Characterization of a Kunitz-type protease inhibitor (MjKuPI) reveals the involvement of MjKuPI positive hemocytes in the immune responses of kuruma shrimp Marsupenaeus japonicus
Serine proteases have been identified in all living organisms from bacteria to mammals. In addition to having roles in food digestion, they play vital roles in serial physiological processes such as blood coagulation, embryogenesis and the immune response. In the immune responses of invertebrates, such as blood clotting and melanization, the serine proteases prevent bleeding and kill pathogens. Despite their benefits, serine proteases can severely damage cellular processes unless they are tightly controlled by protease inhibitors.
Serine protease inhibitors are classified into several families based on sequence homology, location of the reactive domain, structural characteristics and mechanism of action. Serine protease inhibitors have biological roles in physiology and immunity. Regarding their physiological functions, Kunitz-type protease inhibitors have been identified in a wide variety of animals from sea anemone to human and they play vital roles in physiology. An analysis of expressed sequence tags (ESTs) in a nematode found over 100 ESTs with Kunitz domains. Kunitz domains in combi- nation with other domains form multifunctional molecules. For instance, GP300 in the nematodes Dictyocaulus viviparous, Haemonchus contortus and Caenorhabditis elegans has been shown to be a thrombospondin-like protein but it also shows trypsin inhibitor activity because the C-terminus has six closely spaced Kunitz do- mains. GP300 is located in the cuticle and subcuticle of two nematodes, suggesting that GP300 protects host cells from digestive enzymes. Antith- ermostatic molecules, a type of Kunitz-type protease inhibitor, have been identified in saliva glands of several blood-feeding arthropods. In general, Kunitz-type protease inhibitors inhibit the initial reaction of blood clotting by forming a factor Xa and then a factor VIIa-tissue factor complex. Kunitz-type protease inhibitors have been shown to have immunological roles in several studies. For instance, transcripts of Kunitz-type protease inhibitor are upregulated in Drosophila melanogaster following challenge with bacteria or fungi. In addition, a Kunitz-type protease inhibitor (BmSPI-1) protects Bombyx mori from proteases secreted by potential predators and microbes
In this study, we investigated the tissue distribution of MjKuPI mRNA in kuruma shrimp Marsupenaeus japonicus and attempt to determine the role of MjKuPI in shrimp immune system. In this occasion, kuruma shrimp was chosen to be study.
The results showed that mRNA of MjKuPI expressed in all studied tissues but it showed high expression in hemocyte. In challenge test, MjKuPI mRNA level in challenge group showed the up-regulated expression.
Fig. 1. MjKuPI gene expressions in various tissues of M. japonicus. mRNA expression level of MjKuPI in tissues are presented as relative values. mRNA expressions are shown as fold change relative to mRNA expression level of hemocytes using EF-1a as internal control. E, eyestalk; G, gill; H, heart; HE, hemocyte; HP, hepatopancreas; I, intestine; L, lymphoid organ; M, muscle; N, nerve; ST, stomach
Fig. 2. Effect of microbial infection in the response of MjKuPIþ hemocytes. (A) Immunostaining results at 0 and 24 h post injection with V. penaeicida and WSSV. PBS was used as control. (B) An example of hemocyte classes in kuruma shrimp M. japonicus. White asterisks indicate MjKuPI+ hemocytes
In addition, immune staining and flow cytometry analysis showed that the proportion of MjKuPI+ hemocyte increased significantly in challenge group. These finding revealed the role of MjKuPI in shrimp innate immune system.
(Mai et al., 2016)
Mai, H.N., Nguyen, H.T., Koiwai, K., Kondo, H., Hirono, I., 2016. Characterization of a Kunitz-type protease inhibitor (MjKuPI) reveals the involvement of MjKuPI positive hemocytes in the immune responses of kuruma shrimp Marsupenaeus japonicus. Dev Comp Immunol. 63, 121-127
The application of these finding?
--> The result showed that there is one cell type that responds to pathogen infection. So, if recombinant protein of particular pathogen is injected, the shrimp immune system can be stimulate and protect the shrimp from that pathogen.