Dataset Information

Glycoproteomics analysis of Complement Factor H and its effect on complement regulatory function during Streptococcus pneumoniae-associated hemolytic uremic syndrome

ABSTRACT: Hemolytic uremic syndrome caused by an invasive Streptococcus pneumoniae infection (SP-HUS) is a rare and severe disease that primarily affects children under two years of age. The pathophysiology of SP-HUS remains poorly understood, and treatment is largely supportive. Complement factor H (FH) is a key regulator of the alternative pathway of the complement system. It has been hypothesized that loss of sialic acids from FH’s N-glycans may impair its regulatory functions, thereby potentially leading to complement-mediated endothelial cell damage in SP-HUS. In this study, we investigated the N-glycosylation patterns of FH across three N-glycosylation sites for four SP-HUS patients and compared it to healthy controls using LC-MS/MS-based glycopeptide profiling.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

DISEASE(S): Hemolytic-uremic Syndrome

SUBMITTER: Hans Wessels

LAB HEAD: Hans JCT Wessels

PROVIDER: PXD064837 | Pride | 2025-08-21

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Glycoform_fractions_FH.xlsx	Xlsx
	MSFragger_FH_Glycopeptide_identifications.xlsx	Xlsx
	RAW_FH_NHS_Neuraminidase.zip	Other
	RAW_Healthy_controls.zip	Other
	RAW_Patient1.zip	Other

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Project description:Introduction: Altered metabolism is a key hallmark of tumour cells (Pavlova & Thompson, 2016) and has long been associated with relapse and resistance to treatment (Gonçalves et al., 2021; Lv et al., 2022). Recently, we identified a new role for histone deacetylase 6 (HDAC6) in the regulation of glycolytic metabolism (Dowling et al., 2021). HDAC6 is an atypical member of the HDAC family as it is localised in the cytosol, has two deacetylase domains, and contains a ubiquitin-binding domain (Gallinari et al., 2007). HDAC6 has been shown to deacetylate proteins involved in processes including microtubule remodelling and stress responses (Hubbert et al., 2002; Kawaguchi et al., 2003). In a recent publication, we showed that HDAC6 knockdown (K/D) or inhibition, with a compound we discovered called BAS-2, altered the acetylation of glycolytic enzymes and reduced glycolysis in triple-negative breast cancer (TNBC) cells (Dowling et al., 2021). However, we did not investigate whether HDAC6 inhibition altered other metabolic pathways at the time. The mitochondrial tricarboxylic acid (TCA) cycle plays an important role in tumour biology, not alone for energy production through the respiratory chain, but also for metabolic intermediates that serve as building blocks for lipid and nucleic acid synthesis (Eniafe & Jiang, 2021). Some of these intermediates of the TCA cycle have dual roles as ‘oncometabolites’ (Frezza, 2017). Both fumarate hydratase (FH) and succinate dehydrogenase (SDH), two key enzymes in the TCA cycle, are known tumour suppressors (Rustin et al., 2002; Schmidt et al., 2020). Loss of FH is associated with an aggressive form of kidney cancer called hereditary leiomyomatosis and renal cell carcinoma (HLRCC) (Tomlinson et al., 2002). Deficiency of FH causes an increase in fumarate, which has various signalling properties including the non-enzymatic formation of 2-(succino)cysteine (2-SC) adducts that can alter protein activity, a process termed succination (Guberovic & Frezza, 2024; Kinch et al., 2011; Tyrakis et al., 2017). Two recent publications identified that macrophages with FH deficiency show an increase of intracellular fumarate and that this alters mitochondrial structure, causing the release of mitochondrial DNA or RNA to activate inflammatory responses (Hooftman et al., 2023; Zecchini et al., 2023). To date, the majority of studies on FH have been carried out using knockout mice to show the role of FH in tumourigenesis or macrophage inflammation (Valcarcel-Jimenez & Frezza, 2023; Zecchini et al., 2023). However, there are limited studies in tumour cells on the role of FH or on how FH activity is regulated. In this study, we identified that HDAC6 can alter mitochondrial structure, causing cristae damage and the release of mtDNA into the cytosol. We then show by mass spectrometry, immunoprecipitation, and super-resolution imaging that HDAC6 and FH directly interact. Inhibition of HDAC6 with BAS-2 reduces FH activity, resulting in an increase of fumarate, with a downstream increase in protein succination. Utilising stochastic optical reconstruction microscopy (STORM), we pinpoint sites of interaction to the mitochondria, thereby demonstrating a novel regulation of FH in tumour cells.

Project description:Abstract Background Vertebral endplate signal intensity changes visualized by magnetic resonance imaging termed Modic changes (MC) are highly prevalent in low back pain patients. Interconvertibility between the three MC subtypes (MC1, MC2, MC3) suggests different pathological stages. Histologically, granulation tissue, fibrosis, and bone marrow edema are signs of inflammation in MC1 and MC2. However, different inflammatory infiltrates and amount of fatty marrow suggest distinct inflammatory processes in MC2. Aims The aims of this study were to investigate i) the degree of bony (BEP) and cartilage endplate (CEP) degeneration in MC2, ii) to identify inflammatory MC2 pathomechanisms, and iii) to show that these marrow changes correlate with severity of endplate degeneration. Methods Pairs of axial biopsies (n=58) spanning the entire vertebral body including both CEPs were collected from human cadaveric vertebrae with MC2. From one biopsy, the bone marrow directly adjacent to the CEP was analyzed with mass spectrometry. Differentially expressed proteins (DEPs) between MC2 were identified and bioinformatic enrichment analysis was performed. The other biopsy was processed for paraffin histology and BEP/CEP degenerations were scored. Endplate scores were correlated with DEPs. Results Endplates from MC2 were significantly more degenerated. Proteomic analysis revealed an activated complement system, increased expression of extracellular matrix proteins, angiogenic, and neurogenic factors in MC2 marrow. Endplate scores correlated with upregulated complement and neurogenic proteins. Discussion The inflammatory pathomechanisms in MC2 comprises activation of the complement system. Concurrent inflammation, fibrosis, angiogenesis, and neurogenesis indicate that MC2 is a chronic inflammation. Correlation of endplate damage with complement and neurogenic proteins suggest that complement system activation and neoinnervation may be linked to endplate damage. The endplate-near marrow is the pathomechanistic site, because MC2 occur at locations with more endplate degeneration. Conclusion MC2 are fibroinflammatory changes with complement system involvement which occur adjacent to damaged endplates.

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Glycoproteomics analysis of Complement Factor H and its effect on complement regulatory function during Streptococcus pneumoniae-associated hemolytic uremic syndrome

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