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Altered N-glycan composition impacts flagella mediated adhesion in Chlamydomonas reinhardtii

ABSTRACT: For the unicellular alga Chlamydomonas reinhardtii, the presence of N-glycosylated proteins on the surface of two flagella is crucial for both cell-cell interaction during mating and flagellar surface adhesion. However, it is not known whether only the presence or also the composition of N-glycans attached to respective proteins is important for these processes. To this end, we tested several C. reinhardtii insertional mutants and a CRIPSR/Cas9 knockout mutant of xylosyltransferase 1A, all possessing altered N-glycan compositions. Taking advantage of atomic force microscopy and micropipette force measurements, our data revealed that reduction in N-glycan complexity impedes the adhesion force required for binding the flagella to surfaces as demonstrated by force spectroscopy and impairs polystyrene bead binding and transport. Notably, assembly, Intraflagellar Transport and FMG-1B transport into flagella are not affected by altered N-glycosylation. Thus, we conclude that proper N-glycosylation of flagellar proteins is crucial for adhering C. reinhardtii cells onto surfaces, indicating that N-glycans mediate surface adhesion via direct surface contact. Data deposited here include IS-CID data verifying altered N-glycan patterns in flagella of mutants analysed (Ursgal-SugarPy-flagella-IM-results.xls) as well as the N-glycoproteomic characterization of the novel CRISPRXylT1A mutant (Ursgal-SugarPy-CIRSPR-mutant-results.xls). Regarding the latter, PRM measurements verifying the knock out of XylT1A are also deposited here (Results_PRM-measurement_quantification_GT.xls). Further, label free quantification data on isolated flagella of original IM strains verifying that altered N-glycosylation does not impact flagellar protein localization can be found within this dataset (Flagella_IM-WT_ normalized_5_proteins.pdResult).

INSTRUMENT(S): Q Exactive

ORGANISM(S): Chlamydomonas Reinhardtii

SUBMITTER: Lara Hoepfner

LAB HEAD: Michael Hippler

PROVIDER: PXD018353 | Pride | 2020-12-22

REPOSITORIES: Pride

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Source:

			Action	DRS
	AO_IS_wo-lock_80VISF_E8_17042019.raw	Raw
	AO_IS_wo-lock_80VISF_H12_17042019.raw	Raw
	AO_IS_wo-lock_80VISF_SAG-1_17042019.raw	Raw
	AO_IS_wo-lock_80VISF_SAG-2_17042019.raw	Raw
	AO_ManI_flagella_III_23082018_top12.raw	Raw

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Project description:Colorectal cancer, one of the most frequent types of malignancy in the Western world, develops through a multi-step process. The main pathways establishing transformation of normal mucosa to invasive carcinoma include chromosomal instability (CIN), microsatellite instability (MSI) or epigenetic silencing through the CpG Island Methylator Phenotype (CIMP). These pathways have distinct clinical, pathological and genetic characteristics. In general, altered cell surface glycosylation has been linked to colorectal cancer progression, however the impact of MSI-specific pathways on the glycosylation machinery of MSI colon cancer cells has not been investigated yet. In a recent study (Patsos et al., 2009) we have shown that MSI-specific mutations induce marked alterations in cell surface glycosylation, indicating specific changes in the expression of glyco-genes. Therefore the purpose of our experiment is to define these changes by glyco-gene chip analysis. Biallelic mutational inactivation of MSI target genes is believed to drive MSI tumorigenesis. TGFBR2, ACVR2 and AIM2 are among the most frequently mutated genes in MSI colorectal adenomas and carcinomas and functional studies indicate their contribution to MSI-carcinogenesis. A first general screening approach by lectin FACS analysis indicated a correlation between functional inactivation of these genes and cell surface glycosylation of the MSI colon cell line HCT116 (Patsos et al., 2009). For detailed analysis of the altered glycosylation pattern and the underlying molecular mechanisms we have established HCT116 double stable transfectants that allow doxycycline-inducible expression of the three MSI target genes TGFBR2, ACVR2 and AIM2 in a reversible manner. Thus we can experimentally control the expression of the MSI-genes and analyze short and long-term effects on different cell functions, including glycosylation. From the application of the glyco-gene chip for analysis of the transfectants we expect to gain seminal information on the influence of MSI-target genes on glycan expression and function in MSI tumor cells. Correlating loss-of-function of MSI target genes not yet linked with glycosylation to the regulation of the glycophenotype is a novel approach to investigate the glycobiology of tumor cells. In addition it is also suitable to detect new MSI-(glyco)markers for clinical applications, including diagnostic tumor markers and specific targets for immunotherapies.

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Altered N-glycan composition impacts flagella mediated adhesion in Chlamydomonas reinhardtii

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