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Topological analysis of DPY 19L3, a human C ‐mannosyltransferase
Author(s) -
Niwa Yuki,
Nakano Yoshihiko,
Suzuki Takehiro,
Yamagishi Mizuo,
Otani Kei,
Dohmae Naoshi,
Simizu Siro
Publication year - 2018
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.14398
Subject(s) - glycosylation , transmembrane protein , endoplasmic reticulum , in silico , n linked glycosylation , transmembrane domain , chemistry , cytoplasm , mutant , microbiology and biotechnology , biology , topology (electrical circuits) , biochemistry , gene , glycoprotein , receptor , mathematics , combinatorics , glycan
C ‐mannosylation is a rare type of protein glycosylation, the functions and mechanisms of which remain unclear. Recently, we identified DPY 19L3 as a C ‐mannosyltransferase of R‐spondin1 in human cells. DPY 19L3 is predicted to be a multipass transmembrane protein that localizes to the endoplasmic reticulum ( ER ); however, its structure is undetermined. In this study, we propose a topological structure of DPY 19L3 by in silico analysis and experimental methods such as redox‐sensitive luciferase assay and introduction of N ‐glycosylation sites, suggesting that DPY 19L3 comprises 11 transmembrane regions and two re‐entrant loops with the N‐ and C‐terminal ends facing the cytoplasm and ER lumen, respectively. Furthermore, DPY 19L3 has four predicted N ‐glycosylation sites, and we have demonstrated that DPY 19L3 is N ‐glycosylated at Asn 118 and Asn 704 but not Asn 319 and Asn 439 , supporting our topological model. By mass spectrometry, we measured the C ‐mannosyltransferase activity of N ‐glycosylation‐defective mutants of DPY 19L3 and isoform2, a splice variant, which lacks the C‐terminal luminal region of DPY 19L3. Isoform2 does not possess C ‐mannosyltransferase activity, indicating the importance of the C‐terminal region; however, N ‐glycosylations of DPY 19L3 do not have any roles for its enzymatic activity. These novel findings on DPY 19L3 provide important insights into the mechanism of C ‐mannosylation.