Volume 50, Issue 2, Pages 295-302 (April 2013) C. elegans DPY-19 Is a C-Mannosyltransferase Glycosylating Thrombospondin Repeats  Falk F.R. Buettner, Angel Ashikov, Birgit Tiemann, Ludwig Lehle, Hans Bakker  Molecular Cell  Volume 50, Issue 2, Pages 295-302 (April 2013) DOI: 10.1016/j.molcel.2013.03.003 Copyright © 2013 Elsevier Inc. Terms and Conditions

Molecular Cell 2013 50, 295-302DOI: (10.1016/j.molcel.2013.03.003) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 OST and DPY-19 Are Homologous Proteins (A) Structure of the α-mannose in C-C linkage to the C2 of the indole ring of tryptophan. (B) Comparison of the reaction catalyzed by OST and C-mannosyltransferase (CmanT). The common features of the reactions are the dolichol-phosphate of the donor substrate and the transfer of the sugar moiety to protein, although in different linkage. Shown is the typical oligosaccharide transferred by eukaryotic OSTs; the exact structure might vary and is different in bacteria (Schwarz and Aebi, 2011). (C) Prediction of transmembrane helices of C. elegans DPY-19 and OST of Campylobacter lari using the TMHMM prediction program. The numbering of transmembrane domains is based on an alignment between DPY-19 and OST and on the OST crystal structure (Lizak et al., 2011). The alignment of a selection of most distant proteins shows conservation of two luminal loops of DPY-19, which is restricted to Metazoa, Alveolata, and Choanoflagellata, and OST/STT3, found in bacteria and eukaryotes. Amino acids marked red in the C. lari sequence are predicted to bind to Dol-P (Lizak et al., 2011). Three of these amino acids are conserved in DPY-19. Sequences in the alignment are derived from the NCBI protein database (http://www.ncbi.nlm.nih.gov/protein) with accession numbers (top to bottom) NP_498909, NP_056098, XP_001625259, XP_002371434, EGD74485, CAA96722, AAL07040, NP_689926, CAB61569, and YP_002575824. Molecular Cell 2013 50, 295-302DOI: (10.1016/j.molcel.2013.03.003) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 C-Mannosyltransferase Activity of DPY-19 (A) Tritium-labeled Dol-P-Man is generated from labeled GDP-Man and used as donor by the C-mannosyltransferase to mannosylate the synthetic peptide WAKW, presumably on the first tryptophan based on the reported specificity of C-mannosylation in human cells (Doucey et al., 1999). (B) DPY-19 or an empty vector control (pIB) was expressed in S2 cells and assayed for activity. The assay contained ER-enriched vesicles, the radiolabeled substrate GDP-[3H]Man, the intermediate substrate Dol-P, and acceptor peptide WAKW or the nonacceptor peptide WAKA as control. (C) Wild-type C. elegans and mutant dpy-19(e1295) worms were assayed using the same method. All assays were carried out in triplicate and indicated as mean values ± SD. Molecular Cell 2013 50, 295-302DOI: (10.1016/j.molcel.2013.03.003) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Activity of DPY-19 on C. elegans Acceptor Proteins (A) Domain structure of MIG-21 and UNC-5, two C. elegans membrane-linked proteins containing two TSRs. For expression in Drosophila S2 cells, the two TSRs of each protein were cloned in pMT/BiP/V5-His with a copper-inducible promoter, resulting in secretion of V5-His-tagged fragments. (B) Western blot of UNC-5 TSRs expressed in S2 cells with or without coexpression of DPY-19. Protein was detected in cell pellets and cell supernatants using the anti-V5 antibody. (C) Purified secreted UNC-5 fragments were cut from SDS-PAGE gels stained with Coomassie blue and analyzed by LC-MS after tryptic digestion. The peptide of TSR2 containing the C-mannosylation consensus sequence was only observed in the unmannosylated form when expressed without DPY-19 (upper panels). Weak signals around the expected masses of the triple-charged mono- or dimannosylated peptide (inserts in middle and right panel) do not correspond to the exact masses of glycosylated peptides. In cells expressing DPY-19, the unmannosylated peptides were greatly reduced at the expense of peptides with one or two hexoses (green circles), with the doubly glycosylated form being most abundant. TSR1 showed a similar shift in glycosylation upon expression of DPY-19 (Figure S1) MS/MS confirmed the identity of all mass peaks (Figure S2), and signal quantification was carried out by extracted ion chromatograms (Figure S3). (D) MIG-21 was expressed in S2 cells as described for UNC-5. Without DPY-19, MIG-21 is not observed in the medium. Intracellularly, a small increase in apparent MW can be observed as a result of DPY-19 expression. The band at slightly higher MW might be interpreted as the protein still containing the signal sequence (an increase of 1.8 kDa). (E) MS signals corresponding to a mono- or dimannosylated MIG-21 tryptic peptide could be observed in secreted MIG-21 from cells coexpressing DPY-19. The protein contains the WSTWSKW sequence, which is cleaved by trypsin between the second and third W. Signal quantification was determined by extracted ion chromatograms (Figure S3). ∗The peptide mass does not reveal the position of the mannose. Based on the known specificity of C-mannosyltransferases, monomannosylation is expected to occur on the first (as shown) or second tryptophan and dimannosylation on the first and second tryptophan. Molecular Cell 2013 50, 295-302DOI: (10.1016/j.molcel.2013.03.003) Copyright © 2013 Elsevier Inc. Terms and Conditions