A suite of tools for the study of tryptophan C-mannosylation - additional point mutants
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive Plus
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Nichollas Scott
LAB HEAD: Ethan Goddard-Borger
PROVIDER: PXD021612 | Pride | 2020-11-16
REPOSITORIES: Pride
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Project description:Tryptophan C-mannosylation is a co-translational modification unique to the metazoans and apicomplexan parasites. It is the only example of protein C-glycosylation reported to date and appears to play a role in protein folding, trafficking and/or stability. Our knowledge of the prevalence of this modification and its role in biology is limited, and hampered by a lack of tools for studying the modification. Here, we present a simple microbial system for the production of proteins with and without the modification, to facilitate characterisation of the effects of tryptophan mannosylation on protein stability and function. This system facilitates exploration of the C-mannosyltransferase substrate preference and catalytic machinery. The ability to readily produce proteins with this modification facilitated the generation and rigours characterisation of monoclonal antibodies capable of detecting C-mannosyl tryptophan. With these antibodies, and a robust expression system, we were able to identify the first small molecule capable of inhibiting C-mannosyltransferases: a susbtrate-mimicking peptide that is active in cells. Together, these tools provide a solid foundation for interrogating the role of C-mannosyl tryptophan in myriad biological systems.
2020-11-16 | PXD018401 | Pride
Project description:C-mannosylation stabilizes proteins bearing a thrombospondin repeat (TSR) domain in metazoans. Here we show that Plasmodium falciparum expresses a DPY19 C-mannosyltransferase in the endoplasmic reticulum and that DPY19-deficiency abolishes C-glycosylation, destabilizes members of the TRAP adhesin family and inhibits transmission to mosquitoes. P. falciparum gametogenesis was imaged in its entirety in four dimensions using lattice light-sheet microscopy. This revealed defects in egress and exflagellation for DPY19 microgametes. While exflagellation was diminished, DPY19 microgametes still fertilized macrogametes, forming ookinetes but these were abrogated for mosquito infection. The gametogenesis defects corresponded with destabilization of MTRAP, which we show is C-mannosylated in P. falciparum, and the ookinete defect was concordant with defective CTRP secretion on the DPY19 background. Genetic complementation of DPY19 restored ookinete infectivity, sporozoite production and C-mannosylation activity. Therefore, tryptophan C-mannosylation by DPY19 in the early secretory pathway ensures TSR protein quality control at two lifecycle stages for successful transmission of the human malaria parasite.
2022-07-06 | PXD033470 | Pride
Project description:C-mannosylation is a modification of tryptophan residues with a single mannose effecting protein folding, secretion and/or function. To date, only few proteins have been proven to be C-mannosylated and studies aiming at global assessment of protein C-mannosylation from cells or tissues are scarce. In order to interrogate the C-mannosylome of human induced pluripotent stem cells (hiPSCs), we made use of the common finding that C-mannosylation is important for protein secretion. Thus we compared the secretomes of C-mannosyltransferase-deficient DPY19L1 and DPY19L3 mutants to their parental wild-type hiPSCs by mass-spectrometry-based quantitative proteomics. Secretion of numerous proteins was reduced in the mutants.
2021-06-17 | PXD024193 | Pride
Project description: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.
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Project description:Protein O-mannosylation is found in yeast and metazoans and a family of conserved orthologous polypeptide O-mannosyltransferases is believed to initiate this important posttranslational modification. We recently discovered that the large families of cadherins and protocadherins carry highly conserved O-Man glycans in specific EC domains, and it was suggested that the function of E-Cadherin was dependent on the O-Man glycans. Deficiencies in the two human polypeptide O-mannosyltransferases, POMT1 and T2, underlie a subgroup of congenital muscular dystrophies (CMD) designated α-dystroglycanopathies, because deficient O-Man glycans on -dystroglycan impair laminin interaction with -dystroglycan and the dystrophin complex. In order to explore the functions of O-Man glycans on cadherins and protocadherins we used a combinatorial gene editing strategy in multiple cell lines to evaluate to the role of the two POMTs initiation O-Man glycosylation and the major enzyme elongating O-Man glycans, the POMGNT1 2GlcNAc-transferase. Surprisingly, we discovered that O-Man glycans on cadherins and protocadherins do not appear to require POMT1 and T2 and moreover that the O-Man glycans on these proteins are not elongated in contrast to those on dystroglycan.
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Project description:Protein O-mannosylation is found in yeast and metazoans and a family of conserved orthologous polypeptide O-mannosyltransferases is believed to initiate this important posttranslational modification. We recently discovered that the large families of cadherins and protocadherins carry highly conserved O-Man glycans in specific EC domains, and it was suggested that the function of E-Cadherin was dependent on the O-Man glycans. Deficiencies in the two human polypeptide O-mannosyltransferases, POMT1 and T2, underlie a subgroup of congenital muscular dystrophies (CMD) designated α-dystroglycanopathies, because deficient O-Man glycans on -dystroglycan impair laminin interaction with -dystroglycan and the dystrophin complex. In order to explore the functions of O-Man glycans on cadherins and protocadherins we used a combinatorial gene editing strategy in multiple cell lines to evaluate to the role of the two POMTs initiation O-Man glycosylation and the major enzyme elongating O-Man glycans, the POMGNT1 2GlcNAc-transferase. Surprisingly, we discovered that O-Man glycans on cadherins and protocadherins do not appear to require POMT1 and T2 and moreover that the O-Man glycans on these proteins are not elongated in contrast to those on dystroglycan.
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