The evolution of the vertebrate metzincins; insights from Ciona intestinalis and Danio rerio

Background

The metzincins are a large gene superfamily of proteases characterized by the presence of a zinc protease domain, and include the ADAM, ADAMTS, BMP1/TLL, meprin and MMP genes. Metzincins are involved in the proteolysis of a wide variety of proteins, including those of the extracellular matrix. The metzincin gene superfamily comprises eighty proteins in the human genome and ninety-three in the mouse. When and how the level of complexity apparent in the vertebrate metzincin gene superfamily arose has not been determined in detail. Here we present a comprehensive analysis of vertebrate metzincins using genes from both Ciona intestinalis and Danio rerio to provide new insights into the complex evolution of this gene superfamily.

Results

We have identified 19 metzincin genes in the ciona genome and 83 in the zebrafish genome. Phylogenetic analyses reveal that the expansion of the metzincin gene superfamily in vertebrates has occurred predominantly by the simple duplication of pre-existing genes rather than by the appearance and subsequent expansion of new metzincin subtypes (the only example of which is the meprin gene family). Despite the number of zebrafish metzincin genes being relatively similar to that of tetrapods (e.g. man and mouse), the pattern of gene retention and loss within these lineages is markedly different. In addition, we have studied the evolution of the related TIMP gene family and identify a single ciona and four zebrafish TIMP genes.

Conclusion

The complexity seen in the vertebrate metzincin gene families was mainly acquired during vertebrate evolution. The metzincin gene repertoire in protostomes and invertebrate deuterostomes has remained relatively stable. The expanded metzincin gene repertoire of extant tetrapods, such as man, has resulted largely from duplication events associated with early vertebrate evolution, prior to the sarcopterygian-actinopterygian split. The teleost repertoire of metzincin genes in part parallels that of tetrapods but has been significantly modified, perhaps as a consequence of a teleost-specific duplication event.     

Variability in Melanoma Metalloproteinase Expression Profiling

The proteolytic activities of a disintegrin and metalloproteinase (ADAM); a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), and matrix metalloproteinase (MMP) families play important roles in normal and multiple pathological conditions. These metalloproteases have potential roles in the degradation of the ECM and in the processing of bioactive molecules. In the present study, RNA was isolated from multiple normal fibroblast and metastatic melanoma cell lines, as well as the isogenic normal tissue and tumor samples, and the gene expression levels of six ADAMs, eight MMPs, and four ADAMTSs were analyzed by real-time PCR. This approach allowed for detected changes in mRNA expression of the individual metalloproteinase genes to be compared between normal and metastatic states and also between tissue and cultured cells. Increased gene expression of several ADAM and MMP family members (MMP1, MMP8, MMP15, and ADAM15) occurred in melanoma tissue and was replicated in tissue cultures. In general, the level of ADAM and MMP mRNA expression was several-fold higher in cultured cells compared with the isogenic tissue from which they were derived. Passage-dependent expression patterns were observed for MMP8 and MMP9 in in-house-derived metastatic melanoma cell lines. This reiterates earlier suggestions that experiments using cells that have been maintained in culture should be interpreted with great care.     

CUL1 promotes trophoblast cell invasion at the maternal–fetal interface

Human trophoblast progenitor cells differentiate via two distinct pathways, to become the highly invasive extravillous cytotrophoblast (CTB) cells (EVT) or fuse to form syncytiotrophoblast. Inadequate trophoblast differentiation results in poor placenta perfusion, or even complications such as pre-eclampsia (PE). Cullin1 (CUL1), a scaffold protein in cullin-based ubiquitin ligases, plays an important role in early embryonic development. However, the role of CUL1 in trophoblast differentiation during placenta development has not been examined. Here we show that CUL1 was expressed in CTB cells and EVT in the first trimester human placentas by immunohistochemistry. CUL1 siRNA significantly inhibited outgrowth of extravillous explants in vitro, as well as invasion and migration of HTR8/SVneo cells of EVT origin. This inhibition was accompanied by decreased gelatinolytic activities of matrix metalloproteinase (MMP)-9 and increased expression of tissue inhibitors of MMPs (TIMP-1 and -2). Consistently, exogenous CUL1 promoted invasion and migration of HTR8/SVneo cells. Notably, CUL1 was gradually decreased during trophoblast syncytialization and CUL1 siRNA significantly enhanced forskolin-induced fusion of choriocarcinoma BeWo cells. CUL1 protein levels in human pre-eclamptic placental villi were significantly lower as compared to their matched control placentas. Taken together, our results suggest that CUL1 promotes human trophoblast cell invasion and dysregulation of CUL1 expression may be associated with PE.     

ADAMTS: a novel family of extracellular matrix proteases

ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) is a novel family of extracellular proteases found in both mammals and invertebrates. Members of the family may be distinguished from the ADAM (a disintegrin and metalloprotease) family members based on the multiple copies of thrombospondin 1-like repeats they carry. With at least nine members in mammals alone, the ADAMTS family members are predicted by their structural domains to be extracellular matrix (ECM) proteins with a wide range of activities and functions distinct from members of the ADAM family that are largely anchored on the cell surface. ADAMTS2 is a procollagen N-proteinase, and the mutations of its gene are responsible for Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis. ADAMTS4 and ADAMTS5 are aggrecanases implicated in the degradation of cartilage aggrecan in arthritic diseases. Other members of the ADAMTS family have also been implicated in roles during embryonic development and angiogenesis. Current and future studies on this emerging group of ECM proteases may provide important insights into developmental or pathological processes involving ECM remodeling.     

Proteases in eye development and disease

The eye is one of the classical systems in developmental biology. Furthermore, diseases of the eye, many of which have a developmental basis, have devastating effects that often result in blindness. Proteases have diverse roles in ocular physiology and pathophysiology. Here, a broad overview is provided of the recent literature pertaining to the involvement of proteases in various aspects of eye development and disease: lens development (focusing on apoptosis and lens fiber cell denucleation and organelle loss) and cataract progression, cornea development and disease, retina development and degeneration, sclera development and myopia, and the trabecular meshwork and glaucoma. Proteases discussed include caspases, calpains, matrix metalloproteases (MMPs), a disintegrin and metalloproteinases (ADAMs) and ADAM with thrombospondin motifs (ADAMTS), the ubiquitin-proteasome pathway (UPP), tissue plasminogen activator (tPA), and secretases. It is clear that proteases have diverse and important roles in ocular development and disease, and represent, in many cases, useful therapeutic targets for treating ocular conditions, which would otherwise lead to visual impairment.     

Functional evolution of ADAMTS genes: Evidence from analyses of phylogeny and gene organization

Background  

The ADAMTS (A Disintegrin-like and Metalloprotease with Thrombospondin motifs) proteins are a family of metalloproteases with sequence similarity to the ADAM proteases, that contain the thrombospondin type 1 sequence repeat motifs (TSRs) common to extracellular matrix proteins. ADAMTS proteins have recently gained attention with the discovery of their role in a variety of diseases, including tissue and blood disorders, cancer, osteoarthritis, Alzheimer's and the genetic syndromes Weill-Marchesani syndrome (ADAMTS10), thrombotic thrombocytopenic purpura (ADAMTS13), and Ehlers-Danlos syndrome type VIIC (ADAMTS2) in humans and belted white-spotting mutation in mice (ADAMTS20).     

Heparan sulfate regulates ADAM12 through a molecular switch mechanism

The disintegrin and metalloproteases (ADAMs) are emerging as therapeutic targets in human disease, but specific drug design is hampered by potential redundancy. Unlike other metzincins, ADAM prodomains remain bound to the mature enzyme to regulate activity. Here ADAM12, a protease that promotes tumor progression and chondrocyte proliferation in osteoarthritic cartilage, is shown to possess a prodomain/catalytic domain cationic molecular switch, regulated by exogenous heparan sulfate and heparin but also endogenous cell surface proteoglycans and the polyanion, calcium pentosan polysulfate. Sheddase functions of ADAM12 are regulated by the switch, as are proteolytic functions in placental tissue and sera of pregnant women. Moreover, human heparanase, an enzyme also linked to tumorigenesis, can promote ADAM12 sheddase activity at the cell surface through cleavage of the inhibitory heparan sulfate. These data present a novel concept that might allow targeting of ADAM12 and suggest that other ADAMs may have specific regulatory activity embedded in their prodomain and catalytic domain structures.     

Functional classification of ADAMs based on a conserved motif for binding to integrin alpha 9beta 1: implications for sperm-egg binding and other cell interactions

ADAMs (a disintegrin and metalloproteases) are members of the metzincin superfamily of metalloproteases. Among integrins binding to disintegrin domains of ADAMs are alpha(9)beta(1) and alpha(v)beta(3), and they bind in an RGD-independent and an RGD-dependent manner, respectively. Human ADAM15 is the only ADAM with the RGD motif in the disintegrin domain. Thus, both integrin alpha(9)beta(1) and alpha(v)beta(3) recognize the ADAM15 disintegrin domain. We determined how these integrins recognize the ADAM15 disintegrin domain by mutational analysis. We found that the Arg(481) and the Asp-Leu-Pro-Glu-Phe residues (residues 488-492) were critical for alpha(9)beta(1) binding, but the RGD motif (residues 484-486) was not. In contrast, the RGD motif was critical for alpha(v)beta(3) binding, but the other residues flanking the RGD motif were not. As the RX(6)DLPEF alpha(9)beta(1) recognition motif (residues 481-492) is conserved among ADAMs, except for ADAM10 and 17, we hypothesized that alpha(9)beta(1) may recognize disintegrin domains in all ADAMs except ADAM10 and 17. Indeed we found that alpha(9)beta(1) bound avidly to the disintegrin domains of ADAM1, 2, 3, and 9 but not to the disintegrin domains of ADAM10 and 17. As several ADAMs have been implicated in sperm-oocyte interaction, we tested whether the functional classification of ADAMs, based on specificity for integrin alpha(9)beta(1), applies to sperm-egg binding. We found that the ADAM2 and 15 disintegrin domains bound to oocytes, but the ADAM17 disintegrin domain did not. Furthermore, the ADAM2 and 15 disintegrin domains effectively blocked binding of sperm to oocytes, but the ADAM17 disintegrin domain did not. These results suggest that oocytes and alpha(9)beta(1) have similar binding specificities for ADAMs and that alpha(9)beta(1), or a receptor with similar specificity, may be involved in sperm-egg interaction during fertilization. As alpha(9)beta(1) is a receptor for many ADAM disintegrins and alpha(9)beta(1) and ADAMs are widely expressed, alpha(9)beta(1)-ADAM interaction may be of a broad biological importance.     

ADAM 12, a disintegrin metalloprotease, interacts with insulin-like growth factor-binding protein-3

Insulin-like growth factor-binding protein (IGFBP)-3 binds the insulin-like growth factors with high affinity and modulates their actions. Proteolytic cleavage of IGFBP-3 may regulate insulin-like growth factor bioavailability. IGFBP-3 is extensively degraded in serum during pregnancy; however, as yet the pregnancy-specific protease, or proteases, have not been identified. We utilized a yeast two-hybrid assay and a human placental cDNA library to investigate IGFBP-3-interacting proteins. A disintegrin and metalloprotease-12 (ADAM 12), a member of a family of metalloprotease disintegrins that is highly expressed in placental tissue, was identified as interacting with IGFBP-3. This interaction involved the cysteine-rich domain of ADAM 12. Unlike other members of this family of disintegrin metalloproteases that are membrane proteins, ADAM 12 exists as an alternatively spliced soluble secreted protein. To verify the interaction between ADAM 12 and IGFBP-3, an expression construct containing an ADAM 12-S cDNA was transfected into COS-1 cells. Co-precipitation was observed when conditioned medium was analyzed by immunoprecipitation with an antibody against either ADAM 12 or IGFBP-3 followed by Western blotting with anti-IGFBP-3 or anti-ADAM 12. Although minimal proteolysis of IGFBP-3 was observed in conditioned medium from control cells, this was increased approximately 4-fold in conditioned medium from ADAM 12-S-transfected cells. Recombinant ADAM 12-S partially purified from conditioned medium on a heparin-Sepharose column also proteolyzed IGFBP-3. The degradation pattern was similar to that seen with pregnancy serum, and the presence of ADAM 12-S in serum during pregnancy was confirmed. The data suggest that ADAM 12-S has IGFBP-3 protease activity, and it may contribute to the IGFBP-3 protease activity present in pregnancy serum.     

ADAMTS proteins as modulators of microfibril formation and function

The ADAMTS (a disintegrin-like and metalloproteinase domain with thrombospondin-type 1 motifs) protein superfamily includes 19 secreted metalloproteases and 7 secreted ADAMTS-like (ADAMTSL) glycoproteins. The possibility of functional linkage between ADAMTS proteins and fibrillin microfibrils was first revealed by a human genetic consilience, in which mutations in ADAMTS10, ADAMTS17, ADAMTSL2 and ADAMTSL4 were found to phenocopy rare genetic disorders caused by mutations affecting fibrillin-1 (FBN1), the major microfibril component in adults. The manifestations of these ADAMTS gene disorders in humans and animals suggested that they participated in the structural and regulatory roles of microfibrils. Whereas two such disorders, Weill–Marchesani syndrome 1 and Weill–Marchesani-like syndrome involve proteases (ADAMTS10 and ADAMTS17, respectively), geleophysic dysplasia and isolated ectopia lentis in humans involve ADAMTSL2 and ADAMTSL4, respectively, which are not proteases. In addition to broadly similar dysmorphology, individuals affected by Weill–Marchesani syndrome 1, Weill–Marchesani-like syndrome or geleophysic dysplasia each show characteristic anomalies suggesting molecule-, tissue-, or context-specific functions for the respective ADAMTS proteins. Ectopia lentis occurs in each of these conditions except geleophysic dysplasia, and is due to a defect in the ciliary zonule, which is predominantly composed of FBN1 microfibrils. Together, this strongly suggests that ADAMTS proteins are involved either in microfibril assembly, stability, and anchorage, or the formation of function-specific supramolecular networks having microfibrils as their foundation. Here, the genetics and molecular biology of this subset of ADAMTS proteins is discussed from the perspective of how they might contribute to fully functional or function-specific microfibrils.     

Expression of ADAMs and their inhibitors in sputum from patients with asthma

ADAMs (a disintegrin and metalloprotease) constitute a family of cell surface proteins containing disintegrin and metalloprotease domains which associate features of adhesion molecules and proteases. ADAMTSs (a disintegrin and metalloprotease with thrombospondin motifs) bear thrombospondin type I motifs in C-terminal extremity, and most of them are secreted proteins. Because genetic studies have shown that ADAM-33 gene polymorphisms are associated with asthma, we designed this study to assess mRNA expression profile of several ADAM and ADAMTS proteases in sputum from patients with asthma and to investigate the relationship between expression of these proteases and asthma-associated inflammation and airway obstruction. mRNA expression profile of selected ADAM and ADAMTS proteinases (ADAM-8, -9, -10, -12, -15, -17, and -33; ADAMTS-1, -2, -15, -16, -17, -18, and -19), their physiological inhibitors TIMP-1 and TIMP-3, and RECK, a membrane-anchored MMP activity regulator, was obtained by RT-PCR analysis performed on cells collected by sputum induction from 21 patients with mild to moderate asthma and 17 healthy individuals. mRNA levels of ADAM-8, ADAM-9, ADAM-12, TIMP-1, and TIMP-3 were significantly increased, whereas mRNA levels coding for ADAMTS-1, ADAMTS-15, and RECK were significantly decreased in patients with asthma compared with control patients. ADAM-8 expression was negatively correlated with the forced expiratory volume at the first second (FEV(1)) (r = -0.57, P < 0.01), whereas ADAMTS-1 and RECK expressions were positively correlated to FEV(1) (r = 0.45, P < 0.05, and r = 0.55, P = 0.01, respectively). We conclude that expression of ADAMs and ADAMTSs and their inhibitors is modulated in airways from patients with asthma and that these molecules may play a role in the pathogenesis of asthma.     

Eukaryotic expression and antimicrobial spectrum determination of the peptide tachyplesin II

The ADAMs (a disintegrin and metalloproteases) are an important class of enzymes in the regulation of human disease. The pro domains of ADAMs are responsible for the latency and secretion of mature enzymes. Unlike other metzincins, ADAM pro domains remain bound to the mature enzyme after secretion. To understand the functions of human ADAM pro domains and to determine three-dimensional structures, we have screened promising targets for expression and purification properties when using Escherichia coli as the host. The pro domain of ADAM22 (ADAM22-P) expressed in E. coli was folded, as determined by CD and NMR spectroscopy. An ADAM22-P fragment encoding residues 26–199 could be expressed in high amounts, remained soluble above 1 mM, and was suitable for structural studies by NMR spectroscopy. CD spectroscopy and predictions suggest that the secondary structure in ADAM22-P consists of β-strands. Furthermore, our data indicate that the pro domains of ADAMs are expressed as two subdomains. The most N-terminal subdomain (ADAM22-P_N) was found to be susceptible to proteolysis and was required for folding stability of the second subdomain (ADAM22-P_C).     

ADAM12 and PAPP-A: Candidate regulators of trophoblast invasion and first trimester markers of healthy trophoblasts

ABSTRACT

Proper placental development and function is crucial for a healthy pregnancy, and there has been substantial research to identify markers of placental dysfunction for the early detection of pregnancy complications. Low first-trimester levels of a disintegrin and metalloproteinase 12 (ADAM12) and pregnancy-associated plasma protein-A (PAPP-A) have been consistently associated with the subsequent development of preeclampsia and fetal growth restriction. These molecules are both metalloproteinases secreted by the placenta that cleave insulin-like growth factor binding proteins (IGFBPs), although ADAM12 also has numerous other substrates. Recent work has identified ADAM12, and particularly its shorter variant, ADAM12S, as a regulator of the migration and invasion of trophoblasts into the lining of the uterus, a critical step in normal placental development. While the mechanisms underlying this regulation are not yet clear, they may involve the liberation of heparin-binding EGF-like growth factor (HB-EGF) and/or IGFs from IGFBPs. In contrast, there has been relatively little functional work examining PAPP-A or the IGFBP substrates of ADAM12 and PAPP-A. Understanding the functions of these markers and the mechanisms underlying their association with disease could improve screening strategies and enable the development of new therapeutic interventions.     

Infection by Brucella melitensis or Brucella papionis modifies essential physiological functions of human trophoblasts

Brucellosis is a zoonosis caused by bacteria of the Brucella genus. In ruminants, brucellosis causes abortion, followed by chronic infection and secretion of bacteria in milk. In humans, it usually presents as flu‐like symptoms, with serious complications if untreated. Epidemiological studies have only recently established that brucellosis can also cause pregnancy complications in women, but the pathogenic mechanisms are unknown. Pioneering studies in ruminants showed that Brucella infect trophoblasts and then colonise the placenta where they grow to high density. A recent study showed that the main zoonotic Brucella species can infect human cytotrophoblasts (CTB) and extravillous trophoblasts (EVT). In this work, we show that Brucella papionis (associated with stillbirth in primates) also infects human trophoblasts. However, it replicates actively in CTB, whereas its replication is very restricted within EVT. We also observed alteration of several trophoblastic functions upon infection by B. papionis or Brucella melitensis (the most prevalent species in human brucellosis). Infection altered the production of hormones, the ability of CTB to form syncytiotrophoblasts, and the invasion capacity of EVT. We also found that infection can spread between different types of trophoblasts. These findings constitute a new step in understanding how Brucella infection causes adverse pregnancy outcomes.     

Impaired autophagy by soluble endoglin,under physiological hypoxia in early pregnant period,is involved in poor placentation in preeclampsia

In early pregnancy, trophoblasts and the fetus experience hypoxic and low-nutrient conditions; nevertheless, trophoblasts invade the uterine myometrium up to one third of its depth and migrate along the lumina of spiral arterioles, replacing the maternal endothelial lining. Here, we showed that autophagy, an intracellular bulk degradation system, occurred in extravillous trophoblast (EVT) cells under hypoxia in vitro and in vivo. An enhancement of autophagy was observed in EVTs in early placental tissues, which suffer from physiological hypoxia. The invasion and vascular remodeling under hypoxia were significantly reduced in autophagy-deficient EVT cells compared with wild-type EVT cells. Interestingly, soluble endoglin (sENG), which increased in sera in preeclamptic cases, suppressed EVT invasion by inhibiting autophagy. The sENG-inhibited EVT invasion was recovered by TGFB1 treatment in a dose-dependent manner. A high dose of sENG inhibited the vascular construction by EVT cells and human umbilical vein endothelial cells (HUVECs), meanwhile a low dose of sENG inhibited the replacement of HUVECs by EVT cells. A protein selectively degraded by autophagy, SQSTM1, accumulated in EVT cells in preeclamptic placental biopsy samples showing impaired autophagy. This is the first report showing that impaired autophagy in EVT contributes to the pathophysiology of preeclampsia.     

Molecular analysis of ulilysin, the structural prototype of a new family of metzincin metalloproteases

The metzincin clan encompasses several families of zinc-dependent metalloproteases with proven function both in physiology and pathology. They act either as broad spectrum protein degraders or as sheddases, operating through limited proteolysis. Among the structurally uncharacterized metzincin families are the pappalysins, of which the most thoroughly studied member is human pregnancy-associated plasma protein A (PAPP-A), a heavily glycosylated 170-kDa multidomain protein specifically cleaving insulin-like growth factor (IGF)-binding proteins (IGFBPs). Proulilysin is a 38-kDa archaeal protein that shares sequence similarity with PAPP-A but encompasses only the pro-domain and the catalytic domain. It undergoes calcium-mediated autolytic activation, and the mature protein adopts a three-dimensional structure with two subdomains separated by an active site cleft containing the catalytic zinc ion. This structure is reminiscent of human members of the adamalysin/ADAMs (a disintegrin and a metalloprotease) family of metzincins. A bound dipeptide yields information on the substrate specificity of ulilysin, which specifically hydrolyzes IGFBP-2 to -6, insulin, and extracellular matrix proteins but not IGFBP-1 or IGF-II. Accordingly, ulilysin has higher proteolytic efficiency and a broader substrate specificity than human PAPP-A. The structure of ulilysin represents a prototype for the catalytic domain of pappalysins.     

Normal fertility in male mice lacking ADAM32 with testis-specific expression

The a disintegrin and metalloprotease (ADAM) family proteins comprise a group of membrane-anchored proteins. ADAM32 is expressed specifically in testis and is closely related phylogenetically to ADAM2 and ADAM3, which are known to be critical for fertilization in mice. To assess the biological role of ADAM32, we analyzed Adam32-mutant mice. We found that male mice lacking ADAM32 have normal fertility, testicular integrity, and sperm characteristics. ADAM32 was found to exist at lower levels than ADAM2 and ADAM3 in wild-type testis and sperm, respectively. The present study demonstrates that ADAM32 is dispensable for fertility and appears to be functionally unrelated to ADAM2 and ADAM3 in mice.