Atlantic cod (Gadus morhua L.) possesses three homologues of ISG15 with different expression kinetics and conjugation properties.
Two new interferon stimulated gene 15 (ISG15) family members were identified in a subtractive cDNA library constructed from a mixture of head kidney and spleen of Atlantic cod (Gadus morhua) stimulated with polyinosinic:polycytidylic acid (poly I:C). Two full-length Atlantic cod (Ac) ISG15-2 and AcISG15-3 cDNAs were cloned with rapid amplification of cDNA ends (RACE). The cDNA sequence of AcISG15-2 encodes a 16.9kDa protein and AcISG15-3 encodes a 18.4kDa protein, both of which possess the characteristic structural features of two tandem ubiquitin-like domains and the LRGG motif necessary for conjugation. Furthermore, the AcISG15-3 protein is expressed with a C-terminal extension in common with the human ISG15 protein. Gene expression analysis using quantitative reverse transcriptase PCR (RT-qPCR) showed that AcISG15-1, AcISG15-2, and AcISG15-3 transcripts were up-regulated in head kidney after poly I:C stimulation, suggesting that these proteins may be involved in the cod immune response. However, transient expression of myc-tagged AcISG15 proteins revealed differences in their abilities to form conjugates in vitro. We show that AcISG15-2 forms covalent conjugates to a range of cellular protein as a response to poly I:C, recombinant Atlantic salmon IFNa1 (rSasaIFNa1) and infectious pancreatic necrosis virus (IPNV), whereas conjugation was absent for AcISG15-1 and AcISG15-3. Thus, these results suggest there are three ISG15 homologues in Atlantic cod and that the three proteins may play different roles in innate immunity.
Furnes
Clemens
C
Department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsø, Breivika, N-9037 Tromsø, Norway.
Kileng
Øyvind
Ø
Rinaldo
Christine Hanssen
CH
Seppola
Marit
M
Jensen
Ingvill
I
Robertsen
Børre
B
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D016207:Cytokines / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D048389:Gadus morhua / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007113:Immunity, Innate; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D011070:Poly I-C / Q000276:immunology; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014452:Ubiquitins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D016207:Cytokines; D029941:Fish Proteins; D012333:RNA, Messenger; D014452:Ubiquitins; D011070:Poly I-C