Unknown

Dataset Information

0

Protease Inhibition Mechanism of Camelid-like Synthetic Human Antibodies.


ABSTRACT: Macromolecular protease inhibitors and camelid single-domain antibodies achieve their enzymic inhibition functions often through protruded structures that directly interact with catalytic centers of targeted proteases. Inspired by this phenomenon, we constructed synthetic human antibody libraries encoding long CDR-H3s, from which highly selective monoclonal antibodies (mAbs) that inhibit multiple proteases were discovered. To elucidate their molecular mechanisms, we performed in-depth biochemical characterizations on a panel of matrix metalloproteinase (MMP)-14 inhibitory mAbs. Assays included affinity and potency measurements, enzymatic kinetics, a competitive enzyme-linked immunosorbent assay, proteolytic stability, and epitope mapping followed by quantitative analysis of binding energy changes. The results collectively indicated that these mAbs of convex paratopes were competitive inhibitors recognizing the vicinity of the active cleft, with their significant epitopes scattered across the north and south rims of the cleft. Remarkably, identified epitopes were the surface loops that were highly diverse among MMPs and predominately located at the prime side of the proteolytic site, shedding light on the mechanisms of target selectivity and proteolytic resistance. Substrate sequence profiling and paratope mutagenesis further suggested that mAb 3A2 bound to the active-site cleft in a canonical (substrate-like) manner, by direct interactions between 100hNLVATP100m of its CDR-H3 and subsites S1-S5' of MMP-14. Overall, synthetic mAbs carrying convex paratopes can achieve efficient inhibition and thus hold great therapeutic promise for effectively and safely targeting biomedically important proteases.

SUBMITTER: Nam DH 

PROVIDER: S-EPMC7572768 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

altmetric image

Publications

Protease Inhibition Mechanism of Camelid-like Synthetic Human Antibodies.

Nam Dong Hyun DH   Lee Ki Baek KB   Kruchowy Evan E   Pham Henry H   Ge Xin X  

Biochemistry 20200930 40


Macromolecular protease inhibitors and camelid single-domain antibodies achieve their enzymic inhibition functions often through protruded structures that directly interact with catalytic centers of targeted proteases. Inspired by this phenomenon, we constructed synthetic human antibody libraries encoding long CDR-H3s, from which highly selective monoclonal antibodies (mAbs) that inhibit multiple proteases were discovered. To elucidate their molecular mechanisms, we performed in-depth biochemica  ...[more]

Similar Datasets

| S-EPMC7598594 | biostudies-literature
| S-EPMC4424330 | biostudies-literature
| S-EPMC7695701 | biostudies-literature
| S-EPMC2148376 | biostudies-literature
| S-EPMC6282393 | biostudies-literature
| S-EPMC5524736 | biostudies-literature
| S-EPMC6204844 | biostudies-literature
| S-EPMC3268006 | biostudies-literature
| S-EPMC3098910 | biostudies-literature
| S-EPMC7199733 | biostudies-literature