N-glycosylation enhances functional and structural stability of recombinant β-glucuronidase expressed in Pichia pastoris

Zou, Shu-Ping; Huang, Shuxian; Kaleem, Imdad; Chun, Li

doi:10.1016/j.jbiotec.2012.12.015

Cited by 39 publications

(37 citation statements)

References 35 publications

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“…This is similar to previous data showing that glycosylation significantly alters the pI of other similar glycoproteins (20,21). Moreover, glycosylation can provide conformational and structural stability (22,23) and may facilitate correct folding (23,24) and importantly modulate ligand binding and functions (20,25,26). We found that rLILRA3 in its non-glycosylated form required extensive refolding steps, was highly susceptible to aggregation/oxidation, and was non-functional despite being of high purity and high yield (up to 15 mg/liter).…”

Section: Discussionsupporting

confidence: 90%

Glycosylation in a Mammalian Expression System Is Critical for the Production of Functionally Active Leukocyte Immunoglobulin-like Receptor A3 Protein

Lee

Mitchell

Lau

et al. 2013

Journal of Biological Chemistry

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Background: LILRA3 is a soluble receptor with unknown functions that is abundantly present in human serum. Results: Optimally glycosylated recombinant LILRA3 protein produced only in a mammalian system binds potential ligands and suppresses monocyte function. Conclusion: LILRA3 suppresses LPS-mediated TNF production, suggesting that it is a new anti-inflammatory protein.Significance: This work provides first insight into the biochemical characteristics and functions of LILRA3.

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Section: Discussionsupporting

confidence: 90%

Glycosylation in a Mammalian Expression System Is Critical for the Production of Functionally Active Leukocyte Immunoglobulin-like Receptor A3 Protein

Lee

Mitchell

Lau

et al. 2013

Journal of Biological Chemistry

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“…One is to explore protease-resistant feed enzymes (41,42), and the other is to screen engineered feed enzymes with improved protease resistance by rational design (43). Structure-based rational design has been less frequently used than naturally occurring protease-resistant enzymes (24,(44)(45)(46), especially with respect to pepsin resistance. The cleavage of enzymes by proteases involves a nucleophilic attack of the polarized carbonyl group of the substrate peptide bond, followed by stabilization of the oxygen in an oxyanion hole of proteases (47,48).…”

Section: Discussionmentioning

confidence: 99%

“…The cleavage of enzymes by proteases involves a nucleophilic attack of the polarized carbonyl group of the substrate peptide bond, followed by stabilization of the oxygen in an oxyanion hole of proteases (47,48). Therefore, glycans at the N-glycosylation site may sterically protect enzymes against proteases (24). In the present study, we employed a structure-based mutagenesis approach to introduce N-glycosylation sites into Yersinia phytases to determine the underlying mechanism of glycosylation-mediated enzyme resistance to pepsin.…”

Section: Discussionmentioning

confidence: 99%

“…Similarly, bovine pancreatic RNase B occurs naturally as an N-glycoenzyme, and the glycoforms show increased resistance to pronase compared with those of their unglycosylated counterparts (23). The N-glycosylated form of ␤-glucuronidase also exhibits higher resistance to proteolytic degradation by pepsin than is seen with its N-deglycosylated form (24). Site-directed mutagenesis has confirmed that the N-glycosylation of scavenger receptor SREC-I at N289 confers resistance against trypsin digestion (25).…”

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confidence: 99%

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N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Niu

Luo

Shi

et al. 2016

Appl Environ Microbiol

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N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed.A s much as 80% of the total phosphorus in cereal grains, oilseeds, and legumes exists in the form of phytate (myo-inositol hexakisphosphate), one of the most important functional ingredients in animal feed (1). However, phytate usually forms indigestible complexes with mineral cations and proteins, thereby causing reductions in nutrient utilization (2, 3). The dietary phytate undigested by monogastric animals is also released into the environment, where it causes phosphorus pollution (4, 5).The enzyme phytase catalyzes the sequential release of inorganic phosphate and lower myo-inositol phosphates from phytate (6); thus, inclusion of exogenous phytases in animal feeds as enzyme additives can enhance the nutrient uptake, lower the feedstuff production costs, and protect the environment in regions where intensive animal farming is conducted (7,8). Numerous phytases have been identified from microorganisms, animals, and plants (9-11), but only a few of them are commercially produced (12). The extensive application of phytases is limited by enzyme lability and protease sensitivity under high processing temperatures and low physiological pHs. Therefore, improving phytase stability at low pHs and high protease concentrations is desirable.Pepsin, a monomeric protein composed of two ␤-barrel-like domains, represents the main proteolytic enzyme in gastric juice (13). The acidic residues D32 and D215 are mainly responsible for proteoly...

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“…More than half of the proteins found in nature undergo glycosylation, of which three quarters are N-glycosylated [3,4]. The N-linked glycosylation recognizes asparagine (N) residues with a conserved amino acid sequence N-X-S/T/C (where X can be any residue except proline) [5][6][7].…”

Section: Introductionmentioning

confidence: 99%

N-Glycosylation Sited at Residues of Catalytic Center Could Repress The Activity of Endoglucanase from Rhizopus Stolonifer

Tang¹,

Zhang²,

Cheng³

et al. 2017

Proceedings of the 2016 International Conference on Biological Engineering and Pharmacy (BEP 2016)

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Abstract-As a common post-translational modification of proteins, glycosylation has been proven effectively influence the activity of enzyme. To improve the activity of endoglucanase (EGII) from Rhizopus stolonifer TP-02, we studied the effect of glycosylation on the structure and activity of EGII. Two potential glycosylation sites (N84 and N150) were predicted, of which N150 was located in the entrance of catalytic domain (CD). N84D and N150D was obtained by overlapping PCR and expressed in Pichia pastoris for purification. Compared with EGII, the specific activity of N84D was decreased by 28.7%, whereas the activity of N150D was increased by 56.9%. Dynamics simulations results indicated that the N-glycans located in the active center could repress the activity of EGII due to the steric hindrances that effectively hinder the cellulose chains to enter the CD domain.

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N-glycosylation enhances functional and structural stability of recombinant β-glucuronidase expressed in Pichia pastoris

Cited by 39 publications

References 35 publications

Glycosylation in a Mammalian Expression System Is Critical for the Production of Functionally Active Leukocyte Immunoglobulin-like Receptor A3 Protein

Glycosylation in a Mammalian Expression System Is Critical for the Production of Functionally Active Leukocyte Immunoglobulin-like Receptor A3 Protein

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

N-Glycosylation Sited at Residues of Catalytic Center Could Repress The Activity of Endoglucanase from Rhizopus Stolonifer

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