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.

Fig. 1.

The sponge crude extract slows desensitization of AMPA and kainate receptors. Representative whole-cell currents evoked by glutamate (10 mM) before and after treatment with the Cinachyrella extract (25 μg/mL) for several minutes. (A) Quantitation of the mean tau of exponential fits to the current decay before and after brief applications of sponge extract for either 10 s (GluA4 (flip variant)) or 5 s (GluK2a). N ≥ 3; *, P < 0.05; **, P < 0.01 in paired t-tests. (B) The effects on desensitization are long-lasting. Currents evoked by glutamate from GluA4(i) AMPA receptors are shown before (1), during (2) and 20 minutes after a 1 minute application (3) of extract. An example of the percent desensitization during 100 ms glutamate applications over the course of representative experiment is shown.

Fig. 2.

Electrophoretic and chromatographic behaviors of CchGs. SDS–PAGE for CchGs with both heat treatment and mercaptoethanol (line 2), heat treatment but without mercaptoethanol (line 3), no heat treatment without mercaptoethanol (line 4). Standard protein bands (line 1) (A). Protein HPLC chart (B). Gel filtration chromatography analysis (C), and molecular size calibration for standard proteins. Molecular size estimated from the Kav value for CchG is indicated by arrow (D).

Fig. 3.

Hemagglutinating titers for: CchGs and CchGs dialyzed against EDTA (A). CchGs treated with buffers of various pH values (B). CchGs incubated at various temperatures (C) and incubated in boiling water for 30–180 min (D). For all experiments, n = 3, Bars ± SD.

Fig. 4.

Purified CchGs potentiate currents from GluA4 AMPA and GluK2a kainate receptors expressed in HEK293-T/17 cells. Glutamate (10 mM) was fast-applied to evoke currents before and after a 5 min application of CchGs (10 μg/mL). The graphs show weighted mean tau values derived from two-component fits of the current decays; that currents were slowed by 4–8-fold (**P < 0.01 compared with currents before CchGs application).

Fig. 5.

SDS–PAGE for CchG-1 and -2 separated by preparative SDS–PAGE (A), MALDI-TOF-MS of CchGs inset is an expansion of the molecular ion (B), and those of CchG-1 (upper) and 2 (lower) separated by preparative electrophoresis (C).

Fig. 6.

Multiple alignment of amino acid sequences for CchG isolectins and other represented animal galectins. Amino acid sequences for: CchGs deduced from the cDNA sequences (CchGa∼f); determined by Edman degradation (CchG-1 and -2); or X-ray crystallography (CchG crystal), along with other related galectins: Congerin 1 (Conger eel galectins); and human galectin 1 (hGal 1); GCG (51–191 residue, GCG) are aligned. Putative carbohydrate-binding residues are shown by asterisks. Light shadow indicates amino acid residues partly conserved among the proteins. The darker shadow indicates an amino acid residue conserved throughout entire peptides. The alignment of sequences was generated using a software Align X (Life Technologies, Grand Island, NY).

Fig. 7.

SDS–PAGE for standard protein (line 1), CchGs (line 2) and rCchGa (line 3) (A), molecular size calibration for standard proteins and rCchGa in size exclusion chromatography. Molecular size estimated from the Kav value for rCchGa is indicated by an arrow (B).

Fig. 8.

rCchGa potentiates currents from GluK2a kainate receptors expressed in HEK293-T/17 cells. Glutamate (10 mM) was fast-applied to evoke currents before and after a 5-min application of rCchGa (1 μg/mL) in the absence or presence of lactose (20 mM). The graph shows weighted mean tau values derived from two-component fits of 1 second current decays (***P < 0.001 compared with currents before rCchGa application).

Fig. 9.

Rooted phylogenetic tree with branch length generated by ClustalW (online) for CchGa-f, N-terminal forty residues of Cchg-1 and -2, a sequence from X-ray crystal structure, truncated sponge galectin GCG (51–191 residues), congerin I and human galectin 1.