STRUCTURES AND FUNCTIONS OF PENLECTINS AND THEIR INVOLVEMENT IN GASTROINTESTINAL IMMUNE SYSTEM OF BLACK TIGER SHRIMP Penaeus monodon

Abstract

Tachylectin5A and Tachylectin5B, both contain a fibrinogen-related domain (FReD) and have been studied in japanese horseshoe crabs, Tachypleus tridentatus and shown to be involved in host defense. Here, we demonstrate the presence of tachylectin5- like genes in the shrimp, P. monodon, which we have named as Penlectin5-1 (PL5-1), Penlectin5-2 (PL5-2) and Penlectin5-3 (PL5-3). All three lectins contain a signal peptide and a single FReD with an acetyl group and a calcium binding sites. They are structurally similar to japanese horseshoe crab tachylectin5. The PL5-1, PL5-2 and PL5-3 transcripts were expressed in various shrimp tissues in normal shrimp, and its expression was up-regulated in tissues such as hemocytes, posterior intestine or stomach following challenge with pathogenic V. harveyi or V. parahaemolyticus (3HP). The PL5-1, PL5-2 and PL5-3 proteins were expressed in a bacterial expression system and they were expressed as non-soluble proteins. Only the PL5-2 protein was possible to isolate and purify from shrimp plasma using affinity chromatography, and therefore, the PL5-2 protein was used for functional studies of this protein. The PL5-2 protein was detected in various tissues as well as in cell-free hemolymph. The biological function of the PL5-2 protein is to recognize some Gram-positive and Gram-negative bacteria regardless whether they are non-pathogenic or pathogenic. This lectin had hemagglutination activity towards all types of human erythrocytes including A, B and O type, and it had also a bacterial agglutination activity to both Gram-negative and Gram-positive bacteria. Possible binding sites of PL5-2 to bacteria could be at the N-acetyl moiety of the GlcNAc-MurNAc cell wall of the peptidoglycan, since the binding could be inhibited by GlcNAc or GalNAC. To further understand the functions and the involvements of PL5-1, PL5-2 and PL5-3 in response to AHPND in shrimp, knockdown of the PL5 genes using RNAi was performed prior to an oral administration of Vibrio parahaemolyticus (VP3HP) causing AHPND. The results suggest that PL5-1, PL5-2 or PL5-3 silencing in shrimp challenged with VP3HP showed higher increases in mortality as well as more severity of histopathological changes. The presence of PL5-2 protein in both circulating hemolymph and gastrointestinal tract, where host and microbes are usually interacting, may suggest that the physiological function of shrimp tachylectin-like proteins is to recognize and bind to invading bacteria to immobilize and entrap these microbes and subsequently clear them from circulation and the host body. It is also probably possible that the Penlectins are involved in controlling and maintaining the normal flora in the intestine.