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A Bacterial Mannose Binding Lectin as a Tool for the Enrichment of C-and O- Mannosylated Peptides

ABSTRACT: C-mannosylated peptides are easily detected by mass spectrometry (MS) from purified proteins but not from complex biological samples. Enrichment of specific glycopeptides by lectin affinity prior to MS analysis has been widely applied to support glycopeptide identification, but was up till now not available for C-mannosylated peptides. Here we used the α-mannose-specific Burkholderia cenocepacia lectin A (BC2L-A) and show that, in addition to its previously demonstrated high-mannose N-glycan binding capability, this lectin is able to retain C- and O-mannosylated peptides. Next to testing binding abilities to standard peptides, we applied BC2L-A affinity to enrich C-mannosylated peptides from complex samples of tryptic digests of HEK293 and MCF10A whole cell extracts, which led to the identification of novel C-mannosylation sites. In conclusion, BC2L-A enabled specific enrichment of C- and O-mannosylated peptides and might have superior properties over other lectins for binding to α-mannose-terminating glycans.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Adnan Halim

LAB HEAD: Adnan Halim

PROVIDER: PXD031355 | Pride | 2022-05-13

REPOSITORIES: Pride

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			Action	DRS
	210318_Fusion__LC2__NewObj_Lorenzo_HEKWTAC2_Tryp_TCL_BC2LA__080321__2h_ETD_HCD_10_20_ul_1.raw	Raw
	210318_Fusion__LC2__NewObj_Lorenzo_HEKWTAC2_Tryp_TCL_BC2LA__080321__2h_ETD_HCD_10_20_ul_1_6.msf	Msf
	210318_Fusion__LC2__NewObj_Lorenzo_HEKWTAC2_Tryp_TCL_BC2LA__080321__2h_ETD_HCD_10_20_ul_2.raw	Raw
	210318_Fusion__LC2__NewObj_Lorenzo_HEKWTAC2_Tryp_TCL_BC2LA__080321__2h_ETD_HCD_10_20_ul_3.raw	Raw
	210318_Fusion__LC2__NewObj_Lorenzo_HEKWTAC2_Tryp_TCL_BC2LA__080321__2h_ETD_HCD_10_20_ul_4.raw	Raw

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A Bacterial Mannose Binding Lectin as a Tool for the Enrichment of C-and O- Mannosylated Peptides

Project description:C-mannosylated peptides are easily detected by mass spectrometry (MS) from purified proteins but not from complex biological samples. Enrichment of specific glycopeptides by lectin affinity prior to MS analysis has been widely applied to support glycopeptide identification, but was up till now not available for C-mannosylated peptides. Here we used the α-mannose-specific Burkholderia cenocepacia lectin A (BC2L-A) and show that, in addition to its previously demonstrated high-mannose N-glycan binding capability, this lectin is able to retain C- and O-mannosylated peptides. We evaluated the binding abilities of BC2L-A to standard peptides. In conclusion, BC2L-A enabled specific enrichment of C- and O-mannosylated peptides and might have superior properties over other lectins for binding to α-mannose-terminating glycans.

2022-05-31 | PXD031484 | Pride

1. Site-specific O-glycosylation analysis of SARS-CoV-2 spike protein produced in insect and human cells

Project description:2. Enveloped viruses hijack not only host translation processes, but also its glycosylation machinery and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The biosynthetic nature of O-glycosylation is highly variable and is affected by initiating enzyme repertoires in different tissues and cell types, and therefore warrants a more thorough investigation. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S – insect cell or human cell-produced ectodomains, or insect cell-derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complimentary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2

2021-04-07 | PXD024767 | Pride

Counterion Optimization Dramatically Improves Selectivity for Phosphopeptides and Glycopeptides in Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC)

Project description:A well-hydrated counterion can selectively and dramatically increase retention of a charged analyte in hydrophilic interaction chromatography (HILIC). The effect is enhanced if the column is charged, as in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). This combination was exploited in proteomics for the isolation of peptides with certain post-translational modifications (PTMs). The best salt additive examined was magnesium trifluoroacetate. The well-hydrated Mg+2 ion promoted retention of peptides with functional groups that retained negative charge at low pH, while the poorly-hydrated trifluoroacetate counterion tuned down the retention due to the basic residues. The result was an enhancement in selectivity between 6- to 66-fold. These conditions were applied to a tryptic digest of mouse cortex. Gradient elution produced fractions enriched in peptides with phosphate, mannose-6-phosphate, N- and O-linked glycans. The numbers of such peptides identified either equaled or exceeded the numbers afforded by the best alternative methods. This method is a productive and convenient way to isolate peptides simultaneously that contain a number of different PTMs, facilitating study of proteins with “crosstalk” modifications. The fractions from the ERLIC column were desalted prior to C-18-reversed phase (RP) LC-MS/MS analysis. Between 47-100% of the peptides with more than one phosphate or sialyl- residue or with a mannose-6 phosphate group were not retained by a C-18 cartridge but were retained by a cartridge of porous graphitic carbon. This finding implies that the abundance of such peptides may have been significantly underestimated in some past studies.

2021-06-01 | PXD022988 | Pride

O-mannosylated proteins secreted by BCG

Project description:we conducted a ConA lectin-affinity chromatography and mass spectra analysis to identify O-mannosylated proteins in BCG culture filtration.

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TMEM260 is a protein-specific O-mannosyltransferase

Project description:Mutations in the TMEM260 gene cause structural heart defects and renal anomalies syndrome (SHDRA), but the function of the encoded protein remains unknown. We report that TMEM260 is an ER-located protein O-mannosyltransferase that selectively glycosylates defined extracellular immunoglobulin, plexin, transcription factor (IPT) domains of the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors. We demonstrate that disease-causing TMEM260 mutations impair O-mannosylation of IPT domains and that TMEM260 knock out in cells results in receptor maturation defects and abnormal growth of 3D cell models. Thus, our study identifies a new, receptor-specific O-mannosylation pathway that serves critical functions during e.g. epithelial morphogenesis.

2023-05-05 | PXD032328 | Pride

Impaired glycosylation drives golgi stress-mediated gastric tumorigenesis that can be targeted by lectin-drug conjugates

Project description:Downregulation or gene mutation of MUC6, a major component of gastric mucin, is often identified in human gastric cancers. However, the mechanistic role of MUC6 alteration in gastric carcinogenesis remains unclear. Here, using Muc6-deficient mice, we revealed that dysregulated glycosylation in Muc6-deficient gastric epithelium causes aberrant golgi stress responses, resulting in spontaneous gastric cancer development. Muc6-deficient tumor growth is dependent on MAPK activation, which is mediated by golgi stress-induced golph3 upregulation. Glycomic analysis and lectin-binding assays revealed abnormal expression of mannose-rich N-type glycans in Muc6-deficient gastric tumors. Banana lectin-drug conjugates, which bind to mannose-rich glycans, dramatically suppress mannose-rich murine and human gastric cancer growth. Thus, we propose golgi stress responses and aberrant sugar chains as promising therapeutic targets in gastric cancers accompanied with mucin expression disorder.

2024-08-31 | GSE237686 | GEO

Impaired glycosylation drives golgi stress-mediated gastric tumorigenesis that can be targeted by lectin-drug conjugates

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Impaired glycosylation drives golgi stress-mediated gastric tumorigenesis that can be targeted by lectin-drug conjugates II

2024-08-31 | GSE237689 | GEO

Free glycans derived from O-mannosylated glycoproteins suggest the presence of an O-glycoprotein degradation pathway in yeast

Project description:In eukaryotic cells, unconjugated oligosaccharides structurally related to N-glycans (FNGs) are generated either from misfolded N-glycoproteins destined for endoplasmic reticulum (ER)-associated degradation (ERAD), or from lipid-linked oligosaccharides, donor substrates for N-glycosylation of proteins. The mechanism responsible for the generation of FNGs is now well understood, but whether there are other type of free glycans remain unclarified. Here, we report on the accumulation of O-mannosylated free glycans in budding yeast that were cultured in media containing mannose as a carbon source. A structural analysis of these glycans revealed that their structures are identical to those of O-mannosyl glycans that are attached to glycoproteins. Deletion of the cyc8 gene, encoding a general transcription repressor, resulted in the accumulation of excessive amounts of the free O-glycans, concomitant with a severe growth defect, a reduction in the level of cellular O-mannosylated proteins, and compromised cell wall integrity. Our findings provide evidence for a regulated pathway for O-glycoprotein degradation in yeast and offer critical insights into the catabolic mechanisms that control the fate of O-glycosylated proteins.

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Project description:We developed a glycosylation enrichment method based on lectin and single-pot, solid-phase-enhanced sample preparation (SP3) technology, termed lectin-based SP3 technology (LectinSP3). LectinSP3 immobilized lectin on the SP3 beads for N-glycopeptide enrichment. It could identify over 1,100 N-glycosylation sites and 600 N-glycoproteins from 10 μg of mouse testis peptides. Furthermore, using LectinSP3 method, we characterized the N-glycoproteome of 1,000 mouse oocytes in three replicates, and identified a total of 363 N-glycosylation sites from 215 N-glycoproteins.

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