Dataset Information

CD209L/L-SIGN N-Glycosylation and N-Glycosylation Site Occupancy

ABSTRACT: CD209L is a membrane glycoprotein with known glycan-binding properties and it also contains 2 N-glycosylation sequons at sites N92 and N361. Treatment with PNGase F in the presence of H218O, which removes N-linked glycans and isotopically labels the formerly-glycosylated site, confirmed that both unmodified and formerly-glycosylated versions of the peptide spanning the N92 N-glycosylated sequon were present. This suggests that a portion of CD209L protein is N-glycosylated at site N92. nUPLC-MS/MS analyses of CD209L digests enabled detection of a glycopeptide consistent with high-mannose type N-linked glycosylation.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Kevin Chandler

LAB HEAD: Catherine E Costello

PROVIDER: PXD021309 | Pride | 2021-09-09

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20200727_CD209L_CONTROL.raw	Raw
	20200727_CD209L_CONTROL_Mascot_F023124.csv	Csv
	20200727_CD209L_CONTROL_target.raw	Raw
	20200727_CD209L_PNGaseF_18O.raw	Raw
	20200727_CD209L_PNGaseF_18O_Mascot_F023123.csv	Csv

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Publications

CD209L/L-SIGN and CD209/DC-SIGN Act as Receptors for SARS-CoV-2.

Amraei Razie R Yin Wenqing W Napoleon Marc A MA Suder Ellen L EL Berrigan Jacob J Zhao Qing Q Olejnik Judith J Chandler Kevin Brown KB Xia Chaoshuang C Feldman Jared J Hauser Blake M BM Caradonna Timothy M TM Schmidt Aaron G AG Gummuluru Suryaram S Mühlberger Elke E Chitalia Vipul V Costello Catherine E CE Rahimi Nader N

ACS central science 20210630 7

As the COVID-19 pandemic continues to spread, investigating the processes underlying the interactions between SARS-CoV-2 and its hosts is of high importance. Here, we report the identification of CD209L/L-SIGN and the related protein CD209/DC-SIGN as receptors capable of mediating SARS-CoV-2 entry into human cells. Immunofluorescence staining of human tissues revealed prominent expression of CD209L in the lung and kidney epithelia and endothelia. Multiple biochemical assays using a purified reco ...[more]

PMID: 34341769

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Project description:Background Plasma proteins can be modified by addition of sugar residues by non-enzymatic glycation as well as glycosylation. While glycation is a hallmark of hyperglycemia, glycosylation is a complex, enzyme-catalyzed post-translational modification by heterogeneous sugar chains and is present on a majority of plasma proteins. N-linked glycosylation occurs on asparagine residues occurring predominantly on a canonical N-glycosylation motif (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported to be glycosylated less frequently. Albumin is the most abundant protein in human plasma whose glycation has been implicated in end-organ damage in advanced diabetes mellitus, and as such, has both diagnostic and therapeutic implications. However, albumin has long been regarded as a non-glycosylated protein owing to the absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated with two possible attached glycans. Methods To investigate N-linked glycosylation of abundant serum proteins in greater detail, we passed healthy donor samples through a multiple affinity removal spin (MARS14) column followed by trypsin digestion of bound proteins and subsequent glycopeptide enrichment by size-exclusion chromatography (SEC) or mixed-mode anion-exchange (MAX), in parallel. Global analysis of intact glycopeptides was performed by LC-MS/MS to evaluate potential glycosylation at canonical as well as non-canonical sites. PNGase F treatment was carried out to confirm N-linked glycosylation at non-canonical sites Asn-X-Cys/Val. Glycopeptides on albumin at non-canonical sites were further characterized by MS3 fragmentation from immunoprecipitated albumin to confirm the attached glycans. To assess if the observed glycosylation was a general phenomenon, targeted analysis using parallel reaction monitoring (PRM) was carried out on an independent set of twenty human serum samples. Finally, to test whether albumin glycosylation is unique to human albumin or is a general phenomenon, bovine and rabbit serum albumins were subjected to MAX chromatography and LC-MS/MS analysis. Results We report that human albumin is modified by N-linked glycosylation at two sites, Asn68(-Glu-Val) and Asn123-(Glu-Cys), both of which occur in non-canonical N-glycosylation motifs. We detected N-glycopeptides at both sites bearing four complex mono- and bi-antennary N-linked glycans, with Hex5HexNAc4NeuAc2 and Hex5HexNAc4NeuAc1 being the most abundant glycans. These findings were validated by analyzing deglycosylated peptides and MS3-based fragmentation of glycopeptides. Glycosylation at both sites was confirmed by targeted MS in twenty donor samples. Finally, we report that the Asn residue on bovine and rabbit serum albumins that corresponds to the highly conserved Asn123 in human albumin is also N-glycosylated with complex sialylated glycans similar to those observed in human albumin. Conclusions Albumin is a conserved glycoprotein with multiple N-linked glycosylation sites. This glycosylation of albumin has potential functional implications and applications in medicine.