PXD024192

PXD024192 is an original dataset announced via ProteomeXchange.

Title	Thrombospondin repeats secreted by C-mannosyltransferase-deficient mammalian cells
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.
HostingRepository	PRIDE
AnnounceDate	2021-06-18
AnnouncementXML	Submission_2021-06-18_00:00:45.051.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Karsten Cirksena
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	complex glycosylation
Instrument	Q-Tof Ultima

Revision	Datetime	Status	ChangeLog Entry
0	2021-02-15 23:58:50	ID requested
⏵ 1	2021-06-18 00:00:46	announced

Cirksena K, H, ü, tte HJ, Shcherbakova A, Thumberger T, Sakson R, Weiss S, Jensen LR, Friedrich A, Todt D, Kuss AW, Ruppert T, Wittbrodt J, Bakker H, Buettner FFR, The C-Mannosylome of Human Induced Pluripotent Stem Cells Implies a Role for ADAMTS16 C-Mannosylation in Eye Development. Mol Cell Proteomics, 20():100092(2021) [pubmed]

submitter keyword: C-mannosylation, DPY19L1, DPY19L3, TSR, THBS1, ADAMTS16

Falk Büttner
contact affiliation	Hannover Medical School Institute for Clinical Biochemistry (OE 4340) Carl-Neuberg-Strasse 1 Building J03, Level 04, Room 1291 (Office), Room 1250 (Lab) 30625 Hannover
contact email	Buettner.Falk@mh-hannover.de
lab head
Karsten Cirksena
contact affiliation	Hannover Medical School
contact email	cirksena.karsten@mh-hannover.de
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/06/PXD024192

PRIDE project URI

• PRIDE
  1. PXD024192
     1. Label: PRIDE project
     2. Name: Thrombospondin repeats secreted by C-mannosyltransferase-deficient mammalian cells