Photon Factory Activity Report 2014 #32 (2015) B
BL-5A, AR-NW12A, AR-NE3A/2014G537

Crystal structure of a Xenopus laevis skin prototype galectin
Yasuhiro Nonaka1, Takanori Nakamura1, Hiromi Yoshida2 and Shigehiro Kamitori2,*1
Department of Endocrinology, Faculty of Medicine, and 2Life Science Research Center,Kagawa University 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan
1 Introduction
Galectins, a -galactoside-specific soluble lectin family,
are widely distributed in metazoans and fungi. Thegalectin family members show a variety of expressionpatterns, ligand specificities and biological functions.
Mammalian galectin-1, one of the most studied galectins,
is expressed in many tissues and cells, and is involved inmany functions such as induction of cell death, celladhesion and differentiation. Xenopus laevis (Africanclawed frog) has two types of galectins that are similar to
mammalian galectin-1 in amino acid sequence. One type,comprising xgalectin-Ia and -Ib, is regarded as being
equivalent to galectin-1, and the other type, comprisingxgalectin-Va and -Vb, is expected to be a unique galectinsubgroup. The latter is considerably abundant in frog
skin; however, its biological function remains unclear. We
determined and reported the crystal structures ofxgalectin-Ib and -Va [1]. The structures showed that both
galectins formed a mammalian galectin-1-like homodimer,and furthermore, xgalectin-Va formed a homotetramer.
This tetramer structure has not been reported for othergalectins. To elucidate intermolecular interactions in atetramer, more precisely, the structure of xgalectin-Va at ahigher resolution has been determined.

the symmetric association of two dimers (Mol-A/B andMol-C/D) (Fig. 1B). The dimer-dimer interface mainly
involved the F2 and F3 strands, and the S6–F3 and F4–F5
loops. Four carbohydrate-binding sites are located on thesurface of a tetramer. Thus, the tetramer formation withfour carbohydrate-binding sites may be related to thefunction of xgalectin-Va. In the newly determined
structure at 1.36 Å resolution, some amino acid residues
in the dimer-dimer interface were found to adopt
alternative conformations of the side chain, which may
contribute to the intermolecular interactions in a tetramer.

2 Experiment
The recombinant xgalectin-Va was expressed in E. coli
BL21 cells, and purified by affinity chromatography (aglutathione-Sepharose 4B column and a lactose–agarosecolumn). Large single crystals of xgalectin-Va wereobtained with a protein solution (5.0 mg/ml in 20 mM
Tris–HCl (pH 7.2), 50 mM NaCl, 1–2 mM DTT) and areservoir solution (2.4 M sodium malonate (pH 5.0)) by
the sitting drop method at 293 K. Data collection wasdone on PF-AR NW12A (KEK, Japan) using an ADSC
Quantum CCD detector at a wavelength of 1.0 Å. The
structure was solved by an isomorphous replacement
method using the previously reported structure at 1.60 Åresolution (3WUC), and was refined to R-factor of 0.196(Rfree = 0.216), using 1.36 Å resolution data.
3 Results and Discussion
Structure of xgalectin-Va comprised a -sandwich
composed of six-stranded (S1–S6) and five-stranded (F1–F5) -sheets, which is typical of the galectins so far
reported. A lactose molecule, which was introduced
during the protein purification, was found in a
carbohydrate-binding site. Two molecules of xgalectin-Va
formed a homodimer, in which two intermolecular sheets were formed by the F1 and S1 strands, as found ingalectin-1 (Fig. 1A). The tetramer was formed throughFig. 1: Crystal structure of xgalectin-Va. (A) Dimer
structure formed by Mol-A/B is shown with the boundlactose molecules. (B) Tetramer structure is shown.
Acknowledgement
We thank the PF staff for support of data collection.
References[1] Y. Nonaka et al., Glycobiology 25, 792 (2015).
* kamitori@med.kagawa-u.ac.jp