Enzymatic treatment of pork protein for the enhancement of iron bioavailability.

The typical intervention for iron-deficiency anaemia is through oral supplementation with iron salts, which have unpleasant side effects. Therefore, there is a need for the development of supplements which will be absorbed more effectively and may have fewer side effects. This study investigated the effects of partially hydrolysed pork proteins on the bioavailability of non-haem iron. The peptides were derived using either pepsin or a combination of bacterial and fungal proteases, and their ability to deliver iron was evaluated in a rat intestine epithelial tissue model. The greatest iron absorption was achieved with peptides hydrolysed by pepsin of low molecular weight (<6-8 kDa). The peptides hydrolysed with bacterial and fungal enzymes may have bound to the iron too strongly, affecting bioavailability. Finally, hydrolysing proteins using pepsin in the presence of iron produces a complex that resulted in more ferritin expression than mixing the peptides with iron after hydrolysis.

Effects of different proteases on iron absorption property of egg white hydrolysates.

One of the causes of iron deficiency in human is poor absorption of non-heme iron from the diet. While proteins from meats have been reported in the literature to enhance the absorption of non-heme iron, other proteins, such as those from egg, are known to inhibit iron absorption. The objective of this study is to investigate non-heme iron binding property of egg white proteins hydrolyzed using pepsin and a combination of bacterial/fungal proteases. The iron bioavailability of non-heme iron, in the presence of egg white (EW) hydrolysates, was evaluated in vitro using a tissues culture model system - rat intestinal epithelial cells (IEC-6). In the first treatment condition, EW was digested in the presence of ferrous gluconate (FeGluc), producing a peptide-FeGluc complex. In the second treatment, EW was digested in the absence of FeGluc followed by the addition of the non-heme iron. In both treatments, the resulting EW hydrolysates were further separated into >0.1-0.5kDa and >6-8kDa peptide fractions using dialysis. The hydrolysate and FeGluc complex or mixtures were applied to the IEC-6 cells and iron absorption was measured after 2h or 16h. Results showed that the peptide-FeGluc complex digested with a combined proteases from Bacillus licheniformis (SDAY) and from Aspergillus melluss (PP) increased the in vitro iron-binding property but did not enhance iron uptake by the in IEC-6 cells (p<0.05). Peptide-FeGluc complex digested with pepsin alone (>0.1-0.5kDa) resulted in significantly higher iron uptake in IEC-6 cells compared with the higher molecular weight complex (>6-8kDa) produced using the same hydrolysis treatment. Similarly, enhanced iron uptake was observed with the complexes produced with the combined SDAY and PP enzymatic treatments (>0.1-0.5kDa and >6-8kDa) (p<0.05). On the other hand, the enhanced iron absorption effect was not observed when pre-hydrolyzed free peptides were added to FeGluc. Overall, this study suggests that low molecular weight fractions of egg white protein hydrolysates can enhance the bioavailability of non-heme iron. Furthermore, the method by which the egg white proteins are being prepared, i.e., in the presence or absence of FeGluc, can affect the bioavailability of the non-heme iron.

Conserved prosegment residues stabilize a late-stage folding transition state of pepsin independently of ground states.

The native folding of certain zymogen-derived enzymes is completely dependent upon a prosegment domain to stabilize the folding transition state, thereby catalyzing the folding reaction. Generally little is known about how the prosegment accomplishes this task. It was previously shown that the prosegment catalyzes a late-stage folding transition between a stable misfolded state and the native state of pepsin. In this study, the contributions of specific prosegment residues to catalyzing pepsin folding were investigated by introducing individual Ala substitutions and measuring the effects on the bimolecular folding reaction between the prosegment peptide and pepsin. The effects of mutations on the free energies of the individual misfolded and native ground states and the transition state were compared using measurements of prosegment-pepsin binding and folding kinetics. Five out of the seven prosegment residues examined yielded relatively large kinetic effects and minimal ground state perturbations upon mutation, findings which indicate that these residues form strengthened and/or non-native contacts in the transition state. These five residues are semi- to strictly conserved, while only a non-conserved residue had no kinetic effect. One conserved residue was shown to form native structure in the transition state. These results indicated that the prosegment, which is only 44 residues long, has evolved a high density of contacts that preferentially stabilize the folding transition state over the ground states. It is postulated that the prosegment forms extensive non-native contacts during the process of catalyzing correct inter- and intra-domain contacts during the final stages of folding. These results have implications for understanding the folding of multi-domain proteins and for the evolution of prosegment-catalyzed folding.

Understanding the mechanism of prosegment-catalyzed folding by solution NMR spectroscopy.

Multidomain protein folding is often more complex than a two-state process, which leads to the spontaneous folding of the native state. Pepsin, a zymogen-derived enzyme, without its prosegment (PS), is irreversibly denatured and folds to a thermodynamically stable, non-native conformation, termed refolded pepsin, which is separated from native pepsin by a large activation barrier. While it is known that PS binds refolded pepsin and catalyzes its conversion to the native form, little structural details are known regarding this conversion. In this study, solution NMR was used to elucidate the PS-catalyzed folding mechanism by examining the key equilibrium states, e.g. native and refolded pepsin, both in the free and PS-bound states, and pepsinogen, the zymogen form of pepsin. Refolded pepsin was found to be partially structured and lacked the correct domain-domain structure and active-site cleft formed in the native state. Analysis of chemical shift data revealed that upon PS binding refolded pepsin folds into a state more similar to that of pepsinogen than to native pepsin. Comparison of pepsin folding by wild-type and mutant PSs, including a double mutant PS, indicated that hydrophobic interactions between residues of prosegment and refolded pepsin lower the folding activation barrier. A mechanism is proposed for the binding of PS to refolded pepsin and how the formation of the native structure is mediated.

¹H, ¹³C, and ¹5N backbone resonance assignments of the porcine pepsin and porcine pepsin complexed with pepstatin.

Pepsin is formed as the zymogen, pepsinogen, which includes an additional 44 residue prosegment (PS) on the N-terminus. Upon acidification (pH <3) the PS is removed, yielding active pepsin. The PS is critical to such processes as the initiation of correct folding and protein stability. In the present study, the NMR assignments of the 34.6 kDa native porcine pepsin and porcine pepsin complexed with pepstatin are reported in order to obtain structural information regarding PS-catalyzed protein folding. Such information would contribute to a better understanding of the nature of folding/unfolding energy barrier of pepsin and other aspartic proteases.

Crystal structures of the free and inhibited forms of plasmepsin I (PMI) from Plasmodium falciparum.

Plasmepsin I (PMI) is one of the four vacuolar pepsin-like proteases responsible for hemoglobin degradation by the malarial parasite Plasmodium falciparum, and the only one with no crystal structure reported to date. Due to substantial functional redundancy of these enzymes, lack of inhibition of even a single plasmepsin can defeat efforts in creating effective antiparasitic agents. We have now solved crystal structures of the recombinant PMI as apoenzyme and in complex with the potent peptidic inhibitor, KNI-10006, at the resolution of 2.4 and 3.1Å, respectively. The apoenzyme crystallized in the orthorhombic space group P2(1)2(1)2(1) with two molecules in the asymmetric unit and the structure has been refined to the final R-factor of 20.7%. The KNI-10006 bound enzyme crystallized in the tetragonal space group P4(3) with four molecules in the asymmetric unit and the structure has been refined to the final R-factor of 21.1%. In the PMI-KNI-10006 complex, the inhibitors were bound identically to all four enzyme molecules, with the opposite directionality of the main chain of KNI-10006 relative to the direction of the enzyme substrates. Such a mode of binding of inhibitors containing an allophenylnorstatine-dimethylthioproline insert in the P1-P1' positions, previously reported in a complex with PMIV, demonstrates the importance of satisfying the requirements for the proper positioning of the functional groups in the mechanism-based inhibitors towards the catalytic machinery of aspartic proteases, as opposed to binding driven solely by the specificity of the individual enzymes. A comparison of the structure of the PMI-KNI-10006 complex with the structures of other vacuolar plasmepsins identified the important differences between them and may help in the design of specific inhibitors targeting the individual enzymes.

Rational redesign of porcine pepsinogen containing an antimicrobial peptide.

A novel strategy for the controlled release and localization of bioactive peptides within digestive and immunity-related enzymes was developed. The N-terminus of porcine pepsinogen A was fused to the basic amino acid-rich region of bovine lactoferricin B termed 'tLfcB', a cationic antimicrobial/anticancer peptide. Recombinant tLfcB-porcine pepsinogen A was expressed in soluble form in Escherichia coli as a thioredoxin (Trx) fusion protein. Thioredoxin-tLfcB-porcine pepsinogen A was found to activate autocatalytically under acidic conditions. Recombinant pepsin A derived from the activation of the fusion protein had a catalytic rate and substrate affinity similar to that derived from the recombinant thioredoxin-porcine pepsinogen A control. Pepsin-treated thioredoxin-tLfcB-porcine pepsinogen A yielded increased antimicrobial activity against the Gram-negative bacteria E.coli relative to control suggesting that a second function (antimicrobial activity) was successfully engineered into a functional peptidase. The novel design strategy described herein presents a potential strategy for targeted delivery of antimicrobial or therapeutic peptides in transgenic organisms via re-engineering native proteins critical to plant and animal defense mechanisms.

Recombinant prosegment peptide acts as a folding catalyst and inhibitor of native pepsin.

Porcine pepsin A, a gastric aspartic peptidase, is initially produced as the zymogen pepsinogen that contains an N-terminal, 44 residue prosegment (PS) domain. In the absence of the PS, native pepsin (Np) is irreversibly denatured and when placed under refolding conditions, folds to a thermodynamically stable denatured state. This denatured, refolded pepsin (Rp) state can be converted to Np by the exogenous addition of the PS, which catalyzes the folding of Rp to Np. In order to thoroughly study the mechanism by which the PS catalyzes pepsin folding, a soluble protein expression system was developed to produce recombinant PS peptide in a highly pure form. Using this system, the wild-type and three-mutant PS forms, in which single residue substitutions were made (V4A, R8A and K36A), were expressed and purified. These PS peptides were characterized for their ability to inhibit Np enzymatic activity and to catalyze the folding of Rp to Np. The V4A, R8A and K36A mutant PS peptides were found to have nanomolar inhibition constants, Ki, of 82.4, 58.3 and 95.6 nM, respectively, approximately a two-fold increase from that of the wild-type PS (36.2 nM). All three-mutant PS peptides were found to catalyze Np folding with a rate constant of 0.06 min(-1), five-fold lower than that of the wild-type. The observation that the mutant PS peptides retained their inhibition and folding-catalyst functionality suggests a high level of resilience to mutations of the pepsin PS.

Multifunctional aspartic peptidase prosegments.

The structure-function relationships of aspartic peptidases (APs) (EC 3.4.23.X) have been extensively investigated, yet much remains to be elucidated regarding the various molecular mechanisms of these enzymes. Over the past years, APs have received considerable interest for food applications (e.g. cheese, fermented foods) and as potential targets for pharmaceutical intervention in human diseases including hypertension, cancer, Alzheimer's disease, AIDS (acquired immune deficiency syndrome), and malaria. A deeper understanding of the structure and function of APs, therefore, will have a direct impact on the design of peptidase inhibitors developed to treat such diseases. Most APs are synthesized as zymogens which contain an N-terminal prosegment (PS) domain that is removed at acidic pH by proteolytic cleavage resulting in the active enzyme. While the nature of the AP PS function is not entirely understood, the PS can be important in processes such as the initiation of correct folding, protein stability, blockage of the active site, pH-dependence of activation, and intracellular sorting of the zymogen. This review summarizes the current knowledge of AP PS function (especially within the A1 family), with particular emphasis on protein folding, cellular sorting, and inhibition.