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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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MALDI mass spectrum of purified cv-lysozyme 3. Cv-lysozyme 3 was detected as an MH+ ion at m/z 17782.3. The signal at m/z 8879.9 represents the doubly charged intact cv-lysozyme 3 molecule.
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Figure 2: MALDI mass spectrum of purified cv-lysozyme 3. Cv-lysozyme 3 was detected as an MH+ ion at m/z 17782.3. The signal at m/z 8879.9 represents the doubly charged intact cv-lysozyme 3 molecule.

Mentions: Purified cv-lysozyme 3 appeared as a single band with a molecular size of 19.9 kDa as determined by SDS-PAGE under reducing conditions (Figure 1A). However, under non-reducing conditions, a second band with a molecular size of >100 kDa was also detected (Figure 1B). It appeared that the high molecular band represented a cv-lysozyme 3 polymer formed via electrostatic force, a phenomenon reported for other lysozymes [14,59-62]. MALDI mass spectrometry revealed an ion of 17782.3 Da for the purified cv-lysozyme 3 (Figure 2). The MALDI measured molecular weight of the reduced and alkylated cv-lysozyme 3 was 18939 (not shown), indicating the presence of 20 cysteine residues.


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)

MALDI mass spectrum of purified cv-lysozyme 3. Cv-lysozyme 3 was detected as an MH+ ion at m/z 17782.3. The signal at m/z 8879.9 represents the doubly charged intact cv-lysozyme 3 molecule.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: MALDI mass spectrum of purified cv-lysozyme 3. Cv-lysozyme 3 was detected as an MH+ ion at m/z 17782.3. The signal at m/z 8879.9 represents the doubly charged intact cv-lysozyme 3 molecule.
Mentions: Purified cv-lysozyme 3 appeared as a single band with a molecular size of 19.9 kDa as determined by SDS-PAGE under reducing conditions (Figure 1A). However, under non-reducing conditions, a second band with a molecular size of >100 kDa was also detected (Figure 1B). It appeared that the high molecular band represented a cv-lysozyme 3 polymer formed via electrostatic force, a phenomenon reported for other lysozymes [14,59-62]. MALDI mass spectrometry revealed an ion of 17782.3 Da for the purified cv-lysozyme 3 (Figure 2). The MALDI measured molecular weight of the reduced and alkylated cv-lysozyme 3 was 18939 (not shown), indicating the presence of 20 cysteine residues.

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.

Show MeSH