Dataset Information

Thrombospondin repeats secreted by C-mannosyltransferase-deficient mammalian cells

ABSTRACT: C-mannosylation in addition to N- and O-glycosylation is a further but less well studied type of protein glycosylation taking place in the endoplasmic reticulum (ER) characterised by the modification of tryptophan residues with a single mannose effecting protein folding, secretion and/or function. Motivated by an interest in the functional role of C-mannosylation for early developmental processes, we aimed for the functional inactivation of the C mannosyltransferases DPY19L1 and DPY19L3, respectively, and excised parts of their coding sequence from the genome of the hiPSC line CBiPSC2 by applying CRISPR Cas9. To determine the effect of the genomic deletions in DPY19L1 or DPY19L3 on C-mannosylation, TSR2 and TSR3 of human thrombospondin 1 (THBS1) was recombinantly expressed in WT and KO hiPSCs followed by purification an LC-MS analysis. The results are in accordance to our previous findings that DPY19L1 is mainly acting on W1 and W2 whereas DPY19L3 acts on W3 of WxxWxxWxxC motifs of TSRs and proves the functional inactivation of the respective C-mannosyltransferases in the hiPSCs model. A secretome analysis of C mannosyltransferase deficient hiPSCs revealed that secretion of numerous proteins was reduced in the mutants including ADAMTS16, which was previously reported to be essential for optic fissure fusion in zebrafish. In order to analyse C-mannosylation of ADAMTS16, its TSR1 was recombinantly expressed in CHO-K1 WT, DPY19L1 KO and DPY19L3 KO cells, purified and analysed by LC MS analysis. The results revealed that the WSDWSSWSPC motif of TSR1 of ADAMTS16 can be C-mannosylated at all three tryptophan residues. Deletion of DPY19L1 prevented C-mannosylation of the two first tryptophans whereas C-mannosylation of the third tryptophan was not detected in the DPY19L3-mutant.

INSTRUMENT(S): Q-Tof ultima

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Karsten Cirksena

LAB HEAD: Falk Büttner

PROVIDER: PXD024192 | Pride | 2021-06-17

REPOSITORIES: Pride

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Publications

The C-Mannosylome of Human Induced Pluripotent Stem Cells Implies a Role for ADAMTS16 C-Mannosylation in Eye Development.

Cirksena Karsten K Hütte Hermann J HJ Shcherbakova Aleksandra A Thumberger Thomas T Sakson Roman R Weiss Stefan S Jensen Lars Riff LR Friedrich Alina A Todt Daniel D Kuss Andreas W AW Ruppert Thomas T Wittbrodt Joachim J Bakker Hans H Buettner Falk F R FFR

Molecular & cellular proteomics : MCP 20210508

C-mannosylation is a modification of tryptophan residues with a single mannose and can affect protein folding, secretion, and/or function. To date, only a few proteins have been demonstrated to be C-mannosylated, and studies that globally assess protein C-mannosylation are scarce. To interrogate the C-mannosylome of human induced pluripotent stem cells, we compared the secretomes of CRISPR-Cas9 mutants lacking either the C-mannosyltransferase DPY19L1 or DPY19L3 to WT human induced pluripotent st ...[more]

PMID: 33975020

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Project description:Myeloperoxidase (MPO), an important diprotomeric glycoprotein in neutrophil-mediated immunity, produces microbicidal hypohalous acids, but the underpinning glycobiology remains elusive. Deep characterisation of neutrophil-derived MPO (nMPO) using advanced mass spectrometry demonstrated that under-processed oligomannosidic- and hyper-truncated paucimannosidic- and N-acetyl-β-D-glucosamine (GlcNAc) core-type asparagine-linked glycans decorate the protein. Occlusion of Asn355 and Asn391 and sterical hindrance of Asn323- and Asn483-glycans located in the MPO dimerisation zone were found to shape the local glycan processing thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry, and glycopeptide profiling revealed extreme molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants, and a lower-abundance monomer. Longitudinal profiling of maturing, mature, granule-separated, and pathogen-activated neutrophils demonstrated that MPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and rapidly degranulated, but surprisingly carries uniform glycosylation across conditions. Complete proMPO-to-MPO maturation evidently occur during early/mid-stage granulopoiesis. The conserved Asn355- and Asn391-sequons displayed elevated GlcNAc signatures and higher oxidation and chlorination activity of the secretory vesicle/plasma membrane-resident MPO relative to MPO from other granules. Endoglycosidase H-treated nMPO displaying Asn355-/Asn391-GlcNAcylation recapitulated the activity gain and showed increased thermal stability and polypeptide accessibility relative to untreated nMPO as measured by activity assays, circular dichroism and molecular dynamics. Endoglycosidase H-treated nMPO also demonstrated elevated ceruloplasmin-mediated inhibition relative to nMPO. Modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO:ceruloplasmin interface are critical for uninterrupted inhibition. We report on novel bimodal roles of the peculiar MPO glycosylation providing new insight into neutrophil glycobiology.

Dataset Information

Thrombospondin repeats secreted by C-mannosyltransferase-deficient mammalian cells

Publications

The C-Mannosylome of Human Induced Pluripotent Stem Cells Implies a Role for ADAMTS16 C-Mannosylation in Eye Development.

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