Structure–function engineering of novel fish gelatin-derived multifunctional peptides using high-resolution peptidomics and bioinformatics

Latest Research

DP

The DP of protein is an important factor for determining the functional characteristics of peptides. The present study showed that the addition of actinidin significantly increased the DP and improved the functionality of the hydrolysates. Nongonierma, et al.22 showed similar results for the DPP-IV inhibitory activity of peptides derived from milk protein, with the high DPP-IV inhibitory potential of H4 and H11 peptides (with a degree of hydrolysis of 11.27% and 12.19%, respectively) could be due to the long hydrolysis time (≥ 150 min). Another study conducted by Jamdar et al.23 showed that ACE inhibitory activity of peanut protein hydrolysates could be obtained by subtilisin A treatment, which was directly related to the DP. The effect of DP (50–100%) on the antibacterial activity of palm kernel expeller peptides was also investigated by Tan, et al.24. They demonstrated that 70% DP resulted in the highest antibacterial activity against gram-positive bacteria with an IC50 of 200 µg mL−1, whereas no or limited activity against E. coli and other gram-negative bacteria was observed. Taken together, these findings suggest that the herein generated hydrolysates represent a highly active source of multifunctional peptides. Therefore, the hydrolysates underwent further purification and analyses.

Peptidomics approach

Ultrafiltration

The observed higher antibacterial activity after ultrafiltration can be explained by the higher concentration of lower molecular weight peptides in the solution. A previous study in which the peptic hydrolysate of Pacific cod skin was fractionated into different molecular weight solutions using ultrafiltration membranes of 10, 5, and 1 kDa25, revealed that among the obtained four fractions of > 10, 5–10, 1–5, and < 1 kDa peptides, the one of < 1 kDa hold the higher ACE inhibitory activity (70% at 0.5 mg mL−1). Another study performed by Sha et al.26 showed that the porcine skin gelatin hydrolysates with the molecular weight of < 1 kDa obtained from ultrafiltration, with the cutoff of 2.5 and 1 kDa, had the highest DPP-IV inhibitory activity. These results agree with the present findings, based on which the fraction < 3 kDa was chosen for further purification and analyses due to its higher biological activity than the other fractions.

Ion exchange chromatography

The observed reduction of the biological activity of the fractions after anion exchange separation could have resulted from the loss of synergistic peptides. A previous investigation performed on a fraction of higher molecular weight hydrolysates obtained from the same gelatin source and enzymatic reaction, also showed that the antioxidant and anticancer activities of the fraction were decreased after anion exchange chromatography (DEAE-Sephadex A-25 anion exchange column)2. Furthermore, another study performed to understand the effect of bioactive peptides from Oreochromis niloticus skin gelatin on ACE activity and radical scavenging27 demonstrated that the ion exchange chromatography dramatically reduced biological activities of the peptides due to the loss of some synergistic peptides cooperating with their ACE inhibitory and radical scavenging activities. Herein, based on the similar results among samples and enhanced biological activities, the fractions 15, 16, 19, and 21 were pooled and lyophilized for further assessment.

SPE

A positive relationship between the biological activities of the peptides and their ACN concentration was observed upon SPE; hence, their ACE and DPP-IV inhibitory potential, as well as antibacterial activity, could be related to the hydrophobicity of the fractions. Despite the structure–activity relationship of the peptides had not yet been fully elucidated, some structural characteristics of peptides previously determined were shown to impact on their biological activities. In this regard, Lacroix and Li-Chan28 showed that the presence of proline/hydroxyproline in peptide sequences is an important to have a good DPP-IV inhibitory activity. Another study revealed that ACE inhibitory activity is strongly affected by the C-terminal sequence, especially in hydrophobic amino acids, such as proline29. Additionally, the antibacterial activity of bioactive peptides is also affected by several structural parameters such as the number of α-helices, charge, chain length, and hydrophobicity30. Therefore, it is reasonable to assume that fraction with higher ACN concentrations may be more biologically active. However, owing to the low quantity of the recovered fraction of 30% (v/v) ACN after lyophilization and considering no statistical differences in biological activities were detected between these two fractions, this sample was pooled with the fraction of 25% (v/v) ACN.

RP-HPLC and mass spectroscopy

The fractionation by RP chromatography is a good procedure to purify peptides based on their relative hydrophobicity, as more hydrophobic peptides will be retained for longer. Moreover, longer retention time indicates higher amounts of hydrophobic or aromatic amino acid in the peptide structure31. In the present study the chromatogram of the isolated fractions only showed a good ACE inhibitory activity upon fraction 5, which agrees with the literature. In a research on peptides derived from hen egg white lysozyme, 12 fractions were detected after purification by RP-HPLC of which the most active fractions concerning antihypertensive activity were the fractions 2, 7, and 9 (IC50 = 0.01, 0.005, and 0.014 mg mL−1, respectively)32.

As it was mentioned before, ACE and DPP-IV inhibitory potential, and antibacterial activity of the extracts could be related to the hydrophobicity of the fractions; however, the amino acid composition and sequence of the peptides can also significantly alter their biological proprieties. Therefore, the amino acid sequence of the highly active fractions were evaluated next by MALDI-TOF/TOF mass spectroscopy. Three identified peptides were found to be rich in glycine and proline, which could contribute for their biological activity. Thus, the peptides were synthesized for a detailed analysis.

Biological assessment of synthesized peptides

Antibacterial activity

The cell membrane composition could be a very important factor impacting on the action of antibacterial peptides. For example, the existence of an outer membrane, which consists of a lipopolysaccharide layer in gram-negative bacteria, may be the cause of lower antibacterial activity of peptides against them33. However, it has been suggested that the lipopolysaccharide binding ability of peptides is neither directly nor indirectly related to their antibacterial activity. Once the peptides passed through the broken outer membrane, the interaction with the cytoplasmic membrane is the key factor of their antibacterial efficiency, which could in turn be affected by their concentration and sequence34. The present findings are in agreement with those of a previous study, which demonstrated that the antibacterial effect of peptides from oyster (Crassostrea gigas) against S. aureus (with an IC50 of 18.6 ± 20 µg mL−1) was significantly stronger than that against other bacteria strains35.

The protein-binding potential (Boman index) is an important factor to determine the potential of protein interactions based on the peptide sequence. This index is calculated using the sum of free energies of the corresponding side chains for transfer from cyclohexane to water and divided by the total number of the amino acids. This important factor could be used to interpret the ability of a peptide to bind either the microbial cell membrane or other proteins as receptors. A Boman index higher than 2.48, indicates high binding potential36. Therefore, the present results suggest that the antibacterial mechanism of the P1 is not through the interaction with the cell membrane. In contrast, the P2 and P3 showed high Boman index, suggesting that they may interact with membrane portions as receptors. The online database also represents some antibacterial peptides rich in proline (just like P1 in the present study) and tryptophan. Regarding the antibacterial activity of the mixture of three peptides, it is possible to conclude that these peptides may have a synergistic effect on their actions against some particular pathogens such as P. aeruginosa. However, the highest activity against other pathogens was the same as mentioned above. Furthermore, we cannot conclude that mixing the peptides is a good strategy to enhance their antibacterial activity against all bacteria.

Antihypertensive and antidiabetic activity

ACE is responsible for the conversion of the inactive decapeptide (angiotensin I), by cleaving a dipeptide from the C-terminus, into a vasoconstriction octapeptide (angiotensin II), as well as by the inactivation of bradykinin, which is as a vasodilator in the kallikrein-kinin system18. Therefore, ACE inhibition leads to an antihypertensive effect. The results of the present study revealed that all three identified peptides had good ACE inhibitory activities (IC50 < 1 mg mL−1), with the most potent ACE inhibitor being P2 (IC50 = 0.51 mg mL−1). Certainly, the amino acid sequence of the peptides is one of the most important factors affecting their ACE inhibitory activity, which can be strongly affected by the C-terminal sequence, especially by hydrophobic amino acids such as proline29. Indeed, the C-terminal amino acid in P1 and P2 was proline. However, the effect of the peptide structures on ACE inhibitory potential is not fully known. The identified P3 was found to have methionine and arginine at the N- and C-terminus, respectively, but its ACE inhibitory activity was significantly higher than that of P1 with a proline at the C-terminus. In a hexapeptide (VLAQYK) sequenced from beef hydrolysates, despite the presence of aliphatic amino acids at the N- and C-terminus instead of a proline residue, its ACE inhibition was of 30.1%37.

DPP-IV is an enzyme that participated in the incretin hormone system; thus, its inhibition can significantly decrease diabetes mellitus type II4,5. However, synthetic DPP-IV inhibitors have undesired side effects, such as hypoglycemia, weight gain, diarrhea, nausea, and abdominal pain6. Dipeptides with DPP-IV inhibitory IC50 values less than 100 mM usually have tryptophan/threonine/methionine at their N-terminus38, which is in agreement with the sequence of the herein identified P3. As it is mentioned previously, hydrophobic amino acids including alanine, glycine, isoleucine, leucine, phenylalanine, proline, methionine, tryptophan, and valine are responsible for the DPP-IV inhibitory activity of peptides, as these hydrophobic amino acids may facilitate the interaction of peptides with the enzyme active site. Hydrophobic pockets are believed to have an important effect on DPP-IV inhibition by peptides; however, their mechanism of action remains unknown3,38. Zhang et al.39 showed that the dodecapeptide SPTVMFPPQSVL, containing a proline at the second position of the N-terminus, and the octapeptide MHQPPQPL, containing a proline at the second position of the C-terminus, had moderate DPP-IV inhibitory activities. The researchers suggested that not only the peptide sequence, but also its length could affect the DPP-IV-inhibition mechanism. Analysis of the structure–function relationship of various DPP-IV-inhibitory peptides has suggested that the binding to DPP-IV is strongly influenced by the N-terminal amino acid40. Altogether, these findings indicated that a proline located in the N- or C-terminus of peptides, as well as in the penultimate positions, is an important factor regulating its biological activity38,39,40. Of note, the mixture of the peptides showed slightly higher ACE and DPP-IV inhibitory activity than P3 and P1 alone, respectively. However, this result should not encourage the development of the mixture as an opponent to the single peptides since P1 and P2 showed significantly higher ACE and DPP-IV inhibitory activities, respectively.

Cytotoxicity

Bioactive peptides obtained from gelatin hydrolysis are recognized as safe food ingredients with minimal adverse effects20,41,42. Previous studies, both in vitro and in silico15,16, demonstrated that fractions with different molecular weights obtained by ultrafiltration had no toxicity, further supporting the present findings. Nonetheless, to the best of our knowledge, only few studies evaluated the cytotoxicity of fish gelatin hydrolysates in comparison to normal cells. The cytotoxicity of collagen hydrolysates obtained from cartilage was monitored by Schauss, et al.43 as acute oral toxicity in Sprague Dawley rats. This study revealed that the 50% lethal dose was greater than 5000 mg kg−1 body weight. Benjakul et al. also showed that hydrolyzed collagen obtained from seabass skin in Wistar rat was safe, with the 50% lethal dose being higher than 5000 mg kg−1 body weight20. Based on this evidence as well as the present results, it is reasonable to consider that the peptides identified herein are safe for consumption due to their lack of cytotoxicity at different concentrations.

Bioinformatics

The molecular structures of the three peptides were simulated and energy minimized using ChemOffice, and they were docked onto the rigid structure of human ACE and DPP-IV. To confirm the validity of the docking process, the ACE and DPP-IV were separated from their specific ligands (Lisinopril and HL1, respectively) and re-docked onto the rigid structures of the corresponding apoenzymes.

The good binding affinity (more negative) of the synthesized peptides observed for docking onto ACE and DPP-IV pockets could be due to the large surface of the binding interactions and consequent high number of hydrogen bonds formed. The in silico results further confirmed the collected in vitro data. For ACE docking, the highest (6.3 kcal mol−1) and the lowest (8.1 kcal mol−1) binding energies were related to P1 and P2, respectively, which agreed with the ACE inhibitory results. In turn, for DPP-IV docking, the highest (6.6 kcal mol−1) and the lowest (8.1 kcal mol−1) binding energies were related to P3 and P1, respectively, which also confirmed the DPP-IV inhibitory results.

A study performed to identify ACE inhibitory peptides from O. niloticus skin gelatin, two peptides—GPEGPAGAR and GETGPAGPAGAAGPAGPR—were docked onto the rigid pocket of ACE27. The researchers stated that the binding affinity of these peptides was − 9.5 and − 9.3, respectively. However, it is noteworthy that several studies target the rabbit lung ACE, whereas the human ACE was used in the present study.

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