Arg-Ser-775 , 792 and 823 in Spacer Region of ADAMTS-18 Is Critical for Thrombin Cleavage

Cleavage of ADAMTS-18 by thrombin represents a new mechanism of platelet thrombus clearance via the release of active ~45-kDa C-terminal fragments that induces oxidative platelet fragmentation. The exact cleavage sites remain unclear, but Arg (R)775/Ser (S)776 in spacer region of ADAMTS-18 has been shown to be one of the cleavage sites of thrombin. Here, we demonstrate that R792/S793 and R823/S824 are also thrombin cleavage sites by sequence analysis, amino acid mutation and mass spectrometry assay. All these cleavage sites are thrombin-specific and insensitive to other enzymes tested (e.g. cathepsin D or trypsin). Simultaneous mutation of R775, 792, 823 to S775, 792, 823 in ADAMTS-18 completely abrogated the cleavage by thrombin and the generation of active C-terminal 45-kDa fragments. Together with previous study, a total of three thrombin-specific cleavage sites have been identified in spacer region of ADAMTS-18.


Introduction
ADAMTS (a disintegrin and metalloproteinase domain, with thrombo spondin type-1 modules) is a family of 19 secreted Zn-metalloproteinases, which have multidomain structural components in common [1].These include an N-terminal signal peptide, followed by a pro-domain, a metalloproteinase catalytic domain with a zinc binding motif, a disintegrin-like domain, a central thrombospondin type-1-like repeat (TSR), a cysteine rich domain (high sequence homology), a spacer region, and a variable number of C terminal TSR repeats.This family plays important roles in several pathophysiological conditions mainly including arthritis [2], spermatogenesis [3], angiogenesis [4] [5], and thrombosis-related disease [6].Noteworthily, most of these activities are related to proteolytic processing within their C-terminal regions [6]- [9].
ADAMTS-18 has the similar domain organization as other family members.ADAMTS-18 has been shown to be epigenetically silenced in multiple carcinomas and has tumor suppressor activity [10].Mutation of ADAMTS-18 is strongly associated with colorectal cancer [11].The data from National Center for Biotechnology Information (NCBI) subject's gene expression omnibus (GEO) also showed that ADAMTS-18 gene was differentially expressed in subjects with normal skeletal fracture versus subjects with nonunion skeletal fracture [12].Therefore, it is also associated with bone mineral density (BMD) determination in the major human ethnic groups.Recently, some studies indicate that the ADAMTS-18 gene is also play a crucial role in early eye development [13].
Platelet integrin αIIbβ3 (GPIIb/IIIa) is a heterodimeric receptor of the integrin family expressed at high density (50,000 -80,000 copies/cell) on the platelet plasma membrane [14].GPIIIa49-66 (CAPESIEFPVSEAREVLED) is a linear epitope of integrin subunit β3 (GPIIIa) in its extracellular domain.We previously reported that the unique feature of the antibodies (Abs) against GPIIIa49-66 was their ability to induce reactive oxygen species (ROS) through the activation of 12-lipoxygenase and nicotinamide adenine dinucleotide phosphate oxidase (NADPH), leading to complement-independent platelet fragmentation [15] [16].Recently, we revealed that ADAMTS-18 was the physiologic ligand of platelet GPIIIa49-66 [17].Thrombin generated from the endothelium of vessel injury is able to cleave ADAMTS-18.The generated ~45-kDa C-terminal cleavage product of ADAMTS-18 becomes activated.It clusters the β3 integrins and induces oxidative platelet fragmentation as we previously described anti-GPIIIa 49 -66 Ab [17].We have identified that R 775 /S 776 in spacer region of ADAMTS-18 is one of the potential cleavage sites of thrombin [18].However, sequence analysis indicates that there still exist two same sites in spacer region neighboring R 775 /S 776 named R 792 /S 793 and R 823 /S 824 , which also generate similar ~45-kDa C-terminal products in theory when cleaved by thrombin.In this study, we have investigated other thrombin cleavage sites in spacer region of ADAMTS-18 through amino acid mutation and mass spectrometry assay.

In Vitro DNA Translation and Thrombin Cleavage Assay
Biotinylated-methionine-labeled ADAMTS-18 or its mutant was translated using an in vitro Transcend™ Biotinylated Translation Detection Systems following the protocol provided by the manufacturer.All the peptides or translated proteins were then digested by thrombin or cathepsin D or trypsin according to the protocol provided by the manufacture.

Immunoblotting
In vitro translation products were separated by 12% SDS/PAGE gels, transferred to a nitrocellulose membrane, and immunoblotted with horseradish peroxide (HRP) conjugated avidin for 1 hour followed by washing with PBST (0.1% Tween 20).The signal band was detected by chemiluminescence substrate [18].

Mass Spectrometry
Mass spectrometry was performed as previously described [18].Briefly, for analysis of ADAMTS-18 cleavage products, a fresh mixture of enzyme and ADAMTS-18 peptide was submitted to molecular weight determination by Matrix assisted laser desorption ionization quadrupole time of flight (MALDI-QTOF) mass spectrometry (MS) (Applied Biosystems 4700 Proteomics Analyzer).To determine the amino acid sequences of newly observed peaks, MS/MS peptide de novo sequencing using a specific software program (Applied Biosystems DeNovo Explorer) was performed.

Thrombin Cleavage of C-Terminal ADAMTS-18 on Several Sites
Previous study has shown that the full-length ADAMTS-18 is proteolyzed by thrombin and results in ~45-kDa C-terminal fragments releasing [17].The optimal cleavage site for thrombin is R/X [X refers to nonacidic amino acid mainly including R, lys (K), His (H), and Ser (S)] (Figure 1(a)).We have demonstrated that R 775 /S 776 in spacer region of ADAMTS-18 is the potential cleavage site of thrombin [18].However, analysis of the primary amino acid sequence of ADAMTS18 revealed that there exist three similar thrombin cleavage sites in ADAMTS-18 spacer region named R 775 /S 776 , R 792 /S 793 and R 823 /S 824 (Figure 1(b)).The possible molecular weight from these predicted sites to C terminal is ~49-, 47-and 43-kDa, respectively (Figure 1(c)).Therefore, it remained uncertain whether R 792 /S 793 and R 823 /S 824 were also the actual sites of proteolysis.3) Similar results were obtained with P06728 (ELQVSS SYLAVRSLSQKYYLTGGWSID), which covers R 792 /S 793 producing two peptide peaks (~1717.9Da and 1351.52) at R 792 /S 793 site (Table 1).We also incubated these peptides with other enzyme cathepsin D or trypsin, and assayed by mass spectrometry.It demonstrated that these cleavage sites are thrombin-specific, and insensitive to cathepsin D or trypsin (Table 1).

Specificity of Thrombin for Site-Mutated ADAMTS-18 Full-Length Protein
Since R/S 775 , 792 and 823 in spacer region of ADAMTS-18 are critical for thrombin cleavage, we further constructed mammalian expression vector in which all these susceptible sites were mutated to S/S-775 , 792 and 823 (Figure 4(a)).Bio-methionine-labeled ADAMTS18 and site-mutant ADAMTS-18 were synthesized with in vitro translation system using the expression vector of pBudCe 4.1/ADAMTS-18.Both pBudCe 4.1/ADAMTS-18 (lane 2) and its mutant (lane 4) demonstrated two dominant bands of ~135 kDa and ~75 kDa.The ~135 band represented intact ADAMTS-18 (1221 amino acids), and ~75 kDa band does represent the short form of ADAMTS-18 since luciferase control (lane 1) and the empty vector (lane 3) did not transcribe these two bands (Figure 4(b)).The expression of ~75 kDa short form of ADAMTS-18 is consistent with our previous report [18].Figure 4(c) demonstrates wide type ADAMTS-18 is proteolyzed by thrombin, and the cleavage fragment is about ~45-kDa.However, thrombin had no effect on mutated ADAMTS-18 in various concentrations (Figure 4(d)).

Discussion
Despite the similarity shared by ADAMTS family members, most differences among them are found in the Cterminal domains of the protein, suggesting that the C-terminal domains of ADAMTS may determine their in vivo location and substrate specificity [19]- [22].C-terminal processing has been shown in ADAMTS-1 [19] [21], ADAMTS-4 [20] [22], ADAMTS-8 [4], ADAMTS-9 [5], and ADAMTS-13 [6].This splicing will shed light on the biological function of these important proteins.Noteworthily, most of cleavage events occur within the spacer region [19]- [22].We previously reported that cleavage of ADAMTS-18 by thrombin represent a novel mechanism for platelet thrombus clearance [17].The release of the active 45-kDa C-terminal fragment could regulate thrombus size by inducing oxidative platelet fragmentation.In this study, we first revealed that the R 775 /S 776 , R 792 /S 793 and R 823 /S 824 in spacer region of ADAMTS-18 are critical for thrombin cleavage.This cleavage region is similar to those of the ADAMTS family members reported previously [19]- [22].Physiologically, thrombin is generated rapidly, and at high local concentrations during the normal hemostatic response.We found that ADAMTS-18 was proteolyzed by thrombin at a high thrombin concentration, whereas low thrombin concentration had undetectable cleavage effect on ADAMTS-18 which mimics some physiological conditions, especially platelet thrombus formation.It is of interest in this regard that ADAMTS-13 has recently been shown to be inactivated by thrombin contributing to the loss of ADAMTS-13 VWF cleavage function [6].Furthermore, ADAMTS-13 has been reported to limit platelet thrombus formation in a shear rate dependent platelet thrombus model on collagen, by its cleavage of ultra large VWF [23].
In present study, we also find ~75-kDa band which is from ADAMTS-18 cDNA in in vitro translation assays.Thrombin or other enzyme (cathepsin D or trypsin) had no effect on the generation of this band.Thus, it is likely other mechanism has been involved in the ADAMTS-18 processing.

Conclusion
In summary, this report provides a direct proof that the existence of C-terminal proteolytic cleavage sites of ADAMTS-18 by thrombin, has potential drug application in dissolution of arterial thrombi.

Figure 1 .
Figure 1.Putative thrombin cleavage sites in ADAMTS-18 spacer region.(a) The optimal cleave site for thrombin.(b) Diagram of ADAMTS-18 domain structure.Location of predicted thrombin cleavage sites after Arg (R) 775 , R 792 and R 823 are shown above.(c) The possible molecular weight from these predicted cleavage sites to C-terminal of ADAMTS-18.

Figure 2 .
Figure 2. Susceptibility of thrombin for R 823 /S 824 site of ADAMTS18.(a) (b) Synthesized 27-mer ADAMTS-18 peptide P06594 containing R 823 /S 824 (~3134.5 Da) was incubated with PBS (a) or 5 U/ml thrombin (b) for 1 h.The putative thrombin cleavage site was confirmed by MALDI QTOF mass spectrometry.(c) The releasing of 15-mer N terminal peptide (MW 1746.6) when P06594 was cleaved by different concentration of thrombin at R 823 /S 824 site.(d) (e) Mutated 27-mer ADAMTS-18 peptide P06595 (~3064.5 Da, R 823 switches to S 823 ) was incubated with PBS (d) or thrombin (e) in the same condition as P06594 and analyzed by mass spectrometry.Proteolysis after R 823 was prevented by substitution of the arginine (Arg, R) to serine (Ser, S).(f) No 15-mer N terminal peptide (MW 1746.6) was released when P06595 was cleaved by different concentrations of thrombin at S 823 /S 824 site.

Figure 3 .
Figure 3. Inhibition effect of hirudin on thrombin cleavage.Synthesized 27-mer ADAMTS18 peptide P06594 was incubated with 5 U/ml thrombin and equal amount of hirudin for 1 h and analyzed by mass spectrometry.Representative mass spectrometry map showed proteolysis after R 823 was completely inhibited by the addition of hirudin.

Table 1 .
Analysis of thrombin cleavage sites in ADAMTS-18 protein.