Identification and characterization of warm temperature acclimation proteins (Wap65s) in rainbow trout (Oncorhynchus...

Hemopexin is a vital glycoprotein for processing excessive iron in blood and functions as an iron scavenger in mammals. Teleosts however, unlike mammals, have two known hemopexin paralogs called warm temperature acclimation-related 65 kDa protein (Wap65-1 and Wap65-2, collectively termed Wap65s). Although Wap65s in rainbow trout have been considered notable biomarkers with significantly higher and/or lower expression under conditions of stress or disease, the individual roles, similarities and differences between the two paralogs are not well known. The aim of this study was to gain an understanding of the characteristics and functions of trout Wap65s from the perspective of iron-metabolism, physiological roles, and relevant immunological responses. The expression of Wap65-1 and -2 in this study was determined in the face of challenges by Aeromonas salmonicida, infectious hematopoietic necrosis virus (IHNV), and iron-dextran. Immuno-histochemistry (IHC) was employed to localize the major cell types for Wap65-2 expression, and trout leukocytes were isolated and incubated with LPS and OxLDL for comprehending the immunological characteristics of Wap65-2. We demonstrate that Wap65-1 is expressed only in the liver but Wap65-2 is systemically expressed in most organs and tissues. Interestingly, Wap65-1 expression was not significantly changed under A. salmonicida and iron-dextran administration, but was significantly decreased under IHNV. In contrast, Wap65-2 was up-regulated in all challenged groups, however with different expression patterns in the blood and liver. These results suggested that the two paralogs may participate in different biological roles. IHC showed that Wap65-2 antibody had high affinity for leukocyte-like cells, and macrophages but not lymphocytes significantly increased expression under LPS and OxLDL stimulation. These results support the conclusion that trout Wap65-2, not Wap65-1 may have conventional hemopexin functions such as reported in mammals including effects on iron metabolism, inflammation, and acute-phase protein.