The Drosophila GMII gene encodes a Golgi α-mannosidase II

Abstract

In this paper we show the organisation of the Drosophila gene encoding a Golgi alpha-mannosidase II. We demonstrate that it encodes a functional homologue of the mouse Golgi alpha-mannosidase II. The Drosophila and mouse cDNA sequences translate into amino acid sequences which show 41% identity and 61% similarity. Expression of the Drosophila GMII sequence in CHOP cells produces an enzyme which has mannosidase activity and is inhibited by swainsonine and by CuSO(4.) In cultured Drosophila cells and in Drosophila embryos, antibodies raised against a C-terminal peptide localise this product mainly to the Golgi apparatus as identified by cryo-immuno electron microscopy studies and by antibodies raised against known mammalian Golgi proteins. We discuss these results in terms of the possible use of dGMII as a Drosophila Golgi marker.

Manno-epi-cyclophellitols Enable Activity-Based Protein Profiling of Human α-Mannosidases and Discovery of New Golgi Mannosidase II Inhibitors

“…Protein concentration was set to be in excess of the probe concentration to ensure maximum fluorescence polarization signals. Although the pH optimum for dGMII on chromogenic substrates has previously been reported 36 as 5.7, we observed an optimal ABP binding at pH 6.5. This alkaline shift is similar to what we observed for human nonlysosomal glucosylceramidase (GBA2).…”

Comparison of Kifunensine and 1-Deoxymannojirimycin Binding to Class I and II α-Mannosidases Demonstrates Different Saccharide Distortions in Inverting and Retaining Catalytic Mechanisms,

“…The reaction mixture consisted of 25 µL of varying concentrations (1-10 mM) p-nitrophenol-R mannoside (p-NP-mannose) from Sigma, 10 µL of 200 mM buffer, and 10 µL of water or inhibitor. The buffer used was MES pH 5.75, which is optimal for this enzyme (8). The reaction mixture was pre-warmed to 37 °C, and 5 µL of R-mannosidase in 10 mM sodium phosphate pH 6.8, 100 mM NaCl is added to initiate the reaction.…”

“…Golgi R-mannosidase II (mannosyl-oligosaccharide 1,3- (8,9). The cleavage of these two bonds is the committed step of complex N-glycan formation (10).…”

“…Golgi α-mannosidase II (mannosyl-oligosaccharide 1,3-1,6-α-mannosidase, EC 3.2.1.114) is a class II α-mannosidase in the N-glycosylation pathway that catalyzes the hydrolysis of the terminal α1,3- and α1,6-linked mannoses in the high-mannose oligosaccharide GlcNAcMan 5 GlcNAc 2 , converting it to GlcNAcMan 3 GlcNAc 2 ( , ). The cleavage of these two bonds is the committed step of complex N -glycan formation ().…”

Molecular Mechanism of the Glycosylation Step Catalyzed by Golgi α-Mannosidase II: A QM/MM Metadynamics Investigation

“…The available structural data on GMII is from D. melanogaster, which has kinetic and inhibition properties similar to those of mammalian GMII. 25 These two forms also have high sequence identity (41%), 15 their active-site amino acid residues being virtually identical. As observed in many other GHs, 24 it is expected that GMII binds the substrate in a distorted conformation.…”