Short communicationMolecular responses of calreticulin genes to iron overload and bacterial challenge in channel catfish (Ictalurus punctatus)
Introduction
Fish are subject to a host of environmental stressors, particularly in culture conditions where they are regularly exposed to increased crowding, handling, altered water chemistries, and a variety of infectious pathogens. These stressors can upset delicate balances in cellular metabolic responses, increase levels of reactive oxygen species (ROS), and lead to oxidative stress and tissue damage (Victor et al., 2004). Intracellular iron accumulation leads to oxygen radical formation (Toyokuni, 2002) and regulation of cellular iron levels is crucial in mediating oxidative stress responses (Braun, 1998). During bacterial infection, iron metabolism is particularly regulated as host and pathogen compete for control of iron stores (Ganz, 2003, Liu et al., 2010a, Liu et al., 2010b). Iron binding proteins and iron transport and regulatory proteins can play dual roles in both antioxidant and immunomodulatory responses.
Calreticulin is a 46-kDa endoplasmic reticulum (ER) luminal Ca2+-binding chaperone protein (Baumann and Walz, 2001, Corbett and Michalak, 2000, Gelebart et al., 2005, Michalak et al., 2009), which was first characterized in rabbit (Ostwald and MacLennan, 1974). Working together with calnexin and ERp57, it is involved in the chaperoning of nascent polypeptides that traverse through the ER (Hebert and Molinari, 2007). Calreticulin is composed of three distinct structural and functional domains: a globular N-domain, an extended P-domain and an acidic C-domain. In vitro studies indicated that the polypeptide- and oligosaccharide-binding regions are located in the N-domain of calreticulin (Leach et al., 2002). The N-domain contains the double cysteine residues that are involved in the S–S bonds of the secondary structure of the protein. The protein contains an N-terminal cleavable signal sequence that directs it to the ER, and an ER retention/retrieval signal KDEL (Lys-Asp-Glu-Leu) in the C-domain. Between the N- and C-domains of calreticulin is the proline-rich P-domain, which binds Ca2+ with a relatively high affinity (Kd = 1 μM), but low capacity (1 mol of Ca2+ per mol of protein) (Baksh and Michalak, 1991). The calreticulin P-domain contains pairs of triple repeats A (amino acid sequence PXXIXDPDAXKPEDWDE) and B (amino acid sequence GXWXPPXIXNPXYX) (Michalak et al., 2009). The C-domain of calreticulin contains a large number of negatively charged residues that are responsible for the Ca2+ buffering function of the protein. The C-domain binds over 50% of ER luminal Ca2+ (Nakamura et al., 2001) with high capacity (25 mol of Ca2+ per mol of protein) and low affinity (Kd = 2 mM). Calreticulin is implicated in many cellular functions, including lectin-like chaperoning, Ca2+ storage and signaling, regulation of gene expression, cell adhesion, wound healing, cancer and autoimmunity (Gelebart et al., 2005, Michalak et al., 2009). Calreticulin has documented functions in oxidative stress responses caused by hydrogen peroxide, hypoxic injury and iron overload in mammals (Ihara et al., 2006, Jia et al., 2008, Núňez et al., 2001), and was also found to have a protective role in hereditary hemochromatosis, an iron-overload disease (Pinto et al., 2008).
Compared with intensive studies of the gene in mammals, studies of calreticulin gene in teleost fish to date have been limited. It has been identified in zebrafish (Rubinstein et al., 2000) and characterized in rainbow trout (Kales et al., 2004, Kales et al., 2007). Channel catfish (Ictalurus punctatus) is the most important aquaculture species in the United States, accounting for more than 60% of all U.S. aquaculture production (USDA, 2006). In previous microarray studies of catfish transcriptomic responses to enteric septicemia disease (Peatman et al., 2007, Peatman et al., 2008), calreticulin was found to be upregulated three days post infection. To better characterize and analyze catfish calreticulin gene in relation to antioxidant and immune responses, here we have generated full genomic sequences of the calreticulin gene, determined its genomic organization, localized the gene on the catfish physical map, and characterized patterns of calreticulin-related gene expression. Additionally, we have analyzed calreticulin-related gene transcriptional responses to bacterial infection and iron overload.
Section snippets
Identification of ESTs, BAC library screening and genomic sequencing of the catfish calreticulin gene
BLAST searches were used to identify partial cDNAs for calreticulin using channel catfish expressed sequence tags (ESTs) from previous sequencing efforts (Wang et al., 2010). All channel catfish ESTs were assembled into contiguous sequences (contigs) using the sequence assembly program CAP3 (http://deepc2.psi.iastate.edu/aat/cap/cap.html). Three contigs of calreticulin were identified by BLAST analyses, referred to here as calreticulin, calreticulin like and calreticulin like 2 according to the
The channel catfish genome contains at least three calreticulin-related genes
Initially, calreticulin-related cDNAs were identified from ESTs. Sequence analysis indicated the presence of three distinct clusters of cDNAs. To determine the identities of the calreticulin-related genes, phylogenetic analysis was conducted based on amino acid sequences. As shown in Fig. 1, the three catfish cDNAs fell in different clades: one (calreticulin; CALR) is most similar to the salmonid and zebrafish calreticulin genes; the second calreticulin-related cDNA of catfish, herein referred
Acknowledgements
This project was supported by a grant from the USDA AFRI Animal Genome Basic Genome Reagents and Tools Program (USDA/NRICGP award # 2009-35205-05101), and in part by a Specific Cooperative Agreement with USDA ARS Aquatic Animal Health Laboratory, Auburn, AL under Contract Number 58-6420-5-030, and by USDA, ARS CRIS 6402-31000-08-00D.
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