Dataset Information

A broad-specificity O-glycoprotease that enables improved analysis of glycoproteins and glycopeptides containing intact complex O-glycans

ABSTRACT: Analysis of mucin type O-glycans linked to serine/threonine of glycoproteins is technically challenging, in part, due to a lack of effective enzymatic tools that enable their analysis. Recently, several O-glycan-specific endoproteases that can cleave the protein adjacent to the appended glycan have been described. Despite significant progress in understanding the biochemistry of these en-zymes, known O-glycoproteases have specificity constrains, such as inefficient cleavage of glycoproteins bearing sialylated O-glycans, high selectivity for certain type of glycoproteins or protein sequence bias, that limit their analytical application. In this study, we examined the capabilities of an immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa. The peptide substrate sequence selectivity and its impact on IMPa activity was interrogated using an array of synthetic peptides and their glycoforms. We show that IMPa has no specific P1 residue preference and can tolerate most amino acids at the P1 position, except aspartic acid. The enzyme does not cleave between two adjacent O-glycosites, indicating that O-glycosylated serine/threonine is not allowed at position P1. Glycopeptides with as few as two amino acids on either side of an O-glycosite were specifically cleaved by IMPa. Finally, IMPa efficiently cleaved peptides and proteins carrying sialylated and asialylated O-glycans of varying complexity. We present the use of IMPa in a one-step O-glycoproteomics workflow for glycoprofiling of individual purified glycoproteins granulocyte colony-stimulating factor (G-CSF) and receptor-type tyrosine-protein phosphatase C (CD45) without the need for glycopeptide enrichment. In these examples, IMPa enabled identification of O-glycosites and the range of complex O-glycan structures at each site.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hek-293 Cell, Ns-0 Cell

SUBMITTER: Saulius Vainauskas

LAB HEAD: Christopher H. Taron

PROVIDER: PXD029534 | Pride | 2022-01-05

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	List_of_CD45_glycopeptides.xlsx	Xlsx
	List_of_G-CSF_glycopeptides.xlsx	Xlsx
	Sample1_G-CSF_600g_120min_Top10_LM_n1000_QE_Reprosil_Sonation_030421_Run1.raw	Raw
	Sample1_G-CSF_n_600g_120min_Top10_LM_n1000_QE_Reprosil_Sonation_030421_Run2.raw	Raw
	Sample2_CD45_n_600g_120min_Top10_LM_n1000_QE_Reprosil_Sonation_030421_Run1.raw	Raw

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Publications

A Broad-Specificity O-Glycoprotease That Enables Improved Analysis of Glycoproteins and Glycopeptides Containing Intact Complex O-Glycans.

Vainauskas Saulius S Guntz Hélène H McLeod Elizabeth E McClung Colleen C Ruse Cristian C Shi Xiaofeng X Taron Christopher H CH

Analytical chemistry 20211228 2

Characterization of mucin-type O-glycans linked to serine/threonine of glycoproteins is technically challenging, in part, due to a lack of effective enzymatic tools that enable their analysis. Recently, several O-glycan-specific endoproteases that can cleave the protein adjacent to the appended glycan have been described. Despite significant progress in understanding the biochemistry of these enzymes, known O-glycoproteases have specificity constraints, such as inefficient c ...[more]

PMID: 34962767

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Project description:Glycobiology plays a central role in prostate cancer (PCa), as evidenced by the aberrant glycosylation in PCa-derived glycoproteins. Understanding the protein glycosylation changes underling PCa onset and progression may open opportunities for the discovery of novel biomarkers for PCa detection and stratification as well as targets for PCa metastasis. Advances in mass spectrometry-based glycomics and glycoproteomics have opened up exciting avenues for deep characterisation of the immensely, yet poorly understood complex glycoproteome. In this study, we have used integrated glycomics and glycoproteomics to explore the protein, cell and tumour-grade specific N- and O- glycosylation signatures in surgically-removed fresh PCa tissues grouped into five PCa disease grades and tissues from benign prostatic hyperplasia patients (BPH). Porous graphitised carbon–liquid chromatography (PGC–LC)-MS/MS analysis was used to profile the N-and O-glycome, revealing PCa grade specific N and O glycosylation changes, including oligomannosidic and paucimannosidic, bisecting-GlcNAc and highly branched tri- and tetra-antennary fucosylated and sialylated N-glycans as well as sialylated core 1 and 2 type O-glycans. High resolution LC-MS/MS was then employed to capture the micro and macroheterogeneity of 1,085 N-glycosites (>7,400 unique N-glycopeptides) from 540 N-glycoproteins and 360 O-glycosites (>500 unique O-glycopeptides) from 178 O-glycoproteins and reveal protein and cell specific N- and O- glycosylation changes associated with PCa progression. We also showed PCa grade-specific increase in the expression of oligosaccharyltransferase (OST) subunits that correlate with changes in the site occupancy of N-glycoproteins. In conclusion, this study shows that integrated glycomics and glycoproteomics can provide unprecedented insight into key molecular features and site-specific glycan heterogeneity contributing to the highly diverse tumour-microenvironment and allow us to deconstruct the regulation of the protein, cell and tumour-grade-specific glycosylation associated with PCa onset and development.

Project description:A large family of GalNAc transferases (GalNAc-Ts) catalyzes the covalent attachment of N-Acetylgalactosamine (GalNAc) to serine and threonine residues on proteins that pass through the secretory pathway in the first committed step of mucin-type O-glycosylation. Abnormalities in the activity of individual GalNAc-Ts can result in congenital disorders of O-glycosylation (CDG) and influence other biological functions. Although ~85% of proteins that pass through the secretory pathway are modified with O-glycans, only 47 O-glycosylation sites (O-glycosites) from 22 mouse glycoproteins and 17 publications are present on UniProt and an O-glycoprotein database (http://www.oglyp.org/). A compilation of all in vivo O-glycosites (the O-glycoproteome) would be an invaluable step in determining the function of proteins decorated with N-Acetylgalactosamine and what role(s), if any, the O-glycans play. While approaches to delineate O-glycosites have been developed for simple, genetically engineered cell lines, there is no universal approach to mapping the O-glycosites of glycoproteins in complex tissue extracts or biological fluids. We have developed chemical and enzymatic conditions that cleave solitary O-glycans while leaving the modified core protein/peptides assayable by mass spectrometry (MS). The methodology permits the mapping of thousands of O-glycosites decorated with Tn or T antigen from tissues or biological fluids. We then integrated an HCD-pd-EThcD MS workflow and software, including MSFragger-Glyco, pGlyco3, and O-Pair, to study the mouse brain, heart, lung, liver, spleen, kidney, colon, muscle, submandibular gland, and whole blood. The integrated approach identified 2154 solitary O-glycosites from 595 glycoproteins. The O-glycosites and glycoproteins displayed consensus motifs and Gene Ontology (GO) terms for O-glycoproteins. Limited overlap of O-glycosites was observed with protein O-GlcNAcylation and phosphorylation sites. Integrating quantitative glycoproteomics and proteomics revealed a tissue-specific regulation of O-glycosites that the differential expression of Galnt isoenzymes in tissues partly contributes to. We next used established quantitative glycoproteomics and proteomics methods to identify site-specific substrates that may contribute to biologically relevant phenotypes when Galnt2 was genetically ablated in a Galnt2-null mouse model, which presents with lipid and metabolic dysregulation. Our findings suggest networks of Galnt2 direct and indirect interactions that may explain the complex metabolic phenotypes. The mouse O-glycoproteome-map and quantitative glycoproteomics/proteomics approach will provide a valuable foundation for revealing the significance of O-glycosylation biology in CDGs and other diseases and conditions.

Dataset Information

A broad-specificity O-glycoprotease that enables improved analysis of glycoproteins and glycopeptides containing intact complex O-glycans

Dataset's files

Publications

A Broad-Specificity <i>O</i>-Glycoprotease That Enables Improved Analysis of Glycoproteins and Glycopeptides Containing Intact Complex <i>O</i>-Glycans.

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