1. Introduction

von Willebrand factor (VWF) is a plasma glycoprotein that is involved in platelet-dependent haemostasis (Sadler, 1998). VWF is primarily synthesized in vascular endothelial cells and megakaryo­cytes and is released into the plasma as ultralarge multimeric forms (UL-VWF) that are highly active in platelet adhesion and aggregation. A plasma metalloproteinase, ADAMTS13, specifically cleaves the Tyr1605-Met1606 peptidyl bond within the A2 domain of VWF (Dent et al., 1990). Cleavage of UL-VWF into smaller forms by ADAMTS13 limits platelet thrombus formation. A deficiency of ADAMTS13 enzymatic activity caused by either genetic mutations in the ADAMTS13 gene or acquired autoantibodies against ADAMTS13 results in the accumulation of UL-VWF in plasma. This leads to the formation of disseminated platelet-rich microthrombi in arterioles, which is one of the characteristic pathogenic features of thrombotic thrombocytopaenic purpura (TTP), a life-threatening systemic disease (Tsai, 2009). Conversely, excessive cleavage of VWF causes von Willebrand disease type 2A (Sadler, 2005).

Human ADAMTS13 consists of 1427 amino acids and has a modular structure comprising a signal peptide, a short propeptide, a metalloproteinase domain (M), a disintegrin-like domain (D), a thrombospondin-1 type 1 repeat (T1), a Cys-rich domain (C), a spacer domain (S), seven additional type 1 repeats (T2–T8) and two CUB (C1r/C1s, urinary epidermal growth factor, bone morphogenic protein) domains (Levy et al., 2001; Soejima et al., 2001; Zheng et al., 2001). C-terminal truncation of ADAMTS13 after the C but not the S domain results in severe loss of proteolytic activity towards VWF (Soejima et al., 2003; Zheng et al., 2003). Therefore, in addition to the M domain, the ancillary domains including the D, T, C and S domains (DTCS) are necessary for normal ADAMTS13 activity, although the distal C-terminal domains are required for regulation of in vivo thrombus formation under high-shear conditions (Banno et al., 2009). We have previously reported a minimal functional substrate con­sisting of 73 amino-acid residues of the C-terminal region of the VWF A2 domain (Asp1596–Arg1668) and designated VWF73 (Kokame et al., 2004; Miyata et al., 2007). A recent study has shown that the VWF-binding exosites located in the T, C and S domains interact with different segments of VWF73 (Gao et al., 2008). These interactions increased the VWF-binding affinity and rate of substrate cleavage by 300-fold. At least 16 causative missense mutations for congenital TTP and five missense polymorphisms in the ADAMTS13 gene have been identified within the DTCS region (Levy et al., 2001; Kokame et al., 2002; Banno & Miyata, 2008). Although most TTP-causative mutant proteins are likely to show secretion deficiency, a P475S polymorphism variant showed normal secretion but reduced VWF-cleaving activity (Kokame et al., 2002; Akiyama et al., 2008). Detailed structural information on exosite-containing domains will help in understanding the structure-based mechanism of substrate recognition and specificity and the effects of TTP-causative mutations and common polymorphisms. To date, crystal structures of the M and D domains of three human ADAMTS-family proteins, ADAMTS1, ADAMTS4 and ADAMTS5, have been reported (Gerhardt et al., 2007; Mosyak et al., 2008; Shieh et al., 2008). However, no crystal structures of exosite-containing fragments of ADAMTSs have been reported.

Here, we report the expression and purification of the exosite-containing human ADAMTS13-DTCS fragment using mammalian CHO Lec cells with mutations in multiple glycosylation-related genes. Proteins obtained from this cell line are suitable for crystallization because their restricted and homogeneous glycosylation improves the packing of the protein molecules. We also report the results of our crystallization and preliminary X-ray studies of ADAMTS13-DTCS.

2. Methods

2.1. Expression and purification of ADAMTS13-DTCS

An ADAMTS13 cDNA (AB069698) fragment corresponding to amino-acid residues 287–685 (ADAMTS13-DTCS) was amplified by PCR and cloned into a mammalian expression vector based on pcDNA3.1/Myc-His (Invitrogen), which has a mouse Nid1 signal sequence (Yasui et al., 2007). The nucleotide sequence was confirmed by dye-terminator sequencing. The ADAMTS13-DTCS fragment expressed from this vector contains a tobacco etch virus (TEV) proteinase cleavage site (Glu-Asn-Leu-Tyr-Phe-Gln/Gly) followed by tandem His-tag sequences at the C-terminus. We transfected the plasmid into CHO Lec 3.2.8.1 cells (Stanley, 1989) by electroporation and selected colonies resistant to G418 (3 mg ml⁻¹) on 96-well plates in α-minimal essential medium supplemented with 5% foetal bovine serum for 10 d. ADAMTS13-DTCS levels in the media of 48 G418-resistant colonies were examined by Western blotting with anti-6×His antibody (Sigma–Aldrich, St Louis, Missouri, USA). The clone with the highest secretion level of ADAMTS13-DTCS was cultured in the medium containing 0.5 mg ml⁻¹ G418 by the roller-bottle method and the medium was collected every 3 d. The ADAMTS13-DTCS was recovered from the culture medium by 50%(w/v) ammonium sulfate precipitation and was purified by Ni–­NTA agarose chromatography (Sigma–Aldrich). The eluted ADAMTS13-DTCS was incubated with TEV proteinase for 12 h at 297 K to remove the C-­terminal tags. After dialysis in a buffer consisting of 10 mM MES and 100 mM NaCl pH 6.0, the digest was applied onto a Hi-Trap SP HP cation-exchange column (GE Healthcare, Buckinghamshire, England). The column was washed with the same buffer and ADAMTS13-DTCS was eluted with a linear gradient of NaCl (0.1–0.7 M) in 10 mM MES pH 6.0. Fractions were analyzed by SDS–PAGE under reducing conditions (Fig. 1). The fractions rich in ADAMTS13-DTCS (lanes 5 and 6) were combined, dialyzed against 10 mM MES pH 6.0 and concentrated using a Vivaspin-5 separation device (30 kDa molecular-weight cutoff; Sartorius, Edgewood, New York, USA) to a final concentration of ∼10 mg ml⁻¹ for crystallization.

Figure 1 SDS–PAGE analysis of ADAMTS13-DTCS fractions from Hi-Trap SP HP cation chromatography. Proteins were analyzed by SDS–PAGE and stained with Coomassie Brilliant Blue. Lanes 1 and 3, molecular-weight markers (kDa); lane 2, pooled Ni–NTA eluate treated with TEV proteinase; lanes 4–7, eluate fractions Nos. 5–8, respectively, from the Hi-Trap SP HP column. Arrow, ADAMTS13-DTCS. Asterisk, TEV proteinase.

3. Results and discussion

3.2. Crystallization

Of the 288 initial crystallization conditions tested, 20 yielded microcrystals (Fig. 2a). Using solution No. 4 of the PEG/Ion Screen kit [0.2 M lithium chloride, 20%(w/v) PEG 3350 pH 6.8] as a starting condition, the pH of the mother liquor, the concentration and molecular weight of the PEG and the species and concentrations of salts and additives were optimized. The combination of refinement of the crystallization conditions and an increase in the protein concentration (to ∼20 mg ml⁻¹) improved the size of the crystals. Single crystals were obtained from drops made up of 0.5 µl protein solution and 0.5 µl reservoir solution [26%(w/v) PEG 1500, 100 mM MES pH 6.0] supplemented with a one-fifth volume of 40%(w/w) penta­erythritol ethoxylate (3/4 EO/OH; Additive Screen solution No. 52; Hampton Research). Crystals with dimensions of 300 × 100 × 50 µm were formed after 3–7 d at 293 K (Fig. 2b).

Figure 2 Crystals of human ADAMTS13-DTCS grown by the sitting-drop method. (a) Microcrystals obtained from solution No. 4 of the PEG/Ion Screen kit. (b) Crystals obtained using the optimized conditions. The scale bars indicate 0.1 mm.

3.3. X-ray analysis

All diffraction data sets were acquired using the oscillation method on beamline BL41XU at a wavelength of 1.0 Å. The oscillation angle was 1.0° for all data sets. The native data sets for form 1 and form 2 contained 16 867 (2.60 Å resolution) and 12 811 (2.80 Å resolution) unique reflections, respectively. The asymmetric unit was estimated to contain one molecule, with corresponding crystal volume per protein weights of 2.7 and 3.1 ų Da⁻¹ for crystal forms 1 and 2, respectively. Solvent-content estimations based on a single copy of the molecule per asymmetric unit gave values of 53.4% and 48.6% for crystal forms 1 and 2, respectively. The X-ray data showed that the two crystal forms have different unit-cell parameters even when they are obtained under identical conditions. The variation in crystal packing might reflect the mobility of the domains in ADAMTS13-DTCS. The statistics of the data sets are summarized in Table 1.

Table 1 Data-collection statistics for ADAMTS13-DTCS crystals Values in parentheses are for the highest resolution shell. A single crystal was used for measurement for each data set.   Form 1 Form 2 Space group C2 C2 Unit-cell parameters      a (Å) 152.7 138.6  b (Å) 52.9 51.4  c (Å) 76.2 76.4  β (°) 111.4 106.7 Wavelength (Å) 1.0 1.0 Resolution (Å) 50–2.60 (2.69–2.60) 30–2.80 (2.90–2.80) No. of unique reflections 16867 (1272) 12811 (1259) Rmerge† 0.052 (0.176) 0.062 (0.403) I/σ(I) 19.3 (5.7) 13.3 (3.5) Completeness (%) 95.3 (72.7) 99.5 (99.3) Redundancy 3.5 (2.9) 3.7 (3.6) Matthews value (Å3 Da−1) 2.64 2.39 Solvent content (%) 53.4 48.6 †Rmerge = , where Ii(hkl) is the ith intensity measurement of reflection hkl and 〈I(hkl)〉 is its weighted average.