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Aglycone sterics-selective enzymatic glycan remodeling.

ABSTRACT: Precision remodeling of glycans in their native environments is pivotal for understanding glycan-mediated biological events and has important biotechnological implications in fields of clinical diagnosis, glyco-immune checkpoint therapy, and so forth. However, the influence of aglycone-steric diversity on the selectivity of glycan remodeling has been largely overlooked, limiting the application in complex biological scenarios. Here, we report the achievement of aglycone sterics-selective enzymatic glycan remodeling by controlled grafting of functional polymers from glycoenzyme. Through tuning polymer length, a series of enzyme-polymer composites with varying substrate permeability are prepared, which afford an activity pattern-based differentiation strategy for aglycone sterics. This leads to the implementation of glycolipid's partner screening, and aglycone sterics-selective glycan remodeling in a complex biological environment. We further orchestrate the polymer length adjustment with external cues to regulate aglycone-steric selectivity in a multi-faceted fashion, resulting in an unexpected enhancement of glycolipid remodeling, and temporal control of glycan remodeling on live cells.

SUBMITTER: Mao A

PROVIDER: S-EPMC9249669 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Aglycone sterics-selective enzymatic glycan remodeling.

Mao Anwen A Zhang Yan Y Wang Guyu G Zhong Tong T Chen Xinyu X Wang Haiqi H Xie Ran R Wang Xiaojian X Ding Lin L Ju Huangxian H

iScience 20220613 7

Precision remodeling of glycans in their native environments is pivotal for understanding glycan-mediated biological events and has important biotechnological implications in fields of clinical diagnosis, glyco-immune checkpoint therapy, and so forth. However, the influence of aglycone-steric diversity on the selectivity of glycan remodeling has been largely overlooked, limiting the application in complex biological scenarios. Here, we report the achievement of aglycone sterics-selective enzymat ...[more]

PMID: 35789841

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Project description:Functionalization of therapeutic lysosomal enzymes with mannose-6-phosphate (M6P) glycan ligands represents a major strategy for enhancing the cation-independent M6P receptor (CI-MPR)-mediated cellular uptake, thus improving the overall therapeutic efficacy of the enzymes. However, the minimal high-affinity M6P-containing N-glycan ligands remain to be identified and their efficient and site-selective conjugation to therapeutic lysosomal enzymes is a challenging task. We report here the chemical synthesis of truncated M6P-glycan oxazolines and their use for enzymatic glycan remodeling of recombinant human acid α-glucosidase (rhGAA), an enzyme used for treatment of Pompe disease which is a disorder caused by a deficiency of the glycogen-degrading lysosomal enzyme. Structure-activity relationship studies identified M6P tetrasaccharide oxazoline as the minimal substrate for enzymatic transglycosylation yielding high-affinity M6P glycan ligands for the CI-MPR. Taking advantage of the substrate specificity of endoglycosidases Endo-A and Endo-F3, we found that Endo-A and Endo-F3 could efficiently deglycosylate the respective high-mannose and complex type N-glycans in rhGAA and site-selectively transfer the synthetic M6P N-glycan to the deglycosylated rhGAA without product hydrolysis. This discovery enabled a highly efficient one-pot deglycosylation/transglycosylation strategy for site-selective M6P-glycan remodeling of rhGAA to obtain a more homogeneous product. The Endo-A and Endo-F3 remodeled rhGAAs maintained full enzyme activity and demonstrated 6- and 20-fold enhanced binding affinities for CI-MPR receptor, respectively. Using an in vitro cell model system for Pompe disease, we demonstrated that the M6P-glycan remodeled rhGAA greatly outperformed the commercial rhGAA (Lumizyme) and resulted in the reversal of cellular pathology. This study provides a general and efficient method for site-selective M6P-glycan remodeling of recombinant lysosomal enzymes to achieve enhanced M6P receptor binding and cellular uptake, which could lead to improved overall therapeutic efficacy of enzyme replacement therapy.

Dataset Information

Aglycone sterics-selective enzymatic glycan remodeling.

Publications

Aglycone sterics-selective enzymatic glycan remodeling.

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