Interaction of the metalloprotease disintegrins MDC9 and MDC15 with two SH3 domain-containing proteins, endophilin I and SH3PX1.

Metalloprotease disintegrins (a disintegrin and metalloprotease (ADAM) and metalloprotease, disintegrin, cysteine-rich proteins (MDC)) are a family of membrane-anchored glycoproteins that function in diverse biological processes, including fertilization, neurogenesis, myogenesis, and ectodomain processing of cytokines and other proteins. The cytoplasmic domains of ADAMs often include putative signaling motifs, such as proline-rich SH3 ligand domains, suggesting that interactions with cytoplasmic proteins may affect metalloprotease disintegrin function. Here we report that two SH3 domain-containing proteins, endophilin I (SH3GL2, SH3p4) and a novel SH3 domain- and phox homology (PX) domain-containing protein, termed SH3PX1, can interact with the cytoplasmic domains of the metalloprotease disintegrins MDC9 and MDC15. These interactions were initially identified in a yeast two-hybrid screen and then confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells expressing both binding partners. SH3PX1 and endophilin I both preferentially bind the precursor but not the processed form of MDC9 and MDC15 in COS-7 cells. Since rat endophilin I is thought to play a role in synaptic vesicle endocytosis and SH3PX1 has sequence similarity to sorting nexins in yeast, we propose that endophilin I and SH3PX1 may have a role in regulating the function of MDC9 and MDC15 by influencing their intracellular processing, transport, or final subcellular localization.     

Cloning and expression in Pichia pastoris of metalloprotease domain of ADAM 9 catalytically active against fibronectin

ADAM 9 is a member of the cellular metalloprotease/disintegrin/cysteine-rich (MDC) gene family, related to soluble snake venom metalloproteases (SVMP). ADAMs may play important roles in cell-cell fusion, cell-matrix interaction, and other cellular functions. To investigate catalytic activity of human ADAM 9 we have cloned and expressed the metalloprotease domain of human ADAM 9 in Pichia pastoris. The recombinant protein was purified in a three-step purification procedure and activity was detected against gelatin, beta-casein, and fibronectin. In addition we identified five normal and cancer cell lines expressing mRNA of human ADAM 9.     

ADAM13 disintegrin and cysteine-rich domains bind to the second heparin-binding domain of fibronectin

ADAM13 is a member of the disintegrin and metalloprotease protein family that is expressed on cranial neural crest cells surface and is essential for their migration. ADAM13 is an active protease that can cleave fibronectin in vitro and remodel a fibronectin substrate in vivo. Using a recombinant secreted protein containing both disintegrin and cysteine-rich domains of ADAM13, we show that this "adhesive" region of the protein binds directly to fibronectin. Fibronectin fusion proteins corresponding to the various functional domains were used to define the second heparin-binding domain as the ADAM13 binding site. Mutation of the syndecan-binding site (PPRR --> PPTM) within this domain abolishes binding of the recombinant disintegrin and cysteine-rich domains of ADAM13. We further show that the adhesive disintegrin and cysteine-rich domain of ADAM13 can promote cell adhesion via beta(1) integrins. This adhesion requires integrin activation and can be prevented by antibodies to the cysteine-rich domain of ADAM13 and beta(1) integrin. Finally, wild type, but not the E/A mutant of ADAM13 metalloprotease domain, can be shed from the cell surface, releasing the metalloprotease domain associated with the disintegrin and cysteine-rich domains. This suggests that ADAM13 shedding may involve its own metalloprotease activity and that the released protease may interact with both integrins and extracellular matrix proteins.     

Disintegrin-like/Cysteine-Rich Region of ADAM 12 Is an Active Cell Adhesion Domain

ADAM (a disintegrin and metalloprotease) proteins contain structural homology to the P-III class of snake venom metalloproteases (SVMPs) and are postulated to function, by analogy to these SMVPs, as cell adhesion molecules. ADAM 12 has been implicated in fusion of myoblasts, but its mechanism of action is not known. Instead of the RGD-like cell-binding motif present in SVMP disintegrins, the disintegrin domain of ADAM 12 contains a unique SNS sequence and therefore its adhesive potential has been controversial. In this report we demonstrate that the disintegrin-like/cysteine-rich (DC) domain of ADAM 12 constitutes a functional cell adhesion domain. We have expressed the DC domain of mouse ADAM 12 in insect cells and shown that the recombinant protein supported adhesion of C2C12 myoblasts and NIH 3T3 fibroblasts in a divalent cation-dependent manner. A sulfhydryl-specific biotinylation reagent revealed, however, that the overall conformation and flexibility of the cell-binding region of ADAM 12 DC domain may be significantly different from those of the SVMP disintegrins. Moreover, the disulfide bond structure of the DC domain was critical for its function, as incubation of the recombinant protein with reducing agents abolished subsequent cell adhesion. Recombinant DC bound to C2C12 cells with high affinity (K(D) approximately 0.10 microM, total number of binding sites n approximately 4.6 x 10(5)/cell). Adhesive properties of the DC domain of ADAM 12 produced in insect cells were further confirmed by cell surface binding of the DC domain expressed in C2C12 cells and secreted to the medium, consistent with the role of ADAM 12 in cell-cell interactions and myoblast fusion.     

Cooperation of the metalloprotease, disintegrin, and cysteine-rich domains of ADAM12 during inhibition of myogenic differentiation

The extracellular domain of the mature form of ADAM12 consists of the metalloprotease, disintegrin, cysteine-rich, and epidermal growth factor (EGF)-like domains. The disintegrin, cysteine-rich, and EGF-like fragments have been shown previously to support cell adhesion via activated integrins or proteoglycans. In this study, we report that the entire extracellular domain of mouse ADAM12 produced in Drosophila S2 cells supported efficient adhesion and spreading of C2C12 myoblasts even in the absence of exogenous integrin activators. This adhesion was not mediated by beta1 integrins or proteoglycans, was myoblast-specific, and required the presence of both the metalloprotease and disintegrin/cysteine-rich domains of ADAM12. Analysis of the recombinant proteins by far-UV circular dichroism suggested that the secondary structures of the autonomously expressed metalloprotease domain and the disintegrin/cysteine-rich/EGF-like domains differ from the structures present in the intact extracellular domain. Furthermore, the intact extracellular domain (but not the metalloprotease domain or the disintegrin/cysteine-rich/EGF-like fragment alone) decreased the expression of the cell cycle inhibitor p21 and myogenin, two markers of differentiation, and inhibited C2C12 myoblast fusion. Thus, the novel protein-protein interaction reported here involving the extracellular domain of ADAM12 may have important biological consequences during myoblast differentiation.     

ADAMs: key components in EGFR signalling and development

ADAM (a disintegrin and metalloprotease) proteins are membrane-anchored metalloproteases that process and shed the ectodomains of membrane-anchored growth factors, cytokines and receptors. ADAMs also have essential roles in fertilization, angiogenesis, neurogenesis, heart development and cancer. Research on ADAMs and their role in protein ectodomain shedding is emerging as a fertile ground for gathering new insights into the functional regulation of membrane proteins.     

The cysteine-rich domain regulates ADAM protease function in vivo

ADAMs are membrane-anchored proteases that regulate cell behavior by proteolytically modifying the cell surface and ECM. Like other membrane-anchored proteases, ADAMs contain candidate "adhesive" domains downstream of their metalloprotease domains. The mechanism by which membrane-anchored cell surface proteases utilize these putative adhesive domains to regulate protease function in vivo is not well understood. We address this important question by analyzing the relative contributions of downstream extracellular domains (disintegrin, cysteine rich, and EGF-like repeat) of the ADAM13 metalloprotease during Xenopus laevis development. When expressed in embryos, ADAM13 induces hyperplasia of the cement gland, whereas ADAM10 does not. Using chimeric constructs, we find that the metalloprotease domain of ADAM10 can substitute for that of ADAM13, but that specificity for cement gland expansion requires a downstream extracellular domain of ADAM13. Analysis of finer resolution chimeras indicates an essential role for the cysteine-rich domain and a supporting role for the disintegrin domain. These and other results reveal that the cysteine-rich domain of ADAM13 cooperates intramolecularly with the ADAM13 metalloprotease domain to regulate its function in vivo. Our findings thus provide the first evidence that a downstream extracellular adhesive domain plays an active role in regulating ADAM protease function in vivo. These findings are likely relevant to other membrane-anchored cell surface proteases.     

Structures of two elapid snake venom metalloproteases with distinct activities highlight the disulfide patterns in the D domain of ADAMalysin family proteins

The structures of snake venom metalloproteases (SVMPs) are proposed to be useful models to understand the structural and functional relationship of ADAM (a disintegrin and metalloprotease) which are membrane-anchored proteins involved in multiple human diseases. We have purified, sequenced and determined the structures of two new P-III SVMPs – atragin and kaouthiagin-like (K-like) from Naja atra. Atragin exhibits a known C-shaped topology, whereas K-like adopts an I-shaped conformation because of the distinct disulfide pattern in the disintegrin-like (D) domain. K-like exhibits an enzymatic specificity toward pro-TNFα with less inhibition of cell migration, but atragin shows the opposite effect. The specificity of the enzymatic activity is indicated to be dominated mainly by the local structures of SVMP in the metalloprotease (M) domain, whereas the hyper-variable region (HVR) in the cysteine-rich (C) domain is involved in a cell-migration activity. We demonstrate also a pH-dependent enzymatic activity of atragin that we correlate with the structural dynamics of a Zn²⁺-binding motif and the Met-turn based on the structures determined with a pH-jump method. The structural variations between the C- and I-shapes highlight the disulfide bond patterns in the D domain of the ADAM/adamalysin/reprolysins family proteins.     

A novel disintegrin-like domain of a high molecular weight metalloprotease inhibits platelet aggregation

Disintegrin is one of the functionally distinct domains in high molecular weight metalloproteases from various snake venoms and generally has an Arg-Gly-Asp (RGD) sequence that is recognized by specific cell surface integrins. A cDNA encoding the disintegrin-like domain of a snake venom metalloprotease was cloned, expressed in Pichia pastoris, and molecular function of the recombinant protein was characterized. The cDNA sequence indicated that the disintegrin-like domain contains an Asp-Glu-Cys-Asp (DECD) sequence in place of the RGD motif. The expressed disintegrin-like protein was designated as halydin and it was able to inhibit human platelet aggregation in a dose-dependent manner. Unlike other typical RGD-disintegrins, the recombinant non-RGD disintegrin, halydin, inhibited platelet aggregation by suppressing platelet adhesion to collagen rather than by blocking fibrinogen binding to glycoprotein (GP) IIb-IIIa on the platelet surface. Experimental evidence suggests that halydin binds to integrin alpha2beta1 on the platelet surface.     

Role of multiple beta1 integrins in cell adhesion to the disintegrin domains of ADAMs 2 and 3

ADAM disintegrin domains can support integrin-mediated cell adhesion. However, the profile of which integrins are employed for adhesion to a given disintegrin domain remains unclear. For example, we suggested that the disintegrin domains of mouse sperm ADAMs 2 and 3 can interact with the alpha6beta1 integrin on mouse eggs. Others concluded that these disintegrin domains interact instead with the alpha9beta1 integrin. To address these differing results, we first studied adhesion of mouse F9 embryonal carcinoma cells and human G361 melanoma cells to the disintegrin domains of mouse ADAMs 2 and 3. Both cell lines express alpha6beta1 and alpha9beta1 integrins at their surfaces. Antibodies to the alpha6 integrin subunit inhibited adhesion of both cell lines. An antibody that recognizes human alpha9 integrin inhibited adhesion of G361 cells. VLO5, a snake disintegrin that antagonizes alpha4beta1 and alpha9beta1 integrins, potently inhibited adhesion of both cell lines. We next explored expression of the alpha9 integrin subunit in mouse eggs. In contrast to our ability to detect alpha6beta1, we were unable to convincingly detect alpha9beta1 integrin on the surface of mouse eggs. Moreover, treatment of mouse eggs with 250 nm VLO5, which is 250 fold over its approximately IC(50) for inhibition of somatic cell adhesion, had minimal effect on sperm-egg binding or fusion. We did detect alpha9 integrin protein on epithelial cells of the oviduct. Additional studies showed that antibodies to the alpha6 and alpha7 integrins additively inhibited adhesion of mouse trophoblast stem cells and that an antibody to the alpha4 integrin inhibited adhesion of MOLT-3 cells to these disintegrin domains: Our data suggest that multiple integrins (on the same cell) can participate in adhesion to a given ADAM disintegrin domain and that interactions between ADAMs and integrins may be important for sperm transit through the oviduct.     

The ADAMDEC1 (decysin) gene structure: evolution by duplication in a metalloprotease gene cluster on chromosome 8p12

Members of the ADAM superfamily of metalloprotease genes are involved in a number of biological processes, including fertilization, neurogenesis, muscle development, and the immune response. These proteins have been classified into several groups. The prototypic ADAM family is comprised of a pro-domain, a metalloprotease domain, a disintegrin domain, a cysteine-rich region, a transmembrane domain, and a variable cytoplasmic tail. We recently identified a novel member of this superfamily, ADAMDEC1 (decysin). Due to the partial lack of a disintegrin domain and the total lack of a cysteine-rich domain, this protein has been placed in a novel subclass of the ADAM gene family. We have investigated the gene structure of the human and mouse ADAMDEC1 and have revealed a metalloprotease gene cluster on human Chromosome 8p12 comprising ADAMDEC1, ADAM7, and ADAM28. Our results suggest that ADAMDEC1 has arisen by partial gene duplication from an ancestral gene at this locus and has acquired a novel function. ADAMDEC1 is expressed in the immune system, by dendritic cells and macrophages. The relatedness of ADAMDEC1, ADAM7, and ADAM28 suggests that these proteases share a similar function.     

ADAM的遗传和进化

ADAM,又称MDC,分别是去整合蛋白和金属蛋白水解酶（a disintegrin and metalloproteinase,ADAM）及金属蛋白水解酶、去整合蛋白和富半胱氨酸（metalloproteinase/disintegrin/cysteine-rich,MDC）的英文缩写,是近几年在多细胞动物中发现的一类含信号肽区、前调控区、金属蛋白水解酶区、去整合蛋白区、富半胱氨酸区、上皮生长因子区、跨膜区和胞内区的细胞表面糖蛋白,本文简要总结了有关ADAM起源、遗传、进化和亲缘关系的研究结果。 Abstract:ADAM (a disintegrin and metalloproteinase),or named MDC (metalloproteinase/disintegrin/cysteine-rich) is a family of glycoproteins in cell surface,which was found in recent years and consists of a signal peptide,a propetide,a metalloproteinase,a disintegrin,a cysteine-rich domain,and an epidermal growth factor (EGF)-like domain,a transmembrane region,and a cytoplasmic tail.The research results about their origin,heredity,evolution and evolutionary relationship are summaried in this paper.     

Crystal structure of the catalytic domain of human ADAM33

Adam33 is a putative asthma susceptibility gene encoding for a membrane-anchored metalloprotease belonging to the ADAM family. The ADAMs (a disintegrin and metalloprotease) are a family of glycoproteins implicated in cell-cell interactions, cell fusion, and cell signaling. We have determined the crystal structure of the Adam33 catalytic domain in complex with the inhibitor marimastat and the inhibitor-free form. The structures reveal the polypeptide fold and active site environment resembling that of other metalloproteases. The substrate-binding site contains unique features that allow the structure-based design of specific inhibitors of this enzyme.     

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.     

ADAM 23/MDC3, a human disintegrin that promotes cell adhesion via interaction with the alphavbeta3 integrin through an RGD-independent mechanism

ADAM 23 (a disintegrin and metalloproteinase domain)/MDC3 (metalloprotease, disintegrin, and cysteine-rich domain) is a member of the disintegrin family of proteins expressed in fetal and adult brain. In this work we show that the disintegrin-like domain of ADAM 23 produced in Escherichia coli and immobilized on culture dishes promotes attachment of different human cells of neural origin, such as neuroblastoma cells (NB100 and SH-S(y)5(y)) or astrocytoma cells (U373 and U87 MG). Analysis of ADAM 23 binding to integrins revealed a specific interaction with alphavbeta3, mediated by a short amino acid sequence present in its putative disintegrin loop. This sequence lacks any RGD motif, which is a common structural determinant supporting alphavbeta3-mediated interactions of diverse proteins, including other disintegrins. alphavbeta3 also supported adhesion of HeLa cells transfected with a full-length cDNA for ADAM 23, extending the results obtained with the recombinant protein containing the disintegrin domain of ADAM 23. On the basis of these results, we propose that ADAM 23, through its disintegrin-like domain, may function as an adhesion molecule involved in alphavbeta3-mediated cell interactions occurring in normal and pathological processes, including progression of malignant tumors from neural origin.     

Catalytic activity of ADAM28

ADAMs are membrane-anchored glycoproteins containing a disintegrin and metalloprotease domain that have important roles in fertilization, development, and diseases such as Alzheimer's dementia. Here we present the first evidence for catalytic activity of ADAM28, a protein that is highly expressed in the epididymis and lymphocytes. Recombinant ADAM28 cleaves myelin basic protein at two sites. The catalytic activity of ADAM28 is not sensitive to tissue inhibitors of metalloproteases 1 and 2, but can be abolished by a mutation in the catalytic site. Catalytically active ADAM28 will be valuable for further studies of its role in sperm maturation and lymphocyte function.     

cDNA cloning and characterization of vascular apoptosis-inducing protein 1

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification from crude venom of Crotalus atrox of two vascular apoptosis-inducing proteins (VAP1 and VAP2) that specifically induce apoptosis in vascular endothelial cells. We report here the cDNA cloning and characterization of VAP1. VAP1 cDNA encoded a protein with 610 amino acid residues. The amino acid sequence predicted from the cDNA indicated that VAP1 belongs to the metalloprotease/disintegrin family and that it is a multidomain polypeptide with a proprotein domain, a metalloprotease domain, a disintegrin-like domain, and a cysteine-rich domain. In the disintegrin-like domain, the sequence DECD replaces the RGD sequence that has frequently been found in such domains. We demonstrated that VAP1 has Zn(2+)-dependent metalloprotease activity and degrades fibrinogen. After incubation in the presence of either EDTA or EGTA, VAP1 was hardly able to degrade fibrinogen and to induce apoptosis in VEC. Our results indicated that VAP1 is a new type of snake venom metalloprotease/disintegrin and suggest that the metalloprotease activity of VAP1 might be involved in the induction of apoptosis by VAP1 in VEC.     

MDC-9 (ADAM-9/Meltrin gamma) functions as an adhesion molecule by binding the alpha(v)beta(5) integrin

MDC-9 is a widely expressed member of the metalloproteinase/disintegrin/cysteine-rich protein family. The disintegrin domain of MDC-9 lacks an RGD motif but has recently been reported to bind the alpha(6)beta(1) integrin; however, it is unclear whether MDC-9 can bind other integrins. In the present study myeloma cells, but not lymphoblastoid cells, were shown to bind to immobilised, recombinantly expressed MDC-9 disintegrin domain (A9dis). Binding was divalent cation-dependent, being supported by Mn(2+) and Ca(2+). Adhesion of myeloma cells to A9dis was completely inhibited by an antibody to the alpha(v)beta(5) integrin but not by antibodies to other subunits. RGD-containing peptides had no effect on binding, suggesting that MDC-9 interacts with alpha(v)beta(5) in an RGD-independent manner. Flow cytometric analyses demonstrated that myeloma cells, but not lymphoblastoid cells, expressed alpha(v)beta(5) on the cell membrane. These data indicated that the disintegrin domain of MDC-9 can function as an adhesion molecule by interacting with an alpha(v)beta(5) integrin.     

Structural basis of the autolysis of AaHIV suggests a novel target recognizing model for ADAM/reprolysin family proteins

AaHIV, a P-III-type snake venom metalloproteinase (SVMP), consists of metalloproteinase/disintegrin/cysteine-rich (MDC) domains and is homologous to a disintegrin and metalloproteinase (ADAM) family proteins. Similar to brevilysin H6 and jararhagin, AaHIV can easily autolyse to release a stable protein named acucetin, which contains disintegrin-like and cysteine-rich domains. In this study, we determined the crystal structure of AaHIV and investigated the autolysis mechanism. Based on the structure of AaHIV and the results from docking experiments, we present a new model for target recognition in which two protein molecules form a functional unit, and the DC domain of one molecule is used for target recognition while the M-domain of the other is used for target proteolysis. Our results shed new light on the mechanism of target recognition and processing in ADAM/reprolysin family proteins.     

A metalloprotease-disintegrin, MDC9/meltrin-gamma/ADAM9 and PKCdelta are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor.

The ectodomains of many proteins located at the cell surface are shed upon cell stimulation. One such protein is the heparin-binding EGF-like growth factor (HB-EGF) that exists in a membrane-anchored form which is converted to a soluble form upon cell stimulation with TPA, an activator of protein kinase C (PKC). We show that PKCdelta binds in vivo and in vitro to the cytoplasmic domain of MDC9/meltrin-gamma/ADAM9, a member of the metalloprotease-disintegrin family. Furthermore, the presence of constitutively active PKCdelta or MDC9 results in the shedding of the ectodomain of proHB-EGF, whereas MDC9 mutants lacking the metalloprotease domain, as well as kinase-negative PKCdelta, suppress the TPA-induced shedding of the ectodomain. These results suggest that MDC9 and PKCdelta are involved in the stimulus-coupled shedding of the proHB-EGF ectodomain.