Mannosidase II Research Articles

BML-265 and Tyrphostin AG1478 Disperse the Golgi Apparatus and Abolish Protein Transport in Human Cells.

The steady-state localization of Golgi-resident glycosylation enzymes in the Golgi apparatus depends on a balance between anterograde and retrograde transport. Using the Retention Using Selective Hooks (RUSH) assay and high-content screening, we identified small molecules that perturb the localization of Mannosidase II (ManII) used as a model cargo for Golgi resident enzymes. In particular, we found that two compounds known as EGFR tyrosine kinase inhibitors, namely BML-265 and Tyrphostin AG1478 disrupt Golgi integrity and abolish secretory protein transport of diverse cargos, thus inducing brefeldin A-like effects. Interestingly, BML-265 and Tyrphostin AG1478 affect Golgi integrity and transport in human cells but not in rodent cells. The effects of BML-265 are reversible since Golgi integrity and protein transport are quickly restored upon washout of the compounds. BML-265 and Tyrphostin AG1478 do not lead to endosomal tubulation suggesting that, contrary to brefeldin A, they do not target the trans-Golgi ARF GEF BIG1 and BIG2. They quickly induce COPI dissociation from Golgi membranes suggesting that, in addition to EGFR kinase, the cis-Golgi ARF GEF GBF1 might also be a target of these molecules. Accordingly, overexpression of GBF1 prevents the effects of BML-265 and Tyrphostin AG1478 on Golgi integrity.

Non-fucose level as a function of glycoenzyme transcription in a glycoengineered CHO cell line

Background and novelty Antibody-dependent cellular cytotoxicity (ADCC) is one important mode of action for therapeutic mAbs in the field of oncology. It is strongly dependent on the glycan pattern of the Fc N-glycan: low core-fucose levels (= high non-fucose levels) typically result in an increased ADCC. There are several options to reach high non-fucose levels like the selection of the right host that expresses the desired pattern, in vitro glycosylation, use of glycosylation inhibitors, alteration of process parameters and generating glycosylation mutants with modified glycan synthesis activities by cell line engineering. Within Roche Pharma we are working with CHO cell lines designed to produce therapeutic antibodies based on the GlycArt system. These cell lines contain in addition to the recombinant gene for the therapeutic monoclonal antibody (mAb), also recombinant genes for two glycosyltransferases, N-acetylglucosaminyltransferase-III (GntIII) and mannosidase-II (ManII). As a result the CHO cells produce antibodies with a modified glycosylation structure characterized by a low-fucose Fc part. The selection system for the two glycosylation enzymes is based on the use of puromycin whereas for the mAB MSX is used.

MEK1 inactivates Myt1 to regulate Golgi membrane fragmentation and mitotic entry in mammalian cells

The pericentriolar stacks of Golgi cisternae are separated from each other in G2 and fragmented extensively during mitosis. MEK1 is required for Golgi fragmentation in G2 and for the entry of cells into mitosis. We now report that Myt1 mediates MEK1's effects on the Golgi complex. Knockdown of Myt1 by siRNA increased the efficiency of Golgi complex fragmentation by mitotic cytosol in permeabilized and intact HeLa cells. Myt1 knockdown eliminated the requirement of MEK1 in Golgi fragmentation and alleviated the delay in mitotic entry due to MEK1 inhibition. The phosphorylation of Myt1 by MEK1 requires another kinase but is independent of RSK, Plk, and CDK1. Altogether our findings reveal that Myt1 is inactivated by MEK1 mediated phosphorylation to fragment the Golgi complex in G2 and for the entry of cells into mitosis. It is known that Myt1 inactivation is required for CDK1 activation. Myt1 therefore is an important link by which MEK1 dependent fragmentation of the Golgi complex in G2 is connected to the CDK1 mediated breakdown of Golgi into tubules and vesicles in mitosis.

Spatial Regulation of Golgi Phosphatidylinositol-4-Phosphate is Required for Enzyme Localization and Glycosylation Fidelity

The enrichment of phosphatidylinositol-4-phosphate (PI(4)P) at the trans Golgi network (TGN) is instrumental for proper protein and lipid sorting, yet how the restricted distribution of PI(4)P is achieved remains unknown. Here, we show that lipid phosphatase Suppressor of actin mutations 1 (SAC1) is crucial for the spatial regulation of Golgi PI(4)P. Ultrastructural analysis revealed that SAC1 is predominantly located at cisternal Golgi membranes but is absent from the TGN, thus confining PI(4)P to the TGN. RNAi-mediated knockdown of SAC1 caused changes in Golgi morphology and mislocalization of Golgi enzymes. Enzymes involved in glycan processing such as mannosidase-II (Man-II) and N-acetylglucosamine transferase-I (GnT-I) redistributed to aberrant intracellular structures and to the cell surface in SAC1 knockdown cells. SAC1 depletion also induced a unique pattern of Golgi-specific defects in N-and O-linked glycosylation. These results indicate that SAC1 organizes PI(4)P distribution between the Golgi complex and the TGN, which is instrumental for resident enzyme partitioning and Golgi morphology.

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

Golgi alpha-mannosidase II (GMII), a member of glycoside hydrolase family 38, cleaves two mannosyl residues from GlcNAcMan(5)GlcNAc(2) as part of the N-linked glycosylation pathway. To elucidate the molecular and electronic details of the reaction mechanism, in particular the conformation of the substrate at the transition state, we performed quantum mechanics/molecular mechanics metadynamics simulations of the glycosylation reaction catalyzed by GMII. The calculated free energy of activation for mannosyl glycosylation (23 kcal/mol) agrees very well with experiments, as does the conformation of the glycon mannosyl ring in the product of the glycosylation reaction (the covalent intermediate). In addition, we provide insight into the electronic aspects of the molecular mechanism that were not previously available. We show that the substrate adopts an (O)S(2)/B(2,5) conformation in the GMII Michaelis complex and that the nucleophilic attack occurs before complete departure of the leaving group, consistent with a D(N)A(N) reaction mechanism. The transition state has a clear oxacarbenium ion (OCI) character, with the glycosylation reaction following an (O)S(2)/B(2,5) --> B(2,5) [TS] --> (1)S(5) itinerary, agreeing with an earlier proposal based on comparing alpha- and beta-mannanases. The simulations also demonstrate that an active-site Zn ion helps to lengthen the O2'-H(O2') bond when the substrate acquires OCI character, relieving the electron deficiency of the OCI-like species. Our results can be used to explain the potency of recently formulated GMII anticancer inhibitors, and they are potentially relevant in deriving new inhibitors.

Structural Investigation of the Binding of 5‐Substituted Swainsonine Analogues to Golgi α‐Mannosidase II

Golgi alpha-mannosidase II (GMII) is a key enzyme in the N-glycosylation pathway and is a potential target for cancer chemotherapy. The natural product swainsonine is a potent inhibitor of GMII. In this paper we characterize the binding of 5alpha-substituted swainsonine analogues to the soluble catalytic domain of Drosophila GMII by X-ray crystallography. These inhibitors enjoy an advantage over previously reported GMII inhibitors in that they did not significantly decrease the inhibitory potential of the swainsonine head-group. The phenyl groups of these analogues occupy a portion of the binding site not previously seen to be populated with either substrate analogues or other inhibitors and they form novel hydrophobic interactions. They displace a well-organized water cluster, but the presence of a C(10) carbonyl allows the reestablishment of important hydrogen bonds. Already approximately tenfold more active against the Golgi enzyme than the lysosomal enzyme, these inhibitors offer the potential of being extended into the N-acetylglucosamine binding site of GMII for the creation of even more potent and selective GMII inhibitors.

Human lysosomal α‐mannosidases exhibit different inhibition and metal binding properties

Two structurally-related members of the lysosomal mannosidase family, the broad substrate specificity enzyme human lysosomal alpha-mannosidase (hLM, MAN2B1) and the human core alpha-1, 6-specific mannosidase (hEpman, MAN2B2) act in a complementary fashion on different glycosidic linkages, to effect glycan degradation in the lysosome. We have successfully expressed these enzymes in Drosophila S2 cells and functionally characterized them. hLM and hEpman were significantly inhibited by the class II alpha-mannosidase inhibitors, swainsonine and mannostatin A. We show that three pyrrolidine-based compounds designed for selective inhibition of Golgi alpha-mannosidase II (GMII) exhibited varying degrees of inhibition for hLM and hEpman. While these compounds inhibited hLM and GMII similarly, they inhibited hEpman to a lesser extent. Further, the two lysosomal alpha-mannosidases also show differential metal dependency properties. This has led us to propose a secondary metal binding site in hEpman. These results set the stage for the development of selective inhibitors to members of the GH38 family, and, henceforth, the further investigation of their physiological roles.

The molecular basis of inhibition of Golgi alpha-mannosidase II by mannostatin A.

Mannostatin A is a potent inhibitor of the mannose-trimming enzyme, Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan processing pathway. Inhibition of this enzyme provides a route to blocking the transformation-associated changes in cancer cell surface oligosaccharide structures. Here, we report on the synthesis of new Mannostatin derivatives and analyze their binding in the active site of Drosophila GMII by X-ray crystallography. The results indicate that the interaction with the backbone carbonyl of Arg876 is crucial to the high potency of the inhibitor-an effect enhanced by the hydrophobic interaction between the thiomethyl group and an aromatic pocket vicinal to the cleavage site. The various structures indicate that differences in the hydration of protein-ligand complexes are also important determinants of plasticity as well as selectivity of inhibitor binding.

Expression of rat β(1,4)‐N‐acetylglucosaminyltransferase III in Nicotiana tabacum remodels the plant‐specific N‐glycosylation

Plant N-linked glycans differ substantially from their mammalian counterparts, mainly with respect to modifications of the core glycan, which typically contains a beta(1,2)-xylose and an alpha(1,3)-fucose. The addition of a bisecting N-acetylglucosamine residue by beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII) is known to control the processing of N-linked glycans in mammals, for example by preventing alpha(1,6)-fucosylation of the core glycan. In order to outcompete plant-specific beta(1,2)-xylose and alpha(1,3)-fucose modifications, rat GnTIII was expressed either with its native localization domain (GnTIII) or with the cytoplasmic tail, transmembrane domain and stem region (CTS) of Arabidopsis thaliana mannosidase II (ManII) (GnTIII(A.th.)). Both CTSs targeted enhanced yellow fluorescent protein (eYFP) to a brefeldin A-sensitive compartment, indicative of Golgi localization. GnTIII expression increased the fraction of N-glycans devoid of xylose and fucose from 13% +/- 7% in wild-type plants to 60% +/- 8% in plants expressing GnTIII(A.th.). N-Glycans of plants expressing rat GnTIII contained three major glycan structures of complex bisected, complex, or hybrid bisected type, accounting for 70%-85% of the total N-glycans. On expression of GnTIII(A.th.), N-glycans displayed a higher heterogeneity and were of hybrid type. Co-expression of A. thaliana ManII significantly increased the amount of complex bisected structures relative to the plants expressing GnTIII or GnTIII(A.th.), whereas co-expression of human ManII did not redirect the pool of hybrid structures towards complex-type structures. The method described offers the advantage that it can be implemented in any desired plant system for effective removal of beta(1,2)-xylose and alpha(1,3)-fucose from the N-glycan.

Probing the substrate specificity of Golgi alpha-mannosidase II by use of synthetic oligosaccharides and a catalytic nucleophile mutant.

Inhibition of Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the substrate requirements of GMII, oligosaccharides were synthesized that contained an alpha(1,3)- or alpha(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed to alanine (D204A) allowed visualization of soaked oligosaccharides and led to the identification of the binding site for the alpha(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B 2,5 conformation is facilitated by steric hindrance from, and the formation of strong hydrogen bonds to, Asp204. The observation that 1-thio-linked mannosides are not well tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the alpha(1,6)-linked Man of the natural substrate and the beta(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended binding site of the enzyme. A cocrystal structure of this compound with the D204A enzyme revealed the molecular interactions with the beta(1,2)-linked GlcNAc. The structure is consistent with the approximately 80-fold preference of dGMII for the cleavage of substrates containing a nonreducing beta(1,2)-linked GlcNAc. By contrast, the lysosomal mannosidase lacks an equivalent GlcNAc binding site and kinetic analysis indicates oligomannoside substrates without non-reducing-terminal GlcNAc modifications are preferred, suggesting that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.

Evaluation of docking programs for predicting binding of Golgi α‐mannosidase II inhibitors: A comparison with crystallography

Golgi alpha-mannosidase II (GMII), a zinc-dependent glycosyl hydrolase, is a promising target for drug development in anti-tumor therapies. Using X-ray crystallography, we have determined the structure of Drosophila melanogaster GMII (dGMII) complexed with three different inhibitors exhibiting IC50's ranging from 80 to 1000 microM. These structures, along with those of seven other available dGMII/inhibitor complexes, were then used as a basis for the evaluation of seven docking programs (GOLD, Glide, FlexX, AutoDock, eHiTS, LigandFit, and FITTED). We found that small inhibitors could be accurately docked by most of the software, while docking of larger compounds (i.e., those with extended aromatic cycles or long aliphatic chains) was more problematic. Overall, Glide provided the best docking results, with the most accurately predicted binding around the active site zinc atom. Further evaluation of Glide's performance revealed its ability to extract active compounds from a benchmark library of decoys.

Molecular cloning and characterization of Arabidopsis thaliana Golgi α‐mannosidase II, a key enzyme in the formation of complex N‐glycans in plants

N-glycosylation is one of the major post-translational modifications of proteins in eukaryotes; however, the processing reactions of oligomannosidic N-glycan precursors leading to hybrid-type and finally complex-type N-glycans are not fully understood in plants. To investigate the role of Golgi alpha-mannosidase II (GMII) in the formation of complex N-glycans in plants, we identified a putative GMII from Arabidopsis thaliana (AtGMII; EC 3.2.1.114) and characterized the enzyme at a molecular level. The putative AtGMII cDNA was cloned, and its deduced amino acid sequence revealed a typical type II membrane protein of 1173 amino acids. A soluble recombinant form of the enzyme produced in insect cells was capable of processing different physiologically relevant hybrid N-glycans. Furthermore, a detailed N-glycan analysis of two AtGMII knockout mutants revealed the predominant presence of unprocessed hybrid N-glycans. These results provide evidence that AtGMII plays a central role in the formation of complex N-glycans in plants. Furthermore, conclusive evidence was obtained that alternative routes in the conversion of hybrid N-glycans to complex N-glycans exist in plants. Transient expression of N-terminal AtGMII fragments fused to a GFP reporter molecule demonstrated that the transmembrane domain and 10 amino acids from the cytoplasmic tail are sufficient to retain a reporter molecule in the Golgi apparatus and that lumenal sequences are not involved in the retention mechanism. A GFP fusion construct containing only the transmembrane domain was predominantly retained in the ER, a result that indicates the presence of a motif promoting ER export within the last 10 amino acids of the cytoplasmic tail of AtGMII.

Immunocytochemical analysis of Uukuniemi virus budding compartments: role of the intermediate compartment and the Golgi stack in virus maturation.

Previous studies have suggested that Uukuniemi virus, a bunyavirus, matures at the membranes of the Golgi complex. In this study we have employed immunocytochemical techniques to analyze in detail the budding compartment(s) of the virus. Electron microscopy of infected BHK-21 cells showed that virus particles are found in the cisternae throughout the Golgi stack. Within the cisternae, the virus particles were located preferentially in the dilated rims. This would suggest that virus budding may begin at or before the cis Golgi membranes. The virus budding compartment was studied further by immunoelectron microscopy with a pre-Golgi intermediate compartment marker, p58, and a Golgi stack marker protein, mannosidase II (ManII). Virus particles and budding virus were detected in ManII-positive Golgi stack membranes and, interestingly, in both juxtanuclear and peripheral p58-positive elements of the intermediate compartment. In cells incubated at 15 degrees C the nucleocapsid and virus envelope proteins were seen to accumulate in the intermediate compartment. Immunoelectron microscopy demonstrated that at 15 degrees C the nucleocapsid is associated with membranes that show a characteristic distribution and tubulo-vesicular morphology of the pre-Golgi intermediate compartment. These membranes contained virus particles in the lumen. The results indicate that the first site of formation of Uukuniemi virus particles is the pre-Golgi intermediate compartment and that virus budding continues in the Golgi stack. The results raise questions about the intracellular transport pathway of the virus particles, which are 100 to 120 nm in diameter and are therefore too large to be transported in the 60-nm-diameter vesicles postulated to function in the intra-Golgi transport. The distribution of the virus in the Golgi stack may imply that the cisternae themselves have a role in the vectorial transport of virus particles.