zhou et al, 2010). Here, we observed that rcMBL binds two IBV strains, IBV-Beaudette and IBV-M41, in line with a previous observation with an unspecified IBV strain (Kjaerup et al, 2014a).…”

Crosstalk between innate and adaptive immune responses to infectious bronchitis virus after vaccination and challenge of chickens varying in serum mannose-binding lectin concentrations

“…Recently, this was supported in a paper by Zhou et al (2010) [31] who showed a direct MBL-mediated neutralization of severe acute respiratory syndrome-coronavirus (SARS-CoV) by blocking the binding of the virus specific S glycoprotein (SARS-S) to the DC-SIGN receptor (dendritic cell-specific intercellular adhesion molecule 3grabbing nonintegrin-related molecule) suggesting that MBL most likely recognizes an overlapping set of high-mannose-content glycans on the S glycoprotein and thereby competes with DC-SIGN for binding. DC-SIGN is known to bind to different pathogens and triggers intracellular signaling cascades that affect TLR signaling and thereby adaptive immune responses (reviewed in [32]).…”

Cryo-EM analysis of a feline coronavirus spike protein reveals a unique structure and camouflaging glycans

“…2A). The remaining 8 predicted N-glycosylation sites, namely 27 NTSH, 31 NNSK, 482 NYTD, 585 NGSV, and 590 NVTS in the S1 subunit, 1308 NTTH in the S2 subunit, and 1352 NQTK and 1357 NLTA in HR2, were not verified due to the lack of corresponding tryptic peptides. To obtain an unbiased view of the range of N-glycans carried, the pool of released N-glycans was profiled by MALDI-MS after permethylation.…”

“…Site-Specific N-Glycosylation Mapped by MS and Cryo-EM. Glycosylation of CoV S proteins is implicated in protein folding (22), structure stability (22), virus entry, and receptor recognition (23)(24)(25)(26)(27). According to the sequence, 37 N-linked glycosylation sites are predicted for FIPV-UU4 S protein.…”