Two Goose-Type Lysozymes in Mytilus galloprovincialis: Possible Function Diversification and Adaptive Evolution

Abstract

Two goose-type lysozymes (designated as MGgLYZ1 and MGgLYZ2) were identified from the mussel Mytilus galloprovincialis. MGgLYZ1 mRNA was widely expressed in the examined tissues and responded sensitively to bacterial challenge in hemocytes, while MGgLYZ2 mRNA was predominately expressed and performed its functions in hepatopancreas. However, immunolocalization analysis showed that both these lysozymes were expressed in all examined tissues with the exception of adductor muscle. Recombinant MGgLYZ1 and MGgLYZ2 could inhibit the growth of several Gram-positive and Gram-negative bacteria, and they both showed the highest activity against Pseudomonas putida with the minimum inhibitory concentration (MIC) of 0.95–1.91 µM and 1.20–2.40 µM, respectively. Protein sequences analysis revealed that MGgLYZ2 had lower isoelectric point and less protease cutting sites than MGgLYZ1. Recombinant MGgLYZ2 exhibited relative high activity at acidic pH of 4–5, while MGgLYZ1 have an optimum pH of 6. These results indicated MGgLYZ2 adapted to acidic environment and perhaps play an important role in digestion. Genomic structure analysis suggested that both MGgLYZ1 and MGgLYZ2 genes are composed of six exons with same length and five introns, indicating these genes were conserved and might originate from gene duplication during the evolution. Selection pressure analysis showed that MGgLYZ1 was under nearly neutral selection while MGgLYZ2 evolved under positive selection pressure with three positively selected amino acid residues (Y102, L200 and S202) detected in the mature peptide. All these findings suggested MGgLYZ2 perhaps served as a digestive lysozyme under positive selection pressure during the evolution while MGgLYZ1 was mainly involved in innate immune responses.

A defensin from clam Venerupis philippinarum: Molecular characterization, localization, antibacterial activity, and mechanism of action

“…rVpDef was expressed in Escherichia coli Origami (DE3) (Novagen) as a C-terminal His6-tagged fusion protein using the pET-30a(+) system (Novagen). Expression, purification and refolding of rVpDef were performed according to the methods described previously (Wang et al, 2012). In the present study, the rVpDef was expressed as inclusion bodies and purified by HisTrap Chelating Columns under denatured conditions.…”

Molecular characterisation, evolution and expression analysis of g-type lysozymes in Ciona intestinalis

“…These restricted distribution patterns in birds and mammals contrasted with the broad expression patterns of g-type lysozymes in fish and invertebrate species. G-type lysozymes have been detected in the spleen, kidneys, gills, skin, heart, intestines and blood of Japanese flounder, orange-spotted grouper, large yellow croaker, Atlantic cod and grass carp (Hikima et al, 2001;Savan et al, 2003;Yin et al, 2003;Zheng et al, 2007), as well as in the gills, mantle, hepatopancreas, hemocytes and muscles in Mytilus galloprovincialis (Wang et al, 2012). Real-Time analysis has shown that C. intestinalis g-type lysozymes are upregulated in the pharynx after LPS challenge, in particular, CiLys-g1, CiLys-g2 and CiLys-g3 gene expression were significantly boosted at 24e72 h, while CiLys-g4 gene expression was significantly boosted at 1 h and 2e4 h and decreased at 8e48 h, supporting a defensive role for CiLys-g lysozymes.…”

Molecular diversity and evolution of defensins in the manila clam Ruditapes philippinarum

“…For transcript polymorphism detection, two specific primers (P5 and P6, Table 1) designed in the 5 0 UTR and 3 0 UTR were employed to clone the full coding sequence of Rpdefs. The PCR profile and subsequent sequencing were conducted as described previously [33,34]. A total of 126 positive Rpdef clones were bi-directionally sequenced respectively.…”

“…Quantitative real-time PCR (qRT-PCR) was carried out in an ABI 7500 Real-time Detection System by using the SYBR ExScript qPCR Kit (Takara, Japan) as described previously [33]. The PCR amplification was carried out in a total volume of 50 mL, containing 25 mL of 2 Â SYBR Green PCR Master Mix, 20 mL of the diluted cDNA, 1 mL of each of primers (10 mmol/L), and 3 mL of DEPC-treated water.…”

“…The assumption and parameters of each model were as describe previously [33]. The branch model was also conducted using the likelihood ratio test between the oneratio model and the free-ratio model results to detect positive selection acting on particular lineages of the phylogenetic tree.…”