RRC ID 70292
Author Niwa Y, Nakano Y, Suzuki T, Yamagishi M, Otani K, Dohmae N, Simizu S.
Title Topological analysis of DPY19L3, a human C-mannosyltransferase.
Journal FEBS J
Abstract C-mannosylation is a rare type of protein glycosylation, the functions and mechanisms of which remain unclear. Recently, we identified DPY19L3 as a C-mannosyltransferase of R-spondin1 in human cells. DPY19L3 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 DPY19L3 by in silico analysis and experimental methods such as redox-sensitive luciferase assay and introduction of N-glycosylation sites, suggesting that DPY19L3 comprises 11 transmembrane regions and two re-entrant loops with the N- and C-terminal ends facing the cytoplasm and ER lumen, respectively. Furthermore, DPY19L3 has four predicted N-glycosylation sites, and we have demonstrated that DPY19L3 is N-glycosylated at Asn118 and Asn704 but not Asn319 and Asn439 , supporting our topological model. By mass spectrometry, we measured the C-mannosyltransferase activity of N-glycosylation-defective mutants of DPY19L3 and isoform2, a splice variant, which lacks the C-terminal luminal region of DPY19L3. Isoform2 does not possess C-mannosyltransferase activity, indicating the importance of the C-terminal region; however, N-glycosylations of DPY19L3 do not have any roles for its enzymatic activity. These novel findings on DPY19L3 provide important insights into the mechanism of C-mannosylation.
Volume 285(6)
Pages 1162-1174
Published 2018-3-1
DOI 10.1111/febs.14398
PMID 29405629
IF 4.392
Resource
DNA material pCI-neo-DPY19L3(wt)-Gluc(C-term) (RDB19523) pCI-neo-DPY19L3(isoform2)-Gluc (RDB19524) pCI-neo-DPY19L3(wt)-Gluc(N-term) (RDB19525) pCI-neo-DPY19L3(E80)-Gluc (RDB19526) pCI-neo-DPY19L3(K151)-Gluc (RDB19527) pCI-neo-DPY19L3(G183)-Gluc (RDB19528) pCI-neo-DPY19L3(V205)-Gluc (RDB19529) pCI-neo-DPY19L3(T208)-Gluc (RDB19530) pCI-neo-DPY19L3(P237)-Gluc (RDB19531) pCI-neo-DPY19L3(Q262)-Gluc (RDB19532) pCI-neo-DPY19L3(K285)-Gluc (RDB19533) pCI-neo-DPY19L3(N307)-Gluc (RDB19534) pCI-neo-DPY19L3(N339)-Gluc (RDB19535) pCI-neo-DPY19L3(A400)-Gluc (RDB19536) pCI-neo-DPY19L3(T456)-Gluc (RDB19537) pCI-neo-DPY19L3(L486)-Gluc (RDB19538) pCI-neo-DPY19L3(Y515)-Gluc (RDB19539) pCI-neo-DPY19L3(2NQ)-Gluc (RDB19544) pCI-neo-DPY19L3(2NQ+141NV-in)-Gluc (RDB19545)