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Rational and computational design of stabilized variants of cyanovirin-N that retain affinity and specificity for glycan ligands.

ABSTRACT: Cyanovirin-N (CVN) is an 11 kDa pseudosymmetric cyanobacterial lectin that has been shown to inhibit infection by the human immunodeficiency virus by binding to high-mannose oligosaccharides on the surface of the viral envelope glycoprotein gp120. In this work, we describe rationally designed CVN variants that stabilize the protein fold while maintaining high affinity and selectivity for their glycan targets. Poisson-Boltzmann calculations and protein repacking algorithms were used to select stabilizing mutations in the protein core. By substituting the buried polar side chains of Ser11, Ser20, and Thr61 with aliphatic groups, we stabilized CVN by nearly 12 °C against thermal denaturation, and by 1 M GuaHCl against chemical denaturation, relative to a previously characterized stabilized mutant. Glycan microarray binding experiments confirmed that the specificity profile of carbohydrate binding is unperturbed by the mutations and is identical for all variants. In particular, the variants selectively bound glycans containing the Man?(1?2)Man linkage, which is the known minimal binding unit of CVN. We also report the slow denaturation kinetics of CVN and show that they can complicate thermodynamic analysis; in particular, the unfolding of CVN cannot be described as a fixed two-state transition. Accurate thermodynamic parameters are needed to describe the complicated free energy landscape of CVN, and we provide updated values for CVN unfolding.

SUBMITTER: Patsalo V

PROVIDER: S-EPMC3234137 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Rational and computational design of stabilized variants of cyanovirin-N that retain affinity and specificity for glycan ligands.

Patsalo Vadim V Raleigh Daniel P DP Green David F DF

Biochemistry 20111116 49

Cyanovirin-N (CVN) is an 11 kDa pseudosymmetric cyanobacterial lectin that has been shown to inhibit infection by the human immunodeficiency virus by binding to high-mannose oligosaccharides on the surface of the viral envelope glycoprotein gp120. In this work, we describe rationally designed CVN variants that stabilize the protein fold while maintaining high affinity and selectivity for their glycan targets. Poisson-Boltzmann calculations and protein repacking algorithms were used to select sta ...[more]

PMID: 22032696

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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

Rational and computational design of stabilized variants of cyanovirin-N that retain affinity and specificity for glycan ligands.

Publications

Rational and computational design of stabilized variants of cyanovirin-N that retain affinity and specificity for glycan ligands.

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