# Expression of Tailored α-N-Acetylglucosaminidase in Escherichia coli for Synthesizing Mannose-6-Phosphate on N-Linked Oligosaccharides of Lysosomal Enzymes

## TL;DR

Researchers engineered E. coli to produce a modified enzyme that helps create a key sugar phosphate tag on lysosomal enzymes.

## Contribution

The study introduces tailored versions of an α-N-acetylglucosaminidase enzyme expressed in E. coli for synthesizing mannose-6-phosphate.

## Key findings

- Truncated UCE variants with signal peptide, transmembrane domain, propiece, and cytoplasmic tail were successfully expressed and purified in E. coli.
- Fusing maltose-binding protein improved solubility of UCE variants, achieving up to 80.5 mg/L enzyme concentration.
- The UCE variant containing only the core catalytic domain showed the highest enzymatic activity.

## Abstract

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 Man8GlcNAc2 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 trans-Golgi network (TGN). Here, we expressed differently tailored versions of the UCE, including four truncated variants, in Escherichia coli. 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 E. coli. 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.