Accessing the reproducibility and specificity of pepsin and other aspartic proteases

Ahn, Joomi; Cao, Min‐Jie; Yu, Ying; Engen, John R.

doi:10.1016/j.bbapap.2012.10.003

Cited by 124 publications

(120 citation statements)

References 38 publications

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“…The resulting relative deuterium levels were plotted versus the exchange time using Waters DynamX 2.0 TM software. Identification of the peptic peptides was accomplished through a combination of exact mass analysis and MS E [40] using Identity Software (Waters Corp., Milford, MA, USA), as previously described [41,42]. All assignments, deuterated spectra, and data processing were manually checked and verified.…”

Section: Methodsmentioning

confidence: 99%

The Influence of Adnectin Binding on the Extracellular Domain of Epidermal Growth Factor Receptor

Iacob

Chen

Ahn

et al. 2014

J. Am. Soc. Mass Spectrom.

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The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the tenth type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies. The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS). The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography.

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Section: Methodsmentioning

confidence: 99%

The Influence of Adnectin Binding on the Extracellular Domain of Epidermal Growth Factor Receptor

Iacob

Chen

Ahn

et al. 2014

J. Am. Soc. Mass Spectrom.

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“…The protein of interest is usually exposed to D 2 O buffer for various periods of time and the reaction is quenched by adjusting the pH to 2.5 and the temperature to 0 °C. The quenched protein is digested with pepsin or another aspartic protease [50–52], online or offline, and deuterium incorporation into each pepsin fragment is analyzed. Chromatographic separation before MS analysis is accomplished with ultrahigh pressure liquid chromatography (UPLC) that provides high-resolution separations under typical HDX MS experimental conditions [53].…”

Section: Hdx Ms: Tool To Study Protein Conformational Dynamicsmentioning

confidence: 99%

Hydrogen/deuterium exchange mass spectrometry applied to IL-23 interaction characteristics: potential impact for therapeutics

Iacob

Krystek

Huang

et al. 2015

Expert Review of Proteomics

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Interleukin-23 (IL-23) is an important therapeutic target for the treatment of inflammatory diseases. Adnectins are targeted protein therapeutics that are derived from domain III of human fibronectin, and have similar protein scaffold to antibodies. A specific adnectin (Adnectin 2) was identified to bind to IL-23 and compete with IL-23/IL-23R interaction, being a potential protein therapeutic. Hydrogen/deuterium exchange mass spectrometry (HDX MS) and computational methods were applied to probe the binding interactions between IL-23 and Adnectin2 and to determine the correlation between the two orthogonal methods. This review article summarizes the current structural knowledge about Il-23 and it focuses on the applicability of HDX MS to investigate the higher order structure of proteins, which plays an important role for the discovery of new and improved biotherapeutics.

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“…The acidic residues D32 and D215 are mainly responsible for proteolysis of substrates, which occurs by a base catalysis mechanism (14,15). Pepsin is an aspartic protease that catalyzes the hydrolytic cleavage of peptide bonds of a broad range of enzymes, with preference for F and L residues (16). The enzyme exhibits the highest catalytic activity at pH 2.0 and a temperature of 37 to 42°C (17,18).…”

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confidence: 99%

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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Accessing the reproducibility and specificity of pepsin and other aspartic proteases

Cited by 124 publications

References 38 publications

The Influence of Adnectin Binding on the Extracellular Domain of Epidermal Growth Factor Receptor

The Influence of Adnectin Binding on the Extracellular Domain of Epidermal Growth Factor Receptor

Hydrogen/deuterium exchange mass spectrometry applied to IL-23 interaction characteristics: potential impact for therapeutics

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

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