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Molecular cloning, in vitro expression and functional characterization of canine ADAMTS13
Research in Veterinary Science 93 (2012) 213–218
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Molecular cloning, in vitro expression and functional characterization of canine ADAMTS13 H. Maruyama a,⇑, K. Ito a, K. Okabayashi b, M. Sakai c, R. Kano a, T. Watari d, A. Hasegawa a, H. Kamata a a
Laboratory of Veterinary Pathobiology, Department of Veterinary Medicine, Nihon University, Japan Laboratory of Veterinary Biochemistry, Department of Veterinary Medicine, Nihon University, Japan c Laboratory of Veterinary Internal Medicine, Department of Veterinary Medicine, Nihon University, Japan d Laboratory of Comprehensive Veterinary Clinical Studies, Department of Veterinary Medicine, Nihon University, Japan b
a r t i c l e
i n f o
Article history: Received 13 January 2011 Accepted 8 June 2011
Keywords: ADAMTS13 von Willebrand factor Thrombotic thrombocytopenic purpura Dog Hemostasis Cloning
a b s t r a c t A disintegrin and metalloproteinase with thrombospondin type 1 motifs, number 13 (ADAMTS13) is a plasma zinc metalloprotease also known as von Willebrand factor (VWF)-cleaving protease. Deficiency of ADAMTS13 activity is known to cause thrombotic thrombocytopenic purpura (TTP) in humans. We isolated the canine ADAMTS13 cDNA, which encodes 1502 amino acids, and expressed the recombinant protein to evaluate VWF-cleaving ability. Although the propeptide domain was longer and the TSP1 repeat domain was shorter than those in other species, the overall structures were similar to human and mouse ADAMTS13. Recombinant canine ADAMTS13 cleaved the 250-kDa VWF monomer into two fragments of 150 kDa and 120 kDa. Furthermore, high molecular weight VWF multimers were abolished based on the activity of ADAMTS13. These results could facilitate research into hemostatic disorders such as TTP in dogs. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction As a large multimeric plasma glycoprotein, von Willebrand factor (VWF) mediates the adhesion of platelets to the subendothelium of damaged vessels, and also plays a role in the formation of platelet thrombus (Brooks, 2000). VWF is mainly synthesized in the endothelium and secreted into blood as unusually large VWF multimer (UL-VWFM), with a molecular weight greater than 20,000 kDa (Moake et al., 1982). This secreted UL-VWFM is quickly converted to a smaller form, ranging in molecular weight from approximately 500 to 20,000 kDa. High molecular weight VWF multimers are more active in promoting platelet adhesion and aggregation (Arya et al., 2002; Federici et al., 1989; Moake et al., 1986). UL-VWFM remaining in the circulation has thus been reported to cause thrombosis in humans (Moake et al., 1982). A disintegrin and metalloproteinase with thrombospondin type 1 motifs, number 13 (ADAMTS13), is a plasma zinc metalloprotease also known as VWF-cleaving protease (Fujikawa et al., 2001; Gerritsen et al., 2001; Soejima et al., 2001). ADAMTS13 in humans, mice and rats is mainly produced by hepatic stellate cells and released into the circulation (Kume et al., 2007; Uemura et al., ⇑ Corresponding author. Address: Laboratory of Veterinary Pathobiology, Department of Veterinary Medicine, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan. Tel./fax: +81 466 84 3519. E-mail address: [email protected] (H. Maruyama). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.06.006
2005; Zhou et al., 2005). Human ADAMTS13 cleaves a peptide bond between Tyr1605 and Met1606 in the VWF A2 domain, and the VWF monomer is truncated into two fragments. UL-VWFMs are thus converted to a smaller form with appropriate platelet adhesion and aggregation activity (Plaimauer et al., 2002). Human and mouse ADAMTS13 amino acids comprise several domains: a signal peptide; a propeptide; a metalloprotease domain; a disintegrinlike domain; a thrombospondin type 1 (TSP1) motif; a cysteinerich domain; a spacer domain; seven TSP1 repeats; and two C1r/ C1s, urinary epidermal growth factor, bone morphogenic protein (CUB) domains (Bruno et al., 2005; Soejima et al., 2001; Zheng et al., 2001). Thrombotic thrombocytopenic purpura (TTP) refers to a disorder in which widespread platelet- and VWF-rich microthrombi are observed in small blood vessels of multiple organs, with clinical signs such as anemia, thrombocytopenia, renal failure, fever and neurological dysfunction (Asada et al., 1985; Tsai, 2010). TTP is recognized as a life-threatening disease if appropriate treatments, such as plasma exchange or infusion, are not provided (Rock et al., 1991). In human medicine, a key cause of TTP is the accumulation of UL-VWFM by severe reductions in ADAMTS13 activity in circulating blood (Furlan et al., 1997; Levy et al., 2001). TTP can be divided into hereditary and acquired forms. The hereditary form of TTP is known as Upshaw-Schulman syndrome, resulting from a congenital deficiency of ADAMTS13 due to an ADAMTS13 gene mutation (Levy et al., 2001), whereas the acquired form is mainly
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caused by circulating autoantibodies that neutralize ADAMTS13 activity (Rieger et al., 2005). Clinical manifestations similar to TTP seem to be observed in dogs with immune-mediated hemolytic anemia (IMHA), particularly accompanied by thrombocytopenia such as disseminated intravascular coagulation (DIC) or Evan’s syndrome. Whole blood transfusion is performed as one treatment for IMHA, especially in dogs with both severe anemia and thrombocytopenia (Day, 2010). However, transfusion of blood products including platelets is contraindicated in the treatment of human TTP, because infused platelets promote formation of further thrombi and then accelerate ischemic organ failure due to these platelet-rich thrombi (Harkness et al., 1981). In veterinary medicine, insufficient information is currently available regarding canine ADAMTS13 and diagnostic methods for TTP, so many dogs may die without appropriate treatment. To facilitate prospective studies into canine coagulation disorders such as TTP, the present study performed molecular cloning of the canine ADAMTS13 cDNA. Furthermore, recombinant canine ADAMTS13 (rcADAMTS13) was expressed and its ability to cleave VWF was confirmed by detecting changes in both fragments and the multimetric structure of VWF. 2. Materials and methods 2.1. RNA isolation and cDNA preparation Total RNA was isolated from a normal liver specimen obtained from a healthy beagle dog using an RNeasy mini kit (Qiagen, Hilden, Germany), then treated with DNase Turbo (Ambion, TX, USA) to eliminate contaminant genomic DNA. Five micrograms of total RNA was reverse-transcribed into cDNA using SuperScript III (Invitrogen, CA, USA) according to the instructions from the manufacturer. 2.2. Cloning of canine ADAMTS13 cDNA Primer pairs to amplify canine partial ADAMTS13 cDNA were designed based on conserved nucleotide sequences between human (accession No. NM_139025) and mouse (accession No. NM_001001322). Polymerase chain reaction (PCR) was performed using primer pairs (50 -CAGGCTCACCAGGAGGACAC-30 and 50 GACTCCCAGTAGAGCACCTGCTGC-30 ) and the cDNA of canine liver with PrimerSTAR GXL DNA polymerase (Takara Bio, Shiga, Japan) according to the instructions from the manufacturer. The PCR product was cloned into a pMD20-T vector using a Mighty TA-cloning Reagent Set for PrimeSTAR (Takara Bio). Competent INVaF’ cells (Invitrogen) were transformed using the vector with PCR products inserted. Plasmid DNA was extracted from the bacteria cultured in Luria–Bertani (LB) broth using a Quantum prep kit (Bio-Rad, CA, USA). Plasmid DNA was sequenced using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, CA, USA) and SequenceRx Enhancer Solution A (Invitrogen) in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). For further cloning of the more 50 -region, including ATG as the start-codon, primers were designed based on relatively conserved nucleotide sequences between human and mouse ADAMTS13 nucleotide sequences and the canine sequence obtained in this study. Sequences of these primers were 50 -AGGAAGCTCCCAAGAG TAAACAC-30 and 50 -CAGATGCACTTGGAACTGAGC-30 . For the 30 region, 30 -rapid amplification of cDNA ends (RACE) was performed using a 30 -RACE System for rapid amplification of cDNA Ends (Invitrogen) according the instructions from the manufacturer. Based on the nucleotide sequence of canine ADAMTS13 obtained in this study, the following primers were designed: 50 -GAGTGTGAC ATGCAGCTCTTCG-30 for the first PCR; and 50 -CCACTGATGGCACAG-
GAACC-30 for nested PCR. These PCR products were cloned into plasmid vector and sequenced as described above.
2.3. Construction of plasmid vector for transient expression rcADAMTS13 protein To construct the plasmid vector for transient expression of rcADAMTS13, the DNA fragment of the entire open reading frame of rcADAMTS13 was amplified by PCR using the cDNA from a canine normal liver and the primer pair of 50 -aggATGAGGGAGCCTCGCCCC TGGGGGAGATG-30 and 50 -GGGCTCAGCCCAGCCAGGTTCCTGCAC-30 . The small letters indicate the added Kozak sequence. PCR was performed with PrimerSTAR GXL DNA polymerase (Takara Bio). The amplicon was treated using a Mighty TA-cloning Reagent Set for PrimeSTAR (Takara Bio) for A-over hanging, then inserted into a pDNA 3.1/V5-His-TOPO plasmid vector (Invitrogen). The plasmid vector could express fusion protein with tag-proteins at the Cterminal, V5 epitope and 6 histidine. TOP 10 competent cells (Invitrogen) were transformed using the constructed plasmid vector. Plasmid vector was extracted from bacterial cultures grown in LB broth using NucleoBond Xtra Midi Plus EF (Macherey-Nagel, Duren, Germany). The sequence of the plasmid vector was confirmed as described above.
2.4. Transient expression of rcADMTS13 HeLa cells were purchased from Health Science Research Resources Bank (Osaka, Japan) and used for producing rcADAMTS13. The constructed plasmid vector was transfected into HeLa cells using FuGENE HD transfection reagent (Roche, Mannheim, Germany) according to the instructions from the manufacturer. Briefly, 3 106 HeLa cells were plated into each well of a 6-well plate on the day before transfection and cultured in Dulbecco’s modified Eagle’s medium (Sigma–Aldrich, MO, USA) supplemented with 10% fetal bovine serum. These cells were transfected with 2 lg of plasmid DNA and 5 ll of FuGENE HD in 100 ll of serumfree Opti-MEM (Invitrogen). No-template inserted pcDNA 3.1/V5His plasmid vectors were used as a negative control for expression. At 4 h after transfection, culture media were replaced with serumfree Opti-MEM and incubated for 44 h. Media were collected and centrifuged to remove residual cells. Supernatants of media were concentrated using Amicon Ultra-30 k (Millipore, MA, USA). The cells were harvested and washed twice with phosphate-buffered saline (PBS). Concentrated media and cells were then stored at 30 °C until Western blot and functional analyses. 2.5. Western blot of expressed rcADAMTS13 protein The concentrated media and cells were mixed with sodium dodecyl sulfate (SDS) sample buffer (58 mM Tris–Cl pH 6.8, 1.7% sodium dodecyl sulfate, 5% glycerol, 1.5% dithiothreitol, 0.02% bromophenol blue). These samples were electrophoresed on 6% SDS–polyacrylamide gel electrophoresis (PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). After blocking with 1% bovine serum albumin, the membrane was incubated with peroxidase-labeled anti-V5 antibody (1:5000; Invitrogen). The other transferred membrane was incubated with anti-human ADAMTS13 polyclonal antibody to recognize the metalloprotease domain of human ADAMTS13 (1:2500; Santa Cruz Biotechnology, CA, USA), then incubated with peroxidase-linked anti-rabbit immunoglobulin G antibody (1:5000; GE Healthcare, Buckinghamshire, England). Chemiluminescent detection was performed using an ECL Plus Western Blotting Detection System (GE Healthcare) by ChemiDoc XRS (Bio-Rad).
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2.6. Cleaving VWF by rcADAMTS13 protein and plasma
3.2. Deduced amino acid sequence of canine ADAMTS13
The activity of rcADAMTS13 to cleave VWF in concentrated medium was analyzed as previously described (Kokame et al., 2002) with slight modification. In addition, canine plasma was examined for VWF-cleaving activity. Briefly, the concentrated culture medium was serially diluted with 0.05 mM Tris–HCl (pH 7.4) containing 0.15 mM NaCl (TBS) as follows. Canine plasma obtained by mixing citrated plasma collected from five clinically healthy dogs was also serially diluted as the concentrated medium:TBS (percentage of relative activity): 1:0 (100%); 3:1 (75%); 1:1 (50%); and 1:3 (25%). Eight microliters from each dilution was added to 92 ll of reaction buffer (1.5 M urea, 5 mM Tris– HCl, 10 mM BaCl2, 1 mM PMSF, pH 8.0) containing purified human VWF (1 lg; Hematologic Technologies, VT, USA). Since ADAMTS13 needs divalent metal ions to cleave VWF (Furlan et al., 1996), BaCl2 was added to the reaction buffer. After incubation at 37 °C for 24 h, 5 ll of 0.5 M ethylenediaminetetraacetic acid (EDTA) (pH 8.0) as a chelating agent for Ba2+ was added to quench the reaction. Human normal pooled plasma (George King Bio-medical, KS, USA) was used as a positive control. Negative controls comprised the concentrated medium of HeLa cells that were transfected with no-template plasmid (NT-plasmid) and samples pretreated with 5 ll of 0.5 M EDTA.
The deduced amino acid sequences of canine, human and mouse ADAMTS13 were aligned (Fig. 1). The overall deduced amino acid sequence of canine ADAMTS13 was 64.4% and 58.7%, similar to that in human (accession No. NP_620594) and mouse (accession No. NP_001001322) ADAMTS13, respectively. Canine ADAMTS13 had a multidomain structure including a signal peptide, a propeptide, a metalloprotease domain, a disintegrin-like domain, TSP1-1, a cysteine-rich domain, a spacer domain, six TSP1 repeats, and two CUB domains. However, the sequence for the propeptide of canine ADAMTS13 was longer than those from other species. In addition, canine ADAMTS13 lacked TSP1-3 domain and had only six TSP1 repeats. The degree of similarity was summarized for each domain of canine, human and mouse ADAMTS13 (Table 1). Canine ADAMTS13 included motifs found in human and mouse ADAMTS13, such as the putative furin cleavage RXRR (where ‘‘X’’ represents any amino acid residue) site at the end of the propeptide domain, and a zinc-binding motif with HEXXHXXGXXHD in the metalloprotease domain.
2.7. Analysis of the profile of cleaved VWF monomer The reaction samples of crADAMTS13, canine plasma, or human pool plasma (non-diluted with TBS) were electrophoresed on 5% SDS–PAGE and transferred onto PVDF membrane. After blocking with 1% bovine serum albumin, the membrane was incubated with peroxidase-labeled rabbit polyclonal anti-human VWF antibody (1:500; Dako, Glostrup, Denmark). Chemiluminescent detection was performed as mentioned above.
2.8. Analysis of VWF multimetric structure VWF multimetric structures in samples with serially diluted samples were analyzed by SDS-agarose gel electrophoresis as previously described (Baillod et al., 1992), with slight modification. Briefly, the stacking and running gel contained 0.8% and 1.7% SeaKem HGT(P) Agarose (Lonza, ME, USA). Samples were diluted 1:4 with the sample buffer (10 mM Tris–HCl, 2 mM EDTA, pH 8.0, 3% SDS, 10% saccharose, 100 mg/L bromophenol blue) and heated to 60 °C for 15 min before loading to the gel. Samples were run at 100 V until the dye reached the end of the gel and transferred onto PVDF membrane, and subsequent procedures were the same as used in analyzing the profile of cleaved VWF monomer, as described above.
3.3. Transient expression and Western blot analysis of rcADAMTS13 protein The rcADAMTS13 with V5/His fusion protein was expressed in HeLa cells and confirmed by Western blot. Using anti-V5 monoclonal antibody, rcADAMTS13 in both culture medium and cell lysis were detected at a molecular weight of approximately 180 kDa (Fig. 2A). Similarly, using anti-human ADAMTS13 rabbit polyclonal antibody, the band was detected at approximately 180 kDa on the lane for rcADAMTS13 only, and not on any other lanes (Fig. 2B). 3.4. Functional characterization of rcADAMTS13 In analysis of the profile of cleaved VWF monomer by SDS– PAGE and Western blot (Fig. 3), the 250-kDa VWF monomer from rcADAMTS13 was truncated into 150- and 120-kDa fragments. When canine or human plasma was used, signals from these two truncated fragments were also clearly detected. These cleaved VWF fragments were not observed in control cases of buffer, in NT-plasmid medium or in samples pretreated with EDTA. In the analysis of VWF multimetric structure (Fig. 4), although the degradation products of VWF multimers appeared, the high molecular weight VWF multimers disappeared in a rcADAMTS13 concentration-dependent manner. Ablation of VWF multimetric structures was not observed in buffer, NT-plasmid medium or samples treated with EDTA. However, the same phenomenon was also observed in canine and human plasma, and the degree of disappearance of multimetric structures was higher in human plasma than in canine plasma.
3. Results
4. Discussion
3.1. Nucleotide sequence of canine ADAMTS13 cDNA
Human ADAMTS13 is the one of most important factors for regulating hemostasis by cleaving VWF (Fujikawa et al., 2001; Gerritsen et al., 2001; Soejima et al., 2001). Since canine ADAMTS13 had not previously been analyzed, we cloned the entire open reading frame of canine ADAMTS13 cDNA, including the gene encoding 1502 amino acids with several domains corresponding to those of human and mouse ADAMTS13. One characteristic structure of human and mouse ADAMTS13 is reportedly a short propeptide domain at the N-terminus (Bruno et al., 2005; Soejima et al., 2001; Zheng et al., 2001). Lengths of the propeptide domain in human (41 amino acid residues) and
By combining the sequences of partially overlapping cDNA fragments obtained in this study, a linear sequence corresponding to canine ADAMTS13 cDNA was obtained (accession No. AB605619). This sequence contained 4509 bp of the entire open reading frame, encoding 1502 amino acid residues. Comparison of the nucleotide sequence in the open reading frame of canine ADAMTS13 cDNA with those from human (accession No. NM_139025) and mouse (accession No. NM_001001322) ADAMTS13 revealed 70.4% and 65.4% similarity, respectively.
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Fig. 1. Alignment of amino acid sequences for human, mouse and canine ADAMTS13. Conserved amino acid residues are shaded. Each domain is indicated by underlining.
Table 1 Similarity of domains for the canine ADAMTS13 amino acid sequence to those for human and mouse domains. Domain
Signal peptide Propeptide Metalloprotease domain Disintegrin-like domain TSP1-1 Cysteine-rich domain Spacer domain TSP1-2 TSP1-3 TSP1-4 TSP1-5 TSP1-6 TSP1-7 TSP1-8 CUB-1 CUB-2
Similarity (%) Human
Mouse
50.0 12.9 83.0 78.1 87.0 78.5 80.0 69.0 – 51.7 74.1 80.3 81.7 84.5 79.4 68.2
46.0 12.4 73.5 76.0 81.5 78.5 78.5 63.8 – 44.3 69.0 41.3 66.7 77.6 70.1 67.7
mouse (39 amino acid residues) ADAMTS13 are markedly shorter, compared with that in other ADAMTS protease members (around
200 amino acid residues) (Cal et al., 2002). The deduced amino acid sequence of canine ADAMTS13 obtained in this study possessed a long propeptide domain (193 amino acid residues). In addition to the beagle dog sample used in the present study, we confirmed that other breeds of dogs (a golden retriever with liver glycogen degeneration, and a toy poodle with liver nodular hyperplasia) showed the same lengths in this domain (data not shown). In general, the ADAMTS propeptide is required for secretion to perform the common function of maintaining enzyme latency, although humans do not require the ADAMTS13 propteptide (Majerus et al., 2003). The function of the canine ADAMTS13 propeptide should thus be further examined. Two types of mouse ADAMTS13 gene have been reported, depending on the strain: a long-type with a full-length gene; and a short-type without TSP1-7, 8 and two CUB domains (Banno et al., 2004). This difference is caused by the strain-specific insertion of an intracisternal A-particle retrotransposon, including a stop codon. Since cDNA obtained from a beagle dog was used as the template for cloning the canine ADAMTS13 gene in this study, the full-length gene obtained was similar to the long-type mouse gene. However, various types might be present in dogs, as in mice. Further investigations of various breeds of dogs are needed to clarify this issue.
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Medium 1 2
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Fig. 2. Western blot of the expressed rcADAMTS13 in lysed HeLa cells and concentrated culture medium. Monoclonal anti-V5 epitope antibody detected rcADAMTS13 at approximately 180 kDa. (A) Polyclonal anti-human ADAMTS13 antibody also detected rcADAMTS13. (B) Lane 1, NT-plasmid transfection; Lane 2, canine ADAMTS13 plasmid transfection; M, protein standard marker.
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100 Fig. 3. Profile of cleaved VWF monomer by rcADAMTS13 and canine and human plasma in SDS–PAGE following Western blot using anti-human VWF antibody. Fragments of cleaved VWF monomer (150 kDa and 120 kDa), in addition to nontruncated VWF monomer (250 kDa) were detected in rcADAMTS13 (lane 2), canine plasma (lane 4) and human plasma (lane 6), respectively. Signals for these fragments were attenuated in samples treated with EDTA (lanes 1, 3, 5). Lane 7, buffer; Lane 8, medium from NT-plasmid transfection; M, protein standard marker.
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The rcADAMTS13 protein expressed in this study was detected using anti-human ADAMTS13 antibody as a single band in Western blots. The rcADAMTS13 cleaved mature human VWF to approximately 150- and 120-kDa fragments. In addition to this result, the high molecular weight VWF multimetric structure was ablated in a rcADAMTS13 concentration-dependent manner. ADAMTS13 needs divalent metal ions such as Ba2+ or Ca2+ to cleave VWF (Furlan et al., 1996). The VWF-cleaving activity of rcADAMTS13 was inhibited in the presence of EDTA as a chelating agent for Ba2+. These results confirmed that the gene cloned in this study was the canine ADAMTS13 gene. As commercial purified canine VWF was unavailable and the human mature VWF amino acid sequence shows a high degree of homology with the canine sequence (87% identity, data not shown), purified human VWF was used instead of canine VWF as a substrate for ADAMTS13 in this study. Varadi et al. (2009) reported that the degree of human VWF cleavage by dog plasma was weaker than that by human plasma. That result agrees with the present findings. However, TSP1 repeats and the CUB domain within human ADAMTS13 are not necessary for cleaving VWF (Soejima et al., 2003), with these domains acting to allow more efficient recognition and cleaving of VWF (Zhang et al., 2007). Canine ADAMTS13 has a deficient part of the domain that corresponds to TSP1-3 in human and mouse ADAMTS13. This lack of TSP1-3 might have contributed to the decreased ability of canine ADAMTS13 to cleave human VWF.
LMWM
Fig. 4. Activity of VWF multimetric structure abolition in serially diluted rcADAMTS13 (A) and canine and human plasma (B) for SDS-agarose electrophoresis following Western blotting using anti-human VWF antibody. High molecular weight VWF multimetric structures disappeared depending on sample dilution, but these structures remained in samples with EDTA (lane E), buffer (lane 0%) or medium from NT-plasmid transfection (lane NT), respectively. Degradation products are indicated by the areas within the dotted line. HMWM, high molecular weight multimer; LMWM, low molecular weight multimer.
TTP, one of the fatal thromboses, has been shown to be a disorder of VWF regulation due to depressed ADAMTS13 activity (Furlan et al., 1997; Levy et al., 2001). Clinical manifestations of TTP in humans include anemia, thrombocytopenia, renal failure, fever and neurological dysfunction (Asada et al., 1985; Tsai, 2010). In dogs with IMHA, not only hemolytic anemia, but also thrombocytopenia and organ dysfunction due to thromboembolism are observed with high frequency (Carr et al., 2002). The clinical manifestations of TTP are thus similar to those of IMHA, and many dogs might be diagnosed with IMHA rather than TTP. In humans, TTP has two main forms: hereditary; and acquired (Sadler, 2008).
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Hereditary TTP is the result of a congenital deficiency in ADAMTS13 due to a mutation in the ADAMTS13 gene (Levy et al., 2001). The diagnosis of hereditary TTP thus relies on the detection of ADAMTS13 gene mutation, in addition to the measurement of plasma ADAMTS13 activity. Conversely, acquired TTP is mainly caused by circulating autoantibodies that neutralize ADAMTS13 activity (Rieger et al., 2005), and is diagnosed based on measurements of plasma ADAMTS13 activity and detection of autoantibodies to ADAMTS13 antigen (Sadler, 2008). The reason that TTP has not been diagnosed in dogs might be not only the lack of an identified canine ADAMTS13 gene, but also the absence of an assay system for canine plasma ADAMTS13 activity. The present results could thus represent a step towards the establishment of mutation analysis for the canine ADAMTS13 gene and assay methods for canine ADAMTS13 activity to investigate canine TTP. In conclusion, the entire open reading frame of canine ADAMTS13 cDNA sequence was determined and the VWF-cleaving ability of canine ADAMTS13 protein was analyzed. These results could facilitate research into the regulatory mechanisms of normal hemostasis and into hemostatic disorders such as TTP in dogs. Acknowledgement The authors wish to thank Dr. Kazushi Asano (Laboratory of Veterinary Surgery, Nihon University) for providing tissue samples. References Arya, M., Anvari, B., Romo, G.M., Cruz, M.A., Dong, J.F., McIntire, L.V., Moake, J.L., López, J.A., 2002. Ultralarge multimers of von Willebrand factor form spontaneous high-strength bonds with the platelet glycoprotein Ib-IX complex: studies using optical tweezers. Blood 99, 3971–3977. Asada, Y., Sumiyoshi, A., Hayashi, T., Suzumiya, J., Kaketani, K., 1985. Immunohistochemistry of vascular lesion in thrombotic thrombocytopenic purpura, with special reference to factor VIII related antigen. Thrombosis Research 38, 469–479. Baillod, P., Affolter, B., Kurt, G.H., Pflugshaupt, R., 1992. Multimeric analysis of von Willebrand factor by vertical sodium dodecyl sulphate agarose gel electrophoresis, vacuum blotting technology and sensitive visualization by alkaline phosphatase anti-alkaline phosphatase complex. Thrombosis Research 66, 745–755. Banno, F., Kaminaka, K., Soejima, K., Kokame, K., Miyata, T., 2004. Identification of strain-specific variants of mouse Adamts13 gene encoding von Willebrand factor-cleaving protease. The Journal of Biological Chemistry 279, 30896– 30903. Brooks, M., 2000. von Willebrand disease. In: Feldman, B.F., Zinkl, J.G., Jain, N.C. (Eds.), Schalm’s Veterinary Hematology, fifth ed. Lippincott Williams & Wilkins, Philadelphia, pp. 509–515. Bruno, K., Völkel, D., Plaimauer, B., Antoine, G., Pable, S., Motto, D.G., Lemmerhirt, H.L., Dorner, F., Zimmermann, K., Scheiflinger, F., 2005. Cloning, expression and functional characterization of the full-length murine ADAMTS13. Journal of Thrombosis and Haemostasis 3, 1064–1073. Cal, S., Obaya, A.J., Llamazares, M., Garabaya, C., Quesada, V., López-Otín, C., 2002. Cloning, expression analysis, and structural characterization of seven novel human ADAMTSs, a family of metalloproteinases with disintegrin and thrombospondin-1 domains. Gene 283, 49–62. Carr, A.P., Panciera, D.L., Kidd, L., 2002. Prognostic factors for mortality and thromboembolism in canine immune-mediated hemolytic anemia: a retrospective study of 72 dogs. Journal of Veterinary Internal Medicine 16, 504–509. Day, M.J., 2010. In: Weiss, D.J., Wardrop, K.J. (Eds.), Schalm’s Veterinary Hematology, sixth ed. Wiley-Blackwell Publishing, Iowa, pp. 216–225. Federici, A.B., Bader, R., Pagani, S., Colibretti, M.L., De Marco, L., Mannucci, P.M., 1989. Binding of von Willebrand factor to glycoproteins Ib and IIb/IIIa complex: affinity is related to multimeric size. British Journal of Haematology 73, 93–99. Fujikawa, K., Suzuki, H., McMullen, B., Chung, D., 2001. Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family. Blood 98, 1662–1666. Furlan, M., Robles, R., Lämmle, B., 1996. Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis. Blood 87, 4223–4234.
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Kume, Y., Ikeda, H., Inoue, M., Tejima, K., Tomiya, T., Nishikawa, T., Watanabe, N., Ichikawa, T., Kaneko, M., Okubo, S., Yokota, H., Omata, M., Fujiwara, K., Yatomi, Y., 2007. Hepatic stellate cell damage may lead to decreased plasma ADAMTS13 activity in rats. FEBS Letters 581, 1631–1634. Levy, G.G., Nichols, W.C., Lian, E.C., Foroud, T., McClintick, J.N., McGee, B.M., Yang, A.Y., Siemieniak, D.R., Stark, K.R., Gruppo, R., Sarode, R., Shurin, S.B., Chandrasekaran, V., Stabler, S.P., Sabio, H., Bouhassira, E.E., Upshaw, J.D., Ginsburg Jr., D., Tsai, H.M., 2001. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 413, 488–494. Majerus, E.M., Zheng, X., Tuley, E.A., Sadler, J.E., 2003. Cleavage of the ADAMTS13 propeptide is not required for protease activity. The Journal of Biological Chemistry 278, 46643–46648. Moake, J.L., Rudy, C.K., Troll, J.H., Weinstein, M.J., Colannino, N.M., Azocar, J., Seder, R.H., Hong, S.L., Deykin, D., 1982. Unusually large plasma factor VIII: von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura. The New England Journal of Medicine 307, 1432–1435. Moake, J.L., Turner, N.A., Stathopoulos, N.A., Nolasco, L.H., Hellums, J.D., 1986. Involvement of large plasma von Willebrand factor (vWF) multimers and unusually large vWF forms derived from endothelial cells in shear stressinduced platelet aggregation. The Journal of Clinical Investigation 78, 1456–1461. Plaimauer, B., Zimmermann, K., Völkel, D., Antoine, G., Kerschbaumer, R., Jenab, P., Furlan, M., Gerritsen, H., Lämmle, B., Schwarz, H.P., Scheiflinger, F., 2002. Cloning, expression, and functional characterization of the von Willebrand factor-cleaving protease (ADAMTS13). Blood 100, 3626–3632. 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The Journal of Biochemistry (Tokyo) 130, 475–480. Soejima, K., Matsumoto, M., Kokame, K., Yagi, H., Ishizashi, H., Maeda, H., Nozaki, C., Miyata, T., Fujimura, Y., Nakagaki, T., 2003. ADAMTS-13 cysteine-rich/spacer domains are functionally essential for von Willebrand factor cleavage. Blood 102, 3232–3237. Tsai, H.M., 2010. Pathophysiology of thrombotic thrombocytopenic purpura. International Journal of Hematology 91, 1–19. Uemura, M., Tatsumi, K., Matsumoto, M., Fujimoto, M., Matsuyama, T., Ishikawa, M., Iwamoto, T.A., Mori, T., Wanaka, A., Fukui, H., Fujimura, Y., 2005. Localization of ADAMTS13 to the stellate cells of human liver. Blood 106, 922–924. Varadi, K., Rottensteiner, H., Vejda, S., Weber, A., Muchitsch, E.M., Turecek, P.L., Ehrlich, H.J., Scheiflinger, F., Schwarz, H.P., 2009. Species-dependent variability of ADAMTS13-mediated proteolysis of human recombinant von Willebrand factor. Journal of Thrombosis and Haemostasis 7, 1134–1142. Zhang, P., Pan, W., Rux, A.H., Sachais, B.S., Zheng, X.L., 2007. The cooperative activity between the carboxyl-terminal TSP1 repeats and the CUB domains of ADAMTS13 is crucial for recognition of von Willebrand factor under flow. Blood 110, 1887–1894. Zheng, X., Chung, D., Takayama, T.K., Majerus, E.M., Sadler, J.E., Fujikawa, K., 2001. Structure of von Willebrand factor-cleaving protease (ADAMTS13), a metalloprotease involved in thrombotic thrombocytopenic purpura. The Journal of Biological Chemistry 276, 41059–41063. Zhou, W., Inada, M., Lee, T.P., Benten, D., Lyubsky, S., Bouhassira, E.E., Gupta, S., Tsai, H.M., 2005. ADAMTS13 is expressed in hepatic stellate cells. Laboratory Investigation 85, 780–788.
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Molecular cloning, in vitro expression and functional characterization of canine ADAMTS13 H. Maruyama a,⇑, K. Ito a, K. Okabayashi b, M. Sakai c, R. Kano a, T. Watari d, A. Hasegawa a, H. Kamata a a
Laboratory of Veterinary Pathobiology, Department of Veterinary Medicine, Nihon University, Japan Laboratory of Veterinary Biochemistry, Department of Veterinary Medicine, Nihon University, Japan c Laboratory of Veterinary Internal Medicine, Department of Veterinary Medicine, Nihon University, Japan d Laboratory of Comprehensive Veterinary Clinical Studies, Department of Veterinary Medicine, Nihon University, Japan b
a r t i c l e
i n f o
Article history: Received 13 January 2011 Accepted 8 June 2011
Keywords: ADAMTS13 von Willebrand factor Thrombotic thrombocytopenic purpura Dog Hemostasis Cloning
a b s t r a c t A disintegrin and metalloproteinase with thrombospondin type 1 motifs, number 13 (ADAMTS13) is a plasma zinc metalloprotease also known as von Willebrand factor (VWF)-cleaving protease. Deficiency of ADAMTS13 activity is known to cause thrombotic thrombocytopenic purpura (TTP) in humans. We isolated the canine ADAMTS13 cDNA, which encodes 1502 amino acids, and expressed the recombinant protein to evaluate VWF-cleaving ability. Although the propeptide domain was longer and the TSP1 repeat domain was shorter than those in other species, the overall structures were similar to human and mouse ADAMTS13. Recombinant canine ADAMTS13 cleaved the 250-kDa VWF monomer into two fragments of 150 kDa and 120 kDa. Furthermore, high molecular weight VWF multimers were abolished based on the activity of ADAMTS13. These results could facilitate research into hemostatic disorders such as TTP in dogs. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction As a large multimeric plasma glycoprotein, von Willebrand factor (VWF) mediates the adhesion of platelets to the subendothelium of damaged vessels, and also plays a role in the formation of platelet thrombus (Brooks, 2000). VWF is mainly synthesized in the endothelium and secreted into blood as unusually large VWF multimer (UL-VWFM), with a molecular weight greater than 20,000 kDa (Moake et al., 1982). This secreted UL-VWFM is quickly converted to a smaller form, ranging in molecular weight from approximately 500 to 20,000 kDa. High molecular weight VWF multimers are more active in promoting platelet adhesion and aggregation (Arya et al., 2002; Federici et al., 1989; Moake et al., 1986). UL-VWFM remaining in the circulation has thus been reported to cause thrombosis in humans (Moake et al., 1982). A disintegrin and metalloproteinase with thrombospondin type 1 motifs, number 13 (ADAMTS13), is a plasma zinc metalloprotease also known as VWF-cleaving protease (Fujikawa et al., 2001; Gerritsen et al., 2001; Soejima et al., 2001). ADAMTS13 in humans, mice and rats is mainly produced by hepatic stellate cells and released into the circulation (Kume et al., 2007; Uemura et al., ⇑ Corresponding author. Address: Laboratory of Veterinary Pathobiology, Department of Veterinary Medicine, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan. Tel./fax: +81 466 84 3519. E-mail address: [email protected] (H. Maruyama). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.06.006
2005; Zhou et al., 2005). Human ADAMTS13 cleaves a peptide bond between Tyr1605 and Met1606 in the VWF A2 domain, and the VWF monomer is truncated into two fragments. UL-VWFMs are thus converted to a smaller form with appropriate platelet adhesion and aggregation activity (Plaimauer et al., 2002). Human and mouse ADAMTS13 amino acids comprise several domains: a signal peptide; a propeptide; a metalloprotease domain; a disintegrinlike domain; a thrombospondin type 1 (TSP1) motif; a cysteinerich domain; a spacer domain; seven TSP1 repeats; and two C1r/ C1s, urinary epidermal growth factor, bone morphogenic protein (CUB) domains (Bruno et al., 2005; Soejima et al., 2001; Zheng et al., 2001). Thrombotic thrombocytopenic purpura (TTP) refers to a disorder in which widespread platelet- and VWF-rich microthrombi are observed in small blood vessels of multiple organs, with clinical signs such as anemia, thrombocytopenia, renal failure, fever and neurological dysfunction (Asada et al., 1985; Tsai, 2010). TTP is recognized as a life-threatening disease if appropriate treatments, such as plasma exchange or infusion, are not provided (Rock et al., 1991). In human medicine, a key cause of TTP is the accumulation of UL-VWFM by severe reductions in ADAMTS13 activity in circulating blood (Furlan et al., 1997; Levy et al., 2001). TTP can be divided into hereditary and acquired forms. The hereditary form of TTP is known as Upshaw-Schulman syndrome, resulting from a congenital deficiency of ADAMTS13 due to an ADAMTS13 gene mutation (Levy et al., 2001), whereas the acquired form is mainly
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caused by circulating autoantibodies that neutralize ADAMTS13 activity (Rieger et al., 2005). Clinical manifestations similar to TTP seem to be observed in dogs with immune-mediated hemolytic anemia (IMHA), particularly accompanied by thrombocytopenia such as disseminated intravascular coagulation (DIC) or Evan’s syndrome. Whole blood transfusion is performed as one treatment for IMHA, especially in dogs with both severe anemia and thrombocytopenia (Day, 2010). However, transfusion of blood products including platelets is contraindicated in the treatment of human TTP, because infused platelets promote formation of further thrombi and then accelerate ischemic organ failure due to these platelet-rich thrombi (Harkness et al., 1981). In veterinary medicine, insufficient information is currently available regarding canine ADAMTS13 and diagnostic methods for TTP, so many dogs may die without appropriate treatment. To facilitate prospective studies into canine coagulation disorders such as TTP, the present study performed molecular cloning of the canine ADAMTS13 cDNA. Furthermore, recombinant canine ADAMTS13 (rcADAMTS13) was expressed and its ability to cleave VWF was confirmed by detecting changes in both fragments and the multimetric structure of VWF. 2. Materials and methods 2.1. RNA isolation and cDNA preparation Total RNA was isolated from a normal liver specimen obtained from a healthy beagle dog using an RNeasy mini kit (Qiagen, Hilden, Germany), then treated with DNase Turbo (Ambion, TX, USA) to eliminate contaminant genomic DNA. Five micrograms of total RNA was reverse-transcribed into cDNA using SuperScript III (Invitrogen, CA, USA) according to the instructions from the manufacturer. 2.2. Cloning of canine ADAMTS13 cDNA Primer pairs to amplify canine partial ADAMTS13 cDNA were designed based on conserved nucleotide sequences between human (accession No. NM_139025) and mouse (accession No. NM_001001322). Polymerase chain reaction (PCR) was performed using primer pairs (50 -CAGGCTCACCAGGAGGACAC-30 and 50 GACTCCCAGTAGAGCACCTGCTGC-30 ) and the cDNA of canine liver with PrimerSTAR GXL DNA polymerase (Takara Bio, Shiga, Japan) according to the instructions from the manufacturer. The PCR product was cloned into a pMD20-T vector using a Mighty TA-cloning Reagent Set for PrimeSTAR (Takara Bio). Competent INVaF’ cells (Invitrogen) were transformed using the vector with PCR products inserted. Plasmid DNA was extracted from the bacteria cultured in Luria–Bertani (LB) broth using a Quantum prep kit (Bio-Rad, CA, USA). Plasmid DNA was sequenced using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, CA, USA) and SequenceRx Enhancer Solution A (Invitrogen) in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). For further cloning of the more 50 -region, including ATG as the start-codon, primers were designed based on relatively conserved nucleotide sequences between human and mouse ADAMTS13 nucleotide sequences and the canine sequence obtained in this study. Sequences of these primers were 50 -AGGAAGCTCCCAAGAG TAAACAC-30 and 50 -CAGATGCACTTGGAACTGAGC-30 . For the 30 region, 30 -rapid amplification of cDNA ends (RACE) was performed using a 30 -RACE System for rapid amplification of cDNA Ends (Invitrogen) according the instructions from the manufacturer. Based on the nucleotide sequence of canine ADAMTS13 obtained in this study, the following primers were designed: 50 -GAGTGTGAC ATGCAGCTCTTCG-30 for the first PCR; and 50 -CCACTGATGGCACAG-
GAACC-30 for nested PCR. These PCR products were cloned into plasmid vector and sequenced as described above.
2.3. Construction of plasmid vector for transient expression rcADAMTS13 protein To construct the plasmid vector for transient expression of rcADAMTS13, the DNA fragment of the entire open reading frame of rcADAMTS13 was amplified by PCR using the cDNA from a canine normal liver and the primer pair of 50 -aggATGAGGGAGCCTCGCCCC TGGGGGAGATG-30 and 50 -GGGCTCAGCCCAGCCAGGTTCCTGCAC-30 . The small letters indicate the added Kozak sequence. PCR was performed with PrimerSTAR GXL DNA polymerase (Takara Bio). The amplicon was treated using a Mighty TA-cloning Reagent Set for PrimeSTAR (Takara Bio) for A-over hanging, then inserted into a pDNA 3.1/V5-His-TOPO plasmid vector (Invitrogen). The plasmid vector could express fusion protein with tag-proteins at the Cterminal, V5 epitope and 6 histidine. TOP 10 competent cells (Invitrogen) were transformed using the constructed plasmid vector. Plasmid vector was extracted from bacterial cultures grown in LB broth using NucleoBond Xtra Midi Plus EF (Macherey-Nagel, Duren, Germany). The sequence of the plasmid vector was confirmed as described above.
2.4. Transient expression of rcADMTS13 HeLa cells were purchased from Health Science Research Resources Bank (Osaka, Japan) and used for producing rcADAMTS13. The constructed plasmid vector was transfected into HeLa cells using FuGENE HD transfection reagent (Roche, Mannheim, Germany) according to the instructions from the manufacturer. Briefly, 3 106 HeLa cells were plated into each well of a 6-well plate on the day before transfection and cultured in Dulbecco’s modified Eagle’s medium (Sigma–Aldrich, MO, USA) supplemented with 10% fetal bovine serum. These cells were transfected with 2 lg of plasmid DNA and 5 ll of FuGENE HD in 100 ll of serumfree Opti-MEM (Invitrogen). No-template inserted pcDNA 3.1/V5His plasmid vectors were used as a negative control for expression. At 4 h after transfection, culture media were replaced with serumfree Opti-MEM and incubated for 44 h. Media were collected and centrifuged to remove residual cells. Supernatants of media were concentrated using Amicon Ultra-30 k (Millipore, MA, USA). The cells were harvested and washed twice with phosphate-buffered saline (PBS). Concentrated media and cells were then stored at 30 °C until Western blot and functional analyses. 2.5. Western blot of expressed rcADAMTS13 protein The concentrated media and cells were mixed with sodium dodecyl sulfate (SDS) sample buffer (58 mM Tris–Cl pH 6.8, 1.7% sodium dodecyl sulfate, 5% glycerol, 1.5% dithiothreitol, 0.02% bromophenol blue). These samples were electrophoresed on 6% SDS–polyacrylamide gel electrophoresis (PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). After blocking with 1% bovine serum albumin, the membrane was incubated with peroxidase-labeled anti-V5 antibody (1:5000; Invitrogen). The other transferred membrane was incubated with anti-human ADAMTS13 polyclonal antibody to recognize the metalloprotease domain of human ADAMTS13 (1:2500; Santa Cruz Biotechnology, CA, USA), then incubated with peroxidase-linked anti-rabbit immunoglobulin G antibody (1:5000; GE Healthcare, Buckinghamshire, England). Chemiluminescent detection was performed using an ECL Plus Western Blotting Detection System (GE Healthcare) by ChemiDoc XRS (Bio-Rad).
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2.6. Cleaving VWF by rcADAMTS13 protein and plasma
3.2. Deduced amino acid sequence of canine ADAMTS13
The activity of rcADAMTS13 to cleave VWF in concentrated medium was analyzed as previously described (Kokame et al., 2002) with slight modification. In addition, canine plasma was examined for VWF-cleaving activity. Briefly, the concentrated culture medium was serially diluted with 0.05 mM Tris–HCl (pH 7.4) containing 0.15 mM NaCl (TBS) as follows. Canine plasma obtained by mixing citrated plasma collected from five clinically healthy dogs was also serially diluted as the concentrated medium:TBS (percentage of relative activity): 1:0 (100%); 3:1 (75%); 1:1 (50%); and 1:3 (25%). Eight microliters from each dilution was added to 92 ll of reaction buffer (1.5 M urea, 5 mM Tris– HCl, 10 mM BaCl2, 1 mM PMSF, pH 8.0) containing purified human VWF (1 lg; Hematologic Technologies, VT, USA). Since ADAMTS13 needs divalent metal ions to cleave VWF (Furlan et al., 1996), BaCl2 was added to the reaction buffer. After incubation at 37 °C for 24 h, 5 ll of 0.5 M ethylenediaminetetraacetic acid (EDTA) (pH 8.0) as a chelating agent for Ba2+ was added to quench the reaction. Human normal pooled plasma (George King Bio-medical, KS, USA) was used as a positive control. Negative controls comprised the concentrated medium of HeLa cells that were transfected with no-template plasmid (NT-plasmid) and samples pretreated with 5 ll of 0.5 M EDTA.
The deduced amino acid sequences of canine, human and mouse ADAMTS13 were aligned (Fig. 1). The overall deduced amino acid sequence of canine ADAMTS13 was 64.4% and 58.7%, similar to that in human (accession No. NP_620594) and mouse (accession No. NP_001001322) ADAMTS13, respectively. Canine ADAMTS13 had a multidomain structure including a signal peptide, a propeptide, a metalloprotease domain, a disintegrin-like domain, TSP1-1, a cysteine-rich domain, a spacer domain, six TSP1 repeats, and two CUB domains. However, the sequence for the propeptide of canine ADAMTS13 was longer than those from other species. In addition, canine ADAMTS13 lacked TSP1-3 domain and had only six TSP1 repeats. The degree of similarity was summarized for each domain of canine, human and mouse ADAMTS13 (Table 1). Canine ADAMTS13 included motifs found in human and mouse ADAMTS13, such as the putative furin cleavage RXRR (where ‘‘X’’ represents any amino acid residue) site at the end of the propeptide domain, and a zinc-binding motif with HEXXHXXGXXHD in the metalloprotease domain.
2.7. Analysis of the profile of cleaved VWF monomer The reaction samples of crADAMTS13, canine plasma, or human pool plasma (non-diluted with TBS) were electrophoresed on 5% SDS–PAGE and transferred onto PVDF membrane. After blocking with 1% bovine serum albumin, the membrane was incubated with peroxidase-labeled rabbit polyclonal anti-human VWF antibody (1:500; Dako, Glostrup, Denmark). Chemiluminescent detection was performed as mentioned above.
2.8. Analysis of VWF multimetric structure VWF multimetric structures in samples with serially diluted samples were analyzed by SDS-agarose gel electrophoresis as previously described (Baillod et al., 1992), with slight modification. Briefly, the stacking and running gel contained 0.8% and 1.7% SeaKem HGT(P) Agarose (Lonza, ME, USA). Samples were diluted 1:4 with the sample buffer (10 mM Tris–HCl, 2 mM EDTA, pH 8.0, 3% SDS, 10% saccharose, 100 mg/L bromophenol blue) and heated to 60 °C for 15 min before loading to the gel. Samples were run at 100 V until the dye reached the end of the gel and transferred onto PVDF membrane, and subsequent procedures were the same as used in analyzing the profile of cleaved VWF monomer, as described above.
3.3. Transient expression and Western blot analysis of rcADAMTS13 protein The rcADAMTS13 with V5/His fusion protein was expressed in HeLa cells and confirmed by Western blot. Using anti-V5 monoclonal antibody, rcADAMTS13 in both culture medium and cell lysis were detected at a molecular weight of approximately 180 kDa (Fig. 2A). Similarly, using anti-human ADAMTS13 rabbit polyclonal antibody, the band was detected at approximately 180 kDa on the lane for rcADAMTS13 only, and not on any other lanes (Fig. 2B). 3.4. Functional characterization of rcADAMTS13 In analysis of the profile of cleaved VWF monomer by SDS– PAGE and Western blot (Fig. 3), the 250-kDa VWF monomer from rcADAMTS13 was truncated into 150- and 120-kDa fragments. When canine or human plasma was used, signals from these two truncated fragments were also clearly detected. These cleaved VWF fragments were not observed in control cases of buffer, in NT-plasmid medium or in samples pretreated with EDTA. In the analysis of VWF multimetric structure (Fig. 4), although the degradation products of VWF multimers appeared, the high molecular weight VWF multimers disappeared in a rcADAMTS13 concentration-dependent manner. Ablation of VWF multimetric structures was not observed in buffer, NT-plasmid medium or samples treated with EDTA. However, the same phenomenon was also observed in canine and human plasma, and the degree of disappearance of multimetric structures was higher in human plasma than in canine plasma.
3. Results
4. Discussion
3.1. Nucleotide sequence of canine ADAMTS13 cDNA
Human ADAMTS13 is the one of most important factors for regulating hemostasis by cleaving VWF (Fujikawa et al., 2001; Gerritsen et al., 2001; Soejima et al., 2001). Since canine ADAMTS13 had not previously been analyzed, we cloned the entire open reading frame of canine ADAMTS13 cDNA, including the gene encoding 1502 amino acids with several domains corresponding to those of human and mouse ADAMTS13. One characteristic structure of human and mouse ADAMTS13 is reportedly a short propeptide domain at the N-terminus (Bruno et al., 2005; Soejima et al., 2001; Zheng et al., 2001). Lengths of the propeptide domain in human (41 amino acid residues) and
By combining the sequences of partially overlapping cDNA fragments obtained in this study, a linear sequence corresponding to canine ADAMTS13 cDNA was obtained (accession No. AB605619). This sequence contained 4509 bp of the entire open reading frame, encoding 1502 amino acid residues. Comparison of the nucleotide sequence in the open reading frame of canine ADAMTS13 cDNA with those from human (accession No. NM_139025) and mouse (accession No. NM_001001322) ADAMTS13 revealed 70.4% and 65.4% similarity, respectively.
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Fig. 1. Alignment of amino acid sequences for human, mouse and canine ADAMTS13. Conserved amino acid residues are shaded. Each domain is indicated by underlining.
Table 1 Similarity of domains for the canine ADAMTS13 amino acid sequence to those for human and mouse domains. Domain
Signal peptide Propeptide Metalloprotease domain Disintegrin-like domain TSP1-1 Cysteine-rich domain Spacer domain TSP1-2 TSP1-3 TSP1-4 TSP1-5 TSP1-6 TSP1-7 TSP1-8 CUB-1 CUB-2
Similarity (%) Human
Mouse
50.0 12.9 83.0 78.1 87.0 78.5 80.0 69.0 – 51.7 74.1 80.3 81.7 84.5 79.4 68.2
46.0 12.4 73.5 76.0 81.5 78.5 78.5 63.8 – 44.3 69.0 41.3 66.7 77.6 70.1 67.7
mouse (39 amino acid residues) ADAMTS13 are markedly shorter, compared with that in other ADAMTS protease members (around
200 amino acid residues) (Cal et al., 2002). The deduced amino acid sequence of canine ADAMTS13 obtained in this study possessed a long propeptide domain (193 amino acid residues). In addition to the beagle dog sample used in the present study, we confirmed that other breeds of dogs (a golden retriever with liver glycogen degeneration, and a toy poodle with liver nodular hyperplasia) showed the same lengths in this domain (data not shown). In general, the ADAMTS propeptide is required for secretion to perform the common function of maintaining enzyme latency, although humans do not require the ADAMTS13 propteptide (Majerus et al., 2003). The function of the canine ADAMTS13 propeptide should thus be further examined. Two types of mouse ADAMTS13 gene have been reported, depending on the strain: a long-type with a full-length gene; and a short-type without TSP1-7, 8 and two CUB domains (Banno et al., 2004). This difference is caused by the strain-specific insertion of an intracisternal A-particle retrotransposon, including a stop codon. Since cDNA obtained from a beagle dog was used as the template for cloning the canine ADAMTS13 gene in this study, the full-length gene obtained was similar to the long-type mouse gene. However, various types might be present in dogs, as in mice. Further investigations of various breeds of dogs are needed to clarify this issue.
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Fig. 2. Western blot of the expressed rcADAMTS13 in lysed HeLa cells and concentrated culture medium. Monoclonal anti-V5 epitope antibody detected rcADAMTS13 at approximately 180 kDa. (A) Polyclonal anti-human ADAMTS13 antibody also detected rcADAMTS13. (B) Lane 1, NT-plasmid transfection; Lane 2, canine ADAMTS13 plasmid transfection; M, protein standard marker.
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The rcADAMTS13 protein expressed in this study was detected using anti-human ADAMTS13 antibody as a single band in Western blots. The rcADAMTS13 cleaved mature human VWF to approximately 150- and 120-kDa fragments. In addition to this result, the high molecular weight VWF multimetric structure was ablated in a rcADAMTS13 concentration-dependent manner. ADAMTS13 needs divalent metal ions such as Ba2+ or Ca2+ to cleave VWF (Furlan et al., 1996). The VWF-cleaving activity of rcADAMTS13 was inhibited in the presence of EDTA as a chelating agent for Ba2+. These results confirmed that the gene cloned in this study was the canine ADAMTS13 gene. As commercial purified canine VWF was unavailable and the human mature VWF amino acid sequence shows a high degree of homology with the canine sequence (87% identity, data not shown), purified human VWF was used instead of canine VWF as a substrate for ADAMTS13 in this study. Varadi et al. (2009) reported that the degree of human VWF cleavage by dog plasma was weaker than that by human plasma. That result agrees with the present findings. However, TSP1 repeats and the CUB domain within human ADAMTS13 are not necessary for cleaving VWF (Soejima et al., 2003), with these domains acting to allow more efficient recognition and cleaving of VWF (Zhang et al., 2007). Canine ADAMTS13 has a deficient part of the domain that corresponds to TSP1-3 in human and mouse ADAMTS13. This lack of TSP1-3 might have contributed to the decreased ability of canine ADAMTS13 to cleave human VWF.
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Fig. 4. Activity of VWF multimetric structure abolition in serially diluted rcADAMTS13 (A) and canine and human plasma (B) for SDS-agarose electrophoresis following Western blotting using anti-human VWF antibody. High molecular weight VWF multimetric structures disappeared depending on sample dilution, but these structures remained in samples with EDTA (lane E), buffer (lane 0%) or medium from NT-plasmid transfection (lane NT), respectively. Degradation products are indicated by the areas within the dotted line. HMWM, high molecular weight multimer; LMWM, low molecular weight multimer.
TTP, one of the fatal thromboses, has been shown to be a disorder of VWF regulation due to depressed ADAMTS13 activity (Furlan et al., 1997; Levy et al., 2001). Clinical manifestations of TTP in humans include anemia, thrombocytopenia, renal failure, fever and neurological dysfunction (Asada et al., 1985; Tsai, 2010). In dogs with IMHA, not only hemolytic anemia, but also thrombocytopenia and organ dysfunction due to thromboembolism are observed with high frequency (Carr et al., 2002). The clinical manifestations of TTP are thus similar to those of IMHA, and many dogs might be diagnosed with IMHA rather than TTP. In humans, TTP has two main forms: hereditary; and acquired (Sadler, 2008).
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Hereditary TTP is the result of a congenital deficiency in ADAMTS13 due to a mutation in the ADAMTS13 gene (Levy et al., 2001). The diagnosis of hereditary TTP thus relies on the detection of ADAMTS13 gene mutation, in addition to the measurement of plasma ADAMTS13 activity. Conversely, acquired TTP is mainly caused by circulating autoantibodies that neutralize ADAMTS13 activity (Rieger et al., 2005), and is diagnosed based on measurements of plasma ADAMTS13 activity and detection of autoantibodies to ADAMTS13 antigen (Sadler, 2008). The reason that TTP has not been diagnosed in dogs might be not only the lack of an identified canine ADAMTS13 gene, but also the absence of an assay system for canine plasma ADAMTS13 activity. The present results could thus represent a step towards the establishment of mutation analysis for the canine ADAMTS13 gene and assay methods for canine ADAMTS13 activity to investigate canine TTP. In conclusion, the entire open reading frame of canine ADAMTS13 cDNA sequence was determined and the VWF-cleaving ability of canine ADAMTS13 protein was analyzed. These results could facilitate research into the regulatory mechanisms of normal hemostasis and into hemostatic disorders such as TTP in dogs. Acknowledgement The authors wish to thank Dr. Kazushi Asano (Laboratory of Veterinary Surgery, Nihon University) for providing tissue samples. References Arya, M., Anvari, B., Romo, G.M., Cruz, M.A., Dong, J.F., McIntire, L.V., Moake, J.L., López, J.A., 2002. Ultralarge multimers of von Willebrand factor form spontaneous high-strength bonds with the platelet glycoprotein Ib-IX complex: studies using optical tweezers. Blood 99, 3971–3977. Asada, Y., Sumiyoshi, A., Hayashi, T., Suzumiya, J., Kaketani, K., 1985. Immunohistochemistry of vascular lesion in thrombotic thrombocytopenic purpura, with special reference to factor VIII related antigen. Thrombosis Research 38, 469–479. Baillod, P., Affolter, B., Kurt, G.H., Pflugshaupt, R., 1992. Multimeric analysis of von Willebrand factor by vertical sodium dodecyl sulphate agarose gel electrophoresis, vacuum blotting technology and sensitive visualization by alkaline phosphatase anti-alkaline phosphatase complex. Thrombosis Research 66, 745–755. Banno, F., Kaminaka, K., Soejima, K., Kokame, K., Miyata, T., 2004. Identification of strain-specific variants of mouse Adamts13 gene encoding von Willebrand factor-cleaving protease. The Journal of Biological Chemistry 279, 30896– 30903. Brooks, M., 2000. von Willebrand disease. In: Feldman, B.F., Zinkl, J.G., Jain, N.C. (Eds.), Schalm’s Veterinary Hematology, fifth ed. Lippincott Williams & Wilkins, Philadelphia, pp. 509–515. Bruno, K., Völkel, D., Plaimauer, B., Antoine, G., Pable, S., Motto, D.G., Lemmerhirt, H.L., Dorner, F., Zimmermann, K., Scheiflinger, F., 2005. Cloning, expression and functional characterization of the full-length murine ADAMTS13. Journal of Thrombosis and Haemostasis 3, 1064–1073. Cal, S., Obaya, A.J., Llamazares, M., Garabaya, C., Quesada, V., López-Otín, C., 2002. Cloning, expression analysis, and structural characterization of seven novel human ADAMTSs, a family of metalloproteinases with disintegrin and thrombospondin-1 domains. Gene 283, 49–62. Carr, A.P., Panciera, D.L., Kidd, L., 2002. Prognostic factors for mortality and thromboembolism in canine immune-mediated hemolytic anemia: a retrospective study of 72 dogs. Journal of Veterinary Internal Medicine 16, 504–509. Day, M.J., 2010. In: Weiss, D.J., Wardrop, K.J. (Eds.), Schalm’s Veterinary Hematology, sixth ed. Wiley-Blackwell Publishing, Iowa, pp. 216–225. Federici, A.B., Bader, R., Pagani, S., Colibretti, M.L., De Marco, L., Mannucci, P.M., 1989. Binding of von Willebrand factor to glycoproteins Ib and IIb/IIIa complex: affinity is related to multimeric size. British Journal of Haematology 73, 93–99. Fujikawa, K., Suzuki, H., McMullen, B., Chung, D., 2001. Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family. Blood 98, 1662–1666. Furlan, M., Robles, R., Lämmle, B., 1996. Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis. Blood 87, 4223–4234.
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