Biochemical and proteomic characterisation of haemolymph serum reveals the origin of the alkali-labile phosphate (ALP) in mussel (Mytilus galloprovincialis) Caterina Oliveri a , Lorena Peric c , Susanna Sforzini a , Mohammed Banni a,b , Aldo Viarengo a , Maria Cavaletto a , Francesco Marsano a, ⁎ a Department of Science and Technological Innovation, University of Piemonte Orientale “Amedeo Avogadro”, Viale Teresa Michel, 11, 15121 Alessandria, Italy b Laboratory of Biochemistry and Environmental Toxicology, ISA Chott-Mariem, 4042 Sousse, Tunisia c Ruder Bošković Instititute, Center for Marine Research, G. Paliaga 5, 52210 Rovinj, Croatia abstract article info Article history: Received 27 May 2014 Received in revised form 16 July 2014 Accepted 17 July 2014 Available online 24 July 2014 Keywords: Haemolymph serum Mytilus galloprovincialis Alkali-labile phosphate (ALP) Vitellogenin (Vtg)-like proteins Proteomics Mollusc haemolymph proteins are known to play several important physiological roles in the immune system, heavy metal transport and the tissue distribution of lipophilic compounds. In this study, we analysed acetone-extracted proteins from mussel haemolymph by one- and two-dimensional gel electrophoresis. The proteins were identified by comparing mass spectrometry data with the invertebrate EST database, allowing us to establish the mussel haemolymph serum proteome. Extrapallial protein (EP) precur- sor represents the most abundant serum protein; astacin and CuZn superoxide dismutase were also detected. Slight contamination from muscle proteins, due to the sampling method, was also found. No differences were observed in the profiles obtained for male and female serum proteins. One aspect of interest was the previously reported finding that alkali-labile phosphate (ALP) from haemolymph serum may be representative of vitello- genin (vtg)-like protein content in the circulatory fluid of molluscs. In our analysis of mussel haemolymph serum, vitellogenin-like proteins were never found. To confirm these data, a typical methyl-tert-butyl-ether (MTBE) extraction, which is specific for vtg-like proteins, was performed, and the results of the electrophoretic analyses were compared with those obtained by acetonic precipitation. The results showed that the electropho- retic profiles are similar and that vtg-like proteins cannot be identified. Moreover, the main phosphoprotein present in female and male extracts is EP protein precursor. In addition, agarose gel electrophoresis demonstrates that high-molecular-weight forms of vtg-like proteins are not detectable. © 2014 Elsevier Inc. All rights reserved. 1. Introduction Bivalve haemolymph cells (i.e., haemocytes) play an important role in the internal immune defence, and it is hypothesised that these cells can be involved in numerous other processes including wound-healing, shell repair, transport and the intracellular digestion of nutrients (Cheng, 1981, 1984; Fisher, 1986; Wootton and Pipe, 2003; Hong et al., 2006; Hégaret et al., 2011). The proteins present in the haemolymph serum in mussels are also known to be involved in the immune response and to bind a variety of metals (Koutsogiannaki and Kaloyianni, 2010; Itoh et al., 2011; Seufi et al., 2012; Xue et al., 2012). In recent years, although technical advances in proteomics (i.e., two- dimensional gel electrophoresis and mass spectrometry) and its application in the field of comparative biochemistry, physiology and environmental research have increased significantly, few data have been produced regarding the Mytilus sp. haemolymph serum proteome (Riffeser and Hock, 2002). An aspect of great interest was the proposal that in bivalves the alkali-labile phosphate (ALP) from haemolymph serum proteins can be representative of the vitellogenin (vtg)-like protein content (Blaise et al., 1999). This method, based on the determination of labile phos- phates released by proteins after alkali hydrolysis, has been used in plasma and tissues as an indicator of endocrine disruption due to the effects of the presence of xenoestrogens in the environment. Despite genomic research aimed at identifying specific genes associated with maturation and oestrogen exposure in Mytilus edulis (Ciocan et al., 2011), there are still no definitive data regarding the identification of vitellogenin-like proteins in mollusc serum. The aim of the present study is to analyse the haemolymph serum proteome of Mytilus galloprovincialis and to identify the main expressed protein(s). Another important aspect of this research, given the use of phosphorylatable proteins as environmental bio- markers for the assessment of endocrine disruptor chemicals (EDCs), is to identify such protein(s) in the serum. One- and two- Comparative Biochemistry and Physiology, Part D 11 (2014) 29–36 ⁎ Corresponding author. Tel.: +39 131360229; fax: +39 131360243. E-mail address: francesco.marsano@mfn.unipmn.it (F. Marsano). http://dx.doi.org/10.1016/j.cbd.2014.07.003 1744-117X/© 2014 Elsevier Inc. All rights reserved. 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