PMC:6218659 / 1691-7197 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/6218659","sourcedb":"PMC","sourceid":"6218659","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6218659","text":"Introduction\nN-glycosylation of asparagine residues is a common post-translational modification of eukaryotic proteins, required for protein stability, processing, and function. Many diseases are associated with incorrect glycosylation (Freeze et al., 2012). This process requires dolichol-PP-oligosaccharides that provide the oligosaccharides that are transferred (Helenius and Aebi, 2004). The first step in dolichol-PP-oligosaccharide production involves the ER integral membrane enzyme dolichyl-phosphate alpha-N-acetyl-glucosaminyl-phosphotransferase (DPAGT1, E.C. 2.7.8.15, also known as GlcNAc-1-P Transferase [GPT]). It catalyzes the transfer of an N-acetyl-D-glucosamine-1-phosphoryl unit (GlcNAc-1-P) from UDP-N-acetyl glucosamine (UDP-GlcNAc) onto dolichyl phosphate (Dol-P) (Figure 1A) (Heifetz and Elbein, 1977, Lehrman, 1991). The product GlcNAc-PP-Dol is anchored to the ER membrane by its dolichyl moiety and then monosaccharide units are added sequentially to build the N-glycan that is then transferred.\nFigure 1 Structural Features of DPAGT1\n(A) Cartoon of DPAGT1 reaction.\n(B) Michaelis-Menten kinetics, non-titrated substrate at 200 μM. Measurements were performed with three biological and three technical repeats. Data presented are means ± SD.\n(C) Topology of DPAGT1; helices shown as cylinders, strands as arrows and active site in magenta.\n(D) Schematic of DPAGT1 structure. Views shown looking along membrane plane and onto the cytoplasmic face; UDP-GlcNAc in magenta.\n(E) Comparison of DPAGT1 (orange) and MraY (PDB: 5CKR; purple) folds.\n(F) CL9 domain in DPAGT1.\n(G) CL9 strand (loop “E”) and helix in MraY.\nMutations in DPAGT1 impair protein N-glycosylation, leading to at least two syndromes, depending on the extent of loss of activity. Congenital myasthenic syndrome (DPAGT1-CMS OMIM ref: 614750) is a neuromuscular transmission disorder characterized by fatigable weakness of proximal muscles (Basiri et al., 2013, Belaya et al., 2012, Iqbal et al., 2013). Reduced endplate acetylcholine receptors (AChR) and abnormal synaptic structure may be the result of incorrect glycosylation of AChR and other proteins. Mutations in DPAGT1 also cause congenital disorder of glycosylation type Ij (CDG-Ij, OMIM ref: 608093) (Carrera et al., 2012, Selcen et al., 2014, Wu et al., 2003, Würde et al., 2012), a more severe multisystem syndrome that can cause intellectual disability, epilepsy, microcephaly, severe hypotonia and structural brain anomalies.\nInhibition of polyisoprenyl-phosphate N-acetylaminosugar-1-phosphoryl transferases (PNPTs) such as DPAGT1 and the bacterial enzyme MraY, is lethally toxic to many prokaryotic and eukaryotic organisms. Streptomyces bacteria exploit this toxicity by producing the PNPT inhibitor tunicamycin, which blocks MraY, a critical enzyme in biosynthesis of cell walls in many bacterial pathogens (Figures S1A and S1B) (Dini, 2005). Unfortunately, it also inhibits eukaryotic PNPTs, such as DPAGT1 (Heifetz et al., 1979) causing severe toxicity in eukaryotic cells. However, although bacterial (e.g., MraY) and human (e.g., DPAGT1) PNPTs are similar, it should be possible to design synthetically-altered tunicamycin analogues that specifically inhibit bacterial proteins.\nFigure S1 Biochemical and Biophysical Characterization of DPAGT1, Related to Figure 1\n(A) Cartoon of DPAGT1 showing the reaction it performs.\n(B) Cartoon of MraY showing the reaction it performs.\n(C) The identity of the substrate Dol-P and the product, GlcNAc-PP-Dol, was confirmed by mass spectrometry. Top spectrum is DPAGT1 incubated with Dol-P only, bottom spectra are DPAGT1 incubated with both Dol-P and UDP-GlcNAc.\n(D) Comparison of the catalytic activity of DPAGT1 WT and Val264Gly mutant protein (n=9).\n(E) The thermostability of DPAGT1 WT (black) and Val264Gly (grey) mutant proteins tested using label free differential scanning fluorimetry. The effects of addition of the substrates Dol-P and UDP-GlcNAc, and the inhibitor tunicamycin on thermostability of DPAGT1 were also tested (n=9).\n(F) Product inhibition was observed with the product analogue GlcNAc-PP-Und, but not with UMP (n=9).\n(G) DPAGT1 is completely inhibited by a 1:1 ratio of tunicamycin:protein (n=9).\n(H) Lipidomics analysis of OGNG purified DPAGT1 showed the presence of co-purified phospholipid in addition to the supplemented cardiolipin associated with the protein.\n(I) The presence of phosphatidylglycerol is confirmed by tandem mass spectrum of the most intense phospholipid in the lipidomics analysis.\nFor all panels, data presented are means ± SD.\nHere we present structures of human DPAGT1 with and without ligands. The protein production methods, structures, assays and complexes are components of a “target enabling package” developed at the Structural Genomics Consortium (released June 2017, http://www.thesgc.org/tep/DPAGT1), which has already been used by others (Yoo et al., 2018). These structures, combined with mutagenesis and activity analysis, reveal both the mechanism of catalysis by DPAGT1 and the molecular basis of DPAGT1-related diseases. To improve the effectiveness of tunicamycin as a drug, we modified its core scaffold, TUN, using a scalable, semi-synthetic strategy that enabled selective lipid chain addition. These analogues show nanomolar antimicrobial potency, ablated inhibition of DPAGT1, much reduced toxicity, allowed effective treatment of Mycobacterium tuberculosis (Mtb) in mammals, providing leads for tuberculosis (TB) antibiotic development.","divisions":[{"label":"Title","span":{"begin":0,"end":12}},{"label":"Figure caption","span":{"begin":1022,"end":1636}},{"label":"Figure caption","span":{"begin":3238,"end":4573}}],"tracks":[{"project":"2_test","denotations":[{"id":"30388443-22516080-20779456","span":{"begin":252,"end":256},"obj":"22516080"},{"id":"30388443-15189166-20779457","span":{"begin":385,"end":389},"obj":"15189166"},{"id":"30388443-16014-20779458","span":{"begin":819,"end":823},"obj":"16014"},{"id":"30388443-1668306-20779459","span":{"begin":834,"end":838},"obj":"1668306"},{"id":"30388443-23591138-20779460","span":{"begin":1943,"end":1947},"obj":"23591138"},{"id":"30388443-22742743-20779461","span":{"begin":1964,"end":1968},"obj":"22742743"},{"id":"30388443-23249953-20779462","span":{"begin":1984,"end":1988},"obj":"23249953"},{"id":"30388443-22786653-20779463","span":{"begin":2264,"end":2268},"obj":"22786653"},{"id":"30388443-24759841-20779464","span":{"begin":2285,"end":2289},"obj":"24759841"},{"id":"30388443-12872255-20779465","span":{"begin":2302,"end":2306},"obj":"12872255"},{"id":"30388443-22304930-20779466","span":{"begin":2322,"end":2326},"obj":"22304930"},{"id":"30388443-16305528-20779467","span":{"begin":2891,"end":2895},"obj":"16305528"},{"id":"30388443-444447-20779468","span":{"begin":2980,"end":2984},"obj":"444447"},{"id":"30388443-29459785-20779469","span":{"begin":4909,"end":4913},"obj":"29459785"}],"attributes":[{"subj":"30388443-22516080-20779456","pred":"source","obj":"2_test"},{"subj":"30388443-15189166-20779457","pred":"source","obj":"2_test"},{"subj":"30388443-16014-20779458","pred":"source","obj":"2_test"},{"subj":"30388443-1668306-20779459","pred":"source","obj":"2_test"},{"subj":"30388443-23591138-20779460","pred":"source","obj":"2_test"},{"subj":"30388443-22742743-20779461","pred":"source","obj":"2_test"},{"subj":"30388443-23249953-20779462","pred":"source","obj":"2_test"},{"subj":"30388443-22786653-20779463","pred":"source","obj":"2_test"},{"subj":"30388443-24759841-20779464","pred":"source","obj":"2_test"},{"subj":"30388443-12872255-20779465","pred":"source","obj":"2_test"},{"subj":"30388443-22304930-20779466","pred":"source","obj":"2_test"},{"subj":"30388443-16305528-20779467","pred":"source","obj":"2_test"},{"subj":"30388443-444447-20779468","pred":"source","obj":"2_test"},{"subj":"30388443-29459785-20779469","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#9393ec","default":true}]}]}}