Stage-specific activation of MIG-17/ADAMTS controls cell migration in Caenorhabditis elegans

The activation of ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family proteases depends on removal of the prodomain. Although several studies suggest that ADAMTS activities play roles in development, homeostasis and disease, it remains unclear when and where the enzymes are activated in vivo. MIG-17, a Caenorhabditis elegans glycoprotein belonging to the ADAMTS family, is secreted from the body wall muscle cells and localizes to the gonadal basement membrane to control the migration of gonadal distal tip cells. Here, we developed a monoclonal antibody that recognizes the N-terminal neo-epitope of the activated MIG-17. In western blotting, the antibody specifically detected the activated form, the signal for which dramatically increased during the third and fourth larval stages, when MIG-17 is required to direct distal tip cell migration. In in situ staining, the monoclonal antibody recognized the activated form in the basement membrane, whereas it failed to detect a processing-resistant mutant form localized to the basement membrane. MIG-17 was activated in the basement membranes of the muscle, intestine and gonad in the third larval stage, and downregulated in nongonadal basement membranes in young adults and in gonadal basement membranes in older adults. Thus, the activation of MIG-17 is regulated in a spatiotemporal manner during C. elegans development. This is the first report demonstrating the regulated activation of an ADAMTS protein in vivo. Our results suggest that monoclonal antibodies against neo-epitopes have potential as powerful tools for detecting activation of ADAMTSs during development and in disease pathogenesis.     

Control of the basement membrane and cell migration by ADAMTS proteinases: Lessons from C. elegans genetics

The members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of secreted proteins, MIG-17 and GON-1, play essential roles in Caenorhabditis elegans gonadogenesis. The genetic and molecular analyses of these proteinases uncovered novel molecular interactions regulating the basement membrane (BM) during the migration of the gonadal leader cells. MIG-17, which is localized to the gonadal BM recruits or activates fibulin-1 and type IV collagen, which then recruits nidogen, thereby inducing the remodeling of the BM that is required for directional control of leader cell migration. GON-1 acts antagonistically with fibulin-1 to regulate the levels of type IV collagen accumulation in the gonadal BM, which facilitates active migration of the leader cells. The cooperative action of MIG-17 and GON-1 represents an excellent model for understanding the mechanisms of organogenesis mediated by ADAMTS proteinases.     

A fibulin-1 homolog interacts with an ADAM protease that controls cell migration in C. elegans

ADAM (a disintegrin and metalloprotease) family proteins play important roles in animal development and pathogenesis. In C. elegans, a secreted ADAM protein, MIG-17, acts from outside the gonad to control the migration of gonadal distal tip cells (DTCs) that promote gonad morphogenesis. Here, we report that dominant mutations in the fbl-1 gene encoding fibulin-1 spliced isoforms, which are calcium binding extracellular matrix proteins, bypass the requirement for MIG-17 activity in directing DTC migration. Specific amino acid substitutions in the third EGF-like motif of one of the two isoforms, FBL-1C, which corresponds to mammalian fibulin-1C, suppress mig-17 mutations. FBL-1C is synthesized in the gut cells and localizes strongly to the gonadal basement membrane in a MIG-17-dependent manner. Localization of mutant FBL-1C is weaker than that of the wild-type protein and is insensitive to MIG-17 activity, suggesting that it gains a novel function that compensates for its reduced molecular density. We propose that proteolysis by MIG-17 recruits FBL-1C to the gonadal basement membrane, where it is required for the guidance of DTCs, and that mutant FBL-1C acts in a manner that mimics the downstream events of MIG-17-mediated proteolysis.     

The conserved oligomeric Golgi complex acts in organ morphogenesis via glycosylation of an ADAM protease in C. elegans

In C. elegans, the gonad acquires two U-shaped arms through directed migration of gonadal distal tip cells (DTCs). A member of the ADAM (a disintegrin and metalloprotease) family, MIG-17, is secreted from muscle cells and localizes to the gonadal basement membrane where it functions in DTC migration. Mutations in cogc-3 and cogc-1 cause misdirected DTC migration similar to that seen in mig-17 mutants. Here, we report that COGC-3 and COGC-1 proteins are homologous to mammalian COG-3/Sec34 and COG-1/ldlBp, respectively, two of the eight components of the conserved oligomeric Golgi (COG) complex required for Golgi function. Knockdown of any of the other six components by RNA interference also produces DTC migration defects, suggesting that the eight components function in a common pathway. COGC-3 and COGC-1 are required for the glycosylation and gonadal localization of MIG-17, but not for secretion of MIG-17 from muscle cells. Furthermore, COGC-3 requires MIG-17 activity for its action in DTC migration. Our findings demonstrate that COG complex-dependent glycosylation of an ADAM protease plays a crucial role in determining organ shape.     

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.     

Proprotein convertase furin interacts with and cleaves pro-ADAMTS4 (Aggrecanase-1) in the trans-Golgi network

A member of the A disintegrin and metalloproteinase domain with thrombospondin type-1 motifs (ADAMTS-4) protease family can efficiently cleave aggrecan at several sites detected in joints of osteoarthritic patients. Although recent studies have shown that removal of the prodomain of ADAMTS4 is critical for its ability to degrade aggrecan, the cellular mechanisms for its processing and trafficking remain unclear. In this study, by using both furin-specific inhibitor and RNA interference technique, we demonstrate that furin plays an important role in the intracellular removal of ADAMTS4 prodomain. Further, we demonstrate that proADAMTS4 can be processed by means of multiple furin recognition sites: (206)RPRR(209), (209)RAKR(212), or (211)KR(212). The processing of proADAMTS4 was completely blocked by brefeldin A treatment, suggesting that processing occurs in the trans-Golgi network. Indeed, ADAMTS4 is co-localized with furin in trans-Golgi network. Interestingly, the pro form of ADAMTS4, not its mature one, co-precipitates with furin, suggesting that furin physically interacts with the prodomain of ADAMTS-4. In addition, our evidence suggests that a furin-independent pathway may also contribute to the activation of ADAMTS4. These results indicate that the activation mechanism for ADAMTS4 can be targeted for therapeutical intervention against this enzyme.     

C. elegans as a model system to study the function of the COG complex in animal development

The conserved oligomeric Golgi (COG) complex is an octameric protein complex associated with the Golgi apparatus and is required for proper sorting and glycosylation of Golgi resident enzymes and secreted proteins. Although COG complex function has been extensively studied at the cellular and subcellular levels, its role in animal development mostly remains unknown. Recently, mutations in the components of the COG complex were found to cause abnormal gonad morphogenesis in Caenorhabditis elegans. In C. elegans, the COG complex acts in the glycosylation of an ADAM (a disintegrin and metalloprotease) family protein, MIG-17, which directs migration of gonadal distal tip cells to lead gonad morphogenesis. This is the first link between the COG complex and the function of an ADAM protease that is directly involved in organ morphogenesis, demonstrating the potential of C. elegans as a model system to study COG function in animal development.     

ADAMTS7 Cleavage and Vascular Smooth Muscle Cell Migration Is Affected by a Coronary-Artery-Disease-Associated Variant

Recent genome-wide association studies have revealed an association between variation at the ADAMTS7 locus and susceptibility to coronary artery disease (CAD). Furthermore, in a population-based study cohort, we observed an inverse association between atherosclerosis prevalence and rs3825807, a nonsynonymous SNP (A to G) leading to a Ser-to-Pro substitution in the prodomain of the protease ADAMTS7. In light of these data, we sought a mechanistic explanation for this association. We found that ADAMTS7 accumulated in smooth muscle cells in coronary and carotid atherosclerotic plaques. Vascular smooth muscle cells (VSMCs) of the G/G genotype for rs3825807 had reduced migratory ability, and conditioned media of VSMCs of the G/G genotype contained less of the cleaved form of thrombospondin-5, an ADAMTS7 substrate that had been shown to be produced by VSMCs and inhibit VSMC migration. Furthermore, we found that there was a reduction in the amount of cleaved ADAMTS7 prodomain in media conditioned by VSMCs of the G/G genotype and that the Ser-to-Pro substitution affected ADAMTS7 prodomain cleavage. The results of our study indicate that rs3825807 has an effect on ADAMTS7 maturation, thrombospondin-5 cleavage, and VSMC migration, with the variant associated with protection from atherosclerosis and CAD rendering a reduction in ADAMTS7 function.     

Identification of prodomain determinants involved in ADAMTS-1 biosynthesis

The metalloprotease ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin type I motif), similarly to other members of the ADAMTS family, is initially synthesized as a zymogen, proADAMTS-1, that undergoes proteolytic processing at the prodomain/catalytic domain junction by serine proteinases of the furin-like family of proprotein convertases. The goals of this study were to identify residues of the prodomain that play an essential role in ADAMTS-1 processing and to determine the identity of the convertase required for zymogen processing. To gain insight into the putative roles of specific prodomain residues in ADAMTS-1 biosynthesis, we performed biosynthetic labeling experiments in transiently transfected human embryonic kidney 293 cells expressing wild-type and prodomain mutants of proADAMTS-1. Cells expressing wild-type ADAMTS-1 initially produced a 110-kDa zymogen form that was later converted to an 87-kDa form, which was also detected in the media. Although convertases such as PACE4 and PC6B processed proADAMTS-1, we found that furin was the most efficient enzyme at producing the mature ADAMTS-1 87-kDa moiety. Site-directed mutagenesis of the two putative furin recognition sequences found within the ADAMTS-1 prodomain (RRNR173 and RKKR235) revealed that Arg235 was the sole processing site. Use of the Golgi disturbing agent, Brefeldin A, and monensin suggests that the cleavage of proADAMTS-1 takes place in the Golgi apparatus prior to its secretion. Conserved residues within the prodomain of other ADAMTS members hinted that they might act as maturation determinants. Replacement with alanine of selected residues Cys106, Tyr108, Gly110, Cys125, and Cys181 and residues encompassing the 137-144 sequence significantly affected the biosynthetic profile of the enzyme. Our results suggest that conserved residues other than the furin cleavage site in the prodomain of ADAMTS-1 are involved in its biosynthesis.     

Asymmetric localizations of LIN-17/Fz and MIG-5/Dsh are involved in the asymmetric B cell division in C. elegans

LIN-44/Wnt and LIN-17/Frizzled (Fz) function in a planar cell polarity (PCP)-like pathway to regulate the asymmetric B cell division in Caenorhabditis elegans. We observed asymmetric localization of LIN-17/Frizzled (Fz) and MIG-5/Dishevelled (Dsh) during the B cell division. LIN-17::GFP was asymmetrically localized within the B cell prior to and after the B cell division and correlated with B cell polarity. Asymmetric localization of LIN-17::GFP was dependent upon LIN-44/Wnt and MIG-5/Dsh function. The LIN-17 transmembrane domain and a portion of the cysteine-rich domain (CRD) were required for LIN-17 function and asymmetric distribution to the B cell daughters, while the conserved KTXXXW motif was only required for function. MIG-5::GFP was also asymmetrically localized within the B cell prior to and after the B cell division in a LIN-17- and LIN-44-dependent manner. Functions of the MIG-5 DEP, PDZ and DIX domains were also conserved. Thus, a novel PCP-like pathway, in which LIN-17 and MIG-5 are asymmetrically localized, is involved in the regulation of B cell polarity.     

Activation of the proteolytic activity of ADAMTS4 (aggrecanase-1) by C-terminal truncation.

Proteolysis of the hyalectans (aggrecan, versican, brevican) in vivo appears to result from the activity of ADAMTS4 (aggrecanase-1, herein referred to as an hyalectanase). To examine the mode of activation of ADAMTS4, a human chondrosarcoma cell line, JJ012, has been stably transfected with the full-length c-DNA for human ADAMTS4. The cells synthesized a high molecular weight form of the enzyme (p100), which in serum-free culture was processed to three truncated forms, p75, p60, and p50. Treatment of the p100 form with recombinant furin indicated that the p75 form is generated by the removal of the prodomain by a furin-like activity. Analysis with domain-specific antisera showed that the p60 and p50 forms are generated by C-terminal truncation of the p75 form. The appearance of the p60 and p50 forms in culture medium was prevented by inclusion of a furin inhibitor, inhibitors of glycosylphosphatidylinositol synthesis, glucosamine, a hydroxamate-based matrix metalloproteinase (MMP) inhibitor, and TIMP-1, but not by AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride) or E64. Only medium samples containing the p60/p50 forms exhibited aggrecanase activity, and isolation of the p75, p60, and p50 forms by preparative SDS-PAGE showed that only p60 and p50 were active in aggrecanase and versicanase assays. Pig synovium and human cartilages also contained ADAMTS4 in the p75, p60, and p50 forms. We suggest that in vivo production of proteolytically active ADAMTS4 requires not only removal of the prodomain by a furin-like activity but also MMP-mediated removal of a portion of the C-terminal spacer domain.     

ADAMTS1 cleaves aggrecan at multiple sites and is differentially inhibited by metalloproteinase inhibitors

ADAMTS1 is a secreted protein that belongs to the recently described ADAMTS (a disintegrin and metalloprotease with thrombospondin repeats) family of proteases. Evaluation of ADAMTS1 catalytic activity on a panel of extracellular matrix proteins showed a restrictive substrate specificity which includes some proteoglycans. Our results demonstrated that human ADAMTS1 cleaves aggrecan at a previously shown site by its mouse homolog, but we have also identified additional cleavage sites that ultimately confirm the classification of this protease as an 'aggrecanase'. Specificity of ADAMTS1 activity was further verified when a point mutation in the zinc-binding domain abolished its catalytic effects, and latency conferred by the prodomain was also demonstrated using a furin cleavage site mutant. Suppression of ADAMTS1 activity was accomplished with a specific monoclonal antibody and some metalloprotease inhibitors, including tissue inhibitor of metalloproteinases 2 and 3. Finally, we developed an activity assay using an artificial peptide substrate based on the interglobular domain cleavage site (E(373)-A) of rat aggrecan.     

Nuclear translocation of the N-terminal prodomain of interleukin-16

Interleukin-16 (IL-16) is a pleiotropic cytokine that functions as a chemoattractant factor, a modulator of T cell activation, and an inhibitor of human immunodeficiency virus (HIV) replication. These diverse functions are exclusively attributed to the secreted C-terminal peptide of 121 amino acids (mature IL-16), which is cleaved from the precursor protein (pro-IL-16) by caspase-3. Human pro-IL-16 is comprised of 631 amino acids with three PDZ domains, one of which is present in secreted mature IL-16. No cellular localization or biologic functions have been ascribed to the unusually large and highly conserved N-terminal prodomain formed as a result of proteolytic release of the third PDZ domain of pro-IL-16. Here we show that the N-terminal prodomain of pro-IL-16 translocates into the nucleus following cleavage of the C-terminal segment. The nuclear localization signal of pro-IL-16 consists of a classical bipartite nuclear targeting motif. We also show that the nuclear targeting of the IL-16 prodomain induces a G(0)/G(1) arrest in the cell cycle. Taken together, the high degree of conservation of the prodomain among species, the presence of two PDZ motifs, and the nuclear localization and subsequent inhibitory effect on cell cycle progression suggest that pro-IL-16 is cleaved into two functional proteins, a C-terminal-secreted cytokine and an N-terminal product, which affects the cell cycle.     

An NDPase links ADAM protease glycosylation with organ morphogenesis in C. elegans

In the nematode Caenorhabditis elegans, the gonad acquires two U-shaped arms through the directed migration of its distal tip cells (DTCs), which are located at the tip of the growing gonad arms. A member of the ADAM (a disintegrin and metalloprotease) family, MIG-17, regulates directional migration of DTCs: MIG-17 is synthesized and secreted from the muscle cells of the body wall, and diffuses to the gonad where it is required for DTC migration. The mig-23 mutation causes defective migration of DTCs and interacts genetically with mig-17. Here, we report that mig-23 encodes a membrane-bound nucleoside diphosphatase (NDPase) required for glycosylation and proper localization of MIG-17. Our findings indicate that an NDPase affects organ morphogenesis through glycosylation of the MIG-17 ADAM protease.     

The MIG-2/integrin interaction strengthens cell-matrix adhesion and modulates cell motility

Integrin-mediated cell-matrix adhesion plays an important role in control of cell behavior. We report here that MIG-2, a widely expressed focal adhesion protein, interacts with beta1 and beta3 integrin cytoplasmic domains. Integrin binding is mediated by a single site within the MIG-2 FERM domain. Functionally, the MIG-2/integrin interaction recruits MIG-2 to focal adhesions. Furthermore, using alphaIIbbeta3 integrin-expressing Chinese hamster ovary cells, a well described model system for integrin activation, we show that MIG-2 promotes integrin activation and enhances cell-extracellular matrix adhesion. Although MIG-2 is expressed in many cell types, it is deficient in certain colon cancer cells. Expression of MIG-2, but not of an integrin binding-defective MIG-2 mutant, in MIG-2-null colon cancer cells strengthened cell-matrix adhesion, promoted focal adhesion formation, and reduced cell motility. These results suggest that the MIG-2/integrin interaction is an important element in the cellular control of integrin-mediated cell-matrix adhesion and that loss of this interaction likely contributes to high motility of colon cancer cells.     

Molecular signatures of cell migration in C. elegans Q neuroblasts

Metazoan cell movement has been studied extensively in vitro, but cell migration in living animals is much less well understood. In this report, we have studied the Caenorhabditis elegans Q neuroblast lineage during larval development, developing live animal imaging methods for following neuroblast migration with single cell resolution. We find that each of the Q descendants migrates at different speeds and for distinct distances. By quantitative green fluorescent protein imaging, we find that Q descendants that migrate faster and longer than their sisters up-regulate protein levels of MIG-2, a Rho family guanosine triphosphatase, and/or down-regulate INA-1, an integrin α subunit, during migration. We also show that Q neuroblasts bearing mutations in either MIG-2 or INA-1 migrate at reduced speeds. The migration defect of the mig-2 mutants, but not ina-1, appears to result from a lack of persistent polarization in the direction of cell migration. Thus, MIG-2 and INA-1 function distinctly to control Q neuroblast migration in living C. elegans.     

The ADAM10 prodomain is a specific inhibitor of ADAM10 proteolytic activity and inhibits cellular shedding events

ADAM10 is a disintegrin metalloproteinase that processes amyloid precursor protein and ErbB ligands and is involved in the shedding of many type I and type II single membrane-spanning proteins. Like tumor necrosis factor-alpha-converting enzyme (TACE or ADAM17), ADAM10 is expressed as a zymogen, and removal of the prodomain results in its activation. Here we report that the recombinant mouse ADAM10 prodomain, purified from Escherichia coli, is a potent competitive inhibitor of the human ADAM10 catalytic/disintegrin domain, with a K(i) of 48 nM. Moreover, the mouse ADAM10 prodomain is a selective inhibitor as it only weakly inhibits other ADAM family proteinases in the micromolar range and does not inhibit members of the matrix metalloproteinase family under similar conditions. Mouse prodomains of TACE and ADAM8 do not inhibit their respective enzymes, indicating that ADAM10 inhibition by its prodomain is unique. In cell-based assays we show that the ADAM10 prodomain inhibits betacellulin shedding, demonstrating that it could be of potential use as a therapeutic agent to treat cancer.     

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.