Abstract
In this paper, a novel natural influenza A H1N1 virus neuraminidase (NA) inhibitory peptide derived from cod skin hydrolysates was purified and its antiviral mechanism was explored. From the hydrolysates, novel efficient NA-inhibitory peptides were purified by a sequential approach utilizing an ultrafiltration membrane (5000 Da), sephadex G-15 gel column and reverse-phase high-performance liquid chromatography (RP-HPLC). The amino acid sequence of the pure peptide was determined by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) was PGEKGPSGEAGTAGPPGTPGPQGL, with a molecular weight of 2163 Da. The analysis of the Lineweacer–Burk model indicated that the peptide was a competitive NA inhibitor with Ki of 0.29 mM and could directly bind free enzymes. In addition, docking studies suggested that hydrogen binding might be the driving force for the binding affinity of PGEKGPSGEAGTAGPPGTPGPQGL to NA. The cytopathic effect reduction assay showed that the peptide PGEKGPSGEAGTAGPPGTPGPQGL protected Madin–Darby canine kidney (MDCK) cells from viral infection and reduced the viral production in a dose-dependent manner. The EC50 value was 471 ± 12 μg/mL against H1N1. Time-course analysis showed that PGEKGPSGEAGTAGPPGTPGPQGL inhibited influenza virus in the early stage of the infectious cycle. The virus titers assay indicated that the NA-inhibitory peptide PGEKGPSGEAGTAGPPGTPGPQGL could directly affect the virus toxicity and adsorption by host cells, further proving that the peptide had an anti-viral effect with multiple target sites. The activity of NA-inhibitory peptide was almost inactivated during the simulated in vitro gastrointestinal digestion, suggesting that oral administration is not recommended. The peptide PGEKGPSGEAGTAGPPGTPGPQGL acts as a neuraminidase blocker to inhibit influenza A virus in MDCK cells. Thus, the peptide PGEKGPSGEAGTAGPPGTPGPQGL has potential utility in the treatment of the influenza virus infection.
Highlights
The influenza virus remains a highly contagious pathogen that causes high morbidity and mortality [1]
The enzymatic hydrolysates of cod skins were firstly ultrafiltered with an 5 K membrane to obtain the components whose molecular weight were less than 5000 Da
The novel NA-inhibitory peptide PGEKGPSGEAGTAGPPGTPGPQGL was first prepared from cod skin hydrolysates
Summary
The influenza virus remains a highly contagious pathogen that causes high morbidity and mortality [1]. A number of studies have showed that synthetic pyrrolidine-containing compounds exhibited high NA-inhibitory activity [14,15], which suggested that natural pyrrolidine-containing substances (proline and L-hydroxyproline) might play an important role in the inhibition of NA. The preparation of natural NA-inhibitory peptides from cod skin hydrolysates was not reported. The peptide derived from cod skin hydrolysates might have the high potential in the inhibition of NA. To the best of our knowledge, the preparation of natural influenza A H1N1 virus neuraminidase inhibitory peptide from cod skin hydrolysates has seldom been reported. The study aims to prepare efficient NA-inhibitory peptides from cod skin hydrolysates. This study can provide previously unknown information about the effect of the novel NA-inhibitory peptides on influenza A H1N1 virus and alternative approach for antiviral therapy
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