Soluble GlcNAc phosphotransferase

Abstract

The present invention relates to a soluble GlcNAc phosphotransferase, a method of making the same and a method of phosphorylating with the same.

Description

BACKGROUND OF THE INVENTION [0001] 1. Field of the Invention [0002] The present invention relates to a soluble GlcNAc phosphotransferase, a method of making the same and a method of phosphorylating with the same. [0003] 2. Discussion of the Background [0004] Lysosomes are organelles in eukaryotic cells that function in the degradation of macromolecules into component parts that can be reused in biosynthetic pathways or discharged by the cell as waste. Normally, these macromolecules are broken down by enzymes known as lysosomal enzymes or lysosomal hydrolases. However, when a lysosomal enzyme is not present in the lysosome or does not function properly, the enzymes specific macromolecular substrate accumulates in the lysosome as “storage material” causing a variety of diseases, collectively known as lysosomal storage diseases. [0005] Lysosomal storage diseases can cause chronic illness and death in hundreds of individuals each year. There are approximately 50 known lysosomal storage ...

AI Technical Summary

Benefits of technology

The present invention provides a method for phosphorylating a protein by using a soluble GlcNAc-phosphotransferase. This enzyme contains an α subunit, a β subunit, and a proteolytic cleavage site interposed between the α and β subunits. The soluble GlcNAc-phosphotransferase can be isolated and used to phosphorylate glycoproteins in a host cell. The invention also provides methods for producing a polyprotein containing the α and β subunits of the enzyme and methods for treating a patient suffering from a lysosomal storage disease by administering a phosphorylated lysosomal hydrolase.

Problems solved by technology

Lysosomal storage diseases can cause chronic illness and death in hundreds of individuals each year.

In each of these diseases, lysosomes are unable to degrade a specific compound or group of compounds because the enzyme that catalyzes a specific degradation reaction is missing from the lysosome, is present in low concentrations in the lysosome, or is present at sufficient concentrations in the lysosome but is not functioning properly.

However, notwithstanding the ease of purification the recombinant soluble GlcNAc-phosphotransferase was not efficiently subject to post-translational proteolytic cleavage when expressed in mammalian cells such as 293T cells and CHO-K1 cells.

Furthermore, the absence of the γ subunit results in loss of substrate specificity to only those lysosomal enzymes targeted via the mannose-6-phosphate targeting systems, e.g., acid α-glucosidase, acid β-galactosidase, β-hexaminidase, and others as described herein.

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