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Expression and characterization of recombinant human ADAMTS-13
Expression and Characterization of Recombinant Human ADAMTS-13 Barbara Plaimauer and Friedrich Scheiflinger Thrombotic thrombocytopenic purpura (TTP) is a severe disease associated with unusually large, hemostatically hyperactive von Willebrand factor (VWF) and severe deficiency in ADAMTS-13, the protease responsible for the proteolytic degradation of VWF in plasma. ADAMTS-13 prevents inappropriate microvascular platelet aggregation by cleaving VWF between Tyr1605 and Met1606 thereby producing dimers of 176 kd and 140 kd and smaller multimers. Identification of the ADAMTS13 gene and cloning of the corresponding cDNA allowed for the application of recombinant techniques, such as genetic engineering of ADAMTS13 cDNA, cell culture expression, and in vitro activity studies to analyze the functional relationship between ADAMTS-13 and the pathophysiology of ADAMTS-13 deficiency. In vitro expression and characterization of recombinant ADAMTS-13 (rADAMTS-13) clearly established that ADAMTS-13 is deficient in congenital TTP and inhibited in acquired TTP. Recent studies have contributed greatly to our current understanding of the molecular mechanism leading to congenital and acquired TTP. Apart from being a useful tool, availability of rADAMTS-13 raised the prospect of developing a recombinant substitution therapy to improve TTP treatment and allowing present diagnostic assays to be simplified. Here we report on recent advances in cell culture expression and functional characterization of human rADAMTS-13. Semin Hematol 41:24-33. © 2004 Elsevier Inc. All rights reserved.
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LASMA von Willebrand factor (VWF) predominantly originates in Weibel-Palade bodies (WPB) of vascular endothelial cells. Upon stimulation by various agonists, WPB immediately release high-molecular-weight VWF rich in unusually large VWF (ULVWF). Because ULVWF binds avidly to the extracellular matrix of the vascular bed1 and to various platelet surface receptors,2 it is a very effective promoter of platelet adhesion to sites of vascular injury. Interaction of VWF with platelet receptor glycoprotein Ib␣ is of major importance for platelet aggregation and thrombus growth under high shear blood flow.3,4 Platelet aggregation mediated by ULVWF may, however, cause thrombotic microangiopathies by occlusion of vessels, and thus restrict blood supply to vital organs. Regulation of the hemostatic activity of VWF by limiting its multimeric size is achieved by the presence of a specific VWF-cleaving metalloprotease (VWF-cp) in plasma.5,6 This enzyme was shown to cleave in the VWF-A2 domain between Tyr1605 and Met1606, generating homodimers of 176 kd and 140 kd and smaller multimers.7 Severe deficiency of VWF-cp activity (⬍5% of that in normal plasma), which leads to the persistence of ULVWF in the plasma and pathological platelet aggregation in
the microvasculature, is implicated in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), a microangiopathic disorder characterized by systemic microvascular thrombosis associated with thrombocytopenia, hemolytic anemia, and ischemic organ failure.8,9 In 2001 the N-terminal amino acid sequence of purified VWF-cp was determined10,11 and the enzyme rapidly identified as a new member of the ADAMTS (A Disintegrin-like And Metalloprotease with ThromboSpondin type 1 motif) metalloprotease family.12,13 The VWF-cleaving activity of recombinant (r)ADAMTS-13 has been functionally characterized and found to be indistinguishable from plasmaderived VWF-cp.14-17 Here we summarize the recent advances in cell culture expression and functional characterization of rADAMTS-13. The availability of rADAMTS-13 holds the prospect not only for gaining insight into the pathomechanism associated with ADAMTS-13 deficiency, and developing quick and reliable enzyme antigen and activity assays, but also of developing rADAMTS-13 as an alternative therapy to plasma infusion for treating acquired and congenital TTP and perhaps for supportive therapy for other arterial occlusion diseases.
From Baxter BioScience, Biomedical Research Center, Orth/ Donau, Austria. Address correspondence to Friedrich Scheiflinger, PhD, Baxter BioScience, Biomedical Research Center, Uferstrasse 15, Orth/Donau 2304, Austria. © 2004 Elsevier Inc. All rights reserved. 0037-1963/04/4101-0007$30.00/0 doi:10.1053/j.seminhematol.2003.10.006
ADAMTS-13: Identification, Gene Expression, and Domain Structure
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Independently and simultaneously, two groups succeeded in purifying plasma-derived VWF-cp in quantities sufficient to obtain partial N-terminal amino acid sequences.10,11 This enabled a search in the
Seminars in Hematology, Vol 41, No 1 ( January), 2004: pp 24-33
Recombinant Human ADAMTS-13
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Figure 1. Predicted primary structure of ADAMTS-13 polypeptide. S, signal peptide; P, propeptide; the TSP1 type 1 motifs are numbered from 1-8. Potential sites for N-glycosylation are indicated by closed circles. Reprinted with permission.13
genomic and expressed sequence tags databases that identified a new gene on human chromosome 9. Comparative analysis of the translated protein sequence showed that the predicted protein has significant homology to the ADAMTS zinc metalloprotease family and it was therefore designated ADAMTS13.10,12,13,18 An entirely different approach, using positional cloning by genome-wide linkage analysis in pedigrees of families of patients suffering from congenital TTP, yielded essentially the same result.19 The full-length cDNA coding for ADAMTS-13 was cloned quickly.12,13,15,18 The human ADAMTS13 gene contains 29 exons spanning approximately 37 kb on chromosome 9q34 and encodes a precursor polypeptide with 1,427 amino acid residues (Fig 1). Mature, non-posttranslational modified ADAMTS-13 has a calculated molecular mass of 145 kd, whereas protease purified from human plasma has an apparent mass of approximately 180 to 190 kd. The observed difference in the molecular weight is due to posttranslational modifications such as O- and N-linked glycosylation. This is consistent with the presence of 10 consensus sites for N-linked glycosylation, several sites potentially modified by O-linked glycosylation, and one consensus site for C-mannosylation.13 Glycosidase treatment of rADAMTS-13 showed that indeed most, but not all of the mass differences between intracellular rADAMTS-13 and the secreted form can be attributed to N-linked glycosylation14 and to O-linked glycosylation (F.S., unpublished data). Multiple human tissue Northern blotting revealed an approximately 4.7-kb mRNA transcript primarily in the liver and one shorter approximately 2.4-kb mRNA in placenta, in skeletal muscle, and in tumor cell lines derived from colon carcinoma and malignant melanoma.12,13,18 Recent in situ hybridization data revealed ADAMTS-13 expression mainly in the perisinusoidal cells of the liver.20 Semiquantitative polymerase chain reaction (PCR) with normalized first-strand cDNAs indicated moderate expression levels of full-length ADAMTS-13 mRNA in various other human tissues, but the expression was found to be at least 3,000-fold less than -actin.15 Alternatively spliced transcripts were identified that generated internal truncated ADAMTS-13 variants after
the metalloprotease domain.13,19 Smaller forms of ADAMTS-13 (170 kd, 160 kd, and 120 kd) have been isolated from human plasma11 and shown to contain N-terminal amino acid sequences identical to the mature protein. It remains to be determined if these smaller forms represent alternatively spliced isoforms or proteolytic truncation variants. Like many other large extracellular proteins, ADAMTS-13 exhibits a typical multidomain structure (Fig 1).21 Modular structures recognizable in ADAMTS-13 include a signal peptide, propeptide, metalloproteinase domain, disintegrin-like domain, central thrombospondin type 1 (TSP1) repeat, cysteine-rich domain, spacer region, a second set of seven TSP1 repeats, and two CUB domains. ADAMTS-13 contains an unusually short (45 amino acid residues) propeptide region that ends in a potential furin cleavage site (RQRR74). The catalytic domain contains a typical reprolysin-type active site sequence motif, HEXGHXXGXXHD, including three histidine residues coordinating a Zn2⫹ ion, a conserved methionine at position 249 forming the “metturn” common to all “metzincins,”22 and a Ca2⫹binding site motif comprising amino acids Glu83, Asp173, Cys281, and Asp284.13 The presence of metallion binding sites in the catalytic domain is consistent with the reported inhibition of enzymatic activity by EDTA, EGTA or 1,10-phenanthroline.5,6 Further downstream, the metalloprotease domain is followed by a domain with homology to disintegrins, a protein family frequently found in snake venoms. All 19 ADAMTS molecules isolated so far contain a highly conserved cysteine signature of eight cysteine residues in the disintegrin-like domain, but their specific function has not been identified.18,23 The presence of variable numbers of TSP1 (or thrombospondin structural homology repeat TSR) motifs distinguish the ADAMs family of proteases from ADAMTS molecules. TSP1 repeats are structural modules, of roughly 60 –amino acid length, which contain conserved cysteine and tryptophane residues and have been implicated in many biological functions (see below). The central TSP1 repeat has a complete W-XX-W motif that may be modified by C-mannosylation24 and a C-S-X-S/T-C-G sequence that is often O-glycosylated24 and might facilitate interactions
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with CD36.25,26 The first TSP1 repeat is followed by a cysteine-rich domain containing 10 conserved cysteine residues and a RGD sequence possibly important for integrin interactions. Mutation of the RGD sequence to RGE did not show any change in the protease activity in vitro.27 Most likely an assay system more closely mirroring the in vivo situation is necessary to verify experimentally whether the RGD sequence is involved in any cell surface interactions of physiological relevance in vivo. Further downstream, a spacer region, seven sequential TSP1 repeats, and two C-terminal CUB domains can be identified. CUB domains are unique to ADAMTS-1313,19 and are not found in other ADAMTS family members but are common in the related family of astacin metalloproteases.28 Interestingly, the CUB domains as well as TSP1 repeat 7 and 8 seem to be dispensable for in vivo activity in some mouse strains.29 In parallel to the isolation of the human ADAMTS13, the corresponding mouse gene was isolated from a Balb/c mouse-derived liver cDNA library (D. Vo¨ lkl, manuscript in preparation). In general, ADAMTS-13 molecular structure has been found to be conserved across mammals, birds and fish.30 Consistent with this finding, the mouse Adamts-13 protein is organized similarily to that of humans. The overall homology is 69% but a comparison of individual domains reveals some higher conserved regions, including metalloprotease domain (77%), disintegrin domain (81%) TSP1-1 repeat (88%), cysteine-rich domain (80%), and some domains with surprisingly lower conservation, such as propeptide (43%), TSP1-4 (51%), and TSP1-6 (37%). The various degrees of conservation might reflect domains of different importance in vivo or mirror the structural differences in the respective VWF substrate molecules, or both. Mouse Adamts-13 has very little activity against human VWF in vitro (B.P., unpublished data). Thus, the construction of swap mutants might allow the domains involved in substrate recognition to be identified precisely.
Recombinant ADAMTS-13 Expression in Mammalian and Insect Cells Recombinant ADAMTS-1312,13 has been transiently expressed in various mammalian cell lines, including human embryonic kidney cells (HEK 293), Chinese hamster ovary cells (CHO), human liver cells (Chang Liver, SK Hep),15 HeLa cells,14,27 COS cells, and baby hamster kidney (BHK) cells.16,31 Molecular weights reported for rADAMTS-13 are in the range of 180 to 230 kd. No obvious differences have been found in the expression rate and the functional activity of secreted rADAMTS-13 derived from cell lines, indi-
cating that the cell type has no significant effect on rADAMTS-13 synthesis and secretion. Stably transfected COS-7, CHO, and BHK cells express much less rADAMTS-13 than transiently transfected COS-7 cells, suggesting that continuous production of high levels of ADAMTS-13 is disadvantageous for cells in culture.16 ADAMTS-13 is synthesized as a zymogen and most probably secreted as active protease, that is, as protease with the propeptide clipped off. This conclusion is supported by the observation that (1) Nterminal sequences obtained from plasma-derived ADAMTS-13 correspond exactly to the sequences found downstream of the putative furin cleavage site, and (2) rADAMTS-13 cleaves rVWF under standard assay conditions5 without the need to further process or activate the protein (B.P., unpublished data) in a cell-free and plasma-free environment. In addition, evidence of the linkage of the propeptide removal to the secretion process comes from the recent finding that N-terminal His-tagged ADAMTS13, in contrast to C-terminal His-tagged ADAMTS13, is normally synthesized intracellularly but is not secreted in the supernatant (Fig 2). Expression cassettes containing full-length ADAMTS-13 with Histags inserted either N- or C-terminally to the mature ADAMTS-13 sequence were transfected in HEK 293 cells. Cell lysates and conditioned medium were analyzed by Western blotting using a monoclonal antibody raised against the catalytic domain of ADAMTS-13 (242/H1)15 or with an anti–His-tag monoclonal antibody (Fig 2). In cell lysates, both antibodies visualized a single immunoreactive polypeptide of approximately 170 kd (p170). However, analysis of the conditioned medium showed that just the C-terminal His-tagged construct was secreted, and that the N-terminal Histagged ADAMTS-13 was retained within the cell. Apparently the N-terminal His-tag affected the propeptide cleavage in some way or might otherwise have interfered with the secretion pathway. In the supernatants from ADAMTS-13 wild-type and C-terminal His-tagged ADAMTS-13–transfected cells, 242/H1 detected two predominant ADAMTS-13 species migrating at approximately 180 kd (p180) and 160 kd (p160), and an additional faint band at approximately 120 kd (p120) (Fig 2A). On the other hand, the anti–His-tag antibody recognized only p180 (obviously representing full-length ADAMTS13-His) but failed to detect p160 and p120. These results therefore suggested that p160 and p120 are derived from C-terminal proteolytic truncation of full-length ADAMTS-13. The anti–His-tag antibody also detected a faint band comigrating with intracellular ADAMTS-13, which might be derived from cell lysis, and a band at 60 to 70 kd, which could corre-
Recombinant Human ADAMTS-13
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Figure 2. Western blot of transiently expressed rADAMTS-13. Lysates and conditioned medium from transfected HEK 293 cells transiently expressing rADAMTS-13 wild type and N- or C-terminally His-tagged rADAMTS-13 were subjected to sodium dodecyl sulfate (SDS)polyacrylamide gels, blotted, and visualized by monoclonal antibody raised against the catalytic domain of ADAMTS-13 (A) and by anti-His antibody (B). The His-ladder contains 100-, 75-, and 50-kd standard proteins.
spond to the C-terminal processing product leading to the formation of p120 (Fig 2). Full-length rADAMTS-13 expressed in COS-7 and Sf9 insect cells gave rise to a C-terminal processing product of approximately 45 kd.16 It seems likely that apart from propeptide cleavage, ADAMTS-13 can undergo secondary processing events generating C-terminal truncated ADAMTS-13 polypeptides by removal of the CUB domains and probably some of the C-terminal TSP1 repeats as estimated by the size of the generated fragments (Fig 2). For example, C-terminal deletion of approximately 60 kd would lead to a truncation of ADAMTS-13 after TSP1 repeat 4 and removal of 45 kd might lead to a truncation between TSP1 repeats 6 and 8.16 The absence of the processed forms in the cell lysate suggested that processing occurs most likely during or after secretion into the culture medium. Whether truncated ADAMTS-13 species are generated by an autoproteolytic process or by cellular proteases or both, and whether they have the same substrate specificity, remains to be determined. Supposing C-terminal processing is a normal event, it is tempting to speculate that some of the truncated ADAMTS-13 isoforms found in plasma11 correspond to the observed truncated ADAMTS-13 species in cell culture. C-terminal proteolytic processing has been reported for other ADAMTS family
members such as ADAMTS-1, ADAMTS-4, and ADAMTS-12.32-34 Functionally, proteolytic deletion of varying portions of the C-terminus may alter extracellular distribution or modulate the biological property of the protein or both. Preliminary data indicate that ADAMTS-13 might also be subjected to complex proteolytic maturation processes involving C-terminal domains.18 Conceivably ADAMTS-13, particularly with respect to its modular domain organization, fulfills additional biological activities different to the VWF-cleaving activity that are possibly regulated by secondary processing events. The multiple TSP1 repeats of ADAMTS-13 are potential candidates for conferring separate functions. Among the five known members of the thrombospondin family, only TSP1 and TSP2 contain type 1 repeats. TSP1 repeats function as attachment sites for many cell types, and protein and glycosaminoglycan binding sites, and are naturally occurring inhibitors of angiogenesis.35,36 The antiangiogenic activity of TSP1 has been localized to essential residues within its type 1 repeats and is due in part to CD36-mediated inhibition of endothelial cell migration and induction of apoptosis.36-39 TSP1 motifs have been identified in many different protein families40 and also in members of the ADAMTS family. Angioinhibitory activity has been described for METH-1/ADAMTS-1 and METH-2/ADAMTS-8, as well as for their isolated
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C-terminal TSP1 fragments.41,42 It will be of considerable interest to evaluate whether ADAMTS-13 or eventually released fragments containing one or several TSP1 motifs display angioinhibitory activity, particularly with respect to a potential pathophysiological relevance associated with the observed reduced ADAMTS-13 activity in malignant patients.43-46 Somewhat unexpectedly, rADAMTS-13 or truncated fragments had no detectable binding to the cell surface of COS-7 cells and no binding to the extracellular matrix (ECM) of rat primary fibroblasts RFL-6 and COS-1 cells.16 In ADAMTS-1, ECM binding capacity has been attributed to its C-terminal region including the spacer and the TSP1.47 A truncated ADAMTS-1/METH-1 was observed in transfected cells lacking the two C-terminal TSP1 motifs and part of the spacer region, which remained soluble due to its altered affinity to the ECM and was confined to the conditioned medium.32 Nonetheless, truncated ADAMTS-1/METH-1 retained its angioinhibitory property, suggesting a biological significance of the processing event such as providing ADAMTS-1/ METH-1 as a soluble anti-angiogenic factor.32-42 Expression of rADAMTS-13 and some truncation variants by Baculovirus-infected Sf9 cells yielded proteins with apparent lower molecular mass, probably due to altered glycosylation in insect cells. Specific activities of mammalian cell– and insect cell– derived rADAMTS-13 were found to be very similar.16 Baculovirus-derived ADAMTS-13 fragments were also used to characterize ADAMTS-13 inhibitors in patients with acquired TTP.48
Protease Activity of rADAMTS-13 and Substrate Specificity The current mechanistic model to explain the proteolytic degradation of VWF in plasma predicts that unfolding of highly multimeric VWF by arterial shear stress is essential for ADAMTS-13 to access the single cleavage site, located in the VWF-A2 domain. This unique regulatory mechanism fits perfectly to the emerging concept of ADAMTS-13 being secreted as an active protease and regulation of the physiological activity by substrate cleavage site accessibility determined by substrate molecular weight and shear stress conditions. An important step in the biochemical characterization of rADAMTS-13 was to show that the recombinant enzyme cleaves the VWF substrate at the same site as the plasma-derived VWF-cp. In vitro activity of rADAMTS-13 towards the natural substrate VWF was measured in the cell lysate and the conditioned medium of transfected cells using purified human VWF as substrate. Full VWF-cleaving activity was obtained in conditioned cell culture medium.15 Unlike plasma-derived ADAMTS-13,5 we
found that the activation step (BaCl2 preincubation) is not required to obtain activity of rADAMTS-13 (B.P., unpublished data). Addition of a C-terminal His-tag sequence by genetic engineering did not observably affect in vitro activity of rADAMTS-13, but a detailed study comparing specific activities is not available. Insensitivity of the C-terminus to the addition of immune-reactive tag sequences has, however, been shown recently by various groups using wildtype and mutant rADAMTS-13 molecules.14,16,17,27 Although secreted recombinant ADAMTS-13 was found to be active, only modest cleavage activity was recovered from intracellular rADAMTS-13.15 Mixing and spiking experiments led to the conclusion that the cell lysis conditions or a putative intracellular inhibitor or both may block rADAMTS-13 activity or interfere somehow with the assay performance. Another explanation would be that incomplete posttranslational modification of intracellular ADAMTS-13 led to the observed lack of activity. Sequence specificity of VWF cleavage by rADAMTS-13 was verified by N-terminal sequence analysis of the generated VWF cleavage fragments. These sequence data clearly showed that rADAMTS-13 cleaved the peptide bond between Tyr1605 and Met1606 in mature VWF, identical to plasma-derived VWF-cp.15 Further evidence for the substrate specificity of rADAMTS-13 came from the finding that rADAMTS-13 generates the typical triplet structure of plasma-derived VWF when incubated with rVWF, which is multimeric but devoid of any sub-bands.49 Cleavage of rVWF by VWF-cp lead to a VWF banding pattern similar to plasma-derived VWF.49,50 Despite many efforts, no other substrate for ADAMTS-13 apart from VWF could be identified16 (F.S., unpublished data). Metal ion chelators inhibit ADAMTS-13 activity,5,6 but the search for a plasmatic inhibitor has been unsuccessful so far. A candidate molecule, ␣2macroglobulin, binds for example to ADAMTS-151 and to ADAMTS-12,34 but it fails to interact with ADAMTS-13.16 Due to the unique activation mechanism of ADAMTS-13, a specific plasmatic inhibitor might be physiologically unnecessary. The lack of such an inhibitor might also explain the unusually long half-life of 2 to 3 days of VWF-cp.52
ADAMTS-13 Structure–Function Relationship ADAMTS-13 is a multidomain multifunctional protein with a very complex and not yet well understood structure–activity relationship. More insight into the physiological function and the relative importance of domains for specific biological events can be gained from mutational analysis of the ADAMTS13 gene sequence. Genetic engineering allows naturally occurring and purposely designed mutations to be in-
Recombinant Human ADAMTS-13
troduced, as well as the study of the consequences of a mutation by protein expression analysis and biochemical characterization of the resultant protein. Recombinant expression of ADAMTS-13 and its variants is therefore a highly valuable tool for gaining insight into the major pathogenic features of ADAMTS-13 deficiency. A variety of missense, nonsense, deletion, and splice site mutations putatively responsible for hereditary TTP and some single-nucleotide polymorphisms (SNPs) of not yet fully explained in vivo relevance have been detected throughout the ADAMTS13 gene.14,17,19,50,53-57 ADAMTS-13 deficiency caused by missense mutations may lead to either a malfunctioning or a misfolded and therefore not efficiently secreted molecule. Of 26 missense mutations reported so far, more than 50% (n ⫽ 14) are clustered in the catalytic domain (n ⫽ 8) and in the cysteine-rich domain (n ⫽ 6). In the absence of a crystal structure and reliable quantitative assays, recombinant expression of ADAMTS-13 variants allows quick and qualitative estimates of the effect of an individual mutation on ADAMTS-13 activity. For example, consequences of mutations in the highly conserved Zn2⫹ binding motif in the metalloprotease domain are unpredictable and can lead either to an efficient secreted molecule that lacks activity (such as E225A27), or to a molecule that is retained entirely within the cell (such as L232Q54). Mutant L232Q and D235H,56 another mutant mapping to the Zn2⫹-binding domain, are naturally occurring and have been associated with severe ADAMTS-13 deficiency, thereby validating the in vitro results. To gain insight into the functional importance of the individual ADAMTS-13 domains, experimentally truncated fragments were recombinantly expressed and the retained VWF- cleaving activity was determined by two groups.16,27 Both showed that the catalytic domain alone was not able to efficiently cleave VWF, nor were constructs truncated after the disintegrin, the first TSP1 motif, or the cysteine-rich domain, although for some of the constructs, very low activity was detected (including Q449X, truncation in the cysteine-rich domain14,27). Addition of the spacer region regained some activity and further addition of the remaining TSP1 motifs and the CUB domains restored the full activity. The spacer region, and also the more proximal disintegrin- and cysteinerich domain as well as the first TSP1 repeat, was hypothesized to be involved in the substrate recognition process. However, the CUB domains could also be shown to interact with VWF and this interaction can be blocked by a CUB domain– derived peptide.58 Thus, the assembly of the substrate– enzyme complex might involve several domains of ADAMTS-13 and needs to be further analyzed.
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Expression studies also showed that introduction of nonsense mutations in the N-terminal part of the spacer region (G560X, Y570X, T581X, and P590X) destabilizes the protein structure and abolishes secretion of the mutant in the supernatant.27 Although protein expression data have already allowed certain functions to be assigned to some of the protease domains, it should be kept in mind that these conclusions resulted from a rather artificial in vitro ADAMTS-13 activity assay5,59 done under denaturing conditions, which most likely reflect only some aspects of the complex substrate– enzyme interactions in vivo. In addition, the current lack of a validated quantitative ADAMTS-13 antigen assay hampers an exact investigation of the structure–function relationship. On the other hand, sophisticated analysis systems like the use of living cells in parallelplate flow chambers60 might shed light on the multifaceted endothelial cell/VWF/ADAMTS-13 interactions, especially under high velocity and strong shear stress conditions, which are difficult to mimic in static systems. It will be interesting to compare the activity results of recombinant mutant proteins assayed in both the static and parallel-plate flow chamber assay systems.
Recombinant ADAMTS-13 in Acquired and Congenital TTP Congenital and acquired TTP have been associated with the severe lack of VWF-cp activity normally present in plasma.5,6,61-64 Positional cloning of a gene segregating with congenital VWF-cp deficiency provided strong evidence that VWF-cp is identical to ADAMTS-13.19 This assumption was formerly proved by N-terminal sequencing of the purified plasma-derived protein10,11 and ultimately confirmed by protein expression studies using rADAMTS-13. These studies unambiguously showed that ADAMTS-13 is indeed the protease missing in congenital TTP14,55 and inhibited by anti-ADAMTS-13 antibodies in acquired autoimmune TTP.15,65 Using rADAMTS-13, the ADAMTS-13–mediated degradation of VWF multimers was shown to be inhibited by the addition of plasma obtained from a patient with acquired TTP (Fig 3) and the inhibition observed to be attributable to the purified IgG fraction of plasma from TTP patients.15 Neither plasma nor purified IgG from healthy human volunteers was able to inhibit rADAMTS-13 activity, reconfirming previous findings that IgG antibodies from acquired TTP patients are specifically directed against the ADAMTS-13 protein.63,64 Supplementation of congenital deficient TTP plasma (residual ⬍5% VWFcp) with rADAMTS-13 led to a dose-dependent normalization of the VWF-cleaving activity, resulting in the degradation of VWF multimers (Fig 4), which
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Figure 3. Specific inhibition of rADAMTS-13 activity by acquired TTP patient plasma containing neutralizing antibodies. ADAMTS-13 assay mixtures were subjected to SDS–1% agarose gels and the VWF multimer pattern was detected by immunostaining. (A) Inhibitory TTP patient plasma was added (⫹) to the samples before dilution (1:20), activation, and incubation with VWF. Buffer was used instead of TTP plasma in samples marked with (-). Samples used were a normal human plasma (NHP) pool (lanes 2, 3), conditioned medium derived from transfections using the parental vector (lanes 4, 6), and the ADAMTS-13 expression vector (lanes 5, 7). VWF substrate is shown in lane 1. (B) Purified IgG from NHP or acquired TTP patient plasma and buffer as control was mixed with conditioned medium containing rADAMTS-13 either concentrated (lanes 1, 5, 9), or diluted 1:4 (lanes 2, 6, 10), 1:16 (lanes 3, 7, 11), or 1:64 (lanes 4, 8, 12). (C) NHP as source for the plasmatic ADAMTS-13 was incubated directly (lanes 1, 3, 5) or diluted 1:2 (lanes 2, 4, 6) with purified IgG from NHP or inhibiting TTP patient plasma or buffer. From: Plaimauer B, et al. Cloning, expression, and functional characterization of the von Willebrand factor-cleaving protease (ADAMTS-13). Blood 2002;100:3626-3632. Copyright American Society of Hematology, used with permission.
suggests rADAMTS-13 substitution could become an important treatment option in congenitally deficient patients.55
Implications for Treatment and Future Directions Plasma infusion and plasma exchange is currently considered to be the therapy of choice for TTP patients. It is, however, inconvenient and associated with serious complications. The identification of the ADAMTS13 gene and the functional characterization of its expressed cDNA render a replacement therapy with recombinant ADAMTS-13 conceivable. High yield fermentation of a stably producing ADAMTS-13 cell clone and the availability of purified rADAMTS-13 will substantially improve therapeutic interventions
in the upcoming future, at least for congenital and possibly also for acquired TTP patients, eventually in combination with immunosuppressive agents. Further research needs to be done to explore if supra-normal levels of rADAMTS-13 could be of clinical benefit in thrombotic microangiopathies like bone marrow transplantation–associated TTP. Supra-normal levels of rADAMTS-13 might also have applications in the therapy of arterial occlusion diseases such as stroke and acute myocardial infarction or as an adjunctive to thrombolytic therapy by tissue plasminogen activator (t-PA) to prevent restenosis. Furthermore, analytical assays such as for the determination of concentration and functional activity of ADAMTS-13 or assays for specific anti–ADAMTS-13 antibodies will be developed to improve the current diagnostic and monitoring analyses of patients with
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Recombinant Human ADAMTS-13
Figure 4. Reconstitution of congenitally ADAMTS-13 deficient TTP plasma with rADAMTS-13 leads to a dose-dependent normalization of VWF-cleaving protease activity. Conditioned medium from transfected HEK 293 cells containing recombinant ADAMTS-13 (lanes 8, 10, 12) and from transfections with the parental vector (lanes 9, 11, 13) was mixed in various volume ratios with the ADAMTS-13– deficient plasma (lane 7) of a patient with inherited TTP. These mixtures and dilutions of pooled normal human plasma (NHP) for calibration were assayed for their ability to cleave VWF. Buffer was used as control (lane 6). Analysis of VWF multimeric pattern was carried out by SDS–1.4% agarose gel electrophoresis and VWF-specific immunostaining. Reprinted with permission from Blackwell Publishing Ltd.55
clinically suspected TTP. Structural and biochemical analyses of purified ADAMTS-13 and the development of animal models mimicking TTP will aid our understanding of the biological and physiological significance of the ADAMTS-13 molecule.
Note Added in Proof After this article was submitted, a paper, “Cleavage of the ADAMTS13 Propeptide Is Not Required for Protease Activity” by Majerus EM, Zheng X, Tuley EA, and Sadler JE (J Biol Chem. 2003 Sep 15 [Epub ahead of print] PMID: 12975358 [PubMed, as supplied by publisher]), was published. Majerus et al showed that furin-deficient LoVo cells secreted ADAMTS-13 with the propeptide intact and that secreted proADAMTS-13 had normal proteolytic activity towards VWF.
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Willebrand factor to fragments produced by in vivo proteolysis. Blood 87:4223-4234, 1996 Tsai HM: Physiologic cleavage of von Willebrand factor by a plasma protease is dependent on its conformation and requires calcium ion. Blood 87:4235-4244, 1996 Dent JA, Berkowitz SD, Ware J, Kasper CK, Ruggeri ZM: Identification of a cleavage site directing the immunochemical detection of molecular abnormalities in type IIA von Willebrand factor. Proc Natl Acad Sci USA 87:6306-6310, 1990 Bukowski RM: Thrombotic thrombocytopenic purpura: A review. Prog Hemost Thromb 6:287-337, 1982 Moschcowitz E: Hyaline thrombosis of the terminal arterioles and capillaries: A hitherto undescribed disease. Proc NY Pathol Soc 24:21-24, 1924 Fujikawa K, Suzuki H, McMullen B, Chung D: Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family. Blood 98:1662-1666, 2001 Gerritsen HE, Robles R, La¨ mmle B, Furlan M: Partial amino acid sequence of purified von Willebrand factor-cleaving protease. Blood 98:1654-1661, 2001 Soejima K, Mimura N, Hirashima M, Maeda H, Hamamoto T, Nakagaki T, et al: A novel human metalloprotease synthesized in the liver and secreted into the blood: Possibly, the von Willebrand factor-cleaving protease? J Biochem (Tokyo) 130:475-480, 2001 Zheng X, Chung D, Takayama TK, Majerus EM, Sadler JE, Fujikawa K: Structure of von Willebrand factor-cleaving protease (ADAMTS13), a metalloprotease involved in thrombotic thrombocytopenic purpura. J Biol Chem 276:4105941063, 2001 Kokame K, Matsumoto M, Soejima K, Yagi H, Ishizashi H, Funato M, et al: Mutations and common polymorphisms in ADAMTS13 gene responsible for von Willebrand factorcleaving protease activity. Proc Natl Acad Sci USA 99:1190211907, 2002 Plaimauer B, Zimmermann K, Vo¨ lkel D, Antoine G, Kerschbaumer R, Jenab P, et al: Cloning, expression, and functional characterization of the von Willebrand factor-cleaving protease (ADAMTS13). Blood 100:3626-3632, 2002 Zheng X, Nishio K, Majerus EM, Sadler JE: Cleavage of von Willebrand factor requires the spacer domain of the metalloprotease ADAMTS13. J Biol Chem 278:30136-30141, 2003 Motto D, Levy GG, McGee BM, Tsai HM, Ginsburg D: ADAMTS13 mutations identified in familial TTP patients result in loss of VWF-cleaving protease activity. Blood 100: 44, 2002 (abstr) Cal S, Obaya AJ, Llamazares M, Garabaya C, Quesada V, Lopez-Otin C: 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, 2002 Levy GG, Nichols WC, Lian EC, Foroud T, McClintick JN, McGee BM, et al: Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 413:488-494, 2001 Lee T, Bouhassira EE, Lyubsky S, Tsai HM: ADAMTS13, the von Willebrand factor cleaving metalloprotease, is expressed in the perisinusoidal cells of the liver. Blood 100:1938, 2002 (abstr) Tang BL: ADAMTS: A novel family of extracellular matrix proteases. Int J Biochem Cell Biol 33:33-44, 2001 Stocker W, Grams F, Baumann U, Reinemer P, Gomis-Ruth FX, McKay DB, et al: The metzincins—Topological and sequential relations between the astacins, adamalysins, serral-
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41. Iruela-Arispe ML, Vazquez F, Ortega MA: Antiangiogenic domains shared by thrombospondins and metallospondins, a new family of angiogenic inhibitors. Ann NY Acad Sci 886: 58-66, 1999 42. Vazquez F, Hastings G, Ortega MA, Lane TF, Oikemus S, Lombardo M, et al: METH-1, a human ortholog of ADAMTS-1, and METH-2 are members of a new family of proteins with angio-inhibitory activity. J Biol Chem 274: 23349-23357, 1999 43. Mannucci PM, Karimi M, Mosalaei A, Canciani MT, Peyvandi F: Patients with localized and disseminated tumors have reduced but measurable levels of ADAMTS-13 (von Willebrand factor cleaving protease). Haematologica 88:454-458, 2003 44. Blot E, Decaudin D, Veyradier A, Bardier A, Zagame OL, Pouillart: Cancer-related thrombotic microangiopathy secondary to von Willebrand factor-cleaving protease deficiency. Thromb Res 106:127-130, 2002 45. Koo BH, Oh D, Chung SY, Kim NK, Park S, Jang Y, et al: Deficiency of von Willebrand factor-cleaving protease activity in the plasma of malignant patients. Thromb Res 105:471476, 2002 46. Oleksowicz L, Bhagwati N, DeLeon-Fernandez M: Deficient activity of von Willebrand’s factor-cleaving protease in patients with disseminated malignancies. Cancer Res 59:22442250, 1999 47. Kuno K, Matsushima K: ADAMTS-1 protein anchors at the extracellular matrix through the thrombospondin type I motifs and its spacing region. J Biol Chem 273:13912-13917, 1998 48. Luken BM, Turenhout EAM, Fijenheer R, Voorberg J: Novel approaches to determine the characteristics of ADAMTS13 inhibitors in patients with acquired TTP. J Thromb Haemost 2003 (suppl 1, abstr PO309) 49. Turecek PL, Furlan M, La¨ mmle B, Gritsch H, Richter G, Siekmann, et al: Cleavage of recombinant von Willebrand factor by a VWF-depolymerizing protease. Blood 88:326A, 1996 (abstr) 50. Schneppenheim R, Budde U, Oyen F, Angerhaus D, Aumann V, Drewke E, et al: von Willebrand factor cleaving protease and ADAMTS13 mutations in childhood TTP. Blood 101: 1845-1850, 2003 51. Kuno K, Terashima Y, Matsushima K: ADAMTS-1 is an active metalloproteinase associated with the extracellular matrix. J Biol Chem 274:18821-18826, 1999 52. Furlan M, Robles R, Morselli B, Sandoz P, La¨ mmele B: Recovery and half-life of von Willebrand factor-cleaving protease after plasma therapy in patients with thrombotic thrombocytopenic purpura. Thromb Haemost 81:8-13, 1999 53. Kentouche K, Budde U, Furlan M, Scharfe V, Schneppenheim R, Zintl F: Remission of thrombotic thrombocytopenic purpura in a patient with compound heterozygous deficiency of von Willebrand factor-cleaving protease by infusion of solvent/detergent plasma. Acta Paediatr 91:1056-1059, 2002 54. Schneppenheim R, Angerhaus D, Mainusch K, Obser T, Oyen
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F, Budde U: Comparative expression of recombinant wildtype and mutant von Willebrand factor cleaving protease ADAMTS13. J Thromb Haemost (suppl 1, abstr OC117) Antoine G, Zimmermann K, Plaimauer B, Grillowitzer M, Studt JD, La¨ mmle B, et al: ADAMTS13 gene defects in two brothers with constitutional thrombotic thrombocytopenic purpura and normalization of von Willebrand factor-cleaving protease activity by recombinant human ADAMTS13. Br J Haematol 120:821-824, 2003 Assink K, Schiphorst R, Allford S, Karpman D, Etzioni A, Brichard B, et al: Mutation analysis and clinical implications of von Willebrand factor-cleaving protease deficiency. Kidney Int 63:1995-1999, 2003 Veyradier A, Lavergne JM, Obert B, Ribba AS, Fressinaud E, Loirat C, et al: Identification of seven new candidate mutations of ADAMTS13 gene in four French families related to congenital thrombotic thrombocytopenic purpura. J Thromb Haemost (suppl 1, abstr P0310) Dong JF, Moake JL, Bernardo A, Fujikawa K, Ball C, Nolasco L, et al: ADAMTS-13 metalloprotease interacts with the endothelial cell-derivd ultra-large von Willebrand factor. J Biol Chem 278:29633-29639, 2003 Gerritsen HE, Turecek PL, Schwarz HP, La¨ mmle B, Furlan M: Assay of von Willebrand factor (vWF)-cleaving protease based on decreased collagen binding affinity of degraded vWF: A tool for the diagnosis of thrombotic thrombocytopenic purpura (TTP). Thromb Haemost 82:1386-1389, 1999 Dong JF, Moake JL, Nolasco L, Bernardo A, Arceneaux W, Shrimpton CN, et al: ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions. Blood 100: 4033-4039, 2002 Furlan M, Robles R, Solenthaler M, Wassmer M, Sandoz P, La¨ mmle B: Deficient activity of von Willebrand factor-cleaving protease in chronic relapsing thrombotic thrombocytopenic purpura. Blood 89:3097-3103, 1997 Furlan M, La¨ mmle B: Deficiency of von Willebrand factorcleaving protease in familial and acquired thrombotic thrombocytopenic purpura. Baillieres Clin Haematol 11:509-514, 1998 Furlan M, Robles R, Solenthaler M, La¨ mmle B: Acquired deficiency of von Willebrand factor-cleaving protease in a patient with thrombotic thrombocytopenic purpura. Blood 91:2839-2846, 1998 Tsai HM, Lian EC: Antibodies to von Willebrand factorcleaving protease in acute thrombotic thrombocytopenic purpura. N Engl J Med 339:1585-1594, 1998 Gerritsen HE, Robles R, La¨ mmle B, Furlan M: Partial amino acid sequence of purified von Willebrand factor-cleaving protease. Blood 98:1654-1661, 2001 Scheiflinger F, Knoebl P, Trattner B, Plaimauer B, Mohr G, Dockal M, et al: Non-neutralizing IgM and IgG antibodies to von Willebrand factor-cleaving protease (ADAMTS-13) in a patient with thrombotic thrombocytopenic purpura (TTP). Blood 102:3241-3243, 2003
P
LASMA von Willebrand factor (VWF) predominantly originates in Weibel-Palade bodies (WPB) of vascular endothelial cells. Upon stimulation by various agonists, WPB immediately release high-molecular-weight VWF rich in unusually large VWF (ULVWF). Because ULVWF binds avidly to the extracellular matrix of the vascular bed1 and to various platelet surface receptors,2 it is a very effective promoter of platelet adhesion to sites of vascular injury. Interaction of VWF with platelet receptor glycoprotein Ib␣ is of major importance for platelet aggregation and thrombus growth under high shear blood flow.3,4 Platelet aggregation mediated by ULVWF may, however, cause thrombotic microangiopathies by occlusion of vessels, and thus restrict blood supply to vital organs. Regulation of the hemostatic activity of VWF by limiting its multimeric size is achieved by the presence of a specific VWF-cleaving metalloprotease (VWF-cp) in plasma.5,6 This enzyme was shown to cleave in the VWF-A2 domain between Tyr1605 and Met1606, generating homodimers of 176 kd and 140 kd and smaller multimers.7 Severe deficiency of VWF-cp activity (⬍5% of that in normal plasma), which leads to the persistence of ULVWF in the plasma and pathological platelet aggregation in
the microvasculature, is implicated in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), a microangiopathic disorder characterized by systemic microvascular thrombosis associated with thrombocytopenia, hemolytic anemia, and ischemic organ failure.8,9 In 2001 the N-terminal amino acid sequence of purified VWF-cp was determined10,11 and the enzyme rapidly identified as a new member of the ADAMTS (A Disintegrin-like And Metalloprotease with ThromboSpondin type 1 motif) metalloprotease family.12,13 The VWF-cleaving activity of recombinant (r)ADAMTS-13 has been functionally characterized and found to be indistinguishable from plasmaderived VWF-cp.14-17 Here we summarize the recent advances in cell culture expression and functional characterization of rADAMTS-13. The availability of rADAMTS-13 holds the prospect not only for gaining insight into the pathomechanism associated with ADAMTS-13 deficiency, and developing quick and reliable enzyme antigen and activity assays, but also of developing rADAMTS-13 as an alternative therapy to plasma infusion for treating acquired and congenital TTP and perhaps for supportive therapy for other arterial occlusion diseases.
From Baxter BioScience, Biomedical Research Center, Orth/ Donau, Austria. Address correspondence to Friedrich Scheiflinger, PhD, Baxter BioScience, Biomedical Research Center, Uferstrasse 15, Orth/Donau 2304, Austria. © 2004 Elsevier Inc. All rights reserved. 0037-1963/04/4101-0007$30.00/0 doi:10.1053/j.seminhematol.2003.10.006
ADAMTS-13: Identification, Gene Expression, and Domain Structure
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Independently and simultaneously, two groups succeeded in purifying plasma-derived VWF-cp in quantities sufficient to obtain partial N-terminal amino acid sequences.10,11 This enabled a search in the
Seminars in Hematology, Vol 41, No 1 ( January), 2004: pp 24-33
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Figure 1. Predicted primary structure of ADAMTS-13 polypeptide. S, signal peptide; P, propeptide; the TSP1 type 1 motifs are numbered from 1-8. Potential sites for N-glycosylation are indicated by closed circles. Reprinted with permission.13
genomic and expressed sequence tags databases that identified a new gene on human chromosome 9. Comparative analysis of the translated protein sequence showed that the predicted protein has significant homology to the ADAMTS zinc metalloprotease family and it was therefore designated ADAMTS13.10,12,13,18 An entirely different approach, using positional cloning by genome-wide linkage analysis in pedigrees of families of patients suffering from congenital TTP, yielded essentially the same result.19 The full-length cDNA coding for ADAMTS-13 was cloned quickly.12,13,15,18 The human ADAMTS13 gene contains 29 exons spanning approximately 37 kb on chromosome 9q34 and encodes a precursor polypeptide with 1,427 amino acid residues (Fig 1). Mature, non-posttranslational modified ADAMTS-13 has a calculated molecular mass of 145 kd, whereas protease purified from human plasma has an apparent mass of approximately 180 to 190 kd. The observed difference in the molecular weight is due to posttranslational modifications such as O- and N-linked glycosylation. This is consistent with the presence of 10 consensus sites for N-linked glycosylation, several sites potentially modified by O-linked glycosylation, and one consensus site for C-mannosylation.13 Glycosidase treatment of rADAMTS-13 showed that indeed most, but not all of the mass differences between intracellular rADAMTS-13 and the secreted form can be attributed to N-linked glycosylation14 and to O-linked glycosylation (F.S., unpublished data). Multiple human tissue Northern blotting revealed an approximately 4.7-kb mRNA transcript primarily in the liver and one shorter approximately 2.4-kb mRNA in placenta, in skeletal muscle, and in tumor cell lines derived from colon carcinoma and malignant melanoma.12,13,18 Recent in situ hybridization data revealed ADAMTS-13 expression mainly in the perisinusoidal cells of the liver.20 Semiquantitative polymerase chain reaction (PCR) with normalized first-strand cDNAs indicated moderate expression levels of full-length ADAMTS-13 mRNA in various other human tissues, but the expression was found to be at least 3,000-fold less than -actin.15 Alternatively spliced transcripts were identified that generated internal truncated ADAMTS-13 variants after
the metalloprotease domain.13,19 Smaller forms of ADAMTS-13 (170 kd, 160 kd, and 120 kd) have been isolated from human plasma11 and shown to contain N-terminal amino acid sequences identical to the mature protein. It remains to be determined if these smaller forms represent alternatively spliced isoforms or proteolytic truncation variants. Like many other large extracellular proteins, ADAMTS-13 exhibits a typical multidomain structure (Fig 1).21 Modular structures recognizable in ADAMTS-13 include a signal peptide, propeptide, metalloproteinase domain, disintegrin-like domain, central thrombospondin type 1 (TSP1) repeat, cysteine-rich domain, spacer region, a second set of seven TSP1 repeats, and two CUB domains. ADAMTS-13 contains an unusually short (45 amino acid residues) propeptide region that ends in a potential furin cleavage site (RQRR74). The catalytic domain contains a typical reprolysin-type active site sequence motif, HEXGHXXGXXHD, including three histidine residues coordinating a Zn2⫹ ion, a conserved methionine at position 249 forming the “metturn” common to all “metzincins,”22 and a Ca2⫹binding site motif comprising amino acids Glu83, Asp173, Cys281, and Asp284.13 The presence of metallion binding sites in the catalytic domain is consistent with the reported inhibition of enzymatic activity by EDTA, EGTA or 1,10-phenanthroline.5,6 Further downstream, the metalloprotease domain is followed by a domain with homology to disintegrins, a protein family frequently found in snake venoms. All 19 ADAMTS molecules isolated so far contain a highly conserved cysteine signature of eight cysteine residues in the disintegrin-like domain, but their specific function has not been identified.18,23 The presence of variable numbers of TSP1 (or thrombospondin structural homology repeat TSR) motifs distinguish the ADAMs family of proteases from ADAMTS molecules. TSP1 repeats are structural modules, of roughly 60 –amino acid length, which contain conserved cysteine and tryptophane residues and have been implicated in many biological functions (see below). The central TSP1 repeat has a complete W-XX-W motif that may be modified by C-mannosylation24 and a C-S-X-S/T-C-G sequence that is often O-glycosylated24 and might facilitate interactions
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with CD36.25,26 The first TSP1 repeat is followed by a cysteine-rich domain containing 10 conserved cysteine residues and a RGD sequence possibly important for integrin interactions. Mutation of the RGD sequence to RGE did not show any change in the protease activity in vitro.27 Most likely an assay system more closely mirroring the in vivo situation is necessary to verify experimentally whether the RGD sequence is involved in any cell surface interactions of physiological relevance in vivo. Further downstream, a spacer region, seven sequential TSP1 repeats, and two C-terminal CUB domains can be identified. CUB domains are unique to ADAMTS-1313,19 and are not found in other ADAMTS family members but are common in the related family of astacin metalloproteases.28 Interestingly, the CUB domains as well as TSP1 repeat 7 and 8 seem to be dispensable for in vivo activity in some mouse strains.29 In parallel to the isolation of the human ADAMTS13, the corresponding mouse gene was isolated from a Balb/c mouse-derived liver cDNA library (D. Vo¨ lkl, manuscript in preparation). In general, ADAMTS-13 molecular structure has been found to be conserved across mammals, birds and fish.30 Consistent with this finding, the mouse Adamts-13 protein is organized similarily to that of humans. The overall homology is 69% but a comparison of individual domains reveals some higher conserved regions, including metalloprotease domain (77%), disintegrin domain (81%) TSP1-1 repeat (88%), cysteine-rich domain (80%), and some domains with surprisingly lower conservation, such as propeptide (43%), TSP1-4 (51%), and TSP1-6 (37%). The various degrees of conservation might reflect domains of different importance in vivo or mirror the structural differences in the respective VWF substrate molecules, or both. Mouse Adamts-13 has very little activity against human VWF in vitro (B.P., unpublished data). Thus, the construction of swap mutants might allow the domains involved in substrate recognition to be identified precisely.
Recombinant ADAMTS-13 Expression in Mammalian and Insect Cells Recombinant ADAMTS-1312,13 has been transiently expressed in various mammalian cell lines, including human embryonic kidney cells (HEK 293), Chinese hamster ovary cells (CHO), human liver cells (Chang Liver, SK Hep),15 HeLa cells,14,27 COS cells, and baby hamster kidney (BHK) cells.16,31 Molecular weights reported for rADAMTS-13 are in the range of 180 to 230 kd. No obvious differences have been found in the expression rate and the functional activity of secreted rADAMTS-13 derived from cell lines, indi-
cating that the cell type has no significant effect on rADAMTS-13 synthesis and secretion. Stably transfected COS-7, CHO, and BHK cells express much less rADAMTS-13 than transiently transfected COS-7 cells, suggesting that continuous production of high levels of ADAMTS-13 is disadvantageous for cells in culture.16 ADAMTS-13 is synthesized as a zymogen and most probably secreted as active protease, that is, as protease with the propeptide clipped off. This conclusion is supported by the observation that (1) Nterminal sequences obtained from plasma-derived ADAMTS-13 correspond exactly to the sequences found downstream of the putative furin cleavage site, and (2) rADAMTS-13 cleaves rVWF under standard assay conditions5 without the need to further process or activate the protein (B.P., unpublished data) in a cell-free and plasma-free environment. In addition, evidence of the linkage of the propeptide removal to the secretion process comes from the recent finding that N-terminal His-tagged ADAMTS13, in contrast to C-terminal His-tagged ADAMTS13, is normally synthesized intracellularly but is not secreted in the supernatant (Fig 2). Expression cassettes containing full-length ADAMTS-13 with Histags inserted either N- or C-terminally to the mature ADAMTS-13 sequence were transfected in HEK 293 cells. Cell lysates and conditioned medium were analyzed by Western blotting using a monoclonal antibody raised against the catalytic domain of ADAMTS-13 (242/H1)15 or with an anti–His-tag monoclonal antibody (Fig 2). In cell lysates, both antibodies visualized a single immunoreactive polypeptide of approximately 170 kd (p170). However, analysis of the conditioned medium showed that just the C-terminal His-tagged construct was secreted, and that the N-terminal Histagged ADAMTS-13 was retained within the cell. Apparently the N-terminal His-tag affected the propeptide cleavage in some way or might otherwise have interfered with the secretion pathway. In the supernatants from ADAMTS-13 wild-type and C-terminal His-tagged ADAMTS-13–transfected cells, 242/H1 detected two predominant ADAMTS-13 species migrating at approximately 180 kd (p180) and 160 kd (p160), and an additional faint band at approximately 120 kd (p120) (Fig 2A). On the other hand, the anti–His-tag antibody recognized only p180 (obviously representing full-length ADAMTS13-His) but failed to detect p160 and p120. These results therefore suggested that p160 and p120 are derived from C-terminal proteolytic truncation of full-length ADAMTS-13. The anti–His-tag antibody also detected a faint band comigrating with intracellular ADAMTS-13, which might be derived from cell lysis, and a band at 60 to 70 kd, which could corre-
Recombinant Human ADAMTS-13
27
Figure 2. Western blot of transiently expressed rADAMTS-13. Lysates and conditioned medium from transfected HEK 293 cells transiently expressing rADAMTS-13 wild type and N- or C-terminally His-tagged rADAMTS-13 were subjected to sodium dodecyl sulfate (SDS)polyacrylamide gels, blotted, and visualized by monoclonal antibody raised against the catalytic domain of ADAMTS-13 (A) and by anti-His antibody (B). The His-ladder contains 100-, 75-, and 50-kd standard proteins.
spond to the C-terminal processing product leading to the formation of p120 (Fig 2). Full-length rADAMTS-13 expressed in COS-7 and Sf9 insect cells gave rise to a C-terminal processing product of approximately 45 kd.16 It seems likely that apart from propeptide cleavage, ADAMTS-13 can undergo secondary processing events generating C-terminal truncated ADAMTS-13 polypeptides by removal of the CUB domains and probably some of the C-terminal TSP1 repeats as estimated by the size of the generated fragments (Fig 2). For example, C-terminal deletion of approximately 60 kd would lead to a truncation of ADAMTS-13 after TSP1 repeat 4 and removal of 45 kd might lead to a truncation between TSP1 repeats 6 and 8.16 The absence of the processed forms in the cell lysate suggested that processing occurs most likely during or after secretion into the culture medium. Whether truncated ADAMTS-13 species are generated by an autoproteolytic process or by cellular proteases or both, and whether they have the same substrate specificity, remains to be determined. Supposing C-terminal processing is a normal event, it is tempting to speculate that some of the truncated ADAMTS-13 isoforms found in plasma11 correspond to the observed truncated ADAMTS-13 species in cell culture. C-terminal proteolytic processing has been reported for other ADAMTS family
members such as ADAMTS-1, ADAMTS-4, and ADAMTS-12.32-34 Functionally, proteolytic deletion of varying portions of the C-terminus may alter extracellular distribution or modulate the biological property of the protein or both. Preliminary data indicate that ADAMTS-13 might also be subjected to complex proteolytic maturation processes involving C-terminal domains.18 Conceivably ADAMTS-13, particularly with respect to its modular domain organization, fulfills additional biological activities different to the VWF-cleaving activity that are possibly regulated by secondary processing events. The multiple TSP1 repeats of ADAMTS-13 are potential candidates for conferring separate functions. Among the five known members of the thrombospondin family, only TSP1 and TSP2 contain type 1 repeats. TSP1 repeats function as attachment sites for many cell types, and protein and glycosaminoglycan binding sites, and are naturally occurring inhibitors of angiogenesis.35,36 The antiangiogenic activity of TSP1 has been localized to essential residues within its type 1 repeats and is due in part to CD36-mediated inhibition of endothelial cell migration and induction of apoptosis.36-39 TSP1 motifs have been identified in many different protein families40 and also in members of the ADAMTS family. Angioinhibitory activity has been described for METH-1/ADAMTS-1 and METH-2/ADAMTS-8, as well as for their isolated
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C-terminal TSP1 fragments.41,42 It will be of considerable interest to evaluate whether ADAMTS-13 or eventually released fragments containing one or several TSP1 motifs display angioinhibitory activity, particularly with respect to a potential pathophysiological relevance associated with the observed reduced ADAMTS-13 activity in malignant patients.43-46 Somewhat unexpectedly, rADAMTS-13 or truncated fragments had no detectable binding to the cell surface of COS-7 cells and no binding to the extracellular matrix (ECM) of rat primary fibroblasts RFL-6 and COS-1 cells.16 In ADAMTS-1, ECM binding capacity has been attributed to its C-terminal region including the spacer and the TSP1.47 A truncated ADAMTS-1/METH-1 was observed in transfected cells lacking the two C-terminal TSP1 motifs and part of the spacer region, which remained soluble due to its altered affinity to the ECM and was confined to the conditioned medium.32 Nonetheless, truncated ADAMTS-1/METH-1 retained its angioinhibitory property, suggesting a biological significance of the processing event such as providing ADAMTS-1/ METH-1 as a soluble anti-angiogenic factor.32-42 Expression of rADAMTS-13 and some truncation variants by Baculovirus-infected Sf9 cells yielded proteins with apparent lower molecular mass, probably due to altered glycosylation in insect cells. Specific activities of mammalian cell– and insect cell– derived rADAMTS-13 were found to be very similar.16 Baculovirus-derived ADAMTS-13 fragments were also used to characterize ADAMTS-13 inhibitors in patients with acquired TTP.48
Protease Activity of rADAMTS-13 and Substrate Specificity The current mechanistic model to explain the proteolytic degradation of VWF in plasma predicts that unfolding of highly multimeric VWF by arterial shear stress is essential for ADAMTS-13 to access the single cleavage site, located in the VWF-A2 domain. This unique regulatory mechanism fits perfectly to the emerging concept of ADAMTS-13 being secreted as an active protease and regulation of the physiological activity by substrate cleavage site accessibility determined by substrate molecular weight and shear stress conditions. An important step in the biochemical characterization of rADAMTS-13 was to show that the recombinant enzyme cleaves the VWF substrate at the same site as the plasma-derived VWF-cp. In vitro activity of rADAMTS-13 towards the natural substrate VWF was measured in the cell lysate and the conditioned medium of transfected cells using purified human VWF as substrate. Full VWF-cleaving activity was obtained in conditioned cell culture medium.15 Unlike plasma-derived ADAMTS-13,5 we
found that the activation step (BaCl2 preincubation) is not required to obtain activity of rADAMTS-13 (B.P., unpublished data). Addition of a C-terminal His-tag sequence by genetic engineering did not observably affect in vitro activity of rADAMTS-13, but a detailed study comparing specific activities is not available. Insensitivity of the C-terminus to the addition of immune-reactive tag sequences has, however, been shown recently by various groups using wildtype and mutant rADAMTS-13 molecules.14,16,17,27 Although secreted recombinant ADAMTS-13 was found to be active, only modest cleavage activity was recovered from intracellular rADAMTS-13.15 Mixing and spiking experiments led to the conclusion that the cell lysis conditions or a putative intracellular inhibitor or both may block rADAMTS-13 activity or interfere somehow with the assay performance. Another explanation would be that incomplete posttranslational modification of intracellular ADAMTS-13 led to the observed lack of activity. Sequence specificity of VWF cleavage by rADAMTS-13 was verified by N-terminal sequence analysis of the generated VWF cleavage fragments. These sequence data clearly showed that rADAMTS-13 cleaved the peptide bond between Tyr1605 and Met1606 in mature VWF, identical to plasma-derived VWF-cp.15 Further evidence for the substrate specificity of rADAMTS-13 came from the finding that rADAMTS-13 generates the typical triplet structure of plasma-derived VWF when incubated with rVWF, which is multimeric but devoid of any sub-bands.49 Cleavage of rVWF by VWF-cp lead to a VWF banding pattern similar to plasma-derived VWF.49,50 Despite many efforts, no other substrate for ADAMTS-13 apart from VWF could be identified16 (F.S., unpublished data). Metal ion chelators inhibit ADAMTS-13 activity,5,6 but the search for a plasmatic inhibitor has been unsuccessful so far. A candidate molecule, ␣2macroglobulin, binds for example to ADAMTS-151 and to ADAMTS-12,34 but it fails to interact with ADAMTS-13.16 Due to the unique activation mechanism of ADAMTS-13, a specific plasmatic inhibitor might be physiologically unnecessary. The lack of such an inhibitor might also explain the unusually long half-life of 2 to 3 days of VWF-cp.52
ADAMTS-13 Structure–Function Relationship ADAMTS-13 is a multidomain multifunctional protein with a very complex and not yet well understood structure–activity relationship. More insight into the physiological function and the relative importance of domains for specific biological events can be gained from mutational analysis of the ADAMTS13 gene sequence. Genetic engineering allows naturally occurring and purposely designed mutations to be in-
Recombinant Human ADAMTS-13
troduced, as well as the study of the consequences of a mutation by protein expression analysis and biochemical characterization of the resultant protein. Recombinant expression of ADAMTS-13 and its variants is therefore a highly valuable tool for gaining insight into the major pathogenic features of ADAMTS-13 deficiency. A variety of missense, nonsense, deletion, and splice site mutations putatively responsible for hereditary TTP and some single-nucleotide polymorphisms (SNPs) of not yet fully explained in vivo relevance have been detected throughout the ADAMTS13 gene.14,17,19,50,53-57 ADAMTS-13 deficiency caused by missense mutations may lead to either a malfunctioning or a misfolded and therefore not efficiently secreted molecule. Of 26 missense mutations reported so far, more than 50% (n ⫽ 14) are clustered in the catalytic domain (n ⫽ 8) and in the cysteine-rich domain (n ⫽ 6). In the absence of a crystal structure and reliable quantitative assays, recombinant expression of ADAMTS-13 variants allows quick and qualitative estimates of the effect of an individual mutation on ADAMTS-13 activity. For example, consequences of mutations in the highly conserved Zn2⫹ binding motif in the metalloprotease domain are unpredictable and can lead either to an efficient secreted molecule that lacks activity (such as E225A27), or to a molecule that is retained entirely within the cell (such as L232Q54). Mutant L232Q and D235H,56 another mutant mapping to the Zn2⫹-binding domain, are naturally occurring and have been associated with severe ADAMTS-13 deficiency, thereby validating the in vitro results. To gain insight into the functional importance of the individual ADAMTS-13 domains, experimentally truncated fragments were recombinantly expressed and the retained VWF- cleaving activity was determined by two groups.16,27 Both showed that the catalytic domain alone was not able to efficiently cleave VWF, nor were constructs truncated after the disintegrin, the first TSP1 motif, or the cysteine-rich domain, although for some of the constructs, very low activity was detected (including Q449X, truncation in the cysteine-rich domain14,27). Addition of the spacer region regained some activity and further addition of the remaining TSP1 motifs and the CUB domains restored the full activity. The spacer region, and also the more proximal disintegrin- and cysteinerich domain as well as the first TSP1 repeat, was hypothesized to be involved in the substrate recognition process. However, the CUB domains could also be shown to interact with VWF and this interaction can be blocked by a CUB domain– derived peptide.58 Thus, the assembly of the substrate– enzyme complex might involve several domains of ADAMTS-13 and needs to be further analyzed.
29
Expression studies also showed that introduction of nonsense mutations in the N-terminal part of the spacer region (G560X, Y570X, T581X, and P590X) destabilizes the protein structure and abolishes secretion of the mutant in the supernatant.27 Although protein expression data have already allowed certain functions to be assigned to some of the protease domains, it should be kept in mind that these conclusions resulted from a rather artificial in vitro ADAMTS-13 activity assay5,59 done under denaturing conditions, which most likely reflect only some aspects of the complex substrate– enzyme interactions in vivo. In addition, the current lack of a validated quantitative ADAMTS-13 antigen assay hampers an exact investigation of the structure–function relationship. On the other hand, sophisticated analysis systems like the use of living cells in parallelplate flow chambers60 might shed light on the multifaceted endothelial cell/VWF/ADAMTS-13 interactions, especially under high velocity and strong shear stress conditions, which are difficult to mimic in static systems. It will be interesting to compare the activity results of recombinant mutant proteins assayed in both the static and parallel-plate flow chamber assay systems.
Recombinant ADAMTS-13 in Acquired and Congenital TTP Congenital and acquired TTP have been associated with the severe lack of VWF-cp activity normally present in plasma.5,6,61-64 Positional cloning of a gene segregating with congenital VWF-cp deficiency provided strong evidence that VWF-cp is identical to ADAMTS-13.19 This assumption was formerly proved by N-terminal sequencing of the purified plasma-derived protein10,11 and ultimately confirmed by protein expression studies using rADAMTS-13. These studies unambiguously showed that ADAMTS-13 is indeed the protease missing in congenital TTP14,55 and inhibited by anti-ADAMTS-13 antibodies in acquired autoimmune TTP.15,65 Using rADAMTS-13, the ADAMTS-13–mediated degradation of VWF multimers was shown to be inhibited by the addition of plasma obtained from a patient with acquired TTP (Fig 3) and the inhibition observed to be attributable to the purified IgG fraction of plasma from TTP patients.15 Neither plasma nor purified IgG from healthy human volunteers was able to inhibit rADAMTS-13 activity, reconfirming previous findings that IgG antibodies from acquired TTP patients are specifically directed against the ADAMTS-13 protein.63,64 Supplementation of congenital deficient TTP plasma (residual ⬍5% VWFcp) with rADAMTS-13 led to a dose-dependent normalization of the VWF-cleaving activity, resulting in the degradation of VWF multimers (Fig 4), which
30
Plaimauer and Scheiflinger
Figure 3. Specific inhibition of rADAMTS-13 activity by acquired TTP patient plasma containing neutralizing antibodies. ADAMTS-13 assay mixtures were subjected to SDS–1% agarose gels and the VWF multimer pattern was detected by immunostaining. (A) Inhibitory TTP patient plasma was added (⫹) to the samples before dilution (1:20), activation, and incubation with VWF. Buffer was used instead of TTP plasma in samples marked with (-). Samples used were a normal human plasma (NHP) pool (lanes 2, 3), conditioned medium derived from transfections using the parental vector (lanes 4, 6), and the ADAMTS-13 expression vector (lanes 5, 7). VWF substrate is shown in lane 1. (B) Purified IgG from NHP or acquired TTP patient plasma and buffer as control was mixed with conditioned medium containing rADAMTS-13 either concentrated (lanes 1, 5, 9), or diluted 1:4 (lanes 2, 6, 10), 1:16 (lanes 3, 7, 11), or 1:64 (lanes 4, 8, 12). (C) NHP as source for the plasmatic ADAMTS-13 was incubated directly (lanes 1, 3, 5) or diluted 1:2 (lanes 2, 4, 6) with purified IgG from NHP or inhibiting TTP patient plasma or buffer. From: Plaimauer B, et al. Cloning, expression, and functional characterization of the von Willebrand factor-cleaving protease (ADAMTS-13). Blood 2002;100:3626-3632. Copyright American Society of Hematology, used with permission.
suggests rADAMTS-13 substitution could become an important treatment option in congenitally deficient patients.55
Implications for Treatment and Future Directions Plasma infusion and plasma exchange is currently considered to be the therapy of choice for TTP patients. It is, however, inconvenient and associated with serious complications. The identification of the ADAMTS13 gene and the functional characterization of its expressed cDNA render a replacement therapy with recombinant ADAMTS-13 conceivable. High yield fermentation of a stably producing ADAMTS-13 cell clone and the availability of purified rADAMTS-13 will substantially improve therapeutic interventions
in the upcoming future, at least for congenital and possibly also for acquired TTP patients, eventually in combination with immunosuppressive agents. Further research needs to be done to explore if supra-normal levels of rADAMTS-13 could be of clinical benefit in thrombotic microangiopathies like bone marrow transplantation–associated TTP. Supra-normal levels of rADAMTS-13 might also have applications in the therapy of arterial occlusion diseases such as stroke and acute myocardial infarction or as an adjunctive to thrombolytic therapy by tissue plasminogen activator (t-PA) to prevent restenosis. Furthermore, analytical assays such as for the determination of concentration and functional activity of ADAMTS-13 or assays for specific anti–ADAMTS-13 antibodies will be developed to improve the current diagnostic and monitoring analyses of patients with
31
Recombinant Human ADAMTS-13
Figure 4. Reconstitution of congenitally ADAMTS-13 deficient TTP plasma with rADAMTS-13 leads to a dose-dependent normalization of VWF-cleaving protease activity. Conditioned medium from transfected HEK 293 cells containing recombinant ADAMTS-13 (lanes 8, 10, 12) and from transfections with the parental vector (lanes 9, 11, 13) was mixed in various volume ratios with the ADAMTS-13– deficient plasma (lane 7) of a patient with inherited TTP. These mixtures and dilutions of pooled normal human plasma (NHP) for calibration were assayed for their ability to cleave VWF. Buffer was used as control (lane 6). Analysis of VWF multimeric pattern was carried out by SDS–1.4% agarose gel electrophoresis and VWF-specific immunostaining. Reprinted with permission from Blackwell Publishing Ltd.55
clinically suspected TTP. Structural and biochemical analyses of purified ADAMTS-13 and the development of animal models mimicking TTP will aid our understanding of the biological and physiological significance of the ADAMTS-13 molecule.
Note Added in Proof After this article was submitted, a paper, “Cleavage of the ADAMTS13 Propeptide Is Not Required for Protease Activity” by Majerus EM, Zheng X, Tuley EA, and Sadler JE (J Biol Chem. 2003 Sep 15 [Epub ahead of print] PMID: 12975358 [PubMed, as supplied by publisher]), was published. Majerus et al showed that furin-deficient LoVo cells secreted ADAMTS-13 with the propeptide intact and that secreted proADAMTS-13 had normal proteolytic activity towards VWF.
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