Cell surface transglutaminase required for nodavirus entry into freshwater prawn hemocytes.

To identify molecules involved in Macrobrachium rosenbergii nodavirus (MrNV) entry into hemocytes of the giant freshwater prawn M. rosenbergii, biotinylated prawn hemocyte membrane proteins were prepared, purified and separated by SDS-PAGE. The proteins were blotted on the nitrocellulose membrane before incubation with the MrNV capsid protein (MrNV-CP) by a VOPBA technique. Subsequent mass spectrometry and analysis of immune-reactive bands represent putative binding partners including transglutaminase (TG), actin, α2-macroglobulin, α1-tubulin, F1-ATP synthase ß-subunit and a currently uncharacterized protein. The sequence of TG has been characterized and found 5 amino acids differences to a previously reported MrTG (ADX99580), mainly at its N-terminal part and thus, we named it MrTGII (KM008611). Recombinant MrTGII was prepared to produce a polyclonal antibody against it, which was successfully revealed the presence of MrTGII (100 kDa) in prawn hemocyte lysates. Using the pentylamine-biotin incorporation assay, an acyl transfer reaction was observed when hemocyte lysates were added to solutions containing MrNV-CP, suggesting that hemocyte MrTG could use MrNV-CP as the substrate. The expression levels of MrTGII were changed during the course of MrNV infection. By using immunostaining technique, location of MrTGII on the hemocyte surface was confirmed. Specific interaction between MrTGII with MrNV-CP in a dose-dependent manner was confirmed by in vitro ELISA assay. The highest binding activity of MrNV-CP was found with the N-terminal portion of the protein. In vitro neutralization using anti-MrTGII antibody resulted in inhibition of MrNV attachment to the hemocyte surface, accompanied by a dramatic reduction in viral replication. This is the first time that crustacean TG has been shown to be involved in viral entry, in addition to its roles in blood clotting and haematopoiesis.

Reprint of: Virus-binding proteins and their roles in shrimp innate immunity.

Disease outbreaks caused by viral pathogens constitute a major limitation to development of the shrimp aquaculture industry. Many research have been conducted to better understand how host shrimp respond to viral infections with the aim of using the gained knowledge to develop better strategies for disease management and control. One approach has been to study the interactions between host and viral proteins, and particularly host virus-binding proteins that might play an important role in the viral infection process. Within the past five years, increasing numbers of virus-binding proteins (VBPs) have been reported in shrimp. Characterization of these molecules has emphasized on their potential therapeutic applications by demonstrating their activities in inhibition of viral replication via in vivo neutralization assay. However, signaling to induce innate antiviral immune responses as a consequence of binding between viral proteins and VBPs remain to be fully elucidated.

Virus-binding proteins and their roles in shrimp innate immunity.

Disease outbreaks caused by viral pathogens constitute a major limitation to development of the shrimp aquaculture industry. Many research have been conducted to better understand how host shrimp respond to viral infections with the aim of using the gained knowledge to develop better strategies for disease management and control. One approach has been to study the interactions between host and viral proteins, and particularly host virus-binding proteins that might play an important role in the viral infection process. Within the past five years, increasing numbers of virus-binding proteins (VBPs) have been reported in shrimp. Characterization of these molecules has emphasized on their potential therapeutic applications by demonstrating their activities in inhibition of viral replication via in vivo neutralization assay. However, signaling to induce innate antiviral immune responses as a consequence of binding between viral proteins and VBPs remain to be fully elucidated.

Characterization of a novel cyclomaltodextrinase expressed from environmental DNA isolated from Bor Khleung hot spring in Thailand.

A novel gene belonging to the alpha-amylase family was isolated directly from community DNA obtained from soil sediments collected from Bor Khleung hot spring in Thailand. Partial sequences harboring four conserved regions of the alpha-amylase family were amplified by PCR using degenerate primers. Upstream and downstream sequences of these fragments were obtained by a genome walking approach to identify a full-length gene (Env cda13A) encoding 619 amino acids. Amino acid sequence alignments of Env Cda13A with other enzymes suggested that this enzyme was a cyclomaltodextrinase. The Env cda13A gene was expressed in Pichia pastoris as a secreted functional protein of 68 kDa. The partially purified enzyme was shown to be monomeric and hydrolyzed various maltodextrins from maltotriose to maltoheptaose and cyclomaltodextrins to give maltose and glucose as the main products. The enzyme also hydrolyzed pullulan and soluble starch to yield glucose, but the rate of hydrolysis was slow. This study demonstrated the possibility of isolating potentially novel enzymes directly from natural environments and opens an unexplored biodiversity resource in Thailand for future novel gene discoveries.