Abstract
Two ‘trout C-polysaccharide-binding proteins,’ TCBP1 and -2, with relevance to early inflammatory events have been discovered in the last century. The present study characterises the respective cDNA sequences from rainbow trout (Oncorhynchus mykiss), including multiple TCBP1 transcript variants. These variants are generated either by the use of alternative splice sites or the exclusion of exons. The longest mRNA isoform, TCBP1-1, encodes a 245-aa protein with a large signal peptide and a complement component C1q domain. The shortest mRNA isoform, TCBP1-5, contains a premature termination codon and hence fails to encode a functional factor. The 224-aa-long TCBP2 protein consists of a comparably shorter signal peptide and a pentraxin domain. Evolutionary analyses clearly separated TCBP1 and -2 because of distinctive protein motifs. Expression profiling in the liver, spleen, and head kidney tissues of healthy trout revealed that TCBP2 mRNA concentrations were higher than the concentrations of all five TCBP1 mRNA isoforms together. The hepatic levels of these TCBP1 variants increased significantly upon infection with Aeromonas salmonicida, whereas TCBP2 transcript levels rose moderately. As the biological function of TCBP1 is barely understood, we tagged this factor with the green fluorescent protein and visualised its expression in HEK-293 cells. Overexpression of TCBP1 increased the level of active NF-κB factors and induced cell death, indicating its involvement in proapoptotic NF-κB-dependent signalling routes.
Highlights
The acute-phase response (APR) is a fast and non-specific reaction to local or systemic disturbances of homeostasis following an infection or a tissue injury [1]
At least five TCBP1-encoding transcript variants are present in rainbow trout
A reverse-northern analysis applying a TCBP1-specific probe confirmed the presence of three TCBP1 transcripts with lengths between approximately 800 and 1100 bp (Fig. 1a)
Summary
The acute-phase response (APR) is a fast and non-specific reaction to local or systemic disturbances of homeostasis following an infection or a tissue injury [1]. The APR summarizes several physiological changes involving the opsonization and lysis of Abbreviations: aa, amino acids; ADIPOQ, adiponectin; APP, acute-phase proteins; APR, acute-phase response; CRP, C-reactive protein; C1Q, complement component C1q domain; C1QTNF3, C1q and tumour necrosis factor-related protein 3; C1QL2, complement component 1, q subcomponent-like 2; ELAM, endothelial cell-leukocyte adhesion molecule; GFP, green fluorescent protein; hpi, hours postinfection; NF-kB, nuclear factor kappa B; nt, nucleotides; PTX, pentraxin domain; RACE, rapid amplification of cDNA ends; RT-qPCR, quantitative real-time reverse transcription polymerase chain reaction; TCBP, trout C-polysaccharidebinding protein. The C-reactive protein (CRP) was the first acute-phase protein isolated from pneumonia patients in 1930 [9]. CRPs are phylogenetically ancient proteins that belong to the pentraxin family of plasma proteins, which are generally composed of five identical subunits in mammals [10]. The biological functions of CRPs are diverse, comprising the control of cytokine synthesis and peroxide production or the binding to phosphocholine of bacteria and dying cells to eventually activate complement, platelet aggregation and phagocytosis [11,12]
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