Abstract
Dipeptidyl peptidase-III is an important enkephalin degrading enzyme and its inhibitors are expected to be promising in pain management. Some of its inhibitors showed an antinociceptive potential. The present study investigated the evaluation of dietary proteins as potential precursors of dipeptidyl peptidase-III inhibitors by measuring occurrence frequency of dipeptidyl peptidase-III inhibitory peptides. In silico analysis of 69 proteins from 17 food commodities revealed 2659 dipeptidyl peptidase-III inhibitory peptides. β-subunit of hemoglobin and annexin A5 of chicken showed highest dipeptidyl peptidase-III inhibiting potential followed by 12S seed storage globulin 2 of oat, β-conglycinin of soyabean, α-lactalbumin of cow milk, cruciferin CRU4 of rapeseed, and zein-alpha of maize But overall occurrence frequency of dipeptidyl peptidase-III inhibitory peptides was observed highest in maize followed by pumpkin, soyabean, and rapeseed, whereas barley showed the lowest frequency among plant based foods. Chicken and maize had the highest dipeptidyl peptidase-III inhibitory potential in animal and plant derived foods respectively, thus may serve as a natural dietary source for pain modulation.
INTRODUCTION
Both, acute and chronic pains are incapacitating conditions and their improvement is a high priority for patients of cancer and neuropathic disorders.[Citation1] Novel pharmacological entities which act as inhibitors of enkephalin-degrading enzyme(s) reportedly possess antinociceptive potential. Nerve endings of the central nervous system (CNS) and adrenal medulla release some naturally occurring morphine-like substances viz. Leu-enkephalin and Met-enkephalin. Like other neuropeptides, enkephalins are also short lived molecules which are rapidly hydrolyzed after their release at synapsis. In the CNS, several peptidases are involved in enkephalin degradation at different sites but dipeptidyl peptidase-III (DPP-III) is one of the most important enkephalin degrading enzyme with micromolar affinity for encephalin.[Citation2−Citation4] So, DPP-III inhibitors are expected to prolong the action of endogenously released or exogenously applied enkephalins.[Citation3]
DPP-III is a metalloenzyme that removes dipeptide moiety from amino-terminus. It specifically cleaves Arg-Arg from Arg-Arg-4mβNA and prefers dipeptides with hydrophobic residue at P1’ site of oligopeptides. It also hydrolyses various bioactive peptides viz enkephalins, angiotensins, gastrin tetrapeptide, and ACTH suggesting that DPP-III plays a physiologic role in regulating disposition of these bioactive peptides.[Citation5] However, its hydrolysing activity decreases with increase in peptide length.
DPP-III is strongly inhibited by chelating agents and thiol inhibitors.[Citation6] A number of synthetic and modified di-, tri-, and oligo-peptides with DPP-III inhibitory activity have been studied. Spinorphin (Leu-Val-Val-Tyr-Pro-Trp-Thr) and its analogue tynorphin (Val-Val-Tyr-Pro-Trp) were most potent natural peptidyl inhibitors with Ki of 75 nM for DPP-III.[Citation7,Citation8] But both were unstable towards human serum peptidase action.[Citation9] Dipeptides with aromatic pairs such as Tyr-Tyr, Tyr-Phe, and Tyr-dipeptides containing large aliphatic or basic amino acids exhibited greater inhibitory potencies than those having small amino acid in second position.[Citation9,Citation10] Peptides with aromatic and bulky side chains on both amino acids were also potent inhibitors.[Citation11]
Dietary proteins indicate many functionalities by means of biologically active peptides (which are inactive within parent protein) generated after digestion, fermentation, or food processing. Potential of dietary peptides in promoting human health by reducing risk of chronic diseases and boosting natural immune protection has aroused increased scientific and commercial interest over the past decade.[Citation12] Inhibitory peptides against angiotensin converting enzyme (ACE) are well studied food protein-derived bioactive peptides from milk and fish that are being used to treat hypertension.[Citation13−Citation18] DPP-IV inhibitory peptides were found to be effective in regulating diabetes.[Citation19] But sporadic studies correlate the involvement of dietary components in pain regulation. Studies indicated the influence of diet on response of animals and humans to noxious stimuli like heat, pain, and persistent hyperanalgesia (inflammation). Consumption of sucrose, fat rich diet, and highly palatable foods attenuated the pain.[Citation20−Citation23] In human subjects, tryptophan also reduces sensitivity to pain with enhancement of morphine analgesia.[Citation24−Citation26]
Computer-aided techniques have proved to be useful and complementary to empirical methods, in evaluating the potential of proteins as precursors of bioactive peptides.[Citation18,Citation27,Citation28] In present communication, in silico evaluation of potential DPP-III inhibitor precursors from dietary proteins (animal and plant sources) is being reported which can be helpful in developing therapeutic modalities for pain management.
MATERIAL AND METHODS
Major dietary proteins from plant and animal sources were selected. All protein sequences were obtained from the UniProt Knowledge base of ExPASy Proteomics Server available at http://expasy.org. Non-modified peptide based DPP-III inhibitors and their additional information about percent inhibition and Ki values were obtained from published literature. Various dietary proteins were predicted for production of potential DPP-III inhibitory peptides. These peptides were searched from literature and dietary peptide sequences were compared with studied DPP-III inhibitory peptides. Potential of selected dietary proteins was quantified on the basis of occurrence frequency of DPP-III inhibitory peptides using following equation:[Citation29]
TABLE 1 Reported dipeptide inhibitors of DPP-III enzyme
TABLE 2 Studied tri, tetra, and oligopeptide inhibitors of DPP-III enzyme
TABLE 3 Occurrence frequency (A) of DPP-III inhibitory peptides from plant commodities
TABLE 4 Occurrence frequency (A) of DPP-III inhibitory peptides from animal commodities
RESULTS AND DISCUSSION
DPP-III Inhibitory Peptides
Di-, tri-, and oligo-peptides with DPP-III inhibitory activity reported in literature are summarized in and . Inhibition constant (Ki) is commonly used as indicator of the inhibitory potency. The reported efficacy of inhibitory peptides varies greatly and it is difficult to make direct comparison of their absolute inhibitory potency as few studies have reported Ki while others have reported percent inhibition in comparison to control. Differences in reported Ki values for same inhibitory peptide may be attributed differences in experimental conditions such as substrate and source of enzyme used in different studies.
According to literature, highest potency was observed for the dipeptides having aromatic and bulky side chains such as Leu-Arg and Met-Arg causing more than 90% inhibition at 0.5 mM.[Citation6] Phe-Met, Leu-Trp, Trp-Met, Val-Tyr, and Phe-Arg caused 63–85% inhibition.[Citation6] Dipeptides with aromatic pairs such as Tyr-Tyr, Tyr-Phe, and Tyr-dipeptides containing large aliphatic or basic amino acids had greater inhibitory potency than those having small amino acid in second position.[Citation30] For tripeptides, the highest potency (> 90%) was observed for Arg-Phe-Ala and Leu-Trp-Met at 0.5 mM concentration. Among tetrapeptides, Trp-Met-Asp-Phe was most potent inhibitor with > 80% inhibition as compared to other tetrapeptides like Ala-Ala-Ala-Ala, Tyr-Gly-Gly-Phe, and Gly-Gly-Phe-Leu which showed < 60% inhibition.[Citation6] Among oligopeptides Leu-Trp-Met-Arg-Phe-Ala and Tyr-Gly-Gly-Phe-Leu showed more than 90% inhibition. Ile-Val-Tyr-Pro-Trp, Trp-Val-Tyr-Pro-Trp, and Tyr-Pro-Phe-Val-Glu-Pro-Ile also inhibited DPP-III with Ki of 0.100, 0.126, and 0.56 μM, respectively.[Citation31,Citation32]
Potential of Dietary Proteins as Precursors Of DPP-III Inhibitor(s)
In silico analysis of 69 proteins from 17 food commodities revealed 2659 di- and tri-peptides that matched to sequences of DPP-III inhibitory peptides (Appendices A and B, and ). It is important to note that most of the investigated peptides in literature ( and ) were chemically synthesized with the purpose of gaining insight into enzyme’s catalytic and binding properties and also to study the effect of structural characteristics of peptides with respect to their interaction with DPP-III. Thus, reported oligopeptides with DPP-III inhibitory activity might not be found naturally in protein sequences.
DPP-III Inhibitory Peptides in Plant Proteins
Forty-one major protein sequences from 15 plant commodities were investigated (Appendix A in the online supplementary information and ). Among these sequences, storage proteins of Triticum aestivum (wheat), Hordeum vulgare (barley), and Zea mays (maize) were rich in Pro-Phe dipeptide followed by Tyr-Tyr (wheat) and Phe-Leu (maize). Canavalia ensiformis (jack-bean) and Cicer arietinum (chickpea) were found to be rich in Phe-Leu dipeptide with higher frequency in stored protein sequences followed by Avena sativa (oat) and Glycine max (soyabean). Oat and soyabean proteins were rich in Arg-Val dipeptide. Brassica napus (rapeseed), Vicia faba (broad bean), and Cucurbita maxima were rich in Leu-Arg, whereas Pisum sativum (pea), Helianthus annuus (sunflower), and Zamia furfuracea (cardboard palm) had high amounts of Arg-Arg and Pro-Arg (Appendix A in the online supplementary information). In order to quantify the potential of these proteins to serve as bioactive peptide precursors, occurrence frequency (A) was calculated[Citation29] (). Among plant proteins, 12S seed storage globulin 2 from oat showed the highest occurrence frequency value (0.0945) of DPP-III inhibitory peptides followed by β-conglycinin chain from soyabean (0.0933), cruciferin CRU4 (0.0903) from rapeseed and zein-α (0.0901) from maize. Proteins of maize showed highest overall occurrence frequency for DPP-III inhibitory peptides which suggested its best potential amongst the dietary proteins followed by pumpkin, soyabean, and rapeseed. On the other hand, barley proteins were least promising for DPP-III inhibitory peptides. These results are also in agreement to earlier studies of influence of diet on pain regulation. Consumption of Zea mays husk and soy-containing diets suppressed pain in rats up to a significant level.[Citation33,Citation34] These results provide scientific validation for the use of Zea mays husk and Cucurbita maxima seeds for treating pain and inflammatory conditions in Nigerian folk medicine and Brazil, respectively.[Citation34]
DPP-III Inhibitory Peptides in Chicken and Bovine Proteins
Bovine milk and chicken egg proteins are sources of bioactive peptides with various biological activities viz. anti-hypertensive and anti-microbial.[Citation35,Citation36] Among 16 studied chicken proteins, 1342 fragments had DPP-III inhibitory activity with Gly-Glu being the highest occurring dipeptide fragment (Appendix B in the online supplementary information and ). β-subunit of chicken hemoglobin showed highest potential amongst meat proteins with occurrence frequency of 0.108 followed by annexin A5 (0.0996), serum albumin (0.0829), and collagen alpha-3 (VI) (0.0803) (). While collectin-12, with an occurrence frequency of 0.0539 is poorest in DPP-III inhibitory peptides.
Hemoglobin is a major source of bioactive peptides[Citation37] and was also having many DPP-III inhibitory peptides. The present study is also supported by earlier studies on hemoglobin-derived bioactive peptides like kyotrophin which showed analgesic effects. Ovalbumin of chicken egg showed highest DPP-III inhibitory potency with occurrence frequency of 0.0803 followed by ovatransferrin (0.0624) and ovamucoid (0.0619).On the other hand, 384 DPP-III inhibitory fragments were reported in ten investigated proteins from bovine (). In silico results showed that amongst bovine milk proteins, α-lactalbumin (A = 0.0915 for DPP-III inhibitors) had the greatest potential followed by β-casein (0.758) and α-S1-casein (0.747). β-lactoglobulin of milk (whey protein) was limited source (only nine peptides) of DPP-III inhibitory peptides with lowest occurrence frequency of 0.0505.
CONCLUSION
The potential of 69 proteins from 17 food commodities to serve as precursors of DPP-III inhibitory peptides were assessed using an in silico approach. Maize had highest frequency among all studied inhibitors. Apparently, it is the best dietary source for pain regulation. Hemoglobin (β-subunit) and annexin A5 from chicken were found to have highest potential precursors of DPP-III inhibitors. The present study relies entirely on the available data of DPP-III inhibitory peptides, dietary proteins may also contain other putative fragments with DPP-III inhibitory activity, which may be activated and act as antinociceptive. The present in silico study provides first-hand information about potential of proteins carrying bioactive peptide with DPP-III inhibitory activity which can prolong action of endogenously released or exogenously applied enkephalins. These findings also provide the basis of further investigation after in vitro or in vivo assessment of these proteins for generating DPP-III inhibitors.
ACKNOWLEDGMENTS
The authors are thankful to the crew of UniProt Knowledgebase for making protein sequences available to the whole scientific community. The authors would also like to thank Dr. Sunita Siroha, Professor and Head, Department of English, Kurukshetra University, Kurukshetra for checking the language of the manuscript.
SUPPLEMENTAL MATERIAL
Supplemental data for this article can be accessed on the publisher’s website.
Supplementary Material
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