Nanobody-Fused Split OGA for Selective Protein Deglycosylation
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Solution Overview
Problem
Current methods for studying O-GlcNAc modification on proteins are limited by their inability to selectively edit O-GlcNAcylation levels on target proteins, especially when dealing with multiple or unmapped glycosites, and often disrupt cross-talk with other post-translational modifications like phosphorylation and ubiquitylation.
Innovation Solution
Development of a nanobody-fused split O-GlcNAcase (OGA) that selectively removes O-GlcNAc from target proteins, utilizing a split OGA with minimal inherent activity and leveraging nanobody selectivity to restore deglycosidase activity, allowing for precise modulation of O-GlcNAc levels on specific proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If global regulation methods (over-expression, genetic knockdown/knockout, or chemical inhibitors) are used to elevate or reduce O-GlcNAc levels, then O-GlcNAc levels can be globally changed, but wide-spread changes occur requiring additional studies to characterize function on target proteins
Solution Approach 1:
The patent segments the OGA enzyme into two separate fragments (N-terminal fragment and C-terminal fragment) that can be independently expressed and purified. These fragments are then reconstituted in vitro to form an active enzyme complex only when both fragments are present, enabling selective targeting of specific O-GlcNAcylated proteins without globally affecting O-GlcNAc levels on all proteins
Solution Approach 2:
The patent uses an F-box tag as an intermediary element that facilitates selective enrichment of target proteins. The F-box tagged target protein is captured by F-box bound resin, allowing the reconstituted OGA complex to act selectively on the tagged protein while leaving other O-GlcNAcylated proteins unaffected
2Quantity of substance
If chemical inhibitors of OGT or OGA are used, then O-GlcNAc levels can be changed, but inhibitors rapidly induce abnormal expression of OGT or OGA
Solution Approach 1:
By dividing OGA into two inactive fragments that cannot function alone, the patent eliminates the compensatory feedback mechanism that triggers abnormal gene expression. The reconstituted enzyme acts immediately and locally without inducing cellular stress responses that would otherwise lead to abnormal OGT or OGA expression
Solution Approach 2:
The reconstituted OGA complex performs the deglycosylation function directly at the target protein without requiring endogenous OGA expression changes. The system is self-sufficient and does not trigger cellular feedback loops that would alter OGT or OGA gene expression
3Measurement precision
If site-directed mutagenesis is used to target specific glycosites, then specific glycosites can be permanently modified, but it remains challenging for proteins with multiple or unmapped glycosites and may disrupt cross-talk with other PTMs
Solution Approach 1:
The patent segments the enzymatic function from the targeting function. The OGA is divided into fragments that provide the catalytic activity, while separate F-box tags provide the protein-specific targeting. This modular approach allows simultaneous targeting of multiple glycosites on different proteins without the complexity of designing multiple mutagenesis strategies
Solution Approach 2:
The F-box tag serves as an intermediary that bridges the OGA enzyme and the target protein. This intermediary system allows selective enrichment and degradation of F-box tagged proteins while preserving other proteins, enabling study of multiple glycosites without disrupting cross-talk with other PTMs on non-tagged proteins
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables selective removal of O-GlcNAc from target proteins with minimal global perturbation, facilitating the dissection of O-GlcNAc functions and maintaining the integrity of the broader O-GlcNAc proteome, thus providing a tool for understanding the specific contributions of O-GlcNAc to protein function and signaling pathways.
Implementation Method 1
leveraging nanobody selectivity to restore deglycosidase activity, allowing for precise modulation of O-GlcNAc levels on specific proteins
Data Source
AI summary
The present disclosure provides fusion proteins comprising a nanobody and a split glycosyl hydrolase enzyme. Also provided herein are split glycosyl hydrolase enzymes and fusion proteins comprising such enzymes. Further provided herein are polynucleotides, vectors, and cells. The present disclosure also provides methods of deglycosylating a protein and methods of studying the effects of glycosylation on protein function in cells. Also provided herein are methods of treating diseases.