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Isolation of novel prototype galectins from the marine ball sponge Cinachyrella sp. guided by their modulatory activity on mammalian glutamate-gated ion channels.

ABSTRACT: Here we report the bioactivity-guided isolation of novel galectins from the marine sponge Cinachyrella sp., collected from Iriomote Island, Japan. The lectin proteins, which we refer to as the Cinachyrella galectins (CchGs), were identified as the active principles in an aqueous sponge extract that modulated the function of mammalian ionotropic glutamate receptors. Aggregation of rabbit erythrocytes by CchGs was competed most effectively by galactosides but not mannose, a profile characteristic of members of the galectin family of oligosaccharide-binding proteins. The lectin activity was remarkably stable, with only a modest loss in hemagglutination after exposure of the protein to 100°C for 1 h, and showed little sensitivity to calcium concentration. CchG-1 and -2 appeared as 16 and 18 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, whereas matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry indicated broad ion clusters centered at 16,216 and 16,423, respectively. The amino acid sequences of the CchGs were deduced using a combination of Edman degradation and cDNA cloning and revealed that the proteins were distant orthologs of animal prototype galectins and that multiple isolectins comprised the CchGs. One of the isolectins was expressed as a recombinant protein and exhibited physico-chemical and biological properties comparable with those of the natural lectins. The biochemical properties of the CchGs as well as their unexpected activity on mammalian excitatory amino acid receptors suggest that further analysis of these new members of the galectin family will yield further glycobiological and neurophysiological insights.

SUBMITTER: Ueda T

PROVIDER: S-EPMC3581077 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Isolation of novel prototype galectins from the marine ball sponge Cinachyrella sp. guided by their modulatory activity on mammalian glutamate-gated ion channels.

Ueda Takuya T Nakamura Yuka Y Smith Caleb M CM Copits Bryan A BA Inoue Akira A Ojima Takao T Matsunaga Satoko S Swanson Geoffrey T GT Sakai Ryuichi R

Glycobiology 20121204 4

Here we report the bioactivity-guided isolation of novel galectins from the marine sponge Cinachyrella sp., collected from Iriomote Island, Japan. The lectin proteins, which we refer to as the Cinachyrella galectins (CchGs), were identified as the active principles in an aqueous sponge extract that modulated the function of mammalian ionotropic glutamate receptors. Aggregation of rabbit erythrocytes by CchGs was competed most effectively by galactosides but not mannose, a profile characteristic ...[more]

PMID: 23213112

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Project description:Anthelmintic resistance is a major problem in livestock farming, especially of small ruminants, but our understanding of it has been limited by the difficulty in carrying out functional genetic studies on parasitic nematodes. An important nematode infecting sheep and goats is Haemonchus contortus; in many parts of the world this species is resistant to almost all the currently available drugs, including ivermectin. It is extremely polymorphic and to date it has proved impossible to relate any sequence polymorphisms to its ivermectin resistance status. Expression of candidate drug-resistance genes in Caenorhabditis elegans could provide a convenient means to study the effects of polymorphisms found in resistant parasites, but may be complicated by differences between the gene families of target and model organisms. We tested this using the glutamate-gated chloride channel (GluCl) gene family, which forms the ivermectin drug target and are candidate resistance genes. We expressed GluCl subunits from C. elegans and H. contortus in a highly resistant triple mutant C. elegans strain (DA1316) under the control of the avr-14 promoter; expression of GFP behind this promoter recapitulated the pattern previously reported for avr-14. Expression of ivermectin-sensitive subunits from both species restored drug sensitivity to transgenic worms, though some quantitative differences were noted between lines. Expression of an ivermectin-insensitive subunit, Hco-GLC-2, had no effect on drug sensitivity. Expression of a previously uncharacterised parasite-specific subunit, Hco-GLC-6, caused the transgenic worms to become ivermectin sensitive, suggesting that this subunit also encodes a GluCl that responds to the drug. These results demonstrate that both orthologous and paralogous subunits from C. elegans and H. contortus are able to rescue the ivermectin sensitivity of mutant C. elegans, though some quantitative differences were observed between transgenic lines in some assays. C. elegans is a suitable system for studying parasitic nematode genes that may be involved in drug resistance.

Dataset Information

Isolation of novel prototype galectins from the marine ball sponge Cinachyrella sp. guided by their modulatory activity on mammalian glutamate-gated ion channels.

Publications

Isolation of novel prototype galectins from the marine ball sponge Cinachyrella sp. guided by their modulatory activity on mammalian glutamate-gated ion channels.

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