Expression of Tailored α-N-Acetylglucosaminidase in <i>Escherichia coli</i> for Synthesizing Mannose-6-Phosphate on N-Linked Oligosaccharides of Lysosomal Enzymes
Lysosomal enzymes are synthesized as N-glycosylated glycoproteins with mannose-6-phosphate (M6P) moieties, which are responsible for their binding to M6P receptors and transporting to the lysosome. In the M6P biosynthetic pathway, a Man<sub>8</sub>GlcNAc<sub>2</sub> glycoform...
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| description | Lysosomal enzymes are synthesized as N-glycosylated glycoproteins with mannose-6-phosphate (M6P) moieties, which are responsible for their binding to M6P receptors and transporting to the lysosome. In the M6P biosynthetic pathway, a Man<sub>8</sub>GlcNAc<sub>2</sub> glycoform is converted to M6P groups through two consecutive enzymatic reactions, including N-acetylglucosamine (GlcNAc)-1-phosphotransferase (GNPT), transferring GlcNAc-1-phosphate from UDP-GlcNAc to the C6 hydroxyl groups of mannose residues, and then, removal of the covering GlcNAc moiety from the GlcNAc-P-mannose phosphodiester was carried out using an α-N-acetylglucosaminidase (referred to as ‘uncovering enzyme’, UCE) in the <i>trans</i>-Golgi network (TGN). Here, we expressed differently tailored versions of the UCE, including four truncated variants, in <i>Escherichia coli</i>. The four variants with the signal peptide, transmembrane domain, propiece and cytoplasmic tail truncated, respectively, were purified by affinity chromatography, and their enzymatic activities were assayed using a UDP-Glo kit. By fusing a maltose-binding protein (MBP) in the N-terminus of the UCE variants, the fusion proteins could be soluble when expressed in <i>E. coli</i>. The highest concentration of the purified enzyme was 80.5 mg/L of fermentation broth. Furthermore, the UCE with the core catalytic domain exhibited the highest uncovering activity. |
| format | Article |
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| spelling | doaj-art-4fa2f871d52d4d1caaf698b8689b8e232025-08-20T02:24:43ZengMDPI AGBioengineering2306-53542025-04-0112442510.3390/bioengineering12040425Expression of Tailored α-N-Acetylglucosaminidase in <i>Escherichia coli</i> for Synthesizing Mannose-6-Phosphate on N-Linked Oligosaccharides of Lysosomal EnzymesYunsong Cao0Wei Wang1State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, ChinaState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, ChinaLysosomal enzymes are synthesized as N-glycosylated glycoproteins with mannose-6-phosphate (M6P) moieties, which are responsible for their binding to M6P receptors and transporting to the lysosome. In the M6P biosynthetic pathway, a Man<sub>8</sub>GlcNAc<sub>2</sub> glycoform is converted to M6P groups through two consecutive enzymatic reactions, including N-acetylglucosamine (GlcNAc)-1-phosphotransferase (GNPT), transferring GlcNAc-1-phosphate from UDP-GlcNAc to the C6 hydroxyl groups of mannose residues, and then, removal of the covering GlcNAc moiety from the GlcNAc-P-mannose phosphodiester was carried out using an α-N-acetylglucosaminidase (referred to as ‘uncovering enzyme’, UCE) in the <i>trans</i>-Golgi network (TGN). Here, we expressed differently tailored versions of the UCE, including four truncated variants, in <i>Escherichia coli</i>. The four variants with the signal peptide, transmembrane domain, propiece and cytoplasmic tail truncated, respectively, were purified by affinity chromatography, and their enzymatic activities were assayed using a UDP-Glo kit. By fusing a maltose-binding protein (MBP) in the N-terminus of the UCE variants, the fusion proteins could be soluble when expressed in <i>E. coli</i>. The highest concentration of the purified enzyme was 80.5 mg/L of fermentation broth. Furthermore, the UCE with the core catalytic domain exhibited the highest uncovering activity.https://www.mdpi.com/2306-5354/12/4/425lysosomal enzymeuncovering enzymemannose-6-phosphatemaltose-binding protein (MBP) |
| spellingShingle | Yunsong Cao Wei Wang Expression of Tailored α-N-Acetylglucosaminidase in <i>Escherichia coli</i> for Synthesizing Mannose-6-Phosphate on N-Linked Oligosaccharides of Lysosomal Enzymes Bioengineering lysosomal enzyme uncovering enzyme mannose-6-phosphate maltose-binding protein (MBP) |
| title | Expression of Tailored α-N-Acetylglucosaminidase in <i>Escherichia coli</i> for Synthesizing Mannose-6-Phosphate on N-Linked Oligosaccharides of Lysosomal Enzymes |
| title_full | Expression of Tailored α-N-Acetylglucosaminidase in <i>Escherichia coli</i> for Synthesizing Mannose-6-Phosphate on N-Linked Oligosaccharides of Lysosomal Enzymes |
| title_fullStr | Expression of Tailored α-N-Acetylglucosaminidase in <i>Escherichia coli</i> for Synthesizing Mannose-6-Phosphate on N-Linked Oligosaccharides of Lysosomal Enzymes |
| title_full_unstemmed | Expression of Tailored α-N-Acetylglucosaminidase in <i>Escherichia coli</i> for Synthesizing Mannose-6-Phosphate on N-Linked Oligosaccharides of Lysosomal Enzymes |
| title_short | Expression of Tailored α-N-Acetylglucosaminidase in <i>Escherichia coli</i> for Synthesizing Mannose-6-Phosphate on N-Linked Oligosaccharides of Lysosomal Enzymes |
| title_sort | expression of tailored α n acetylglucosaminidase in i escherichia coli i for synthesizing mannose 6 phosphate on n linked oligosaccharides of lysosomal enzymes |
| topic | lysosomal enzyme uncovering enzyme mannose-6-phosphate maltose-binding protein (MBP) |
| url | https://www.mdpi.com/2306-5354/12/4/425 |
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