Development and characterization of recombinant ADP-ribose binding reagents that allow simultaneous detection of mono and poly ADP-ribose.
J Biol Chem
; 300(9): 107609, 2024 Sep.
Article
in En
| MEDLINE
| ID: mdl-39074634
ABSTRACT
ADP-ribosylation (ADPRylation) is a post-translational modification (PTM) of proteins mediated by the activity of a variety of ADP-ribosyltransferase (ART) enzymes, such as the Poly (ADP-ribose) Polymerase (PARP) family of proteins. This PTM is diverse in both form and biological functions, which makes it a highly interesting modification, but difficult to study due to limitations in reagents available to detect the diversity of ADPRylation. Recently we developed a set of recombinant antibody-like ADP-ribose (ADPR) binding proteins using naturally occurring ADPR binding domains (ARBDs), including macrodomains and WWE domains, functionalized by fusion to the constant "Fc" region of rabbit immunoglobulin. Herein, we present an expansion of this biological toolkit, where we have replaced the rabbit Fc sequence with the sequence from two other species, mouse and goat. These new reagents are based on a previously characterized set of naturally occurring ARBDs with known specificity. Characterization of the new reagents demonstrates that they can be detected in a species-dependent manner by secondary immunological tools, recognize specific ADPR moieties, and can be used for simultaneous detection of mono ADPR and poly ADPR at single-cell resolution in various antibody-based assays. The expansion of this toolkit will allow for more multiplexed assessments of the complexity of ADPRylation biology in many biological systems.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Adenosine Diphosphate Ribose
Limits:
Animals
/
Humans
Language:
En
Journal:
J Biol Chem
Year:
2024
Document type:
Article
Affiliation country:
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