Analysis of Litopenaeus vannamei hemocyanin interacting proteins reveals its role in hemolymph clotting.

Hemocyanin is the main component of hemolymph plasma proteins and possesses diverse immunological properties and immunomodulatory functions. However, the interacting networks of hemocyanin in shrimp immune response remain poorly understood. In this study, 39 potential hemocyanin interacting partners were identified from Litopenaeus vannamei plasma by co-immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Gene Ontology (GO) analysis showed that most of the identified interactors were cell proteins involved in metabolic process and binding. Among these identified proteins, transglutaminase (TGase), a crucial regulator in hemolymph clotting cascade, was chosen for further studies. Far-Western blot and His-pull down assays revealed that hemocyanin directly interacted with TGase. Further analysis demonstrated that hemocyanin and TGase followed similar expression patterns upon pathogen infection. Moreover, in vivo knockdown of hemocyanin led to a significant decrease in TGase expression, as well as inhibited hemolymph clotting. Taken together, these data suggest that hemocyanin might positively regulate hemolymph clotting by modulating TGase in shrimp. SIGNIFICANCE: The interaction networks among immune-related factors is critical for the innate immune response in invertebrates. We report for the first time, proteins that potentially interact with hemocyanin, which led to the identification of 39 possible hemocyanin-proteins including the clotting-related factor TGase. Further studies demonstrated that hemocyanin directly interacted with TGase and modulated its expression, therefore affecting the formation of hemolymph clotting. These findings not only extend our knowledge of the immune interaction networks but also contribute to shrimp disease control and prevention.