Affinity Proteomic Approach for Identification of an IgA-like Protein in Litopenaeus vannamei and Study on Its Agglutination Characterization

Abstract

An unknown protein reacted with anti-human IgA, namely, IgA-like protein, has been reported in shrimp, but information regarding its identification is not available. In the present study, an affinity proteomic strategy was applied to identify the IgA-like protein of shrimp Litopenaeus vannamei. The protein of 75 kDa was isolated and confirmed by affinity chromatography and Western blotting with goat anti-human IgA, respectively, and then identified as hemocyanin, a member of IgSF, by mass spectrometry. Moreover, our results showed that human IgA and L. vannamei hemocyanin could separately react with goat anti-human IgA or rabbit anti-shrimp affinity hemocyanin (a-hemocyanin). Further evidences indicated that the recombinant protein of the Ig-like conserved domain could react with anti-human IgA. Interestingly, our results indicated that L. vannamei hemocyanin could aggregate with eight species of shrimp pathogenic bacteria and four types of animal erythrocytes directly. These results indicate that L. vannamei hemocyanin, an IgA-like protein, has dual function of reaction with anti-human IgA as an antigen and of activity binding to bacteria and animal erythrocytes as an agglutinin, suggesting its characteristic role as an IgSF molecule. In addition, our approach suggests that affinity proteomics based on heterogeneous antibody can speed up the identification of Fossman antigens.

White Spot Syndrome Virus Proteins and Differentially Expressed Host Proteins Identified in Shrimp Epithelium by Shotgun Proteomics and Cleavable Isotope-Coded Affinity Tag

“…Our cICAT results show for the first time that the protein level of hemocyanin is up-regulated from 12 hpi. Hemocyanin functions as an immunoglobulin superfamily molecule (57) and is one of the important host factors against pathogenic invasion (2). WSSV infection could induce the aggregation of hemocytes to sites of infection (42).…”

SNPs of hemocyanin C‐terminal fragment in shrimp Litopenaeus vannamei

“…Adult penaeid shrimps L. vannamei (Guolian shrimp 1 # bred by Zhanjian Guolian Aquatic Products Co. Ltd., China), length [8][9][10][11][12] …”

Identification and agglutination properties of hemocyanin from the mud crab (Scylla serrata)

“…We subsequently found that hemocyanin from L. vannamei could bind to eight bacterial pathogens and four animal erythrocytes [23,24]. This suggested that S. serrata hemocyanin may have agglutinative properties.…”

“…Identification of the proteins eluted from the column was carried out using polyacrylamide gel electophoresis (PAGE), SDS-PAGE, immunoblotting and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis as described previously [23]. In brief, PAGE and SDS-PAGE were performed using a 3% stacking gel (pH 6.8) and a 10% separating gel (pH 8.9) in Tris-Glycine buffer (pH 8.3).…”