An IAP retrotransposon in the mouse ADAMTS13 gene creates ADAMTS13 variant proteins that are less effective in cleaving von Willebrand factor multimers

Abstract

Severe deficiency of ADAMTS13, a von Willebrand factor (VWF)-cleaving metalloprotease, causes thrombotic thrombocytopenic purpura. When analyzed with VWF multimers, but not with an abbreviated VWF peptide (VWF73) as the substrate, the plasma ADAMTS13 activity levels of mouse strains segregated into a high and a low group that differed by approximately 10 fold. Low ADAMTS13 activity was detected in mice containing 2 alleles of intracisternal A-type particle (IAP) retrotransposon sequence in the ADAMTS13 gene. Molecular cloning of mouse ADAMTS13 identified 2 truncated variants (IAP-a and IAP-b) in the low-activity mice. Both of the IAP variants lacked the 2 carboxyl terminus thrombospondin type 1 repeat (TSR) and CUB domains of full-length ADAMTS13. The IAP-b variant also had splicing abnormalities affecting the spacer domain sequence and had miniscule enzymatic activity. Compared with full-length ADAMTS13, the IAP-a variant was approximately one ninth as active in cleaving VWF multimers but was only slightly less active in cleaving VWF73 peptide. Recombinant human ADAMTS13 was also less effective in cleaving VWF multimers than VWF73 when the C-terminal TSR sequence was deleted. In summary, the carboxyl terminus TSR sequence is important for cleaving VWF multimers. Assay results should be interpreted with caution when peptide substrates are used for analysis of variant ADAMTS13 proteins.

Figure 1

Comparison of ADAMTS13 activity in 2 mouse strains. (A) SDS-PAGE and immunoblotting depicting VWF proteolytic fragments (176-kDa and 140-kDa fragment dimmers) generated from cleavage of exogenous VWF multimers by ADAMTS13 in a normal human plasma, a FVB/NJ plasma, and a C57BL/6J plasma. VWF multimers were incubated with each plasma sample at the indicated dilution in the presence or absence of EDTA. The increased optic density in the absence of EDTA represents the product of proteolysis. (B) A plotting of the level of the 176-kDa fragment dimer against the plasma concentration. At each dilution, the FVB/NJ plasma was more active than the normal human plasma or the C57BL/6J plasma in generating the proteolytic fragment. (C) A dot plot of the ADAMTS13 activity levels in 18 FVB/NJ and 18 C57BL/6J mice. The mean and standard deviation are also shown for each group (P < .001).

Figure 2

A composite of agarose gels depicting the products of mouse ADAMTS13 mRNA by RT-PCR. mRNA isolated from the livers of a FVB/NJ mouse and a C57BL/6J mouse was reverse transcribed and amplified with the indicated primer pairs. The analysis yielded similar levels of products with primer pair PP2 but much lower levels of products with primer pairs PP3 or PP4 in C57/BL/6J mice, suggesting that most of the mRNA was truncated. The approximate locations of the primers are illustrated in the bottom panel.

Figure 3

Mapping of coding sequences of mouse ADAMTS13 cDNA and the domain structures of the predicted proteins. (A) A full-length coding sequence consisting of 29 exons was cloned from a FVB/NJ mouse, whereas 2 coding sequences (IAP-a and IAP-b) were cloned from a C57BL/6J mouse. Both IAP variants ended with an exon consisting of sequences from the LTR of IAP and its 5′ flanking intronic sequence. IAP-b differed from IAP-a in that its exon 15 contained extra residues from intron 15 and lacked the exon 16 of IAP-a. As estimated by real-time RT-PCR using 2 primer pairs indicated in this panel, IAP-b accounted for 26% of the total ADAMTS13 mRNA. (B) The predicted domain structures of the 3 forms of ADAMTS13 cloned from mouse livers. Both IAP variants ended with a 16-residue sequence derived from the aberrant exon 24. A 61-residue sequence in the spacer domain of IAP-a was replaced by a novel sequence of 52 residues encoded by the extraneous sequence of exon 15.

Figure 4

Synthesis and secretion of mouse ADAMTS13. Mouse ADAMTS13 cDNA constructs were used to transfect HEK 293T cells. The synthesized proteins were separated by SDS-PAGE and visualized by immunoblotting with ant-V5 tag sequence. A representative composite gel of ADAMTS13 proteins in the cell lysates and culture media for each cDNA. The cell lysates were diluted by 2-fold for the analysis.

Figure 5

Substrate-dependent variation of VWF cleaving activity of mouse plasma samples and recombinant mouse ADAMTS13 proteins. (A) Activity levels of FVB/NJ and C57BL/6J plasma samples against VWF multimers (multimers), human VWF73 fusion peptide (hVWF73), and mouse VWF73 fusion peptide (mVWF73). Mouse plasma samples were assayed for activity of cleaving the indicated substrates, and all values were expressed using normal human plasma as the reference. Each column in the graph represents the mean and standard deviation of results from 23 FVB/NJ or 21 C57BL/6J different mouse plasma samples for VWF multimers and 5 different plasma samples each for the VWF peptide substrates. FVB/NJ plasma ADAMTS13 was much more effective than C57BL/6J plasma in cleaving VWF multimers (P < .001). This difference diminished from 11- to 1.6-fold when either human or mouse VWF73 was used as the substrate. (B) Activity levels (means and standard deviations) of recombinant full-length ADAMTS13 protein and its IAP-a variant against various types of substrates. FRETS indicates FRETS-VWF73. Recombinant proteins were assayed for enzymatic activity against the indicated substrates. All values were expressed against normal human plasma as the reference. Each column represents the mean and standard deviation of results obtained with 3 lots of the protein. The IAP-a protein was less effective than full-length mouse ADAMTS13 in cleaving VWF multimers (P < .001). The difference diminished from 9- to 1.4- to 2.0-fold when VWF73 peptides were used as the substrates.

Figure 6

Comparison of the activity levels of recombinant human ADAMTS13 and its truncated variants against VWF multimers or human VWF73 fusion peptide. (A) The lengths of the recombinant proteins used in the experiment are depicted against the domain structure of ADAMTS13. (B) The activity levels of each protein against VWF multimers (■) or human VWF73 fusion peptide (▩) are depicted. Recombinant proteins were assayed for activity of cleaving either VWF multimers or human VWF73 fusion peptide. All values were expressed using normal human plasma as the reference. Each column represents the mean and standard deviation of results obtained from 3 lots of the protein. The inset shows a magnification of the results for AD2. AD2 and AD5 were relatively less effective in cleaving VWF73 multimers than cleaving VWF73 peptide (P < .05 and < .001, respectively). No such substrate-dependent difference was observed for AD6 or AD7.