At least three human ADAMTS13 splice variants involving combinations of exons 8 and 25 are currently known and sequenced (isoform 1 (NM_139025), isoform 2 (NM_139027) and isoform 3 (NM_139026)). In order to better understand ADAMTS13 splicing patterns and guide our investigation of unidentified splicing patterns, we first computationally examined the underlying mechanisms and characterized another previously unidentified splicing pattern. We specifically analyzed: (i) splice site strengths (using MAXENT (13
)), (ii) evolutionary conservation, (14
), (iii) splicing regulators, (iv) published splicing-related mutations (1
) and (v) SNP databases.
We first calculated, using MaxEntScan, the splice site strengths by scoring ADAMTS13 exon-intron boundaries. Notably, the 5′ ss (splice site) in the 25th
intron of wild type (isoform 1) showed a large score reduction (Supplement Fig. 1
), suggesting that alternative splicing is likely at exon 25 and could contribute to transcript heterogeneity in this area due to a weak 5′ intronic ss. In fact, an upstream alternative 5′ ss was identified (Supplement Fig. 2
, 5′ ss (i)) which gives rise to a shorter 25th
exon (longer corresponding intron) seen in isoforms 2 and 3. This second splice site has a higher MAXENT score relative to its counterpart (Supplement Fig. 1
, compare 25_L to 25_S). We next analyzed the conservation of intron 25 in ADAMTS13, which is very weak (Supplement Fig. 2
), suggesting that either the intron retention event is not regulated through intronic sequences or that this event is unique to humans. Because splicing regulatory sequences participate in defining exon-intron boundaries, we then asked whether intron 25 or its flanking exons contain splicing signals that impact the use of intron 25 splice sites. We analyzed known regulatory sequences from high-throughput studies (15
). Within the human mRNA transcript, splicing regulatory sequences in the retained intron were not relatively more frequent than in any other intron of the transcript. We did find several insertions in the human intron that might lead to an intron inclusion event. In particular, in Supplement Fig. 3
, the regions marked as 2, 3 and 5 exhibit 6, 6, and 4 potential exonic enhancers, respectively, in the human intron, compared to none in the mouse intron.
In order to validate our computational analysis, we systematically scanned 11 tissues and 3 cell lines for the existence of intron 25 splicing isoforms. Interestingly, splice variants 2 and 3 were not detected in whole tissue screens (). However, an ensuing study of hepatic stellate cells (LX2), a cell line that represents an important non-parenchymal population of liver cells (10
) which significantly contributes to plasma ADAMTS13 (9
), revealed a new splice isoform in which the entire 25th ADAMTS13 intron is retained (termed here IR-25) (). Because hepatic stellate cells represent a minor portion (5–8%) of the cells that comprise the liver, the apparent lack of IR-25 in total liver tissue likely represents dilution of this isoform amidst a large amount of RNA derived from hepatocytes and other resident populations. A hepatoma cell line, Hep3B, also displayed this novel transcript (). At least two other shorter, less abundant transcripts were observed in .. One of these transcripts was identified as an additional new variant that not only retains intron 25, but also lacks exon 28 (data not shown). The resulting protein is biologically indistinct from IR-25 at the protein level due to a premature stop codon.
Sequencing the amplified exonic boundary confirmed that the entire 25th
intron (465 bps) is included in IR-25. The intron retention is unique in that the retained intron includes a premature stop codon in the open reading frame following exon 25. As a result, IR-25 is truncated, lacking both C-terminal CUB domains and instead carries 64 novel amino acids, starting from amino acid #1190: SYVLSSFLSG SCCRRGGQRH LPLGRTGTST WSLGCVPGRP GSGLALFLPG KAKPPFYYYQ GEVT. There are two possible scenarios following this intron retention event: (i) mRNA degradation through Nonsense Mediated mRNA Decay; (ii) synthesis of a truncated protein. If the IR-25 mRNA is translated, a shortened protein product would be generated which lacks both CUB domains and instead carries 64 intron-encoded amino acids (the first intronic amino acid matches the first amino acid in exon 26) and bears 134 kDa. To test these possibilities, isoform 1 (WT) or IR-25 were transiently transfected into HEK293 cells, and harvested 24 hours later. We have demonstrated earlier that Western blotting can not detect endogenous expression of ADAMTS13 in Hek293  and therefore we have use a specific monoclonal antibody to detect the recombinant forms in these cells. Western blotting revealed that cells transduced with IR-25 maintain most of ADAMTS13 intracellularly (, Whole cell). In fact, IR-25 intracellular expression is only slightly lower (~15%) than its WT counterpart according to band densitometry. The aberrant splicing form, however, is not secreted at appreciable levels into culture media (, Media). Similar expression patterns were obtained when using Chinese hamster ovary cells for transfection, indicating that this phenomenon is generally applicable. The intracellularly accumulated protein does retain its ability to recognize and specifically cleave VWF substrate (). These findings are consistent with previous studies of a frameshift mutation within the first CUB domain of ADAMTS13 as well as another mutation in the second CUB domain, both of which produce truncated forms of ADAMTS13 that are not sorted nor secreted properly yet display specific activity against a VWF substrate (1
). In accordance with the literature and the results presented here, an absence of both CUB domains in IR-25 may result in impaired structural integrity and render it non-processive within the cellular secretory system. The additional 64 amino acids found on the C-terminus of IR-25 are unlikely to adversely affect production or impart abnormal cellular localization. Indeed, an application that predicts subcellular localization signals, PSORT (www.psort.org
), indicates that the 64 novel amino acids are unlikely to include a new sorting motif (data not shown).
Fig. 2 Expression and functional characterization of the human ADAMTS13 wild type (WT) and intron retention isoforms (IR-25). (A) Western blot analysis of WT and IR-25 protein expression 24 hours following transient transfection of HEK293 cells. Thirty μg (more ...)
IR-25 ADAMTS13’s extracellular exclusion underscores its potential clinical relevance. A decreased level of plasma ADAMTS13 stemming from changes to the relative abundance of WT ADAMTS13 and IR-25 could induce thrombotic conditions such as TTP. An imbalance towards increasing amounts of IR-25 could arise in two ways. It could develop under the influence of mutations or SNPs in ADAMTS13
that promote intron retention at the exon 25–26 junction, but those have yet to be identified. The imbalance could also occur via trans-factors affecting splicing characteristics, namely pre-mRNA splicing machinery. We were able to detect IR-25 within human lung cancer cell line, but not in normal lung cells () as well as in three other cancer tissues (glioma, breast and weak signal in sarcoma) (). It is therefore probable that a characteristic inherent to many types of cancer could contribute to the manifestation of IR-25. Indeed, cancerous cells are specifically known to produce a higher number of aberrant intron retention events (17
). This feature is conjectured to result from changes in the abundance of splicing factors or their abnormal phosphorylation. The appearance of IR-25 in hepatoma cells can thus be reasonably inferred, but its presence in hepatic stellate cells remains mechanistically unclear.
Twenty percent of cancer patients experience thrombosis at some time during the course of their disease (18
). The risk of venous thromboembolic events is particularly higher in cancers of the liver, pancreas, ovary and lung. The exact mechanism underlying the cancer-associated hypercoagulable state is not fully understood. In this report we describe in vitro
experiments which show a decreased secretion of IR-25 ADAMTS13 form. It is possible to speculate that this altered form of ADAMTS13 may contribute to the hypercoagulable state in cancer patients in different mechanisms, such as competitive inhibition of the normal form at various levels. A physiologically dominate defected ADAMTS13 which appears in various cancers might explain the broad reports regarding high chances of cancer patients developing blood clots. Further clinical studies will be needed to assess the correlation between the presence of IR-25 ASAMTS13 form and venous thromboembolic events.