Membrane Topological Model of Glycosyltransferases of the GT-C Superfamily

Abstract

Glycosyltransferases that use polyisoprenol-linked donor substrates are categorized in the GT-C superfamily. In eukaryotes, they act in the endoplasmic reticulum (ER) lumen and are involved in N-glycosylation, glypiation, O-mannosylation, and C-mannosylation of proteins. We generated a membrane topology model of C-mannosyltransferases (DPY19 family) that concurred perfectly with the 13 transmembrane domains (TMDs) observed in oligosaccharyltransferases (STT3 family) structures. A multiple alignment of family members from diverse organisms highlighted the presence of only a few conserved amino acids between DPY19s and STT3s. Most of these residues were shown to be essential for DPY19 function and are positioned in luminal loops that showed high conservation within the DPY19 family. Multiple alignments of other eukaryotic GT-C families underlined the presence of similar conserved motifs in luminal loops, in all enzymes of the superfamily. Most GT-C enzymes are proposed to have an uneven number of TDMs with 11 (POMT, TMTC, ALG9, ALG12, PIGB, PIGV, and PIGZ) or 13 (DPY19, STT3, and ALG10) membrane-spanning helices. In contrast, PIGM, ALG3, ALG6, and ALG8 have 12 or 14 TMDs and display a C-terminal dilysine ER-retrieval motif oriented towards the cytoplasm. We propose that all members of the GT-C superfamily are evolutionary related enzymes with preserved membrane topology.

Keywords: C-mannosylation; GPI-anchor; N-glycosylation; O-mannosylation; dolichol-phosphate; endoplasmic reticulum; glycosyltransferase; mannose; mannosyltransferase; oligosaccharyltransferase.

Figure 1

Human enzymes using dolichol-linked donor substrates. The official protein symbols (www.genames.org) of all known human enzymes are indicated. PIG (phosphatidylinositol glycan anchor) enzymes generate the GPI (glycosylphosphatidylinositol)anchor. ALG (asparagine-linked glycosylation) enzymes transfer four mannoses and three glucoses to the lipid-linked oligosaccharide before it is transferred to proteins by the oligosaccharyltransferase complex. STT3 (staurosporine- and temperature-sensitive) is the catalytic subunit of the oligosaccharyltransferase and occurs in two forms. Two POMTs (protein O-mannosyltransferases) and four TMTC (transmembrane and tetratricopeptide repeat containing) proteins transfer O-mannose to protein. Finally, four homologues of the Caenorhaditis elegans C-mannosyltransferase DPY-19 are present in human.

Figure 2

(A) Transmembrane probability of STT3A and DPY19L1. Transmembrane domain prediction was carried out using the algorithm PolyPhobius with a multiple alignment comprising 25 most diverse members of the two families, as described in experimental procedures. Proposed transmembrane domain (TMD) numbering is based on the structure of bacterial Campylobacter lari oligosaccharyltransferase [21]. (B) Upper panel: Conserved residues involved in catalysis in STT3. Lower panel: Conserved areas (arrows in Figure 2A) in extracytoplasmic (luminal) loops (EL) of the STT3 and DPY19 family. *E in EL5 is part of the catalytic pocket, but due to the large size of this loop and the presence of multiple D (aspartic acid) and E (glutamic acid) residues, no clear equivalent can be observed in DPY19 enzymes (see supplemental Figure S1A,B). The asparagine (N) in EL3 is highly conserved in STT3, but has not been associated with the catalytic pocket.

Figure 3

Secretion and C-mannosylation of an UNC-5 fragment modified by native and mutated C. elegans DPY-19. (A) Secretion of UNC-5 from S2 cells grown at 21 and 28 °C. A V5-His₆-tagged UNC-5 construct encompassing the two TSRs was transfected in S2 cells without (−) or with (+) C. elegans DPY-19 or with C. elegans DPY-19 carrying the mutation E63A, E65A, R211A, E220A, E400A, or R471A. A plasmid expressing V5-tagged epidermial growth factor (EGF)-repeats 16–20 (EGF) of Drosophila melanogaster Notch was used as a transfection and secretion control. Transfected cells were grown at 21 or 28 °C, and secreted proteins were detected on Western blot using an anti-V5 antibody. (B) Extracted ion chromatograms of the unmodified, mono-C-mannosylated, and di-C-mannosylated TSR2 tryptic peptide LDGGWSSWSDWSACSSSCHR. All graphs are shown with equal intensities (13,000 counts per second).

Figure 4

Predicted membrane topology and amino acid conservation of human POMT1 and TMTC1. Transmembrane domain prediction was carried out using the algorithm PolyPhobius, as described in experimental procedures. The position of the predicted extracytoplasmic loops EL1, EL2, EL3 and EL5 is indicated by arrows and amino acid conservation in these loops is shown in the sequence logos.

Figure 5

Membrane topology prediction of other eukaryotic glycosyltransferases of the GT-C superfamily. Arrows indicate the positions of the conserved areas that are depicted in Figure 6, corresponding to EL1, EL2, EL3, and EL5 (EL6 for ALG10, due to the two additional TMDs). The dilysine motif at the C-terminus of proteins with even numbers of predicted TMDs is shown.

Figure 6

Conserved domains in extracytoplasmic loops of GT-C enzymes. Conserved domains in EL1, EL2, EL3, and EL5 (EL6 for ALG10) of indicated GT-C members, presented as logos. The positions correspond to arrows in Figure 5.

Figure 7

General membrane topology model of glycosyltransferase-C members. Location of the four conserved extracytoplasmic loops in the GT-C families are indicated by color-coded balls representing acidic amino acids. The 11 TMDs (numbered in black) present in all GT-C members are depicted. Inserted TMDs (in broken lines) increase the number of TMDs to 13 (blue) or to 12/14 with the C-terminus in the cytoplasm (red).