Cloning, expression and characterization of the pig liver GDP-mannose pyrophosphorylase. Evidence that GDP-mannose and GDP-Glc pyrophosphorylases are different proteins

Abstract

GDP-Man, the mannosyl donor for most Man-containing polymers is formed by the transfer of Man-1-P to GTP to form GDP-Man and PP i . This reaction is catalyzed by the widespread and essential enzyme, GDP-Man pyrophosphorylase (GMPP). The pig liver GMPP consists of an a subunit (43 kDa) and a b subunit (37 kDa). Purified pig GMPP catalyzes the synthesis of GDP-Glc (from Glc-1-P and GTP) and GDP-Man (from Man-1-P and GTP), but has higher activity for the formation of GDP-Glc than for synthesis of GDP-Man. In the present study, we report the cloning of the cDNA for the b subunit of GMPP, and its expression in a bacterial system resulting in the formation of active enzyme. The full length cDNA encoding the b subunit was isolated from a porcine cDNA library, and its predicted gene product showed high amino-acid sequence homology to GMPPs from other species. The gene was expressed in Escherichia coli cells, and a 37-kDa protein was over-produced in these cells. This gene product reacted strongly with antibody reactive to the native b subunit of pig GMPP. Most interestingly, this recombinant protein had high activity for synthesizing GDP-Man (from Man-1-P and GTP), but very low activity for the formation of GDP-Glc (from Glc-1-P and GTP). Other properties of the recombinant protein were also analyzed. This study suggests that the b subunit is the GMPP, whereas the a subunit, or a combination of both subunits, may have the GDP-Glc pyrophosphorylase activity.Keywords: GDP-Man; GDP-Glc; N-linked; pyrophosphorylase; cloning.At least 48 different sugar nucleotides (i.e. nucleoside diphosphate sugars) have been isolated, and many of these have been shown to be intermediates in the biosynthesis of various types of complex carbohydrates [1±3]. One such nucleotide, GDP-dMan, was first isolated from yeast in 1954 [4]. This activated form of Man is the major mannosyl donor for the synthesis of glycoproteins [5,6], glycosylphosphatidylinositol (GPI) membrane anchors [7], and various bacterial and lower eukaryotic cell wall polymers [8,9].GDP-d-Man plays a key role in the biosynthetic pathway that produces the N-linked oligosaccharides of many membrane and secretory glycoproteins of eukaryotic cells [10]. This biosynthetic pathway is initiated in the ER with the formation of a lipid-linked oligosaccharide, i.e Man 9 GlcNAc 2 -PP-Dol, which ultimately donates its oligosaccharide to specific asparagine residues on the newly synthesized polypeptide chain. The first five Man residues are derived directly from GDP-Man to give Man 5 GlcNAc 2 -PP-Dol, while the next four Man residues are contributed by Dol-P-Man to form Man 9 GlcNAc 2-PP-Dol [11±14]. The Man component of Dol-P-Man is also derived from GDP-Man. Man residues also occur in GPI anchors which have a common`core' glycan structure. Those Man residues are also derived from Dol-P-Man [15,16]. In addition, GDP-lfucose can be derived from GDP-d-Man through a series of oxidation, reduction and epimerization reactions [17].Thus, the enzyme, GDP-Man pyrophosphorylase or GTP:a-dMan-1-P g...

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

“…Importantly, the native complex had GGPP activity higher than GMPP activity, but recombinant subunit b expressed in E. coli showed high GMPP activity and very low GGPP activity. This finding suggests that the subunit a has GGPP activity or confers the activity to the complex (Ning and Elbein, 2000). In this work, consistent with its activities in Arabidopsis seedlings, rVTC1 showed relatively low GGPP activity compared with its GMPP activity, even in the presence of rKJC1.…”

“…Importantly, besides GMPP activity, the animal GMPP complex has high GGPP activity, while subunit b alone had very weak GGPP activity (Szumilo et al, 1993;Ning and Elbein, 2000). In this context, substrate specificity of the mixture of rVTC1 and rKJCs was compared with that of rVTC1 alone using various nucleotide sugars and PPi as the substrates.…”

“…Proteins corresponding to KJCs are widely distributed in eukaryotes including mammals (Supplemental Figure 12), but this does not imply conserved functionality. A native GMPP was purified from pig liver comprising a complex of subunits a and b, which correspond to KJC1 and VTC1, respectively (Szumilo et al, 1993;Ning and Elbein, 2000). Importantly, the native complex had GGPP activity higher than GMPP activity, but recombinant subunit b expressed in E. coli showed high GMPP activity and very low GGPP activity.…”

“…While in mammals, GDP-Glc is also synthesized from Glc 1-P by a GMPP complex (Szumilo et al, 1993;Ning and Elbein, 2000), a mechanism for the generation of GDP-Glc remains to be clarified in vascular plants.…”

Gènes impliqués dans les alpha-dystroglycanopathies

“…de comprendre le lien entre CDG et -DGpathies, deux groupes de pathologies associées à des anomalies de glycosylation. Le dernier gène connu impliqué dans les -DGpathies est le gène GMPPB dont la protéine participe à la synthèse du GDP-Man à partir de GTP et Man-1-phosphate ( Figure 1D) [53]. Le GDP-Man est à l'origine du Dol P-Man, substrat des mannosyltransférases qui vont initier la synthèse des cores M1, M2 et M3.…”