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Structure-Based High-Efficiency Homogeneous Antibody Platform by Endoglycosidase Sz Provides Insights into Its Transglycosylation Mechanism.
- Source :
-
JACS Au [JACS Au] 2024 Apr 11; Vol. 4 (6), pp. 2130-2150. Date of Electronic Publication: 2024 Apr 11 (Print Publication: 2024). - Publication Year :
- 2024
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Abstract
- Monoclonal antibodies (mAbs) have gradually dominated the drug markets for various diseases. Improvement of the therapeutic activities of mAbs has become a critical issue in the pharmaceutical industry. A novel endo-β- N -acetylglucosaminidase, EndoSz, from Streptococcus equi subsp. zooepidemicus Sz105 is discovered and applied to enhance the activities of mAbs. Our studies demonstrate that the mutant EndoSz-D234M possesses an excellent transglycosylation activity to generate diverse glycoconjugates on mAbs. We prove that EndoSz-D234M can be applied to various marketed therapeutic antibodies and those in development for antibody remodeling. The remodeled homogeneous antibodies (mAb-G2S2) produced by EndoSz-D234M increase the relative ADCC activities by 3-26-fold. We further report the high-resolution crystal structures of EndoSz-D234M in the apo -form at 2.15 Å and the complex form with a bound G2S2-oxazoline intermediate at 2.25 Å. A novel pH-jump method was utilized to obtain the complex structure with a high resolution. The detailed interactions of EndoSz-D234M and the carried G2S2-oxazoline are hence delineated. The oxazoline sits in a hole, named the oxa-hole, which stabilizes the G2S2-oxazoline in transit and catalyzes the further transglycosylation reaction while targeting Asn-GlcNAc (+1) of Fc. In the oxa-hole, the H-bonding network involved with oxazoline dominates the transglycosylation activity. A mobile loop2 (a.a. 152-159) of EndoSz-D234M reshapes the binding grooves for the accommodation of G2S2-oxazoline upon binding, at which Trp154 forms a hydrogen bond with Man (-2). The long loop4 (a.a. 236-248) followed by helix3 is capable of dominating the substrate selectivity of EndoSz-D234M. In addition, the stepwise transglycosylation behavior of EndoSz-D234M is elucidated. Based on the high-resolution structures of the apo -form and the bound form with G2S2-oxazoline as well as a systematic mutagenesis study of the relative transglycosylation activity, the transglycosylation mechanism of EndoSz-D234M is revealed.<br />Competing Interests: The authors declare no competing financial interest.<br /> (© 2024 The Authors. Published by American Chemical Society.)
Details
- Language :
- English
- ISSN :
- 2691-3704
- Volume :
- 4
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- JACS Au
- Publication Type :
- Academic Journal
- Accession number :
- 38938812
- Full Text :
- https://doi.org/10.1021/jacsau.4c00004