1 ISJ 10: 77-83, 2013 ISSN 1824-307X REVIEW The molluscan HSP70s and their expression in hemocytes L Wang, C Yang, L Song Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China Accepted September 23, 2013 Abstract The heat shock protein 70s (HSP70s) are a class of functionally related proteins involved in the folding and unfolding, translocation of other proteins, and stress responses in almost all organisms. As the most analyzed heat shock proteins, numerous HSP70s have been identified and characterized from bacteria, plants and animals. Molluscan HSP70 is one of the largest and most important groups in the invertebrate HSP70 family. Accumulating evidences have demonstrated the relevant physiological and ecological importance of HSP70 in response to pathogen infection and environmental stressors. This chapter reviews the interest arose around HSP70s in molluscan animals, mainly the recent research progress about the diversity of molluscan HSP70 family members, their sequence characters and expression profiles in hemocytes under various stressors. Key Words: Mollusc; Heat shock protein 70; expression profile; immune challenge; environmental stressor Introduction Heat shock protein 70 (HSP70) is one of the most abundant HSP families involved in the folding and unfolding, translocation of other proteins, and stress responses in almost all organisms. They consist of a class of functionally related proteins including HSP68, HSP70, HSC70, HSP75 and HSP78 (GRP78), which are localized to distinct subcellular compartments including cytoplasm, mitochondria, and endoplasmic reticulum (ER) (Boorstein et al., 1994; Mayer and Bukau, 2005). The amino acid sequences of HSP70 family members are highly conservative from archaebacteria to humans, and there are two major functional domains, the N-terminal ATPase domain and the C-terminal peptide binding domain (Sung et al., 2001; Mayer and Bukau, 2005). The HSP70s are involved in a variety of physiological processes and perform complex functions, such as serving as molecular chaperones (Gething & Sambrook, 1992), involved in the regulation of apoptosis (Böttger et al., 2008), and playing important roles in response to bacterial challenge (Cellura et al., 2006), oxidative stress (Golli-Bennour and Bacha, 2011) and various environmental stressors (Cellura et al., 2006). Recent studies in land snails Sphincterochila species ___________________________________________________________________________ Corresponding author: Linsheng Song Institute of Oceanology Chinese Academy of Sciences 7 Nanhai Rd., Qingdao 266071, China E-mail: lshsong@qdio.ac.cn suggested that HSP70 was also involved in the natural annual cycle of activity and aestivation and the survival strategy during desiccation and heat stress, and the adaptation of land snails to different habitats engenders the development of distinct strategies of HSP70 expression in response to stress (Mizrahi et al., 2012). The mollusc phylum is one of the largest and most important groups in the animal kingdom, and around 130,000 extant species are described (Haszprunar and Wanninger, 2012). Most of them live in freshwater or seawater, and they have to survive environmental perturbation from homeostasis, a situation generically described as stress. The production of acute phase proteins, such as the HSPs, is regarded as a classical response against stressors. This chapter reviews the interest arose around molluscan HSP70s in the last 5 years, mainly in the diversity, sequence characters and their expression profiles in hemocytes under various stresses. The HSP70 family members in mollusc Due to the important roles of HSP70s in the response against environmental stressors and the maintenance of homeostasis in molluscs, they have been studied extensively and the amount of their nucleotide sequences has increased noticeably during the past decades. There are totally 213 nucleotide sequences of molluscan HSP70 so far available in the database of NCBI, including 124 77 http://en.wikipedia.org/wiki/Extant_taxon http://en.wikipedia.org/wiki/Species http://www.sciencedirect.com/science/article/pii/S0960982212005921 http://www.sciencedirect.com/science/article/pii/S0960982212005921 Table 1 The full length of cDNA sequences encoding HSP70 in mollusc species Species gene Accession Number Reference snail Biomphalaria glabrataembryonic HSP70.1 L44127 Laursen et al., 1997 Pomacea canaliculata HSC70 Not released Zheng et al., 2012 sea hare Aplysia californica BiP/GRP78 NM_001204652 Kuhl et al., 1992 oyster Crassostrea gigas HSC70 AJ305315 Boutet et al., 2003b HSP70 AJ318882 Boutet et al., 2003b GRP78 BAD15288 Yokoyama et al., 2006 GRP94 AB262084 Kawabe and Yokoyama, 2009 Other HSP70s See the reference Zhang et al., 2012 Ostrea edulis HSC70 AJ305316 Boutet et al., 2003a HSP70 AF144646 Boutet et al., 2003a Oedcl5 AF416608 Piano et al., 2005 OedclD2 AF416609 Piano et al., 2005 Crassostrea hongkongensis HSP70 FJ157365 Zhang and Zhang, 2012 mussel Mytilus galloprovincialis HSP70 DQ178174, DQ178175 Franzellitti and Fabbri, 2005 HSC70 DQ178176, DQ178177 Franzellitti and Fabbri, 2005 HSP70 AY861684 Cellura et al., 2006 HSC70, HSC71 AJ783714, AJ783715 Kourtidis et al., 2006 HSP70-2, HSP70-3, HSP70-4 AJ783711, AJ783712, AJ783713 Kourtidis et al., 2006 scallop Argopecten irradians HSP70 AY485261 Song et al., 2006 Chlamys farreri HSP70 AY206871 Song et al., 2006 Mizuhopecten. yessoensis HSP70 AY485262 Song et al., 2006 Pinctada fucata HSP70 EU822509 Wang et al., 2009 abalone Haliotis discus hannai HSP70 DQ324856 Cheng et al., 2007 Haliotis diversicolor HSP70 ACO36048 unpublished HSC70 ACO36047 unpublished clam Laternula elliptica HSP70 EF198332. Park et al., 2007 Meretrix meretrix HSC71 HQ256748 Yue et al., 2011 Tegillarca granosa HSP70 N936877 Zhou et al., 2013 from bivalves, 77 from gastropods and 12 from cephalopods. The information about the full length cDNA sequences encoding HSC70 and HSP70 in mollusc is summarized in Table 1, and the species include snail (Laursen et al., 1997; Zheng et al., 2012), scallop (Song et al., 2006; Wang et al., 2009), oyster (Boutet et al., 2003a and 2003b; Piano et al., 2005; Zhang and Zhang, 2012; Zhang et al., 2012), mussel (Franzellitti and Fabbri, 2005; Cellura et al., 2006; Kourtidis et al., 2006), abalone (Cheng et al., 2007), and clam (Park et al., 2007; Yue et al., 2011). Other cDNA sequences encoding GRP78 and GRP94, the representatives of the GRP members in the molluscan HSP70 family, have also reported in sea hare (Kuhl et al., 1992) and oyster (Yokoyama et al., 2006; Kawabe and Yokoyama, 2009; Zhang et al., 2012). Though only one HSP70 has been reported in some molluscan species, all eukaryotes are believed to have more than one gene encoding HSP70 proteins in their genomes. For example, there are at least 11 unique HSP70 genes in human (Tavaria et al., 1996), 39 putative HSP70s in sea urchin (Sodergren et al., 2006), and 10 putative HSP70s in fungus Blastocladiella emersonii (Georg and Gomes, 2007). It is noteworthy that HSP70 gene family is remarkably expanded in C. gigas (Zhang et al., 2012). A search of the genome sequence revealed that there were 88 members of HSP70 family in C. gigas, which were believed to play crucial roles in protecting cells against heat and other stressors (Zhang et al., 2012). Structural features of molluscan HSP70s The molluscan HSP70s share common structural and evolutionary features with homologues from other species (Piano et al., 2005; Kourtidis et al., 2006), including the highly conserved N-terminal domain and the diverse C-terminal 78 http://www.ncbi.nlm.nih.gov/nuccore http://www.ncbi.nlm.nih.gov/nuccore http://www.ncbi.nlm.nih.gov/nuccore domains (Demand et al., 1998; Fuertes et al., 2004; Kourtidis et al., 2006). The highly conserved N-terminal domains of molluscan HSP70s usually shared three signature motifs (IDLGTTYS, IFDLGGGTFDVSIL, and IVLVGGSTRIPKIQK) and one ATP/GTP-binding motif (AEAYLGKT) (Wang et al., 2009; Zhou et al., 2013). In spite of high conservation, there are still some small variations in the N-terminal domains of molluscan HSP70s. For example, there is an extra NQSQ tetrapeptide in the ATPase domain of HSC70s from O. edulis (Boutet et al., 2003a) and C. gigas (Boutet et al., 2003b), and there are two nonsynonymous mutations, Y406I and G413E, in the ATP/GTP-binding motif of HSP70 from different geographical populations of A. irradians (Yang et al., unpublished data). Congruous with the difference in their subcellular localizations and functions, the C-terminal domains of different HSP70s usually display low sequence homology with each other (Demand et al., 1998; Fuertes et al., 2004; Piano et al., 2005), especially between HSP70 and HSC70 (Fabbri et al., 2008). The tetrapeptide motif GGMP is an important element mediating cofactor binding to the HSP molecule by forming a structural entity together with the helical subdomain and the EEVD motif (Demand et al., 1998), and it has been once regarded as the peculiar sequence of HSC70s (Fuertes et al., 2004; Piano et al., 2005; Fabbri et al., 2008). However, in some species, both of HSP70 and HSC70 contain GGMP tetrapeptide with variable numbers. For example, there are one, two, three and five GGMP tetrapeptides in the HSP70s from pearl oyster Pinctada fucata, blood clam Tegillarca granosa, Pacific abalone Haliotis discus hannai and Argopecten irradians respectively (Wang et al., 2009; Zhou et al., 2013; Cheng et al., 2007; Song et al., 2006). Therefore, it is necessary to investigate the effects of such structural variations on the expression profiles of HSP70 and HSC70 (Fuertes et al., 2004), and these information could also provide insights into functional specificities of HSP70s (Wang et al., 2009). Moreover, there is a large amino acid deletion about 60 residues encompassing the end of the peptide-binding domain and a part of the C-terminal domain of HSC70 from O. edulis (Kourtidis et al., 2006; Fabbri et al., 2008). The molluscan HSP70s located in cytosolism, ER, nuclear and mitochondrion always have the specific localization motifs GP(T/K)(V/I)EE(V/M)D, KDEL, NUCDISC and MITDISC, respectively. Multiple alignments revealed that most of molluscan HSP70s localized in the cytosolism sharing the cytosolic localization motif GP(T/K)(V/I)EE(V/M)D (Boorstein et al., 1994; Demand et al., 1998; Zhang and Zhang, 2012; Zhou et al., 2013). For example, 76 out of 88 HSP70s from C. gigas shared the motifs of GP(T/K)(V/I)EE(V/M)D, and they were predicted locating in the cytoplasm (Yang et al., unpublished data). Though EEVD and EEMD are both regarded as the cytosolic localization motifs, the effect of their sequence difference on structure and function still need further confirmation (Zhang and Zhang, 2012). Besides, GRP78 (Yokoyama et al., 2006) and other seven HSP70s from C. gigas (Yang et al., unpublished data) located in the ER also contain the motif KDEL. It is noteworthy that one HSP70 in oyster possessed a mitochondrial localization motif MITDISC, and this is the first mitochondrial HSP70 found in mollusc (Yang et al., unpublished data). Molluscan HSP70s are also classified into two groups of inducible HSP70s and cognate HSC70s at the present time, and they are closely matched to the corresponding HSP groups of other phylum in the phylogenetic analysis (Fabbri et al., 2008). However, it is not always accurate to assign a HSP70 into a specific group according to the phylogeny relationship. For example, several HSP70s from oysters (Boutet et al., 2003a and 2003b; Kourtidis et al., 2006) and scallops (Song et al., 2006) identified as inducible HSP70 proteins were clustered into HSC70 according to the phylogenetic analysis (Fabbri et al., 2008). Since there is limited information about the functions or activities of molluscan HSP70s, their classification is still not available currently. It has been reported that divergent evolution usually predominates when the members within one gene family acquire different functions (Ohta and Nei, 1994), and this is confirmed by inducible and cognate HSP70s, which belong to one family but display different expression patterns and functions. The phylogenetic reconstruction of molluscan HSP70s also indicates the occurrence of multiple duplication events in the evolution of HSP70 family, which is in agreement with the presence of multiple copies of the heat-inducible gene in molluscs. A phylogenetic analysis of 169 molluscan HSP70 proteins, including 88 from C. gigas, 12 from L. gigantean and 68 from other molluscs showed that 71 out of 88 C. gigas HSP70s were clustered together (Zhang et al., 2012). It suggested that these genes were likely received significant positive selection and derived from oyster-specific expansions, and they might play major roles in oyster’s adaptation to heat and other stressors (Zhang et al., 2012). Expression of molluscan HSP70s in hemocytes under various stressors As the most abundant and well studied HSPs, HSP70s are considered to play important roles in various physiological processes and protect organisms against various stressors. There are numerous studies to recognize the relevant physiological and ecological importance of molluscan HSP70s expression in response to the stresses resulted from changes of season and other environmental factors, such as temperature (Cellura et al., 2006), heavy metal (Boutet et al., 2003b; Thompson et al., 2012; Taylor et al., 2013), hypoxia (Clark and Peck, 2009; Clark et al., 2013), pH (Cummings et al., 2011), pollutants of PAHs (Song et al., 2006) and toxins (Mello et al., 2012; Mello et al., 2013), pharmaceuticals (Gust et al., 2013) and bacteria challenge (Cellura et al., 2006; Song et al., 2006; Cheng et al., 2007; Xu and Faisal, 2009). Most of the information on HSP70 expression in molluscs was mainly obtained from five tissues including gill, digestive gland, muscle, mantle and hemocytes. In the gill of C. gigas, the expression pattern of HSP70s altered significantly at different temperatures. There were some HSP70 genes highly expressed at 79 normal temperature, and some genes were highly expressed at low temperature, while some other genes were highly expressed at high temperature (Zhang et al., 2012). Regardless of the expression in other tissues, the research progress about the expression of molluscan HSP70s in hemocytes under various stressors is summarized in this chapter based on the reports in the past 5 years. Most molluscs have an open circulatory system composing of heart, blood vessels, sinusoids and hemolymph. As the major part of the hemolymph, hemocytes comprise the major component of the non-specific defense mechanisms, and they are involved in a series of cellular immune reactions (Song et al., 2010; Mello et al., 2012). The circulating hemocytes are able to migrate from the hemolymph to connective tissues, promote localized responses following injury or microorganism invasion (Mello et al., 2012), and discriminate pathogenic and non-pathogenic bacteria. For example, the expression of HSP70 gene in mussel hemocytes increased significantly after V. anguillarum challenge, while V. splendidus and M. lysodeikticus could not induce the expression of HSP70 (Cellura et al., 2006). Recently, the expression of HSP70s in molluscan hemocytes have been investigated extensively against several environment stressors, such as high temperature (Yang et al., unpublished data), heavy metal (Taylor et al., 2013), pollutants of toxins (Mello et al., 2012; Mello et al., 2013), pharmaceuticals (Gust et al., 2013), bacterial infections (Wang et al., 2009) and seasonal changes (Li et al., 2009), and their expression profiles are generally divided into three cases, up-regulated, invariable and down-regulated. When exposed to different stressors, up-regulated expression of HSP70s mRNA was the general case observed in molluscan hemocytes. For example, the mRNA expression of HSP70 in pond snail Lymnaea stagnalis increased (2.6-fold) after they were exposure to the mixtures of four pharmaceuticals (Gust et al., 2013). After incubation with the purified paralytic toxin of dinoflagellate Alexandrium minutum, saxitoxin (STX), the mRNA level of HSP70 in oyster hemocytes increased 2-fold (Mello et al., 2013). Moreover, the up-regulation of HSP70s expression in molluscan hemocytes usually displays a clearly time-dependent and dose-dependent pattern. The mRNA level of HSP70 in hemocytes of C. gigas increased at 4 h after the hemocytes were incubated with 1000 μg/L of PbTx-2 (Mello et al., 2012). At 6 h, 12 h and 24 h post heat stress treatment, the expression of HSP68 in C. gigas was up-regulated and relative mRNA level was 3.78-, 16.11- and 112.16- fold of that in the control group, respectively (Yang et al., unpublished data). After challenged by V. alginolyticus, the mRNA expression of HSP70 in hemocytes of pearl oyster P. fucata increased to the maximum level at 4 h, and returned to control level at 32 h (Wang et al., 2009). The mRNA expression of HSP70 in hemocytes of zebra mussel Dreissena polymorpha reached the highest level (2.8-fold) at 1 h post LPS stimulation, and decreased at 2 h, and then increased again from 3 h to 6 h post-stimulation (Xu and Faisal, 2009). The mRNA expression of HSP70 in hemocytes of blood clam T. granosa were all significantly up-regulated at 6 h after Pb2+, Cd2+ and Cu2+ treatments, and peaked at 12 h after treatments (Zhou et al., 2013). Except for the frequently up-regulation of HSP70s, it is interesting that the expression of HSP70 could also be down-regulated under some stressors. For example, the expression of HSP70 was significantly down-regulated when the Sydney Rock oyster Saccostrea glomerata was exposed to some heavy mental, such as zinc and copper (Taylor et al., 2013), cadmium and lead (Thompson et al., 2012). The excretory-secretory products (ESPs) from the larva of parasite Schistosoma mansoni could reduce the HSP70 protein levels in hemocytes of its snail intermediate host Biomphalaria glabrata, and the reduction in hemocytes of S. mansoni-resistant strain was less marked, while that in hemocytes of S. mansoni-susceptible snails was remarkable (approximately 70%) after infected by S. mansoni for 35 days (Zahoor et al., 2010). Regulation of molluscan HSP70 expression The up-regulation and down-regulation, as well as the dose-dependent and time-dependent expression pattern of HSP70 in the hemocytes of mollusks exposed to various stressors strongly suggested that the regulation mechanism of HSP70 expression was indeed complicated. Generally, the expression of HSP70 genes is mainly regulated at the transcription level (Park et al., 2007), and the regulation is mediated by direct interaction of heat shock transcription factors (HSFs) and their corresponding heat shock elements (HSEs) in the promoters of HSP70s (Wu, 1995; Buckley et al., 2001), and other indirect signaling pathways (Buckley et al., 2001; Park and Liu, 2001; Gourgou et al., 2010; Zahoor et al., 2010). The interaction of HSFs and HSEs in the promoters of HSP70s is the prime strategy to regulate HSP70 expression. Though molluscan HSF1s have been identified in the genome of M. trossulus, C. gigas and Haliotis asinina, the relevant study on the regulation mechanism of molluscan HSP70 expression is at the very beginning. It has been reported that HSF1 of intertidal mussels (genus Mytilus) releases from HSP70 and translocates into the nucleus in response to small increase of temperature, and remains inactive on the promoter until the mussels encounter a higher temperature (Buckley et al., 2001). The regulation of HSP70 expression also involves other cell proteins and signaling pathways after HSF1 has been bound to the promoter, including the mitogen-activated protein kinases (MAPK) signaling cascade (Buckley et al., 2001; Park and Liu, 2001; Gourgou et al., 2010) and the extracellular signal-regulated kinase (ERK) signaling pathway (Zahoor et al., 2010). In M. galloprovincialis, the increased phosphorylation of p38-MAPK and c-Jun N-terminal kinase (JNK) paralleled with the increased expression of HSP70, strongly supporting the involvement of MAPK signaling cascade in the induction of HSP70 genes under various stressors (Malagoli et al., 2004; Kefaloyianni et al., 2005; Anestis et al., 2007; Gourgou et al., 2010). After M. galloprovincialis was exposed to 30 °C acute thermal 80 http://www.ncbi.nlm.nih.gov/protein/BAK61501.1 http://www.ncbi.nlm.nih.gov/protein/ABR15461.1 stress, the activation profile of p38-MAPK phosphorylation was sustained and significant, while that of JNKs was transient and relatively moderate (Gourgou et al., 2010). This direct evidence demonstrated the principal roles of p38-MAPK and JNKs in transducing the stress signal via mobilization of specific transcription factors and the transcriptional up-regulation of HSP70 genes (Gourgou et al., 2010). The ERK signaling pathway has also been reported to regulate HSP70 expression in ESP-challenged hemocytes of B. glabrata, in which the mitogen-activated protein-ERK kinase 1/2 (MEK1/2) inhibitor could significantly reduce HSP70 protein levels, and this might be a strategy employed by the parasite to manipulate the immune response of the intermediate snail host (Zahoor et al., 2010). Conclusion Molluscan HSP70 is one of the largest and most important groups in the invertebrate HSP70 family, with consequential specializations in member diversity and sequence characteristics. The expanded family of oyster HSP70 offers an explanation for extensive repertoire of HSPs as well as the sophisticated strategies in response to stresses. Accumulating evidences have demonstrated the relevant similar expression profiles of molluscan HSP70s responding against pathogen infection and environmental stressor, which could be mainly regulated at the transcription level and be mediated by the interaction of HSFs and corresponding HSEs in the promoters of HSP70s. Acknowledge The authors would like to thank the lab members for helpful discussion. Some results cited in this review was supported by grants (No. 30925028 to LS) from Natural Science Foundation of China (NSFC). Reference Anestis A, Lazou A, Portner HO, Michaelidis B. Behavioral, metabolic, and molecular stress responses of marine bivalve Mytilus galloprovincialis during long-term acclimation at increasing ambient temperature. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293: 911-921, 2007. Becker J, Craig EA. Heat-shock proteins as molecular chaperones. Eur. J. Biochem. 219: 11-23, 1994. Boutet I, Tanguy A, Moraga D. 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