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

Abstract

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...

Effects of N-Glycosylation on the Structure, Function, and Stability of a Plant-Made Fc-Fusion Anthrax Decoy Protein

“…On the other hand, for drugs that treat chronic conditions, the absence of glycosylation is desired to avoid effector functions and associated inflammatory responses. Another important consideration is that glycosylated proteins are less susceptible to proteases, such as pepsin, compared with aglycosylated counterparts (Niu et al, 2016), which should be considered to maximize protein yield.…”

Engineering the residual side chains of HAP phytases to improve their pepsin resistance and catalytic efficiency

“…In our previous studies, Y. kristensenii YkAPPA and Y. enterocolitica YeAPPA were sensitive to pepsin after expression in E. coli , but E. coli -produced Y. rohdei YrAPPA was highly pepsin-resistant 21 30 36 37 . In this study, we employed a structure-based rational design approach to engineer a residual side chain in the surface pepsin cleavage site of Yersinia phytases to increase enzyme performance.…”

“…The surface positive charge of Photinus pyralis firefly luciferase was reduced to generate a mutant enzyme with an increased trypsin digestion half-life of about 4-fold at 23 °C 29 . Site-directed mutagenesis demonstrated that the N -glycosylation conferred pepsin resistance on Yersinia phytases and stability at acidic pH 30 . Other engineered phytase mutants also showed increased resistance to proteolysis and improved thermostability 31 32 33 .…”

Temporin-SHa and Its Analogs as Potential Candidates for the Treatment of Helicobacter pylori

“…The effect of mucins on peptides activity was evaluated by MIC and by time killing assay as explained below. The resistance of the peptides to pepsin was tested as previously described with some changes [28]. Briefly, temporin-SHa and its analogs (at 1 mg/mL, final concentration) were incubated for 4 h at 37 °C with 1 mg/mL (final concentration) of pepsin from porcine gastric mucosa (Sigma-Aldrich) in HCl 10mM, pH 2.…”