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A new lysozyme from the eastern oyster, Crassostrea virginica, and a possible evolutionary pathway for i-type lysozymes in bivalves from host defense to digestion.

Xue Q, Hellberg ME, Schey KL, Itoh N, Eytan RI, Cooper RK, La Peyre JF - BMC Evol. Biol. (2010)

Bottom Line: The topology of a phylogenetic analysis of cv-lysozyme 3 cDNA (full length 663 bp, encoding an open reading frame of 187 amino acids) is also consistent with a transitional condition, as cv-lysozyme 3 falls at the base of a monophyletic clade of bivalve lysozymes identified from digestive glands.Rates of nonsynonymous substitution are significantly high at the base of this clade, consistent with an episode of positive selection associated with the functional transition from defense to digestion.The pattern of molecular evolution accompanying the shift from defensive to digestive function in the i-type lysozymes of bivalves parallels those seen for c-type lysozymes in mammals and suggests that the lysozyme paralogs that enhance the range of physiological conditions for lysozyme activity may provide stepping stones between defensive and digestive forms.

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Affiliation: Department of Veterinary Science, Louisiana State University Agricultural Center, Baton Rouge, LA 70830, USA. qxue@lsu.edu

ABSTRACT

Background: Lysozymes are enzymes that lyse bacterial cell walls, an activity widely used for host defense but also modified in some instances for digestion. The biochemical and evolutionary changes between these different functional forms has been well-studied in the c-type lysozymes of vertebrates, but less so in the i-type lysozymes prevalent in most invertebrate animals. Some bivalve molluscs possess both defensive and digestive lysozymes.

Results: We report a third lysozyme from the oyster Crassostrea virginica, cv-lysozyme 3. The chemical properties of cv-lysozyme 3 (including molecular weight, isoelectric point, basic amino acid residue number, and predicted protease cutting sites) suggest it represents a transitional form between lysozymes used for digestion and immunity. The cv-lysozyme 3 protein inhibited the growth of bacteria (consistent with a defensive function), but semi-quantitative RT-PCR suggested the gene was expressed mainly in digestive glands. Purified cv-lysozyme 3 expressed maximum muramidase activity within a range of pH (7.0 and 8.0) and ionic strength (I = 0.005-0.01) unfavorable for either cv-lysozyme 1 or cv-lysozyme 2 activities. The topology of a phylogenetic analysis of cv-lysozyme 3 cDNA (full length 663 bp, encoding an open reading frame of 187 amino acids) is also consistent with a transitional condition, as cv-lysozyme 3 falls at the base of a monophyletic clade of bivalve lysozymes identified from digestive glands. Rates of nonsynonymous substitution are significantly high at the base of this clade, consistent with an episode of positive selection associated with the functional transition from defense to digestion.

Conclusion: The pattern of molecular evolution accompanying the shift from defensive to digestive function in the i-type lysozymes of bivalves parallels those seen for c-type lysozymes in mammals and suggests that the lysozyme paralogs that enhance the range of physiological conditions for lysozyme activity may provide stepping stones between defensive and digestive forms.

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Phylogenetic relationships among bivalve i-type lysozymes. The phylogenetic tree was inferred based on a Bayesian analysis of amino acid sequences from the core alignment. Numbers above branches indicate Bayesian posterior probabilities, the first for the analysis based on the BAli-Phy alignment and the second for the analysis based on the ClustalW analysis. Asterisks indicate nodes supported by maximum likelihood bootstrap values ≥ 0.75. Branch lengths are shown below branches. The novel C. virginica lysozyme isolated here (cv-lysozyme 3) is circled. The larger box indicates the 13 sequences used for the PAML analysis. The smaller box indicates the clade containing the digestive cv-lysozyme 2 (cv2C). Isoelectric points (pI) and trypsin cutting site numbers (TCS#) predicted from the aligned core sequences are shown.
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Figure 6: Phylogenetic relationships among bivalve i-type lysozymes. The phylogenetic tree was inferred based on a Bayesian analysis of amino acid sequences from the core alignment. Numbers above branches indicate Bayesian posterior probabilities, the first for the analysis based on the BAli-Phy alignment and the second for the analysis based on the ClustalW analysis. Asterisks indicate nodes supported by maximum likelihood bootstrap values ≥ 0.75. Branch lengths are shown below branches. The novel C. virginica lysozyme isolated here (cv-lysozyme 3) is circled. The larger box indicates the 13 sequences used for the PAML analysis. The smaller box indicates the clade containing the digestive cv-lysozyme 2 (cv2C). Isoelectric points (pI) and trypsin cutting site numbers (TCS#) predicted from the aligned core sequences are shown.

Mentions: Despite differences in the alignment, the phylogenetic trees based on the two core alignments produced identical topologies with similar Bayesian support values (Figure 6). The common tree united cv-lysozyme 1, cv-lysozyme 3, and a cDNA of unknown function from C. gigas (referred to here as cg-lysozyme 4) with a clade of lysozymes that includes the digestive cv-lysozyme 2 with strong support (BPP ≥ 0.99). The defensive cv-lysozyme 1 is positioned basally, with a well-supported cv-lysozyme 3/cg-lysozyme 4 clade coming off next as the sister to the clade including cv-lysozyme 2. The maximum likelihood tree (not shown) did not differ significantly from the Bayesian tree, with the only topological differences being the monophyly of Calyptogena and whether the Tapes/Calyptogena or Cg-1/Ostrea clade is sister to the crown group including cv-lysozyme 1, although support values for this tree are lower than for the Bayesian tree (Figure 6).


A new lysozyme from the eastern oyster, Crassostrea virginica, and a possible evolutionary pathway for i-type lysozymes in bivalves from host defense to digestion.

Xue Q, Hellberg ME, Schey KL, Itoh N, Eytan RI, Cooper RK, La Peyre JF - BMC Evol. Biol. (2010)

Phylogenetic relationships among bivalve i-type lysozymes. The phylogenetic tree was inferred based on a Bayesian analysis of amino acid sequences from the core alignment. Numbers above branches indicate Bayesian posterior probabilities, the first for the analysis based on the BAli-Phy alignment and the second for the analysis based on the ClustalW analysis. Asterisks indicate nodes supported by maximum likelihood bootstrap values ≥ 0.75. Branch lengths are shown below branches. The novel C. virginica lysozyme isolated here (cv-lysozyme 3) is circled. The larger box indicates the 13 sequences used for the PAML analysis. The smaller box indicates the clade containing the digestive cv-lysozyme 2 (cv2C). Isoelectric points (pI) and trypsin cutting site numbers (TCS#) predicted from the aligned core sequences are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3020801&req=5

Figure 6: Phylogenetic relationships among bivalve i-type lysozymes. The phylogenetic tree was inferred based on a Bayesian analysis of amino acid sequences from the core alignment. Numbers above branches indicate Bayesian posterior probabilities, the first for the analysis based on the BAli-Phy alignment and the second for the analysis based on the ClustalW analysis. Asterisks indicate nodes supported by maximum likelihood bootstrap values ≥ 0.75. Branch lengths are shown below branches. The novel C. virginica lysozyme isolated here (cv-lysozyme 3) is circled. The larger box indicates the 13 sequences used for the PAML analysis. The smaller box indicates the clade containing the digestive cv-lysozyme 2 (cv2C). Isoelectric points (pI) and trypsin cutting site numbers (TCS#) predicted from the aligned core sequences are shown.
Mentions: Despite differences in the alignment, the phylogenetic trees based on the two core alignments produced identical topologies with similar Bayesian support values (Figure 6). The common tree united cv-lysozyme 1, cv-lysozyme 3, and a cDNA of unknown function from C. gigas (referred to here as cg-lysozyme 4) with a clade of lysozymes that includes the digestive cv-lysozyme 2 with strong support (BPP ≥ 0.99). The defensive cv-lysozyme 1 is positioned basally, with a well-supported cv-lysozyme 3/cg-lysozyme 4 clade coming off next as the sister to the clade including cv-lysozyme 2. The maximum likelihood tree (not shown) did not differ significantly from the Bayesian tree, with the only topological differences being the monophyly of Calyptogena and whether the Tapes/Calyptogena or Cg-1/Ostrea clade is sister to the crown group including cv-lysozyme 1, although support values for this tree are lower than for the Bayesian tree (Figure 6).

Bottom Line: The topology of a phylogenetic analysis of cv-lysozyme 3 cDNA (full length 663 bp, encoding an open reading frame of 187 amino acids) is also consistent with a transitional condition, as cv-lysozyme 3 falls at the base of a monophyletic clade of bivalve lysozymes identified from digestive glands.Rates of nonsynonymous substitution are significantly high at the base of this clade, consistent with an episode of positive selection associated with the functional transition from defense to digestion.The pattern of molecular evolution accompanying the shift from defensive to digestive function in the i-type lysozymes of bivalves parallels those seen for c-type lysozymes in mammals and suggests that the lysozyme paralogs that enhance the range of physiological conditions for lysozyme activity may provide stepping stones between defensive and digestive forms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Science, Louisiana State University Agricultural Center, Baton Rouge, LA 70830, USA. qxue@lsu.edu

ABSTRACT

Background: Lysozymes are enzymes that lyse bacterial cell walls, an activity widely used for host defense but also modified in some instances for digestion. The biochemical and evolutionary changes between these different functional forms has been well-studied in the c-type lysozymes of vertebrates, but less so in the i-type lysozymes prevalent in most invertebrate animals. Some bivalve molluscs possess both defensive and digestive lysozymes.

Results: We report a third lysozyme from the oyster Crassostrea virginica, cv-lysozyme 3. The chemical properties of cv-lysozyme 3 (including molecular weight, isoelectric point, basic amino acid residue number, and predicted protease cutting sites) suggest it represents a transitional form between lysozymes used for digestion and immunity. The cv-lysozyme 3 protein inhibited the growth of bacteria (consistent with a defensive function), but semi-quantitative RT-PCR suggested the gene was expressed mainly in digestive glands. Purified cv-lysozyme 3 expressed maximum muramidase activity within a range of pH (7.0 and 8.0) and ionic strength (I = 0.005-0.01) unfavorable for either cv-lysozyme 1 or cv-lysozyme 2 activities. The topology of a phylogenetic analysis of cv-lysozyme 3 cDNA (full length 663 bp, encoding an open reading frame of 187 amino acids) is also consistent with a transitional condition, as cv-lysozyme 3 falls at the base of a monophyletic clade of bivalve lysozymes identified from digestive glands. Rates of nonsynonymous substitution are significantly high at the base of this clade, consistent with an episode of positive selection associated with the functional transition from defense to digestion.

Conclusion: The pattern of molecular evolution accompanying the shift from defensive to digestive function in the i-type lysozymes of bivalves parallels those seen for c-type lysozymes in mammals and suggests that the lysozyme paralogs that enhance the range of physiological conditions for lysozyme activity may provide stepping stones between defensive and digestive forms.

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