1. Introduction

Variable lymphocyte receptors (VLRs) recognize foreign antigens to achieve adaptive immunity in jawless vertebrates, in contrast to the immunoglobulin (Ig) type antigen receptors of jawed vertebrates (Pancer et al., 2004; Alder et al., 2005). In lamprey and hagfish, the only two surviving jawless vertebrates, VLRs have been isolated by repeated injections with a cocktail of particulate antigens and mitogens (Pancer et al., 2004, 2005). VLRs recognize antigens using leucine-rich repeat (LRR) modules and a highly variable insert (HVI) composing the ectodomain of the functional receptor of VLRs (Han et al., 2008; Velikovsky et al., 2009; Kim et al., 2007). For diverse antigen recognition by VLRs, various numbers of LRR modules are generated by recombinatorial DNA assembly into the vlr loci, which differs from the analogous process for antibody V, D and J gene segments (Alder et al., 2005; Pancer et al., 2004; Tasumi et al., 2009).

VLRs further enhance antigen-binding specificity through an HVI in the C-terminal leucine-rich repeat (LRRCT), which adopts various secondary structures and conformations (Han et al., 2008; Velikovsky et al., 2009). The HVI amino-acid sequence and length are important for antigen-specific interaction by VLRs (Han et al., 2008; Deng et al., 2010; Velikovsky et al., 2009). The amino-acid sequence of the HVI is highly diverse among all known VLRs. It is therefore a rare occurrence for the HVI of VLR2913 to be identical to that of VLR RBC36, which recognizes the H-trisaccharide from human blood type O erythrocytes (Han et al., 2008). Owing to the rare identity of the HVIs between VLR2913 and VLR RBC36, H-trisaccharide or a similar glycan are potential antigen candidates for VLR2913, the specific antigen of which remains undetermined.

We are currently attempting to discover the antigen for VLR2913 and characterize the modes of VLR-antigen recognition through hypothetical interactions between VLR2913 and H-trisaccharide. To elucidate the crucial factor for antigen recognition by VLRs and to obtain a high level of soluble VLR2913, we designed key-residue mutant forms of VLR2913 fused with internalin B (InlB-VLR2913-ECD), which displays high thermodynamic and pH stabilities (Han et al., 2008; Lee et al., 2012). VLR RBC36 recognizes H-trisaccharide via hydrogen bonds to three key residues and van der Waals interactions with several residues on the concave surface (Han et al., 2008). Since hydrogen bonds would contribute to antigen recognition more than van der Waals interactions, we mutated the three key residues on the concave surface of the LRR modules of InlB-VLR2913-ECD to mimic hydrogen bonds based on the sequence alignment and the crystal structure of VLR RBC36 in complex with H-trisaccharide (Han et al., 2008). We overexpressed the soluble InlB-VLR2913-ECD with three mutations (A70D, N118D and D119Q) in the LRR modules using an Escherichia coli expression system. In this report, we describe the cloning, overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of InlB-VLR2913-ECD.

2. Materials and methods

2.3. Crystallization and X-ray data collection

InlB-VLR2913-ECD was crystallized at 295 K using the sitting-drop vapour-diffusion method by mixing equal volumes (1.5 µl each) of protein solution and reservoir solution. Crystals with approximate dimensions of 100 × 100 × 150 µm (Fig. 1) appeared after one week using a reservoir solution consisting of 0.17 M ammonium sulfate, 25.5%(w/v) PEG 4000, 15%(v/v) glycerol (Wizard III condition No. 30; Emerald BioSystems, Bainbridge Island, Washington, USA). No further cryoprotectant was required for cryo-cooling in liquid nitrogen. X-ray diffraction data were collected at 100 K using a MicroMax-007 HF microfocus X-ray generator and an R-AXIS IV⁺⁺ imaging-plate area detector (Rigaku, Tokyo, Japan) at the Korea Basic Science Institute (KBSI), Republic of Korea. The statistics of data collection are summarized in Table 1. For each image, the crystal was rotated by 1° and the raw data were processed using the HKL-2000 program suite (Otwinowski & Minor, 1997).

Table 1 Data-collection statistics for InlB-VLR2913-ECD Values in parentheses are for the highest resolution shell. X-ray wavelength (Å) 1.54178 Temperature (K) 100 Space group P41212 or P43212 Unit-cell parameters (Å) a = 91.12, b = 91.12, c = 62.87 Resolution range (Å) 50-2.04 (2.08-2.04) No. of total/unique reflections 132286/17451 Rmerge# (%) 5.2 (32.1) Data completeness (%) 99.9 (100) Multiplicity 7.6 (7.3) Average I/(I) 44.6 (8.2) #Rmerge = , where Ii(hkl) is the intensity of the ith measurement of reflection hkl and <I(hkl)> is the average intensity of reflection hkl.

Figure 1 Crystal of InlB-VLR2913-ECD. The dimensions of the crystal are approximately 100 × 100 × 150 µm.

3. Results and discussion

VLR2913-ECD was constructed as a fusion protein with internalin B (InlB-VLR2913-ECD) to facilitate its structural characterization. The protein was successfully overexpressed using an E. coli system and was purified to 99% homogeneity by affinity chromatography, size-exclusion chromatography and anion-exchange chromatography. InlB-VLR2913-ECD crystals were obtained using the sitting-drop vapour-diffusion method at 295 K with reservoir solution consisting of 0.17 M ammonium sulfate, 25.5%(w/v) PEG 4000, 15%(v/v) glycerol (Fig. 1). X-ray diffraction data were collected to 2.04 Å resolution (Fig. 2) and indexed in a tetragonal space group. A total of 132 286 measured reflections were merged into 17 451 unique reflections, giving an R_merge of 5.2% and a completeness of 99.9%. The space group was determined to be P4₁2₁2 (or P4₃2₁2) on the basis of systematic absences and symmetry. The unit-cell parameters were a = 91.12, b = 91.12, c = 62.87 Å (Table 1). If one monomer molecule is assumed to be present in the crystallographic asymmetric unit, the calculated Matthews coefficient (V_M) is 2.75 Å³ Da^-1 and the solvent content is 55.2%. We are currently attempting to solve the structure of InlB-VLR2913-ECD using molecular replacement. The structural study of InlB-VLR2913-ECD is expected to provide structural insight to elucidate the different modes of antigen recognition by VLRs.

Figure 2 An X-ray diffraction image from an InlB-VLR2913-ECD crystal. The edge of the detector corresponds to a resolution of 2.04 Å and is represented as a circle. The crystal-to-detector distance was 150 mm and the exposure time for data collection was 3 min per image.