Dataset Information

Mapping sites of O-fucose modification on mouse NOTCH2

ABSTRACT: The O-fucose modification sites on mouse NOTCH2 were determined by mass spectral glycoproteomic site mapping. The Extracellular Domain of mouse NOTCH2 was produced in HEK293T cells in the presence or absence of the three Fringe enzymes: Lunatic fringe, Manic fringe, or Radical fringe. The protein was purified from the medium, reduced and alkylated, digested with trypsin, chymotrypsin or V8 protease, and the resulting peptides were analyzed by nano-LC mass spectrometry. The majority of the sites were identified using an Agilent model 6340 Ion Trap mass spectrometer. A few sites were identified using a Q-Exactive Plus mass spectrometer.

INSTRUMENT(S): 6340 Ion Trap LC/MS, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Robert Haltiwanger

LAB HEAD: Robert S. Haltiwanger

PROVIDER: PXD020431 | Pride | 2020-09-11

REPOSITORIES: Pride

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Publications

Canonical Notch ligands and Fringes have distinct effects on NOTCH1 and NOTCH2.

Kakuda Shinako S LoPilato Rachel K RK Ito Atsuko A Haltiwanger Robert S RS

The Journal of biological chemistry 20200819 43

Notch signaling is a cellular pathway regulating cell-fate determination and adult tissue homeostasis. Little is known about how canonical Notch ligands or Fringe enzymes differentially affect NOTCH1 and NOTCH2. Using cell-based Notch signaling and ligand-binding assays, we evaluated differences in NOTCH1 and NOTCH2 responses to Delta-like (DLL) and Jagged (JAG) family members and the extent to which Fringe enzymes modulate their activity. In the absence of Fringes, DLL4-NOTCH1 activation was mo ...[more]

PMID: 32820046

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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

Mapping sites of O-fucose modification on mouse NOTCH2

Publications

Canonical Notch ligands and Fringes have distinct effects on NOTCH1 and NOTCH2.

Similar Datasets

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