Molecular characterization,expression patterns,and subcellular localization of a classical and a novel nonclassical MHC class Iαmolecules from Japanese eel Anguilla japonica

Major histocompatibility complex(MHC)molecules play a critical role in the immune response of vertebrate animals by presenting foreign antigens to T lymphocytes.In this study,we first cloned and identified a classical and a novel nonclassical MHC Iαmolecules from Japanese eel(Anguilla japonica),named as AjMHC I-UBA and AjMHC I-L,respectively.The full-length cDNA of AjMHC I-UBA contains an open reading frame(ORF)of 1047 bp encoding a predicted protein of 348 amino acids,while AjMHC I-L 1089 bp encodes 362 amino acids.The multiple alignment of the amino acid sequence showed that AjMHC I-UBA and AjMHC I-L consist of an N-terminal MHC I superfamily domain withinα1 andα2 helices,an IgC-MHC Iα3 domain,and a transmembrane region close to the C-terminal,which are similar to other fish and mammal species.Molecular polymorphism analysis showed that eight different major alleles of AjMHC I-UBA,named as AjMHC I-UBA*0101~1001,were identified from six Japanese eel individuals.Furthermore,a distinguishing signature of the nonclassical L-lineage genes specific motif“HINMTL”,including an N-glycosylation site(NXS/T),was present in theα3 domain of AjMHC I-L sequences.Although the predicted three-dimensional structures of AjMHC I-UBA and AjMHC I-L are similar to that of human MHC Iα,phylogenetic analysis showed that these two protein molecules belong to classical MHC I UBA gene of U-lineage and nonclassical MHC I gene of L-lineage,respectively.Quantitative real-time polymerase chain reaction(qRT-PCR)analysis revealed that the highest expression of AjMHC I-UBA and AjMHC I-L was found in the intestine and the expression level of AjMHC I-UBA in all of the examined tissues was significantly higher than that of AjMHC I-L.The expressions of AjMHC I-UBA and AjMHC I-L in liver,kidney and spleen were significantly induced following injection with the viral mimic poly I:C,LPS,and Aeromonas hydrophila infection.In vitro,the AjMHC I-UBA and AjMHC I-L transcripts of Japanese eel liver cells were significantly enhanced by the treatment of poly I:C or the stimulation of the high concentration of A.hydrophila.Subcellular localization showed that under natural conditions,AjMHC I-UBA and AjMHC I-L were uniformly distributed in the cytoplasm,and aggregated into spots or flakes in the cytoplasm after the stimulation of poly I:C or LPS.These results collectively suggested that AjMHC I-UBA and AjMHC I-L are important components possibly involved in Japanese eel defense against viral and bacterial infection.Taken together,these findings provide valuable insight into the immune function of MHC class I in the immune system of teleost.