Purification, Biochemical Characterization and Digestive Stability Analysis of the Major Allergen Paramyosin From Haliotis discus hannai

Abstract

Paramyosin (PM) is a structural protein in invertebrates, playing an important role in maintaining the texture of muscle. Although PM has been identified as a major allergen, its characteristics have not been well investigated. In this study, PM with a molecular mass of 97 kDa was purified to homogeneity from Haliotis discus hannai using ammonium sulfate fractionation and hydroxyapatite column chromatography. Liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis identified 11 peptide fragments with a total of 66 amino acid residues, which were 100% identical to PM from Haliotis discus discus. The sequence of the PM gene, including its complete open reading frame of 2583 bp encoding 860 amino acid residues, was determined by molecular cloning. PM is almost a long-rod chain protein, and α-helix is the dominant secondary structure. Simulated gastrointestinal fluids digestion showed that PM is highly tolerant to digestive proteinases. A specific polyclonal antibody against PM was prepared. Western blot analysis revealed that PM was the most abundant in H. discus hannai, followed by Rapana venosa, Argopecten irradians, and Uroteuthis chinensis, while PM could not be detected in shrimp Penaeus merguiensis and freshwater fish Hypophthalmichthys molitrix, strongly suggesting the unique existence in shellfish.