Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts

42-POS Board 42 Enzymatic Manipulation of Antibody Fc-Mediated Effector Functions Eric Sundberg . University of Maryland School of Medicine, Baltimore, USA.

In order to evade host immune mechanisms, many bacteria secrete a diversity of immunomodulatory enzymes. Streptococcus pyogenes, one of the most common human pathogens, secretes a large endoglycosidase, EndoS, which removes carbohydrates in a highly specific manner from IgG antibodies. This renders antibodies incapable of eliciting host effector functions through either the complement pathway or via Fc γ receptor signaling, providing the bacteria with a survival advantage. On account of this antibody-specific modifying activity, EndoS is currently being developed as a promising injectable therapeutic for autoimmune diseases that rely on autoantibodies. Additionally, EndoS is a key enzyme used in the chemoenzymatic synthesis of homogenously glycosylated antibodies with tailored Fc γ receptor- mediated effector functions. Despite the tremendous utility of this enzyme, the molecular basis of EndoS specificity for, and processing of, IgG antibodies has remained poorly understood. We have recently determined the high-resolution X-ray crystal structure of EndoS, which provides the first mechanistic insight into its unique enzymatic properties. Based on this structure, we rationally designed chimeric endoglycosidases in which we exchanged the glycosidase domain of EndoS with that of EndoF1 in order to create enzymes that exhibit high specificity for antibody substrates while changing their glycan specificity to that of EndoF1 (high mannose type) from that of EndoS (complex biantennary type). Using mass spectrometry and surface plasmon resonance assays, we found these engineered enzymes to be highly specific and efficient for the glycoprotein substrates for which we designed them. This novel glycoprotein engineering strategy for constructing chimeric endoglycosidases that are able to manipulate the glycan composition on IgG antibodies provides new opportunities to engineer antibodies with unique glycan compositions for previously unachievable therapeutic applications.

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