Dataset Information

Glycoproteomics and proteomics analysis of PBMCs from Colorectal Cancer patients

ABSTRACT: Non-invasive prognostic markers are needed to improve survival of colorectal cancer (CRC) patients. Towards this goal, we here apply integrative systems glycobiology approaches to tumour tissues and PBMCs from CRC patients and matching controls as well as a CRC patient-derived cell line. The untargeted -omics-driven approaches revealed that non-canonical paucimannosidic proteins from monocytic and cancer cell origins are prominent signatures in CRC tumour tissues, and that their expression associates with CRC progression. Guided by these novel relationships, we then show in vitro that N-acetyl-β-D-hexosaminidase (Hex) drives paucimannosidic protein biosynthesis in CRC cells, and is intimately involved in processes underpinning CRC metastasis (adhesion, migration, invasion). Importantly, Hex activity was elevated in PBMCs and plasma from patients with advanced CRC relative to those with early-stage disease. Notably, we show that plasma Hex activity accurately informs on CRC patient survival. Our study opens new avenues for effective prognostication and therapeutic intervention in CRC.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Whole Body

DISEASE(S): Colon Cancer

SUBMITTER: Rebeca Kawahara

LAB HEAD: Morten Thaysen-Andersen

PROVIDER: PXD051907 | Pride | 2025-05-06

REPOSITORIES: Pride

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Publications

HEXB Drives Raised Paucimannosylation in Colorectal Cancer and Stratifies Patient Risk.

Kawahara Rebeca R Kautto Liisa L Bansal Naaz N Dipta Priya P Chau The Huong TH Liquet-Weiland Benoit B Ahn Seong Beom SB Thaysen-Andersen Morten M

Molecular & cellular proteomics : MCP 20250211 3

Noninvasive prognostic markers are needed to improve the survival of colorectal cancer (CRC) patients. Toward this goal, we applied untargeted systems glycobiology approaches to snap-frozen and formalin-fixed paraffin-embedded tumor tissues and peripheral blood mononuclear cells from CRC patients spanning different disease stages and matching controls to faithfully uncover molecular changes associated with CRC. Quantitative glycomics and immunohistochemistry revealed that noncanonical paucimanno ...[more]

PMID: 39947398

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Project description:We recently discovered that human neutrophils express immunomodulatory glycoproteins carrying unusual and highly truncated paucimannosidic N-glycans (Man1-3GlcNAc2Fuc0-1), but their biosynthesis remains elusive. Guided by the well-characterised truncation pathway in invertebrates and plants in which the N-acetyl-β-D-hexosaminidase (Hex) isoenzymes catalyse paucimannosidic protein (PMP) formation, we here set out to test if the homologous human Hex α and β subunits encoded by HEXA and HEXB drive a similar truncation pathway in human neutrophils. To this end, we performed quantitative glycomics and glycoproteomics of several CRISPR-Cas9-edited Hex-disrupted neutrophil-like HL-60 mutants (HEXA-/- and HEXB-/-) and matching unedited cell lines. Hex disruption was validated using next-generation sequencing, ELISA, proteomics and Hex activity assays. Excitingly, all Hex-disrupted mutants displayed significantly reduced levels of paucimannosylation, particularly of Man2-3GlcNAc2Fuc1, relative to unedited HL-60 suggesting that both HEXA and HEXB contribute to PMP formation via a hitherto unexplored truncation pathway in neutrophils. Quantitative N-glycomics indeed demonstrated reduced utilisation of a putative non-canonical truncation pathway in favour of the canonical elongation pathway in all Hex-disrupted mutants relative to unedited controls. Quantitative glycoproteomics recapitulated the truncation-to-elongation switch in all Hex-deficient mutants and showed a pronounced switch for N-glycoproteins co-trafficking with Hex to the azurophilic granules of neutrophils such as myeloperoxidase. Finally, we supported the Hex-PMP relationship by documenting that primary neutrophils isolated from an early-onset Sandhoff disease patient (HEXB-/-) displayed dramatically reduced paucimannosylation relative to neutrophils from an age-matched unaffected donor. We conclude that both human Hex α and β mediate PMP formation via a putative non-canonical truncation pathway in neutrophils.

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:Neutrophils are short-lived yet vital innate immune cells equipped with bioactive glycoproteins packed in cytosolic granules that can readily be mobilised to elicit an effective and timely response against invading pathogens. Since the dynamic glycosylation processes underpinning the dramatic metamorphosis associated with myeloid progenitor-to-neutrophil differentiation remain unmapped, we here perform a comprehensive spatiotemporal profiling of the complex N-glycoproteome of isolated granule populations from blood-derived neutrophils and during maturation of bone marrow-derived progenitors using glycomics-assisted glycoproteomics. Firstly, glycomics indicated that the granule populations of mature (resting) neutrophils exhibit distinctive glycophenotypes including, most strikingly, unusual paucimannosidic- and monoantennary complex-type N-glycans in azurophilic granules. The granule-specific N-glycosylation features were recapitulated by glycoproteomics, which also uncovered extensive site- and protein-specific N-glycosylation decorating 4,772 N-glycopeptides from 352 N-glycoproteins across the neutrophil granules. Excitingly, comprehensive mining of proteomics and transcriptomics data collected from discrete myeloid progenitor stages for glycopeptide and glyco-enzyme expression revealed that dramatic glycoproteome remodelling underpins the promyelocytic-to-metamyelocyte transition and that remodelling is driven predominantly by proteome expression changes rather than modulation of the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of N-glycoprotein biosynthesis that were strongly reduced with myeloid differentiation leading to reduced N-glycosylation efficiency in late-stage neutrophil maturation and in corresponding granules. Our study provides new spatiotemporal insights into the dynamic neutrophil N-glycoproteome, which exhibits intriguingly complex site-, protein- and granule-specific N-glycosylation features and which undergoes strong remodelling during myeloid progenitor-to-neutrophil metamorphosis.

Project description:Changes in IgG glycosylation have been reported to be associated with colorectal cancer (CRC), but the alteration in specific subclass of IgG is unknown. Herein, we optimized five common IgG glycopeptides enrichment methods to acquire comprehensive profiling of IgG glycoproteomics in CRC. Notably, incomplete tryptic digestion of IgG occurred when using ordinary ratio of protease to protein, which significantly impacted the final statistical analysis of association between IgG glycosylation and CRC. We introduced a method based on two-step enzymatic digestion, enabling the complete digestion of IgG glycopeptides and further improving the detection intensity of the target glycopeptides. By this approach, total 47 IgG glycopeptides can be identified by nanoLC-ESI-MS/MS. Following rapid and automatic data processing through the self-developed program, we observed that IgG1_H3N4F1 and IgG1_H3N4 were substantially increased in CRC, while IgG1_H5N5F1, IgG1_H5N4F1S1and IgG2_H5N4F1 were markedly decreased. Strikingly, these four glycopeptides were continually significantly changed in early and late stages of CRC. Further evaluation of the diagnostic performance showed they all obtained a fair performance in discriminating the patients from the normal. In terms of the glycan features, it demonstrated that CRC progression associates with the increased agalactosylation, and the decreased digalactosylation and galactosylation per antenna on diantennay glycans of IgG1 and IgG2. Concurrently, the decreased sialylation of IgG1 was strongly correlated with CRC. Moreover, tumor-specific glycosylation analysis showed that the alterations of subclass-specific IgG glycosylation were more significant in colon cancer, and no obvious difference between colon and rectal cancer was found. More importantly, all these findings were validated in an independent cohort. This two-step enzymatic approach is crucial to explore the relationship between the disease progression and IgG glycosylation.

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