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.

Qinggang Xue,Richard K Cooper,Jerome F La Peyre,Naoki Itoh,Kevin L Schey,Ron I Eytan,Michael E Hellberg

doi:10.1186/1471-2148-10-213

Abstract

BackgroundLysozymes 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.ResultsWe 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.ConclusionThe 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.

Highlights

Lysozymes are enzymes that lyse bacterial cell walls, an activity widely used for host defense and modified in some instances for digestion
Lysozymes identified from organisms ranging from bacteriophage to humans share a muramidase activity cleaving the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine of peptidoglycan, a major component of the bacterial cell wall [2,3,4], but have different amino acid sequences
While we have previously proposed that i-type lysozymes may have followed a comparable adaptive path between host defense and digestion as seen in some vertebrate c-type lysozymes [57], the chain of evolutionary steps involved in the shift between functions remains unknown

Summary

Introduction

Lysozymes are enzymes that lyse bacterial cell walls, an activity widely used for host defense and modified in some instances for digestion. Structural studies reveal that a phylogenetically broad sample of lysozymes (bacteriophage T4 lysozyme, i-type Japanese littleneck clam (Tapes japonica or Venerupis philippinarum in the NCBI Taxonomy database) lysozyme, c-type chicken egg-white lysozyme, and the g-type goose egg-white lysozyme) all have a similar conformation surrounding the enzyme catalytic center, even though their amino acid sequences bear little similarity [11,12,13,14] Given their shared activities and structure, these different lysozymes appear to have been derived from a common ancestor [11,12,13].

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