Molecular responses of calreticulin genes to iron overload and bacterial challenge in channel catfish (Ictalurus punctatus)

Abstract

Infection and inflammation are often accompanied by oxidative stress caused by the accumulation of reactive oxygen species which can be deleterious to the health of the host. Antioxidant defense mechanisms and components are crucial in limiting cellular and tissue-level damage and restoring homeostasis. In mammals, calreticulin is a 46-kDa multifunctional calcium binding protein of the endoplasmic reticulum that has many critical functions in the eukaryotic cell including regulation of intracellular calcium homoeostasis, lectin binding and chaperoning, and oxidative stress responses. In previous studies from our lab, the calreticulin gene was observed to be strongly upregulated in catfish during challenge with infectious Gram-negative bacteria. However, little is known about the function of this gene in teleost fish. The objective of this study, therefore, was to characterize the calreticulin gene from channel catfish, to determine its genomic organization, to profile its patterns of tissue expression, and to establish its potential for physiological antioxidant and immune responses in catfish after bacterial infection with Edwardsiella ictaluri and iron treatment. Our results indicate that there are at least three calreticulin related genes in the catfish genome. The three calreticulin genes are widely expressed in various tissues under homeostatic conditions and their expression showed significant upregulation following infection and/or iron level changes.

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.