Development and Characterisation of a Versatile Single-Domain Antibody Specific for M1-linked Ubiquitin Chains

Area of Science:

• Molecular Biology
• Biochemistry
• Immunology

Background:

• Ubiquitin signalling relies on diverse polyubiquitin chains encoding specific cellular functions.
• Limited availability of chain type-specific reagents hinders the study of less abundant ubiquitin chain types.

Purpose of the Study:

• To develop versatile, ubiquitin linkage-specific affinity reagents using single-domain antibodies (sdAbs).
• To generate and characterize an sdAb that specifically recognizes methionine-1 (M1)-linked ubiquitin chains.

Main Methods:

• Phage display and synthetic human sdAb libraries were used to identify specific binders.
• Characterization involved binding affinity measurements, specificity testing (diubiquitin and polyubiquitin chains), AlphaFold3 modelling, and saturation mutagenesis.
• Functional validation was performed using ELISA, immunoblotting, immunoprecipitation, ubiquitination analysis, and immunofluorescence microscopy.

Main Results:

• Identified 2A6, a human sdAb with high affinity (nanomolar) and specificity for M1-linked ubiquitin chains.
• 2A6 demonstrated specificity for M1-linked chains at both diubiquitin and polyubiquitin chain levels.
• Functional assays confirmed 2A6's utility for detecting and enriching M1-linked ubiquitin in various biological applications.

Conclusions:

• Single-domain antibodies serve as a versatile scaffold for creating ubiquitin linkage-specific affinity reagents.
• The developed 2A6 sdAb provides a cost-effective and tractable tool for investigating M1-linked ubiquitin signalling.
• This work establishes a framework for developing sdAbs targeting other ubiquitin linkages and architectures.