Novel Compounds & Methods for Producing N-Glycans

Abstract

A novel deglycosylating enzyme (and methods of using same) that removes a broad range of N-glycans from N-glycosylated proteins while leaving a saccharide marker on the protein site is currently available for commercialization. The enzyme is heat stable and capable of cleaving high mannose, complex, and hybrid N-glycans regardless of core fucosylation or sialylation. This enzyme is food grade and will find applications in design of suitable prebiotics and food products as well as in proteomic and glycoproteomic methods applied to biomarker discovery.

Full Description

An endoglycosidase is reported that is more effective than similar commercial enzymes or any of those previously reported in literature.  De-glycosylation with endoglycosidases remove N-glycans leaving a core N-acetylglucosamine.  Currently, the only method to achieve this cleavage is through sequential multiple enzyme exposure.  No previously available enzyme is capable of cleaving all three types of naturally occurring N-glycans (high mannose, complex, and hybrid).  Furthermore, existing enzymes do not efficiently and effectively cleave N-glycans if they are fucosylated or sialylated.

Researchers at the University of California Davis have discovered and characterized a single enzyme (and methods of using same) that overcomes the inherent limitations of previous methods and products.  The invention can be used to deglycosylate high mannose N-glycans, as well as complex N-glycans, such as those found in human milk proteins and in serum.  This invention can successfully cleave proteins that are highly fucosylated and/or sialylated - conditions which render those glycans virtually inaccessible to alternative endoglycosidases.  In addition to its unique specificity, the enzyme has characteristics that make it ideal for industrial applications including robustness (long extended use, high turnover numbers, and high heat stability).  Furthermore, the enzyme is of food grade origin, making it ideal for large-scale food and pharmaceutical industry usage.

Applications

Rapid, efficient production of free N-glycans, possibly for:

• use in a food product, e.g., as a prebiotic to stimulate growth of beneficial gut bacteria;
• use in a pharmaceutical composition, e.g., for immune system stimulation or pathogen protection; and
• use for glycan characterization and glycoproteomic studies.

Rapid, efficient production of denuded proteins, possibly for:

• use in a food or consumer product with reduced allergenic potential;
• use in a pharmaceutical composition with reduced allergenic potential and more predictable chemical properties;
• use in a food produce with increased digestibility; and
• use for protein characterization and proteomic studies.

Enzyme use in food products:

• to increase digestibility of glycoproteins;
• to stimulate growth of beneficial gut bacteria;
• to reduce allergenic responses to food glycoproteins; and
• to induce satiety.

Enzyme use in pharmaceutical products:

• to reduce allergenic potential;
• to improve activity of therapeutic glycoproteins; and
• to improve immune response to free glycans.

Analytical applications such as high throughput proteomics and glycoproteomics.

Features/Benefits

This enzyme has several key features which make it an excellent candidate for research and industrial uses:

• Heat stable
• Food-grade origin
• Capable of cleaving all types of N-glycans
• Can cleave core-fucosylated glycoproteins
• Can cleave in the presence of sialic acids and fucoses
• Can fully denude glycosylated proteins in a single step, instead of sequential multiple enzyme exposure

Patent Status