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RESEARCH ARTICLE
Mechanisms of the Interaction Between Two ADAMTS13 Gene Mutations Leading to Severe Deficiency of Enzymatic Activity Flora Peyvandi,1 Silvia Lavoretano,1 Roberta Palla,1 Carla Valsecchi,1 Giuliana Merati,1 Raimondo De Cristofaro,2 Edoardo Rossi,3 and Pier Mannuccio Mannucci1 1 Angelo Bianchi Bonomi Hemophilia and Thrombosis Center and Fondazione Luigi Villa, IRCCS Maggiore Hospital, Mangiagalli, Regina Elena Foundation and University of Milan, Milan, Italy; 2Hemostasis Research Center, Institute of Internal Medicine and Geriatrics, Catholic University School of Medicine, Rome, Italy; 3Immunohematology and Blood Transfusion Service, Luigi Sacco Hospital, Milan, Italy
Communicated by Arupa Ganguly The inherited deficiency of the von Willebrand factor-cleaving protease ADAMTS13 is associated with rare forms of thrombotic thrombocytopenic purpura (TTP). We investigated a woman with a family history of chronic recurrent TTP and undetectable plasma levels of ADAMTS13 activity. Genetic analysis revealed two missense mutations in the heterozygous state: p.Val88Met substitution in the metalloprotease domain and p.Gly1239Val substitution in the first CUB domain of ADAMTS13. To explore the mechanism of ADAMTS13 deficiency in this patient, the wild type (WT; ADAMT13WT) and each mutant construct (ADAMTS13Val88Met, ADAMTS13Gly1239Val) were transiently expressed in HEK 293 and COS-7 cells. To recapitulate the compound heterozygous state of the patient, both mutant ADAMTS13 proteins were also expressed together. The p.Val88Met mutation led to a defect of secretion of the protease associated with a reduction of enzymatic activity, the p.Gly1239Val mutation led to a secretion defect causing intracellular accumulation of the protease. The mechanistic effects of the mutations were further explored by means of differential immunofluorescence, that demonstrated an homogeneous distribution of ADAMTS13WT in the Cis-Golgi and endoplasmic reticulum (ER) compartments, a reduction of ADAMTS13Val88Met in both compartments, while ADAMTS13Gly1239Val failed to reach the Cis-Golgi compartment r 2006 Wiley-Liss, Inc. and remained in the ER. Hum Mutat 27(4), 330–336, 2006. KEY WORDS:
ADAMTS13; thrombotic thrombocytopenic purpura (TTP); mutations expression study
INTRODUCTION Thrombotic thrombocytopenic purpura (TTP) is an inherited or acquired disorder characterized by the massive formation of platelet thrombi in the microcirculation accompanied by hemolytic anemia, thrombocytopenia, and clinical and laboratory signs of renal and neurological failure. In many cases the trigger of TTP is the presence in plasma of ultralarge multimers of the adhesive glycoprotein von Willebrand factor (ULVWF; MIM] 193400), due to the inherited or acquired deficiency of the plasma metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin 1 repeats; MIM] 604134) that physiologically reduces the size of VWF multimers [Moake, 2002]. Unlike other members of the ADAMTS family of proteases, ADAMTS13 consists of a relatively short propeptide, of several thrombospondin-1 (TSP1) repeats and 2 CUB (complement components C1r/C1s, urinary Epidermal Growth factor and bone morphogenetic protein-1) domains at the Cterminus. The enzymatic activity of ADAMTS13 depends on divalent metal ions in the following order of potency: Zn21oCa21 oSr21oBa21 ions [Furlan et al., 1996; Zheng et al., 2001]. Inherited TTP is a rare autosomal recessive disorder due to homozygous or double heterozygous mutations in the ADAMTS13 gene [Assink et al., 2003; Bestetti et al., 2003; Kokame and Miyata, 2004; Licht et al., 2004; Studt et al., 2005; Uchida et al., 2004; Veyradier et al., 2004]. As many as 53 mutations causing inherited ADAMTS13 deficiency have been identified so far in r 2006 WILEY-LISS, INC.
regions of the gene encoding different domains [Assink et al., 2003; Bestetti et al., 2003; Kokame and Miyata, 2004; Licht et al., 2004; Studt et al., 2005; Uchida et al., 2004; Veyradier et al., 2004], with only a few of them characterized by in vitro expression studies [Kokame and Miyata, 2004; Uchida et al., 2004]. We carried out a molecular investigation in an Italian woman from a previously reported family with chronic recurrent TTP [Noris et al., 2005], who had her first episode of TTP at the age of 23 years. DNA analysis identified two missense mutations, a G to A substitution of nt 262 (c.262G4A, p.Val88Met), located in the metalloprotease domain [Bestetti et al., 2003] and a G to T substitution of nt 3717 (c.3717G4T, p.Gly1239Val), located in the first CUB domain of ADAMTS13. The metalloprotease
Received 26 April 2005; accepted revised manuscript 25 August 2005. Correspondence to: Dr. Flora Peyvandi, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, IRCCS Maggiore Hospital, University of Milan,Via Pace, 9^20122, Milan, Italy. E-mail: £
[email protected] Grant sponsor: Fondazione Cariplo; Grant sponsor: Italian Ministry of Instruction, University, and Research (MIUR). DOI 10.1002/humu.20267 Published online 1 February 2006 in Wiley InterScience (www. interscience.wiley.com).
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domain is known to have an important role for Zn21 binding and protease activity whereas the physiological role of the CUB domains located at the C-terminus of ADAMTS13 is less certain. In order to explain the patient’s phenotype of severe ADAMTS13 deficiency, we chose to explore the mechanistic effect of each separate mutation and of their combination by means of expression studies in mammalian cells. MATERIALS AND METHODS Patient An Italian woman had her first episode of TTP at the age of 23 years during her first pregnancy. Ten additional episodes subsequently occurred, usually in association with such precipitating events as pregnancy, first-trimester miscarriages, or infections. She was successfully treated for each episode with plasma infusions, antiplatelet agents, and anticoagulants. The patient’s sister died at the age of 55 years because of a cerebrovascular event during a severe episode of TTP; her brother has had no clinical manifestation of TTP so far in spite of ADAMTS13 deficiency at the same degree of severity of the propositus [Noris et al., 2005]. This study was conducted with the patient’s consent and approval by the Institutional Review Board, IRCCS Maggiore Hospital, Mangiagalli and Regina Elena Foundation. Measurement of ADAMTS13 Activity, Anti-ADAMTS13 Activity, and VWF Multimers in Plasma Venous blood was collected into 0.1 vol 0.129 M trisodium citrate during a remission period. Platelet-poor plasma was obtained by centrifugation at 3,000g for 20 min, snap frozen, and stored at 801C until tested. ADAMTS13 activity was measured in plasma with the collagen binding assay (CBA) [Gerritsen et al., 1999]. Antibodies neutralizing ADAMTS13 were detected using a previously described method and residual ADAMTS13 activity was measured by CBA [Peyvandi et al., 2004a]. Plasma ADAMTS13 activity was also measured with a modification [Allford et al., 2000] of an immunoblotting assay [Furlan et al., 1998], tailored to be more sensitive to very low levels of protease activity. Plasma was diluted in 10 mM Tris, 150 mM NaCl, 1 mM Pefabloc (pH 7.4), and incubated at 371C for 5 min with 10 mM barium chloride (SigmaAldrich, St. Louis, MO, www.sigma-aldrich.com). A total of 100 ml of the incubation mixture was added to 50 ml of a 5 U/ml solution of recombinant VWF (kindly provided by Dr. Turecek, Baxter Bioscience, Vienna, Austria). The reaction mixture was overlaid on a circular dialysis membrane (Millipore VSWP, diameter 25 mM; Millipore, Billerica, MA; www.millipore.com) floating on the surface of 50 ml of dialysis buffer (1.5 M urea, 5 mM Tris-HCl, pH 8) and incubated for 24 hr in a dry oven at 371C. The reaction was stopped by the addition of 10 ml of 0.2 M EDTA, pH 7.4. A calibration curve was obtained by serial dilutions (1:20–1:1280) of pooled normal plasma. Proteolysis of VWF was visualized using 0.6% vertical SDS agarose gel electrophoresis according to Warren et al. [2003]. The discontinuous buffer system was modified using the method of Raines et al. [1990]; molecular sieving was obtained with three different agarose layers, including 30% glycerol in the running gel. Before electrophoresis each sample was diluted in sample buffer and incubated for 20 min at 601C. Proteins were electrotransferred to an Immobilon Polyvinylidene Fluoride (PVDF) membrane (Millipore, Billerica, MA) overnight (O/N) and ULVWF multimers were visualized using a rabbit polyclonal anti-human VWF antibody (DAKO Cytomation, Glostrup, Denmark; www.dako.com) and a goat anti-rabbit IgG (BioRad
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Laboratories, Hercules, CA; www.bio-rad.com) peroxidase-conjugated antibody. For visualization the filters are stained with a luminol-iodophenol solution and immediately expose to X-ray films (Amersham Bioscences, Uppsala, Sweden; www.amersham. com). Residual VWF multimers were analyzed by Typhoon 8600 Variable Mode Imager (Image QuantTM software; Amersham Biosciences, Uppsala, Sweden). The lower limit of sensitivity of the assay with these modifications is 1.6%. The VWF multimeric pattern was analyzed in platelet poorplasma by discontinuous low-resolution SDS-agarose gel electrophoresis (0.9% LGT-agarose) and autoradiography [Ruggeri and Zimmerman, 1981]. Genetic Analysis DNA was extracted from peripheral blood leukocytes and all the exons and intron–exon boundaries of the ADAMTS13 gene (NT–017539) were amplified by PCR and sequenced using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit on an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Monza, Italy; www.appliedbiosystems.com). The primer sequences and amplification parameters are available on request. The secondary structure prediction of the effect of each mutation on ADAMTS13 structure was obtained using the predict protein program available on line (www.aber.ac.uk/compsci/research/biol/ dss/prof/ or http://us.expasy.org). Transient Transfection of Wild-Type (WT) and Mutant ADAMTS13 Expression Vectors A pCDNA 3.1 mammalian expression vector (kindly provided by Dr. F. Scheiflinger, Baxter Bioscience, Vienna, Austria) containing the entire coding region and a small part of the 30 untranslated region of the ADAMTS13 gene (pCDNA 3.1ADAMTS13WT) was used as a template for site directed mutagenesis. The p.Val88Met and p.Gly1239Val mutations were introduced into pCDNA3.1-ADAMTS13WT using the QuickChangeTMSite-directed Mutagenesis Kit (Stratagene, La Jolla, CA; www.stratagene.com). A forward primer (50 -CTGGAGCT GCTGGTGGCCATGGGCCCCGATGTCTTCC-30 ) and a reverse primer (50 -GGAAGACATCGGGGCCCATGGCCACCAG CAGCTCCAG-30 ) were used to introduce c.262G4A (signed as A) in human ADAMTS13 cDNA (NM–017587), to obtain pCDNA 3.1-ADAMTS13Val88Met. A forward primer (50 -TCT CTCAACTGCAGTGCGGTGGACATGTTGCTGCTTTGGG-30 ) and a reverse primer (50 -CCCAAGCAGCAACATGTCCACCG CACTGCAGTTGAGAGA-30 ) were used to introduce c.3717G4T (signed as T) in human ADAMTS13 cDNA, to obtain pCDNA 3.1-ADAMTS13Gly1239Val. The WT and mutant constructs were also subcloned into the mammalian expression vector pCDNA 3.1D/V5-His TOPO [Cheng and Shuman, 2000]. To explore the functional significance of the mutations, 50 mg of each vector were transiently transfected in COS-7 and HEK293 cells by electroporation according to the manufacture’s instructions (EQUIBIO/Easyject Plus; Thermo Electron Corp., Needham Heights, MA; www.thermo.com). Serum-free conditioned medium was added to equivalent numbers of confluent cells at 24 hr and the conditioned medium collected at 72 hr was clarified by centrifugation and concentrated 10-fold using AMICON Centricons YM-30 Column (Millipore, Billerica, MA). Cells were washed with phosphate-buffered saline, pH 7.2–7.4, and lysed with 1 ml of buffer (1% NONIDET NP40, 150 mM NaCl, 5 mM Tris). To recapitulate the compound heterozygous state of the patient, both mutant constructs were also transiently transfected together (ADAMTS13Val88Met1Gly1239Val). Human Mutation DOI 10.1002/humu
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The transfection efficiency was evaluated by luciferase system (Renilla Luciferase Assay System; Promega, Madison, WI; www.promega.com). Recombinant ADAMTS13 enzymatic activity was measured in the cell conditioned media using the modification of the immunoblotting assay as described above. Serial dilutions of WT cell supernatants (1:10–1:640) were used for the calibration curve. Western Blot Analysis To measure the levels of expression of WT and mutant recombinant ADAMTS13 in transfected cell lines, amounts of conditioned media and cell lysates according to the transfection efficiency were solubilized in gel-loading buffer (50 mM Tris-HCl pH6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol), separated by SDS-polyacrylamide electrophoresis (7% polyacrylamide) and transferred on Pure Nitrocellulose membrane (Bio-Rad, Hercules, CA). After blocking with skim milk, membranes were incubated with 1 mg/ml anti-V5 monoclonal antibody against the C-terminal tag of recombinant ADAMTS13 (Invitrogen, Carlsbad, CA; www.invitrogen.com) and then with peroxidase-labeled anti-mouse immunoglobulin G (Amersham Bioscences, Uppsala, Sweden). Detection was carried out using a chemiluminescent substrate (ECL Blotting System; Amersham Bioscences, Uppsala, Sweden) followed by exposure on autoradiographic film. Immuno£uorescence Studies COS-7 cells were transfected with ADAMTS13WT and with each mutant construct and plated in six-well cluster plates containing coverslips. At 24 hr after transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 for intracellular staining. Permeabilized cells were then incubated with the anti-V5 monoclonal antibody against the Cterminal tag of recombinant ADAMTS13 followed by anti-mouse IgG fluorescein isothiocyanate (FITC) conjugated antibody (BioSource International, Camarillo, CA; www.biosource.com). To detect the cellular localization of WT and mutant recombinant ADAMTS13, transfected cells were stained simultaneously with anti-V5 antibody and mouse polyclonal IgG1 and IgG2 antibodies against the proteins GM130 (a Cis-Golgi marker) and BipGRP78 (a chaperon protein involved in Golgi–endoplasmic reticulum [ER] transport) (BD Biosciences, Franklin Lakes, NJ; www.bdbiosciences.com). Recombinant ADAMTS13 stained with FITC conjugated antibody was detectable as green color, identifying the presence of the protein but not its localization. GM130 and Bip-GRP78 stained with Cy3-conjugated antibody (Chemicon International, Temecula, CA; www.chemicon.com) were detectable as red color, identifying the different cell compartments. Image acquisition using both fluorescent signals (green and red) identified the localization of the ADAMTS13 proteins in each cell compartment, visible as yellow color. Coverslips were mounted onto slides and examined with a Leica DMR epifluorescence microscope (Leica Imaging System, Cambridge, United Kingdom; www.leica.com) equipped with a CCD camera (Cohu, San Diego, CA; www.cohu-cameras.com) using a specific filter. The images were recorded using a QFISH software (Leica Imaging System, Cambridge, United Kingdom). RESULTS Laboratory and Genetic Data During periods of clinical and laboratory remission the patient had on several occasions less than 6% of ADAMTS13 activity as Human Mutation DOI 10.1002/humu
measured by CBA and less than 1.6% as measured by the more sensitive immunoblotting assay that is able to measure as little as 1.6% of protease activity. ULVWF multimers were repeatedly detected in plasma whereas no anti-ADAMTS13 neutralizing activity was detectable. Genetic analysis (nucleotides numbered from the A of the initiation Met codon) identified 2 previously reported missense mutations and a SNP, G to C substitution at nt 1342 (c.1342G4C, p.Gln448Glu) [Levy et al., 2001]. The first missense mutation was a G to A substitution at nt 262 in exon 3 (c.262G4A), leading to the substitution of Valine 88 to a Methionine [Bestetti et al., 2003] in the metalloprotease domain of the ADAMTS13 protein (p.Val88Met); the second missense mutation was a G to T substitution at nt 3717 in exon 27 (c.3717G4T), leading to the substitution of Glycine 1239 to a Valine in the first CUB domain (p.Gly1239Val) [Noris et al., 2005; Peyvandi et al., 2004b]. The patient was hetero zygous for both mutations and for the p.Gln448Glu polymor phism, that has an allelic frequency of 32% in Italians. The novel mutation p.Gly1239Val was not found in 100 normal Italians. Secondary structure prediction indicated that there should be no significant change in the helical region encompassing Glycine 1239 on replacement with Valine, but that the p.Val88Met mutation, located on the metalloprotease domain, should enhance the conformational instability of the 80–87 region that contains a Glutamate 83 involved in a Ca21 ion coordination. Western Blot Analysis Both the conditioned medium and lysate of cells transfected with the ADAMTS13WT construct contained a single immunoreactive band with a molecular mass of approximately 190 kDa (Fig. 1). The band was not detectable in the medium of untransfected cells used as a negative control. In the conditioned medium ADAMTS13Val88Met appeared as a band with the same molecular mass of the ADAMTS13WT, but chemiluminescence intensity was estimated to be approximately 40 to 50% of that of ADAMTS13WT. In cell lysate an increased amount of the mutant protease compared with ADAMTS13WT was seen. For ADAMTS13Gly1239Val a secretion defect causing intracellular accumulation with no detectable protein in supernatant was observed. In the conditioned medium and the lysate of cells transfected with both the mutant constructs (ADAMTS13Val88Met1Gly1239Val) an intermediate pattern of results was observed (Fig. 1). ADAMTS13 Activity in Cell-Conditioned Media The enzymatic activity of recombinant ADAMTS13 was measured by immunoblotting assay 72 hr after transfection in the conditioned media of cells transfected with WT and mutant constructs. The enzymatic activities of ADAMTS13Val88Met, ADAMTS13Gly1239Val, and ADAMTS13Val88Met1Gly1239Val were reduced on average to 24%, 6%, and 18%, respectively, in comparison with ADAMTS13WT (Fig. 2). Immuno£uorescence Studies ADAMTS13WT was mainly localized in the perinuclear area, ADAMTS13Val88Met was diffusely present throughout the cytoplasm with a perinuclear enhancement, and ADAMTS13Gly1239Val was diffusely present throughout the cytoplasm with no perinuclear enhancement (Fig. 3). An anti-V5 monoclonal antibody recognizing recombinant ADAMTS13 proteins and monoclonal antibodies recognizing
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FIGURE 1. Western Blot analysis of the recombinant ADAMTS13 proteins. The ADAMTS13 WT and mutants were transiently expressed in HEK293 cells.The culture media (A) and cell lysates (B) were analyzed byWestern Blot analysis using an anti-V5 monoclonal antibody against the C-terminal tag of recombinant ADAMTS13.
FIGURE 2. Recombinant ADAMTS13 enzymatic activity measured by immunoblotting assay using recombinant VWF as substrate. From left to right: Lane 1^5: standard curve of ADAMTS13WT protease activity based on di¡erent dilutions (1:10 shows 100% of protease activity and 1:640 shows 1.6%). Lanes 6^8: the protease activity in the conditioned medium of cells transfected by ADAMTS13Val88Met, ADAMTS13Gly1239Val and ADAMTS13 Val88Met1Gly1239Val constructs, respectively.
markers of Cis-Golgi and ER were used to characterize the cellular localization of recombinant ADAMTS13 detected as yellow fluorescence. ADAMTS13Val88Met was visualized in both compart-
ments, but with lesser intensity than the WT protein, while ADAMTS13Gly1239Val was visualized only in the ER and not in the Golgi compartment (Fig. 4). Human Mutation DOI 10.1002/humu
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FIGURE 3. Immuno£uorescence studies of recombinant ADAMTS13 WT and mutant proteins in transfected COS-7 cells. Transfected COS-7 cells were stained with anti-V5 monoclonal antibody against C-terminal tag of recombinant ADAMTS13 and revealed with a secondary antimouse-FITC-conjugated antibody. ADAMTS13WT was mainly localized in the perinuclear area, ADAMTS13 Val88Met showed a di¡usion in the cytoplasm but also a perinuclear enhancement whereas ADAMTS13 Gly1239Val was di¡usely present throughout the cytoplasm with no perinuclear enhancement. [Color ¢gure can be viewed in the online issue, which is available at www. interscience.wiley.com.]
DISCUSSION Congenital TTP is a rare disorder characterized by undetectable or very low plasma levels of the VWF-cleaving protease ADAMTS13 associated with an array of mutations in the ADAMTS13 gene [Assink et al., 2003; Bestetti et al., 2003; Kokame and Miyata, 2004; Licht et al., 2004; Studt et al., 2005; Uchida et al., 2004; Veyradier et al., 2004]. Until now, only few of these mutations have been characterized by in vitro expression studies [Kokame and Miyata, 2004; Uchida et al., 2004] in order to explain the mechanisms underlying phenotypes. We chose to investigate a patient who was peculiar because she developed the first episode of TTP only when she was 23 years old and in concomitance with her first pregnancy, and because undetectable levels of ADAMTS13 enzymatic activity were associated with compound heterozygosity for two missense mutations, one located in the metalloprotease domain and the other in the first CUB domain of ADAMTS13. As previously reported by Noris et al. [2005], her sister, who developed chronic renal failure and died, also showed the heterozygous mutation p.Ser890Ile in complement factor H (MIM] 134370). Now 15 mutations have been identified in the metalloprotease domain and nine of them have been characterized by in vitro expression studies [Kokame and Miyata, 2004; Uchida et al., 2004]. Most of them led to an alteration of the secretion pathway and/or affected the specific activity of the enzyme, sometimes resulting in the absence of enzymatic activity due to Zn21 binding site alterations [Uchida et al., 2004]. In this study, another mutation in the metalloprotease domain of ADAMTS13, p.Val88Met, was characterized by in vitro expression studies. Recombinant ADAMTS13Val88Met was synthesized in normal amounts in cell lysates, but its secretion was impaired by approximately 40 to 50% compared to ADAMTS13WT. This pattern was confirmed by immunofluorescence studies, showing that ADAMTS13Val88Met was visualized in both the ER and Golgi compartments, but with less intensity than the WT recombinant protease. The mutant protease also had reduced enzymatic activity, the most sensitive immunoblotting assay measuring 24% of ADAMTS13 activity in comparison with the WT protease. These data are consistent with the analysis of the secondary structure of ADAMTS13, that predicted a rearrangement of the region between residues 61–70 and a slight increase of the sheet content over residues 80–90 compared to ADAMTS13WT. We surmise that the p.Val88Met mutation alters the Ca21-binding site Human Mutation DOI 10.1002/humu
and leads to partial secretion defect due to intracellular accumulation. On the whole, these expression studies corroborate the available knowledge on the key role of the metalloprotease domain on the secretion and enzymatic activity of the protease. The role of the CUB domains at the C-terminus of the protease, where the p.Gly1239Val mutation is located, is of uncertain physiological relevance. A few studies support the views that the seventh and eighth TSP-1 repeats and the CUB domains are dispensable for protease activity [Banno et al., 2004]. On the other hand, studies based upon peptides from the CUB domains that inhibited the VWF-cleaving protease activity under flow suggested a functional role for these domains [Bernardo et al., 2003]. Moreover Majerus et al. [2005] have recently showed that the binding of ADAMTS13 to immobilized VWF appeared to be modulated by the most C-terminal TSP-1 and CUB domains. Until now, six different mutations on the first and second CUB domains of ADAMTS13 were identified. p.Cys1213Tyr and p.Arg1219Trp, located on the first CUB domain, were associated with reduced enzymatic activity but normal secretion [Kokame and Miyata, 2004; Motto et al., 2003]. The c.4143–4144insA mutation, located in the second CUB domain, had little effect on the specific activity of the enzyme but secretion was impaired, conserving only 15% of activity in comparison with the WT protease. The defect may be due to the removal of the central b- strands present in the CUB domain, resulting in the destruction of its architecture [Pimanda et al., 2004]. The missense mutation p.Gly1239Val, located in the first CUB domain, when analyzed by secondary structure prediction, was expected to produce no significant change in the helical region encompassing Glycine 1239 on replacement with Valine. However, our in vitro expression studies showed reduced enzymatic activity of recombinant ADAMTS13Gly1239Val associated with increased immunofluorescence staining throughout the cytoplasm, with no specific quantitative detection. No perinuclear enhancement and a retention of the mutant protein in the ER compartment were observed, suggesting an important role of the first CUB domain in the secretion pathway of ADAMTS13. The constructs carrying both p.Val88Met and p.Gly1239Val and hence mimicking the compound heterozygous state of the patient were characterized by a reduction of the specific activity of the protease but failed to accurately reflect the undetectable levels measured in patient. The presence of a strong CMV promoter (human cytomegalovirus immediate-early promoter) in the expression vector used for in vitro expression studies may be
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FIGURE 4. Di¡erential immuno£uorescence staining studies. A: Panels 1, 2, and 3 show cells transfected with WT and mutant constructs stained simultaneously with anti-V5 monoclonal antibody against recombinant ADAMTS13 (green) and anti-Bip-GRPp78 monoclonal antibody (red) against a chaperon protein of ER compartment. B: Panels 1, 2, and 3 show cells transfected with WTand mutant constructs stained simultaneously with anti-V5 monoclonal antibody against recombinant ADAMTS13 (green) and antiGM130 monoclonal antibody (red) against a Cis-Golgi matrix protein. The yellow color demonstrates the localization of ADAMTS13WT in both compartments whereas ADAMTS13 Gly1239Val are located only in ER. Cell expressing ADAMTS13 Val88Met showed a very faint dotted yellow color in both compartments.
responsible for an overexpression of ADAMTS13 enzyme activity. This promoter is usually employed to permit a high level of expression of recombinant proteins in a wide range of mammalian cells [Boshart et al., 1985; Nelson et al., 1987]. It should also be considered that the patient, besides the p.Val88Met and p.Gly1239Val mutations, is also heterozygous for the common p.Gln448Glu polymorphism. We did not produce a construct containing also the p.Gln448Glu polymorphism, because previous expression studies have convincingly shown that this polymorphism does not affect ADAMTS13 activity [Kokame et al., 2002]. However, it cannot be ruled out that the mechanistic interaction between the two mutations and the polymorphism may explain the lower levels of ADAMTS13 activity measured in this patient’s plasma.
ACKNOWLEDGMENTS The authors thank Dr. Isabella Garagiola and Dr. Maria Rita Balestrieri (Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione Luigi Villa, IRCCS Maggiore Hospital, Mangiagalli, Regina Elena Foundation, and University of Milan, Italy) for their help in immunofluorescence studies; Dr. Maria Teresa Canciani and Dr. Rossana Lombardi (Angelo Bianchi Bonomi Hemophilia and Thrombosis Centre, Maggiore Hospital Mangiagalli, Regina Elena Foundation, and University of Milan, Milan, Italy) for ADAMTS13 enzymatic activity measurement; Dr. Antonella Lattuada (Immunohematology and Blood Transfusion Service, L.Sacco Hospital, Milan, Italy) for the clinical identification of the proband, family history, and sample collection. Human Mutation DOI 10.1002/humu
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HUMAN MUTATION 27(4), 330^336, 2006
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