A glycosyl hydrolase family 16 gene is responsible for the endogenous production of β-1,3-glucanases within decapod crustaceans

Abstract

To identify the gene responsible for the production of a β-1,3-glucanase (laminarinase) within crustacea, a glycosyl hydrolase family 16 (GHF16) gene was sequenced from the midgut glands of the gecarcinid land crab, Gecarcoidea natalis and the freshwater crayfish, Cherax destructor. An open reading frame of 1098 bp for G. natalis and 1095 bp for C. destructor was sequenced from cDNA. For G. natalis and C. destructor respectively, this encoded putative proteins of 365 and 364 amino acids with molecular masses of 41.4 and 41.5 kDa. mRNA for an identical GHF16 protein was also expressed in the haemolymph of C. destructor. These putative proteins contained binding and catalytic domains that are characteristic of a β-1,3-glucanase from glycosyl hydrolase family 16. The amino acid sequences of two short 8-9 amino acid residue peptides from a previously purified β-1,3-glucanase from G. natalis matched exactly that of the putative protein sequence. This plus the molecular masses of the putative proteins matching that of the purified proteins strongly suggests that the sequences obtained encode for a catalytically active β-1,3-glucanase. A glycosyl hydrolase family 16 cDNA was also partially sequenced from the midgut glands of other amphibious (Mictyris platycheles and Paragrapsus laevis) and terrestrial decapod species (Coenobita rugosus, Coenobita perlatus, Coenobita brevimanus and Birgus latro) to confirm that the gene is widely expressed within this group. There are three possible hypothesised functions and thus evolutionary routes for the β-1,3-glucanase: 1) a digestive enzyme which hydrolyses β-1,3-glucans, 2) an enzyme which cleaves β-1,3-glycosidic bonds within cell walls to release cell contents or 3) an immune protein which can hydrolyse the cell walls of potentially pathogenic micro-organisms.

Functional Characterization of the Novel Laminaripentaose-Producing β-1,3-Glucanase MoGluB and Its Biocontrol of Magnaporthe oryzae

“…4 β-1,3-Glucanases are widely distributed in various organisms, including bacteria, 5 fungi, 6 plants, 7 and invertebrates. 8 They can be classified into two categories depending on their substrate specificities and catalytic patterns. 9 The first class is the exo-β-1,3-glucanase, which cleaves β-1,3-bonds from the nonreducing end of a substrate and sequentially releases glucose monomers.…”

“…β-1,3-Glucanases cleave the β-1,3-bonds in a variety of β-1,3-glucans such as curdlan, pachyman, and laminarin . β-1,3-Glucanases are widely distributed in various organisms, including bacteria, fungi, plants, and invertebrates . They can be classified into two categories depending on their substrate specificities and catalytic patterns .…”

Analysis of Molecular Modeling and Molecular Docking of Beta-glucanase from Metagenomic Expression Library as Candida Antibiofilm Candidate

“…This residue is possible to form disulfide bonds which are very important for the thermal stability of enzymes. 15 The MKAFGlu1 sequence has a low homology when compared to the conservation region in the GH16 member sequences such as Haliotis tuberculata (GenBank access code No. AFQ98375.1), Homoeocera gigantea (GenBank access code No.…”

“…Multiple alignments of amino acid sequences of endo-1,3-β-glucanase GH16 showed that the most conservative amino acid residue feature was "Glu-xxx-Asp-xxx-xxx-Glu" (in the red box Figure 3). 15,16 The first glutamate residue acts as a nucleophilic residue, while the second glutamate acts as an acid-base catalyst residue. 16 However, the most conserved amino acid residue was not found in MKAFGlu1, so it can be concluded that MKAFGlu1 is a new representation of endo-1,3-β-glucanase GH16.…”

Presence and activity of endo-β-1,4-mannase, an important digestive carbohydrase within the digestive fluid of terrestrial crustaceans

“…Christmas Island red crabs, Gecarcoidea natalis (Pocock 1888) and terrestrial hermit crabs, Coenobita brevimanus (Dana 1852) and Coenobita perlatus (Milne Edwards et al 1837) were collected from the rainforest on Christmas Island, and humanely euthanised. Tissues (midgut gland, muscle and gill), were also taken from G. natalis and preserved in RNA later (ThermoFisher Scientific AM7020) as described by Linton et al (2015).…”