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Study of Self Catalyzed Protein Glycation on Monoclonal Antibody A (MAb A)
Author(s) -
Liu Hong,
Yang Yi,
Flores Heather,
Zhang Boyan,
Swartz Trevor,
Patapoff Tom
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.553.8
Subject(s) - glycation , chemistry , amadori rearrangement , lysine , biochemistry , glycosylation , maillard reaction , catalysis , monoclonal antibody , antibody , amino acid , receptor , immunology , biology
Protein glycation is known as a nonenzymatic reaction between reducing sugars, and amine groups. It may lead to functional changes of a protein. Recently, a site specific glycation on lysine Kn of MAb A was reported, with a hypothesis that MAb A self catalysis in reaction with glucoses at specific site. To elucidate the mechanism of the site specific glycation, an in vitro forced glycation study was employed to generate fully glycated MAb A. Subsequently, characterization of the glycated MAb A and its thermal degradation products were performed, through boronate affinity HPLC, ESI‐Mass spectrometry, Ion‐exchange HPLC, and peptide map. Finally, molecular dynamics (MD) simulations were used to map the local electrostatic environment around Kn under glycation conditions. The in vitro study showed that Kn is preferably glycated up to 90%, with four other lysine sites glycated at various low levels. Thermal stress study demonstrated that the main degradation pathway of highly glycated MAb A is hydrolysis of the Schiff base, the reverse reaction of glycation. Glycated Kn was preferably hydrolyzed as compared to the other glycated lysine sites. These results demonstrated the site specific glycation at Kn is self catalyzed. MD analysis suggested the observed self catalysis is caused by a strong electron‐donating environment surrounding Kn with three aspartates potentially involved in Kn reaction with a glucose.