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Cross-linking mass spectrometry uncovers interactions between high-density lipoproteins and the SARS-CoV-2 spike glycoprotein

ABSTRACT: Multiple studies to date have shown that high-density lipoprotein (HDL) levels are reduced in patients with coronavirus disease 2019 (COVID-19), and that the extent of this reduction is associated with poor clinical outcomes. While lipoproteins are known to play a key role during the lifecycle of hepatitis C virus, the direct influence they have upon coronavirus (CoV) infections remains poorly understood. In this study we utilise cross-linking mass spectrometry (XL-MS) to determine circulating protein interactors of the severe acute respiratory syndrome (SARS)-CoV-2 spike glycoprotein. Spike was revealed to interact with HDL, through direct interactions with apolipoprotein (Apo)-A1, ApoC3 and ApoD, highlighting multiple modalities of how SARS-CoV-2 and HDL may interact. XL-MS of plasma isolated from COVID-19 patients uncovered HDL protein interaction networks, dominated by acute phase serum amyloid proteins (SAA), whereby serum amyloid A2 (SAA2) was shown to bind to ApoD. ApoD was confirmed to also interact with core apolipoproteins of both LDL and VLDL, suggesting that SARS-CoV-2 may bind multiple lipoprotein species. The interaction between ApoD and spike was further validated in cells using immunoprecipitation-MS, which uncovered a novel interaction between both ApoD and spike with membrane-associated progesterone receptor component 1 (PGRMC1). Mechanistically, XL-MS coupled with data-driven structural modelling determined that ApoD may interact within the receptor-binding domain (RBD) of spike, posing the hypothesis that ApoD may interfere with binding to the angiotensin-converting enzyme 2 (ACE2) receptor. However, utilising multiple cell-based assays we determined ApoD to have no effect upon viral replication or infectivity.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Sean Burnap

LAB HEAD: Manuel Mayr

PROVIDER: PXD036640 | Pride | 2023-07-20

REPOSITORIES: Pride

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Source:

			Action	DRS
	ApoD_FullLength_Spike_DSSO_SCX_HPLC_F20.msf	Msf
	ApoD_FullLength_Spike_DSSO_SCX_HPLC_F20.raw	Raw
	ApoD_FullLength_Spike_DSSO_SCX_HPLC_F21.msf	Msf
	ApoD_FullLength_Spike_DSSO_SCX_HPLC_F21.raw	Raw
	ApoD_FullLength_Spike_DSSO_SCX_HPLC_F22.msf	Msf

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Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes an extensively glycosylated spike protein that protrudes from the viral envelope to bind to angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor to mediate host-cell entry. Currently, the predominant recombinant spike protein production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, recombinant full-size spike protein was produced transiently in CHO and HEK cell suspensions. To further evaluate the sialic acid linkages presenting on spike glycans, a two-step amidation process, employing dimethylamine and ammonium hydroxide reactions in a solid support system, was developed to differentially modify the sialic acid linkages on glycans and glycopeptides from spike protein. We determined global and site-specific N-linked glycosylation patterns in soluble SARS-CoV-2 spike using MALDI-TOF and LC-MS/MS with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation. We identified the glycan compositions at 21 and 19 out of the 22 predicted N-glycosylation sites of the SARS-CoV-2 spike proteins produced in CHO and HEK, respectively. The N-glycan site at 1158 position (N1158) and the N-glycan sites at 122 ,282 and 1158 positions (N122, N282 and N1158) were found to be unoccupied on spike secreted from CHO and HEK cells, respectively. The structural mapping of glycans of recombinant human spike proteins revealed that CHO-Spike presented more complex and higher sialylation (α2,3-linked) content while HEK-Spike displayed more high-mannose and minor content of α2,3- and α2,6-linked sialic acids. The N74 site represents the most heavily N-glycosylated site on both spike proteins while some high-mannose abundant sites (N17, N234, N343, N616, N709, N717, N801 and N1134) on HEK-Spike may differentially shield the virus compared to CHO-spike and provide a different host immune system interaction strategy. Collectively, these data underscore the importance of characterizing site-specific glycosylation on recombinant human spike protein from HEK and CHO cells in order to better understand the impact of the production host on this complex and important protein used in diagnostics and vaccines.

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Cross-linking mass spectrometry uncovers interactions between high-density lipoproteins and the SARS-CoV-2 spike glycoprotein

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