Design of Glyco-Linkers at Multiple Structural Levels to Modulate Protein Stability.

N-glycosylation has critical roles in regulating protein stability, but the molecular basis is poorly understood. In this study, we integrated experimental and computational techniques to investigate the mechanism by which full-length N-glycans modulate protein stability from quaternary structure perspective. We found the two inherent N-glycans of β-glucuronidase expressed in Pichia pastoris function as "glyco-linkers" that hold spatially proximal motifs together to compact the local protein structure. We further designed and placed glyco-linkers in the unusual form of glyco-bridge and glyco-hairpin at the interfaces between domains and monomers with higher structural level, respectively, which conferred dramatically higher kinetic stability and thermodynamic stability than the inherent N-glycans. Our study not only provides unique insight into the interactions between glycans and proteins from a quaternary structure perspective but also facilitates the rational design of N-glycans as general tools that can enhance protein stability.

• Publication year

  2018

• Venue

  Journal of Physical Chemistry Letters

• Publication date

  2018-07-30

• Fields of study

  Biology, Medicine, Materials Science, Chemistry

• Identifiers
  DOI 10.1021/acs.jpclett.8b01570PMID 30060662
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Structure-guided engineering of the substrate specificity of a fungal β-glucuronidase toward triterpenoid saponins

  2017cited by this paper
• Comparative thermal inactivation analysis of Aspergillus oryzae and Thiellavia terrestris cutinase: Role of glycosylation

  2017cited by this paper
• Comprehensive analysis of protein glycosylation by solid-phase extraction of N-linked glycans and glycosite-containing peptides

  2016cited by this paper
• N‐linked glycosylation of recombinant cellobiohydrolase I (Cel7A) from Penicillium verruculosum and its effect on the enzyme activity

  2016cited by this paper
• Stabilizing the CH2 Domain of an Antibody by Engineering in an Enhanced Aromatic Sequon.

  2016cited by this paper
• The Dependence of Carbohydrate-Aromatic Interaction Strengths on the Structure of the Carbohydrate.

  2016cited by this paper
• Role of N-linked glycosylation in the secretion and enzymatic properties of Rhizopus chinensis lipase expressed in Pichia pastoris

  2015cited by this paper
• Enhanced Aromatic Sequons Increase Oligosaccharyltransferase Glycosylation Efficiency and Glycan Homogeneity.

  2015cited by this paper
• Glycosylation May Reduce Protein Thermodynamic Stability by Inducing a Conformational Distortion.

  2015cited by this paper
• The intrinsic and extrinsic effects of N-linked glycans on glycoproteostasis.

  2014cited by this paper
• Specificity of O-glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

  2014cited by this paper
• Emerging Principles for the Therapeutic Exploitation of Glycosylation

  2014cited by this paper
• N-Glycosylation Is Required for Matriptase-2 Autoactivation and Ectodomain Shedding*

  2014cited by this paper
• Systematic optimization of interface interactions increases the thermostability of a multimeric enzyme.

  2013cited by this paper
• Knockout of an endogenous mannosyltransferase increases the homogeneity of glycoproteins produced in Pichia pastoris

  2013cited by this paper
• Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

  2013cited by this paper
• Engineering the Pattern of Protein Glycosylation Modulates the Thermostability of a GH11 Xylanase*

  2013cited by this paper
• Exploring the molecular mechanism of stabilization of the adhesion domains of human CD2 by N-glycosylation.

  2012cited by this paper
• N‐glycosylation of enhanced aromatic sequons to increase glycoprotein stability

  2012cited by this paper
• Specific and nonspecific effects of glycosylation.

  2012cited by this paper
• Protein Native-State Stabilization by Placing Aromatic Side Chains in N-Glycosylated Reverse Turns

  2011cited by this paper
• Glycosylation of the enhanced aromatic sequon is similarly stabilizing in three distinct reverse turn contexts

  2011cited by this paper
• Context-dependent effects of asparagine glycosylation on Pin WW folding kinetics and thermodynamics.

  2010cited by this paper
• The N-X-S/T consensus sequence is required but not sufficient for bacterial N-linked protein glycosylation.

  2005cited by this paper
• Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding.

  2003cited by this paper
• Assembly of asparagine-linked oligosaccharides.

  1985cited by this paper

• Tetramerization of GH2 β-Glucuronidases is Essential for Catalyzing the Hydrolysis of the Large Substrate Glycyrrhizin.

  2022cites this paper
• Enhancing the Thermostability of Phytase to Boiling Point by Evolution-Guided Design

  2022cites this paper
• Engineered microorganisms and enzymes for efficiently synthesizing plant natural products

  2021cites this paper
• Recombinant protein production in the filamentous fungus Trichoderma

  2020cites this paper
• Bioengineering oligomerization and monomerization of enzymes: learning from natural evolution to matching the demands for industrial applications

  2020cites this paper
• Prion protein post-translational modifications modulate heparan sulfate binding and limit aggregate size in prion disease.

  2020cites this paper
• Efficient Display of Aspergillus niger β-Glucosidase on Saccharomyces cerevisiae Cell Wall for Aroma Enhancement in Wine.

  2019cites this paper
• Enhanced yeast surface display of β‐glucuronidase using dual anchor motifs for high‐temperature glycyrrhizin hydrolysis

  2019cites this paper