ADAMTS Proteases Research Articles

256. Ovarian lymphatic vascular development is hormonally regulated and Adamts1-dependent

The lymphatic system is important for return of extra-vascular fluid to the blood circulation, conductance of hormones and immune cell trafficking. Delicate hormonal control of fluid conductance during reproductive cycles is exemplified by the ovarian hyperstimulation syndrome, a dangerous condition of hypovolemia caused by fluid accumulation in the abdomen and reproductive tissues, in response to hormonal hyperstimulation. This study is the first to investigate the relationship between ovarian lymphatic development and follicle growth. Quantitative morphometric analysis of vessel size and number in mouse ovary revealed, for the first time, that the ovarian lymphatic vasculature develops postnatally and in synchrony with the induction of ovarian CYP19a1 (Aromatase); the time when secondary follicles become FSH-responsive and estrogenic. Mechanistically, we found that the FSH-analogue eCG mediates induction of lymphatic vascular endothelial growth factor Vegfd and the receptor Vegfr3 (Flt4) in granulosa cells. Importantly, stimulation with eCG also enhanced ovarian lymphatic vessel number and size. However, formation of ovarian lymphatics also required the matrix-remodelling protease Adamts1, since ovaries from Adamts1−/− mice failed to undergo normal lymphatic vascular development. Treatment of Adamts1 null mice with eCG significantly increased the number and size of ovarian lymphatic vessels, however, the vessels were still smaller and fewer in number than wildtypes. These combined results indicate that the ovarian lymphatic system develops in response to hormonal signals, which promote folliculogenesis, through induction of lymphangiogenic factors in granulosa cells; as well as involving Adamts1-dependent mechanisms. This study is the first demonstration of the novel principle of hormonal regulation of lymphangiogenesis in any tissue and suggests a requirement for functional lymphatics during normal folliculogenesis. In addition our results inform the elucidation of the tightly regulated processes that control fluid dynamics and immune cell surveillance within reproductive tissues.

Altered composition of the cumulus-oocyte complex matrix during in vitro maturation of oocytes

In vitro maturation (IVM) of mammalian oocytes has potential health benefits for patients undergoing assisted reproduction as an alternative to gonadotrophin treatment. This procedure is also useful for studying the process of oocyte and early embryo development. However, oocytes undergoing IVM have much lower competence than in vivo matured oocytes. Efforts to optimize IVM success have focused on replicating in vivo timing, hormonal milieu and cumulus cell responses associated with maturing oocytes. We have previously identified two extracellular matrix proteins, the protease Adamts1 and hyaluronan-binding proteoglycan Versican, produced by mural granulosa cells that selectively incorporate into the periovulatory cumulus-oocyte complex (COC). Murine COC were cultured in the presence of epidermal growth factor and/or FSH. mRNA and protein were measured by real time PCR and Western blot and compared to in vivo derived COC. COCs from mice that underwent IVM for 6 or 20 h in the presence of epidermal growth factor, FSH or in combination had a > 10-fold reduction in mRNA (P < 0.05) for Adamts1 and Vcan when compared with in vivo matured COCs. Hyaluronan synthase 2 expression was up-regulated up to 8-fold (P < 0.05) over the unstimulated control, demonstrating successful induction of cumulus gene expression by the IVM conditions. While in vivo matured COCs showed abundant levels of these proteins, COCs that underwent IVM had neither detectable Adamts1, nor intact or Adamts1-cleaved Vcan. Human cumulus and granulosa cells matured in vivo contained abundant mRNA for Adamts1 and Vcan, demonstrating the potential relevance to human IVM. These results indicate that extensively altered COC matrix composition is present during IVM and may contribute to the observed poorer competence of the derived oocytes.

O-Fucosylation of Thrombospondin Type 1 Repeats in ADAMTS-like-1/Punctin-1 Regulates Secretion: IMPLICATIONS FOR THE ADAMTS SUPERFAMILY

The ADAMTS superfamily contains several metalloproteases (ADAMTS proteases) as well as ADAMTS-like molecules that lack proteolytic activity. Their common feature is the presence of one or more thrombospondin type-1 repeats (TSRs) within a characteristic modular organization. ADAMTS like-1/punctin-1 has four TSRs. Previously, O-fucosylation on Ser or Thr mediated by the endoplasmic reticulum-localized enzyme protein-O-fucosyltransferase 2 (POFUT2) was described for TSRs of thrombospondin-1, properdin, and F-spondin within the sequence Cys-Xaa(1)-Xaa(2)-(Ser/Thr)-Cys-Xaa-Xaa-Gly (where the fucosylated residue is underlined). On mass spectrometric analysis of tryptic peptides from recombinant secreted human punctin-1, the appropriate peptides from TSR2, TSR3, and TSR4 were found to bear either a fucose monosaccharide (TSR3, TSR4) or a fucose-glucose disaccharide (TSR2, TSR3, TSR4). Although mass spectral analysis did not unambiguously identify the relevant peptide from TSR1, metabolic labeling of cells expressing TSR1 and the cysteine-rich module led to incorporation of [(3)H]fucose into this construct. Mutation of the putative modified Ser/Thr residues in TSR2, TSR3, and TSR4 led to significantly decreased levels of secreted punctin-1. Similarly, expression of punctin-1 in Lec-13 cells that are deficient in conversion of GDP-mannose to GDP-fucose substantially decreased the levels of secreted protein, which were restored upon culture in the presence of exogenous l-fucose. In addition, mutation of the single N-linked oligosaccharide in punctin-1 led to decreased levels of secreted punctin-1. Taken together, the data define a critical role for N-glycosylation and O-fucosylation in the biosynthesis of punctin-1. From a broad perspective, these data suggest that O-fucosylation may be a widespread post-translational modification in members of the ADAMTS superfamily with possible regulatory consequences.

Cell-surface Processing of Pro-ADAMTS9 by Furin

Processing of polypeptide precursors by proprotein convertases (PCs) such as furin typically occurs within the trans-Golgi network. Here, we show in a variety of cell types that the propeptide of ADAMTS9 is not excised intracellularly. Pulse-chase analysis in HEK293F cells indicated that the intact zymogen was secreted to the cell surface and was subsequently processed there before release into the medium. The processing occurred via a furin-dependent mechanism as shown using PC inhibitors, lack of processing in furin-deficient cells, and rescue by furin in these cells. Moreover, down-regulation of furin by small interference RNA reduced ADAMTS9 processing in HEK293F cells. PC5A could also process pro-ADAMTS9, but similarly to furin, processed forms were absent intracellularly. Cell-surface, furin-dependent processing of pro-ADAMTS9 creates a precedent for extracellular maturation of endogenously produced secreted proproteins. It also indicates the existence of a variety of mechanisms for processing of ADAMTS proteases.

Identification of substrates of the extracellular protease ADAMTS1 by DIGE proteomic analysis

Proteolytic modification of components of the extracellular milieu by metalloproteinases plays important roles in the regulation of multiple cellular and physiological processes and pathological conditions. ADAMTS1 is a secreted enzyme of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteases, which is related to angiogenesis and inflammation processes. Here, we describe a proteomic screening for putative ADAMTS1 substrates by analyzing the protein profiles obtained from cultures of transfected cells overexpressing the protease as compared to parental cells. Conditioned medium proteins of cultures of the two cell lines have been quantitatively compared by DIGE. Proteins showing differential levels have been identified by MS techniques leading to the finding of five potential new substrates of ADAMTS1: the basement membrane proteins nidogen-1 and -2, the desmosomal protein desmocollin-3, and the extracellular glycoproteins dystroglycan 1 and Mac-2-binding protein. Nidogen-1 and -2 have been further validated as substrates by immunochemical analysis. Our results demonstrate the utility of the DIGE proteomic technique for the discovery of specific substrates of matrix proteases.

The characterisation of six ADAMTS proteases in the basal chordate Ciona intestinalis provides new insights into the vertebrate ADAMTS family

ADAMTS, constituting a recently discovered family of secreted zinc-dependent metalloproteases, have been shown to have critical physiological roles through identification of a number of natural animal and human gene mutations. The identification of six ADAMTS genes in the basal chordate Ciona intestinalis provides new insight into how, when and in what order the vertebrate orthologues have evolved. The phylogenetic assignments, based on sequences conserved across all genes, are supported by conserved domain structures within defined sub-families. The phylogeny and the frequent localisation of ADAMTS genes in paralogous regions of the genome are consistent with the vertebrate lineages having arisen by large scale or genome duplication. The high level of conservation in the protease active site of vertebrate orthologues within some sub-families suggests subfunctionalisation, whereas the greater divergence in others would favour the evolution of novel substrate specificities and these observations are borne-out where substrate-specificity is known. The expansion and sub-specialization of the ADAMTS family is a component of the increased complexity of extracellular matrix that is associated with the evolution of vertebrates.

026. The cumulus matrix in ovulation: inert packaging or active delivery vehicle for the oocyte?

Preovulatory follicles respond to the LH-surge with a cascade of molecular events. The ovulatory signal initially impinges on the mural granulosa layers triggering rapid tissue morphogenesis and ultimately terminal differentiation of these cells. Mural granulosa cells transiently produce a suite of transcriptional regulators, EGF-like ligands as well the extracellular matrix (ECM) proteoglycan, versican and the protease ADAMTS-1. These act in concert with permissive oocyte signals to induce and organise a complex hyaluronan (HA) rich ECM surrounding the cumulus cells and oocyte. This expanded cumulus matrix is analogous in composition to an extensive form of pericellular matrices actively associated with cell migration. During ovulation the cumulus matrix becomes anti-adhesive to the intra-follicular environment but is strongly pro-adhesive for the oviductal fimbria. When the follicle apex is perforated the COC is released binds to the fimbria and transports into the oviduct where fertilisation occurs. Success of ovulation and fertilisation is sensitive to the appropriate production and assembly of cumulus matrix components that are in turn dependent on an appropriate balance of oocyte and granulosa derived signals. Production of these cumulus matrix components is thus a potential checkpoint that assures ovulation of competent oocytes. The HA matrix is cross-linked by organiser molecules and also is enriched in proteases ADAMTS-1, 4, 5. Although these have potentially redundant functionality, ADAMTS-1 null female mice are profoundly sub-fertile and have reduced ovulation rate. Specific components of the cumulus matrix are disorganised in ADAMTS-1 null mice and cleavage of versican in these cumulus complexes is reduced. Thus ADAMTS-1 and versican have unique roles in normal cumulus matrix expansion that is important for ovulation. Altered interaction of the cumulus complex with neighbouring tissues alters transport through the oviduct, while abnormal persistence of COC matrix structure after ovulation is also likely to impair sperm interaction and penetration. Evidence thus indicates that the expanded cumulus matrix plays several active roles in oocyte release, transport and sperm interaction.

The ADAMTS Proteases, Extracellular Matrix, and Vascular Disease

The importance of proteases as mediators of extracellular matrix (ECM) degradation and vascular cell phenotype in the pathogenesis of vascular disease is indisputable. In fact, excellent cases can be made for the matrix metalloproteinases (MMPs) (see review1), the serine proteases (see review2), and the cysteine and aspartic proteases (see review3) being involved in many of the events in vascular disease. In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Jonsson-Rylander and her colleagues show us that ADAMTS1, a member of another family of proteases, is also involved.4 Descendants from the ADAM family of proteases, the ADAMTS members (a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs), currently numbering 19, evolved as nonintegral membrane proteins that associate with the cell surface and ECM through specific protein domains (see reviews5,6). Like the ADAM family, the ADAMTS proteases are multidomain proteins with common structural motifs that include an N-terminal signal sequence, a prodomain, a catalytic domain with Zn binding site, an ancillary domain, a disintegrin-like domain, a central thrombospondin repeat domain (TSR), a cysteine rich domain, a cysteine free spacer domain, and usually one or more TSRs. Some of the ADAMTS members also contain unique C-terminal domains that can contain PLAC and/or CUB sequences. These proteases are synthesized as zymogens that undergo processing by convertases such as furin and by metalloproteinases such as MT-4 MMP.7,8 Like the MMPs, the ADAMTS members can be inhibited by tissue inhibitors of matrix metalloproteinases or TIMPs. For example TIMP-3 is a potent inhibitor of ADAMTS4 and 5.9 Other inhibitors exist as well. For example, C-terminal truncation of ADAMTS4 can be blocked by TIMP-1.7 Proteins that share structural homology to the ADAMTS members also may function as ADAMTS inhibitors. One example is papilin, a protein produced by the fruit …

230. Altered matrix composition of cumulus oocyte complexes following in vitro maturation

The luteinizing hormone (LH) surge initiates cumulus expansion, through synthesis of hyaluronan and cross-linking proteins including versican, which stabilise the cumulus oocyte complex (COC) matrix. Versican is a substrate for the protease ADAMTS-1 and mRNA for each are localised to granulosa cells (GCs) and greatly induced following the LH surge. In humans, the use of in vitro maturation (IVM) of oocytes is an appealing option, reducing costs and risk of side effects associated with in vitro fertilisation. IVM oocytes are of poorer quality, likely resulting from altered gene expression and environmental conditions during oocyte maturation. Real-time PCR showed that IVM and immature COCs from Balb/c mice have 12 and 13 fold-reduced levels of ADAMTS-1 and versican expression respectively compared to in vivo matured COCs (PMSG+hCG 12h). Ovulated COCs (PMSG+hCG 15h) had similar low levels of ADAMTS-1 and versican. Samples isolated from F1 C57Bl/6xCBA mice showed similar reduced versican and ADAMTS-1 mRNA. Western blot analysis revealed that full length and cleaved versican, from ADAMTS-1/4 activity, was not detected in immature COCs, was present in in vivo matured COCs isolated from follicles, but strongest in ovulated COCs. IVM COCs had no detectable versican protein, supporting the mRNA data. Full-length versican was also present in GCs after PMSG+hCG 12h or 15h. ADAMTS-1 protein was most abundant in in vivo matured COCs with reduced levels seen in ovulated COCs, but was absent from IVM and immature COCs. These results indicate that ADAMTS-1 and versican are secreted products of granulosa cells that bind and incorporate into the COC matrix. The presence of versican and ADAMTS-1 is not essential for cumulus matrix expansion in vitro, but may contribute to oocyte maturation, ovulation of the COC and/or interaction with sperm during fertilisation.

Discovery and Characterization of a Novel, Widely Expressed Metalloprotease, ADAMTS10, and Its Proteolytic Activation

We describe the discovery and characterization of ADAMTS10, a novel metalloprotease encoded by a locus on human chromosome 19 and mouse chromosome 17. ADAMTS10 has the typical modular organization of the ADAMTS family, with five thrombospondin type 1 repeats and a cysteine-rich PLAC (protease and lacunin) domain at the carboxyl terminus. Its domain organization and primary structure is similar to a novel long form of ADAMTS6. In contrast to many ADAMTS proteases, ADAMTS10 is widely expressed in adult tissues and throughout mouse embryo development. In situ hybridization analysis showed widespread expression of Adamts10 in the mouse embryo until 12.5 days of gestation, after which it is then expressed in a more restricted fashion, with especially strong expression in developing lung, bone, and craniofacial region. Mesenchymal, not epithelial, expression in the developing lung, kidney, gonad, salivary gland, and gastrointestinal tract is a consistent feature of Adamts10 regulation. N-terminal sequencing and treatment with decanoyl-Arg-Val-Lys-Arg-chloromethylketone indicate that the ADAMTS10 zymogen is processed by a subtilisin-like proprotein convertase at two sites (Arg64/Gly and Arg233/Ser). The widespread expression of ADAMTS10 suggests that furin, a ubiquitously expressed proprotein convertase, is the likely processing enzyme. ADAMTS10 expressed in HEK293F and COS-1 cells is N-glycosylated and is secreted into the medium, as well as sequestered at the cell surface and extracellular matrix, as demonstrated by cell surface biotinylation and immunolocalization in nonpermeabilized cells. ADAMTS10 is a functional metalloprotease as demonstrated by cleavage of alpha2-macroglobulin, although physiological substrates are presently unknown.

ADAMTS7B, the Full-length Product of the ADAMTS7 Gene, Is a Chondroitin Sulfate Proteoglycan Containing a Mucin Domain

We have characterized ADAMTS7B, the authentic full-length protein product of the ADAMTS7 gene. ADAMTS7B has a domain organization similar to that of ADAMTS12, with a total of eight thrombospondin type 1 repeats in its ancillary domain. Of these, seven are arranged in two distinct clusters that are separated by a mucin domain. Unique to the ADAMTS family, ADAMTS7B is modified by attachment of the glycosaminoglycan chondroitin sulfate within the mucin domain, thus rendering it a proteoglycan. Glycosaminoglycan addition has potentially important implications for ADAMTS7B cellular localization and for substrate recognition. Although not an integral membrane protein, ADAMTS7B is retained near the cell surface of HEK293F cells via interactions involving both the ancillary domain and the prodomain. ADAMTS7B undergoes removal of the prodomain by a multistep furin-dependent mechanism. At least part of the final processing event, i.e. cleavage following Arg(220) (mouse sequence annotation), occurs at the cell surface. ADAMTS7B is an active metalloproteinase as shown by its ability to cleave alpha(2)-macroglobulin, but it does not cleave specific peptide bonds in versican and aggrecan attacked by ADAMTS proteases. Together with ADAMTS12, whose primary structure also predicts a mucin domain, ADAMTS7B constitutes a unique subgroup of the ADAMTS family.

A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motifs: the ADAMTS family

ADAMTS proteases are complex secreted enzymes containing a prometalloprotease domain of the reprolysin type attached to an ancillary domain with a highly conserved structure that includes at least one thrombospondin type 1 repeat. Known functions of ADAMTS proteases include processing of procollagens and von Willebrand factor as well as catabolism of aggrecan, versican and brevican. They have been demonstrated to have important roles in connective tissue organization, coagulation, inflammation, arthritis, angiogenesis and cell migration. ADAMTS can be grouped into distinct clades within which there is conservation of modular organization, protein sequence, gene structure and possibly, of substrate preference. ADAMTS proteases are synthesized as zymogens, with constitutive proprotein convertase removal of the propeptide occurring prior to secretion. Their enzymatic specificity is heavily influenced by their ancillary domain, which plays a critical role in directing these enzymes to their substrates, the cell surface and the extracellular matrix.

Expression pattern of the ADAMTS proteinases in human prostate cancer cell lines

Expression pattern of the ADAMTS proteinases in human prostate cancer cell lines

The role of ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) in a model of cartilage degradation

Introduction Cleavage of aggrecan between residues Glu373–Ala374, which is believed to be a key event in aggrecan destruction in arthritic diseases, has been attributed to an enzymatic activity, aggrecanase. Two cartilage aggrecanases have been identified, aggrecanase-1 (ADAM-TS4) and aggrecanase-2 (ADAM-TS5) and both enzymes have been shown very efficiently to cleave soluble aggrecan at the Glu373–Ala374site.Objective To determine whether ADAM-TS4 and/or ADAM-TS5 are the aggrecanases responsible for aggrecan catabolism following interleukin-1 (IL-1) and tumor necrosis factor (TNF) treatment of bovine articular cartilage.Results (1) IL-1- and TNF-stimulated release of aggrecan was associated with cleavage of aggrecan within the C-terminus at the ADAM-TS4 and ADAM-TS5-sensitive sites, Glu1480–Gly1481, Glu1667–Gly1668, and Glu1871–Leu1872. (2) The order of cleavage following IL-1 stimulation of cartilage explants was the same as when soluble aggrecan is digested with recombinant human ADAM-TS4 and ADAM-TS5. (3) Both constitutive and stimulated cleavage of aggrecan at the ADAM-TS4 and ADAM-TS5-sensitive sites in cartilage was blocked by a general metalloproteinase inhibitor but not by a MMP-specific inhibitor, and this inhibition correlated with inhibition of aggrecan release from cartilage. (4) PCR and Western blot analysis indicated that both ADAM-TS proteases are expressed in cartilage explants; ADAM-TS5 is constitutively expressed whereas ADAM-TS4 is induced following IL-1 and TNF treatment. (5) Immunodepletion of both ADAM-TS4 and ADAM-TS5 from bovine articular cartilage cultures following IL-1 stimulation resulted in a 90% reduction of aggrecanase activity in the culture medium.

Expression and activity of ADAMTS-5 in synovium.

ADAMTS proteinases, belonging to the adamalysin subfamily of metalloproteinases, have been implicated in a variety of cellular events such as morphogenesis, cell migration, angiogenesis, ovulation and extracellular matrix breakdown. Aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5) have been identified in cartilage and are largely responsible for cartilage aggrecan breakdown. We have shown previously that synovium, the membrane lining diarthrodial joints, generates soluble aggrecanase activity. We report here the expression, localization and activity of ADAMTS-5 from human arthritic and bovine synovium. ADAMTS-5 was expressed constitutively in synovium with little or no transcriptional regulation by recombinant human interleukin-1 alpha or all-trans-retinoate, factors previously shown to upregulate aggrecanase activity in cartilage. Aggrecanase activity generated by synovium in vitro and recombinant ADAMTS-5 cleaved aggrecan extensively, resulting in aggrecan fragments similar to those generated by chondrocyte-derived aggrecanases, and the activity was inhibited by heparin. ADAMTS-5 was immunolocalized in human arthritic synovium, where staining was mostly pericellular, particularly in the synovial lining and around blood vessels; some matrix staining was also seen. The possibility that synovium-derived ADAMTS-5 may play a role in cartilage aggrecan breakdown is discussed.