Karakostis K et al. (2015), Heterologous expression of newly identified gal...

ECB-ART-44377

Sci Rep 2015 Dec 07;5:17665. doi: 10.1038/srep17665.

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Heterologous expression of newly identified galectin-8 from sea urchin embryos produces recombinant protein with lactose binding specificity and anti-adhesive activity.

Karakostis K , Karakostis K , Costa C , Zito F , Matranga V .

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Galectin family members specifically bind beta-galactoside derivatives and are involved in different cellular events, including cell communication, signalling, apoptosis, and immune responses. Here, we report a tandem-repeat type galectin from the Paracentrotus lividus sea urchin embryo, referred to as Pl-GAL-8. The 933nt sequence encodes a protein of 34.73 kDa, containing the conserved HFNPRF and WGxExR motifs in the two highly similar carbohydrate-recognition domains (CRD). The three-dimensional protein structure model of the N-CRD confirms the high evolutionary conservation of carbohydrate binding sites. The temporal gene expression is regulated during development and transcripts localize at the tip of the archenteron at gastrula stage, in a subset of the secondary mesenchyme cells that differentiate into blastocoelar (immune) cells. Functional studies using a recombinant Pl-GAL-8 expressed in bacteria demonstrate its hemo-agglutinating activity on human red blood cells through the binding to lactose, as well as its ability in inhibiting the adhesion of human Hep-G2 cells to the substrate. The recent implications in autoimmune diseases and inflammatory disorders make Gal-8 an attractive candidate for therapeutic purposes. Our results offer a solid basis for addressing the use of the new Pl-GAL-8 in functional and applicative studies, respectively in the developmental and biomedical fields.

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Species referenced: Echinodermata
Genes referenced: LOC100887844 LOC115919910 LOC583082 LOC594261 SpZ12-1

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	Figure 1. Sequence analysis of Paracentrotus lividus Galectin-8.(A) Graphic representation of Pl-GAL-8 protein structure composed of a 23 aa-long N-terminal region and two tandem repeat CRD domains (24–158 and 178–311), joined by a linker region (159–177). The positions of putative serine, threonine and tyrosine residues are indicated. (B) Alignment of the Pl-GAL-8 deduced sequence with homologs from: Sp, Strongylocentrotus purpuratus (XP_781871); Bf, Branchiostoma floridae (XP_002610290.1); Sk, Saccoglossus kowalevskii (XP_002731587); Gg, Gallus gallus (NP_001010843.1); Rn, Rattus norvegicus (AAH72488); Hs, Homo sapiens (AAF19370.1); Ci, Ciona intestinalis (XP_002126450.1). Numbers after the species acronyms refer to the size in amino acids of the deduced proteins. Identical amino acids are shaded in black, conservative amino acids substitutions are shaded in grey. Hyphenation indicates: (−) gaps inserted for maximizing the match, (*) Cysteine residues; (#) HFNPRF conserved motifs, (+) WGxExR conserved motifs; (≫≫) glycosylation motif. (C) Phylogenetic relationships among members of the galectin-8 family. Rooted phylogenetic tree was derived from (B). Branch lengths are proportional to the evolutionary distance showing the divergence among different species. The scale bar indicates an evolutionary distance of 0.1 aa substitutions per position in the sequence.
	Figure 2. Three dimensional structure model of Pl-GAL-8 N-terminal domain.Ribbon model of Pl-GAL-8 N-terminal domain obtained by homology modelling using as template the X-Ray structure of the human Gal-8 N-terminal domain (pdb file: 2yv8;). The indicated amino acid residues are carbohydrates-binding sites known23 to interact with lactose via hydrogen bonding (R50, H70, N72, R74, N83, W90 and E93) or water-mediated bonds (H52 and R64) and with the galactose ring by van der Waals interactions (W90). N-ter, N-terminal; C-ter, C-terminal.
	Figure 3. Temporal and spatial expression of Pl-gal-8 during P. lividus embryo development.(A) Temporal expression monitored by comparative qPCR analysis, of the Pl-Gal-8 transcription levels in sea urchin embryos at different developmental stages: Cl, cleavage; B, blastula; G, gastrula; Pl, pluteus. The Pl-z12-1 mRNA was used as an internal endogenous reference gene49; cDNA from cleavage stage was used as the reference sample and was set as 1 in arbitrary units. The y-axis represents the relative quantity values (RQ or ΔΔCt value, see Methods). (B) Spatial expression profile studied by whole mount in situ hybridization, using a DIG-antisense Pl-gal-8 RNA probe on embryos fixed at different developmental stages: (a), blastula; (b), mesenchyme blastula stage; (c), (d), middle gastrula; (e), prism; (f), pluteus. Captions: bl, blastocoel cavity; vp, vegetal plate; ar, archenteron; f, foregut; m, midgut; h, hindgut. Arrows in (c,d) point to the secondary mesenchyme cells; asterisks in (e) to the gut sphincters.
	Figure 4. Purification of recombinant Pl-GAL-8 protein.(A) Schematic representation of the recombinant Pl-GAL-8 fusion protein of 799 aa, including a N-terminal His-tag, the trigger factor chaperon protein, a 45 aa spacer and the Pl-GAL-8 protein. Numbers on top of the bar indicate amino acids of the construct. The calculated sizes of the protein segments expressed in kDa are indicated underneath. (B) SDS-PAGE (7.5%) of washes and eluted fractions from the Ni-nitrilotriacetate affinity chromatography of extracts containing the fusion protein from E. coli transformed by the recombinant pcold-TF-Pl-gal-8 plasmid. Molecular weight markers are indicated on the left. In the separated lane on the right, the purified fusion protein obtained; molecular weight markers on the right.
	Figure 5. Carbohydrates-binding activity of recombinant Pl-GAL-8.Hemoagglutination assays on human red blood cells run in duplicates in U-shaped wells. (A) Inhibition of hemo-agglutination was tested by the addition of increasing micromolar (µM) concentrations of: recombinant Pl-GAL-8; BSA, serum bovine albumin; TF, trigger factor; 100 °C, 1 μM of recombinant Pl-GAL-8 denatured at 100 °C for 5 min before testing (negative control). (B) Interaction of mono- or di-sacharide sugars with recombinant Pl-GAL-8 (0.25 µM) at the indicated millimolar concentrations (mM). Captions: lact, lactose; galact, galactose; mann, mannose. Wells showing spread RBC exhibit agglutination activity, wells with small red dots lack agglutination ability. Results shown in (A,B) describe a representative experiment; (C,D) report the mean of 3 replicates from 3 different experiments, +/− SD, expressed in arbitrary units.
	Figure 6. Inhibition of HepG2 cells adhesion to culture plates by soluble Pl-GAL-8.Cells were incubated at 37 °C for 3 h in the presence of increasing micromolar concentrations of soluble recombinant human Galectin-8 (rHs-GAL-8, red line) or recombinant P. lividus-GAL-8 (rPl-GAL-8, blue line). The percentage of cells that remained bound to the wells bottom was determined by measuring the intensity of auto fluorescence developed after several PBS washings and 0.5% glutaraldehyde fixation. Measurements of fluorescence intensities obtained in arbitrary units, are expressed as percentage of adherent cells. Values are presented as the mean of three replicates from a representative experiment and ± SD, standard deviation.

References [+] :

Annunziata, Pattern and process during sea urchin gut morphogenesis: the regulatory landscape. 2014, Pubmed, Echinobase