Dolichol phosphate mannose synthase: a Glycosyltransferase with Unity in molecular diversities

Abstract

N-glycans provide structural and functional stability to asparagine-linked (N-linked) glycoproteins, and add flexibility. Glycan biosynthesis is elaborative, multi-compartmental and involves many glycosyltransferases. Failure to assemble N-glycans leads to phenotypic changes developing infection, cancer, congenital disorders of glycosylation (CDGs) among others. Biosynthesis of N-glycans begins at the endoplasmic reticulum (ER) with the assembly of dolichol-linked tetra-decasaccharide (Glc₃Man₉GlcNAc₂-PP-Dol) where dolichol phosphate mannose synthase (DPMS) plays a central role. DPMS is also essential for GPI anchor biosynthesis as well as for O- and C-mannosylation of proteins in yeast and in mammalian cells. DPMS has been purified from several sources and its gene has been cloned from 39 species (e.g., from protozoan parasite to human). It is an inverting GT-A folded enzyme and classified as GT2 by CAZy (carbohydrate active enZyme; http://www.cazy.org ). The sequence alignment detects the presence of a metal binding DAD signature in DPMS from all 39 species but finds cAMP-dependent protein phosphorylation motif (PKA motif) in only 38 species. DPMS also has hydrophobic region(s). Hydropathy analysis of amino acid sequences from bovine, human, S. crevisiae and A. thaliana DPMS show PKA motif is present between the hydrophobic domains. The location of PKA motif as well as the hydrophobic domain(s) in the DPMS sequence vary from species to species. For example, the domain(s) could be located at the center or more towards the C-terminus. Irrespective of their catalytic similarity, the DNA sequence, the amino acid identity, and the lack of a stretch of hydrophobic amino acid residues at the C-terminus, DPMS is still classified as Type I and Type II enzyme. Because of an apparent bio-sensing ability, extracellular signaling and microenvironment regulate DPMS catalytic activity. In this review, we highlight some important features and the molecular diversities of DPMS.

Keywords: Asparagine-linked glycoprotein; Carbohydrate active enzyme; Dolichol phosphate mannose synthase; Glycosyltransferase; Phosphoprotein.

Figure 1. ER Localization of Dol-P and DPMS

(a) Detection of Dol-P: Fixed capillary endothelial cells were incubated with amphomycin in a buffer containing Ca²⁺ and stained with anti-amphomycin mouse monoclonal antibody [8] followed by FITC-conjugated rabbit anti-mouse IgG secondary antibody before collecting images in a fluorescence microscope. (b) Detection of DPMS: Fixed capillary endothelial cells were incubated with anti-DPMS rabbit polyclonal antibody [9] followed by Rhodamin-conjugated goat anti-rabbit IgG before collecting images in a fluorescence microscope. (c) Detection of Dol-P and DPMS: Fixed capillary endothelial cells were incubated with amphomycin in a buffer containing Ca²⁺ followed by anti-amphomycin antibody (mouse monoclonal) and then with anti-DPMS antibody (rabbit polyclonal) as above. This followed staining with FITC-conjugated rabbit anti-mouse IgG and Rhodamin-conjugated goat anti-rabbit IgG antibodies prior to collecting images in a fluorescence microscope. (d) Detection of Dol-P by electron microcopy: Thin (60nm–100nm) sections of capillary endothelial cells on copper grids treated with amphomycin in a buffer containing Ca²⁺ followed by anti-amphomycin antibody. The immune complexes then treated with 20nm gold conjugated-rabbit anti-mouse IgG before collecting images in a Phillips −200 electron microscope at a magnification of 20,000.

Figure 2. Unrooted phylogenetic tree with branch lengths for DPM1 sequences from 39 species

The sequence sources of DPM1 from different species are from Table 1. After alignment the dendogram was generated using the CLUSTAL W program [43] on the GenomeNet server (http://www.genome.jp/tools/clustalw/). GenomeNet is a Japanese network of database and computational services for genome research operated by the Kyoto University is Bioinformatics Center.

Figure 3. Hydropathy profile of DPMS from four different species

For scanning the polypeptide sequences of DPMS, 19 amino acids faulted as one window and hydropathy index was calculated based on one window. (a) Bovine capillary endothelial cells; (b) human; (c) S. cerevisiae; and (d) A. thaliana. The hydropathic character of DPMS is computed under ExpASy Bioinformatics Resource Portal (http://web.expasy.org/portscale/) [42] on the logarithm established [43] with a window of 19.

Figure 4d. Alignment of amino acid sequence of DPMS

The alignment of DPMS sequences from all 39 species used the CLUSTAL W program [43] (http://www.genome.jp/tools/claustalw/). The PKA-motif is highlighted in blue and is present in all DPMS sequences except in C. parapsilosis. In addition, the sequences from S. cerevisiae, G. glabrata and U. maydis each has a second serine residue following the primary phosphorylation site and is absent from the rest. The significance of this discrepancy may be evolution derived. The experimental results suggest that serine-142 in S. cerevisiae could partially fulfil the need of serine-141 if mutated [30]. The metal (Mn²⁺ or Mg²⁺) binding DAD motif is present in DPMS sequence from all 39 species and highlighted in magenta. The complete alignment of all DPMS sequences is in the Supplement Figure sf4.