The interaction between ADAM22 and 14-3-3β

ADAM family consists of a number of transmembrane proteins that contain a disintegrin and metalloprotease domain. ADAMs are involved in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis and inflammatory response. The ADAM proteins have both cell adhesion and protease activities. Adam22 is highly expressed in human brain. The adam22-/- mice presented severe ataxia and died before weaning, but the function of ADAM22 is still unknown. 14-3-3 β interacting with ADAM22 was detected by using yeast two-hybrid assay. The specificity of interaction between ADAM22 and 14-3-3β was proved by in vitro binding assay and immunoprecipitation. The major 14-3-3β binding site was located in the last 28 amino acid residues of ADAM22 cytoplasmic tail. Protein 14-3-3β is abundant and plays an important role in mediating cell diffusion, migration and cell cycle control. The interaction of ADAM22 and 14-3-3β suggests that the ADAM22 may play a crucial role in neural function a     

Transcriptomic analysis of IgG4 Fc-fusion protein degradation in a panel of clonally-derived CHO cell lines using RNASeq

In this study, we report an investigation of a panel of clonally-derived Chinese hamster ovary (CHO) cell lines exhibiting variability in the proportion of full-length IgG4 Fc-fusion protein produced. The recombinant protein was found to be degraded during cell culture into four shorter “clipped” species (three of the four cleavage sites occurred at arginine residues) and preliminary analyses suggested that a host cell enzyme was responsible for proteolysis. To identify the specific enzyme responsible, RNA sequencing was used to identify gene expression differences between the cell lines with a “high” and “low” clipping phenotype. From this analysis, six protease-encoding genes were found to be significantly upregulated in those cell lines yielding the lowest proportion of full-length IgG4 Fc-fusion protein. Four of these protease candidates were deprioritized after examination of their cleavage site specificity. The remaining enzymes, Adam19 and Furin, were found to be capable of cleavage at arginine residues, and inhibitors for both proteases were added to cell-free media to determine if the product degradation could be reduced. While the Adam19 inhibitor had no impact, Furin inhibitor I (specific for the proprotein convertase family of enzymes) was found to result in a 33–39% increase in complete IgG4 Fc-fusion protein when compared with untreated samples.     

The interaction between ADAM22 and 14-3-3β

ADAM family consists of a number of transmembrane proteins that contain a disintegrin and metalloprotease domain. ADAMs are involved in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis and inflammatory response. The ADAM proteins have both cell adhesion and protease activities. Adam22 is highly expressed in human brain. The adam22-/- mice presented severe ataxia and died before weaning, but the function of ADAM22 is still unknown. 14-3-3β interacting with ADAM22 was detected by using yeast two-hybrid assay. The specificity of interaction between ADAM22 and 14-3-3β was proved by in vitro binding assay and immunoprecipitation. The major 14-3-3β binding site was located in the last 28 amino acid residues of ADAM22 cytoplasmic tail. Protein 14-3-3β is abundant and plays an important role in mediating cell diffusion, migration and cell cycle control. The interaction of ADAM22 and 14-3-3β suggests that the ADAM22 may play a crucial role in neural function and development.     

The interaction between ADAM22 and 14-3-3β

ADAM family consists of a number of transmembrane proteins that contain a disintegrin and metalloprotease domain. ADAMs are involved in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis and inflammatory response. The ADAM proteins have both cell adhesion and protease activities.Adam22 is highly expressed in human brain. Theadam22-/- mice presented severe ataxia and died before weaning, but the function of ADAM22 is still unknown. 14-3-3 β interacting with ADAM22 was detected by using yeast two-hybrid assay. The specificity of interaction between ADAM22 and 14-3-3β was proved byin vitro binding assay and immunoprecipitation. The major 14-3-3β binding site was located in the last 28 amino acid residues of ADAM22 cytoplasmic tail. Protein 14-3-3β is abundant and plays an important role in mediating cell diffusion, migration and cell cycle control. The interaction of ADAM22 and 14-3-3β suggests that the ADAM22 may play a crucial role in neural function and development.     

Characterization and comparative genomic analysis of intronless Adams with testicular gene expression

ADAM (a disintegrin and metalloprotease) family members with testis-specific or -predominant gene expression are divided phylogenically into two groups: ADAMs 2, 3, 5, 27, and 32 (the first group) and ADAMs 4, 6, 20, 21, 24, 25, 26, 29, 30, and 34 (the second group). We cloned and sequenced cDNAs for previously unidentified mouse Adams that belong to the second group. We found that all the Adam genes in the second phylogenic group are transcribed by both somatic and germ cells in mouse testis, representing a unique expression pattern different from that of the first-group Adams. Genomic analyses revealed that all the second-group Adam genes lack introns interrupting protein-coding sequences and many of them are present as multicopy genes, resulting in total of 14 functional mouse genes in this phylogenic group. Comparing the mouse and human ADAM genes, we found that a number of these mouse Adam genes do not have human orthologues and, even if they exist, some orthologues are pseudogenes in human. These results suggest the differential expansion of the second-group Adam genes in the mouse genome during evolution and a relationship between these Adams and male reproduction unique to mouse.     

Identification of heat shock protein 5, calnexin and integral membrane protein 2B as Adam7-interacting membrane proteins in mouse sperm

In mammals, sperm acquire their motility and ability to fertilize eggs in the epididymis. This maturation process involves the acquisition of particular proteins from the epididymis. One such secretory protein specifically expressed in the epididymis is Adam7 (a disintegrin and metalloprotease 7). Previous studies have shown that Adam7 that resides in an intracellular compartment of epididymal cells is transferred to sperm membranes, where its levels are dependent on the expression of Adam2 and Adam3, which have critical roles in fertilization. Here, using a proteomics approach based on mass spectrometry, we identified proteins that interact with Adam7 in sperm membranes. This analysis revealed that Adam7 forms complexes with calnexin (Canx), heat shock protein 5 (Hspa5), and integral membrane protein 2B (Itm2b). Canx and Hspa5 are molecular chaperones, and Itm2b is a type II integral membrane protein implicated in neurodegeneration. The interaction of Adam7 with these proteins was confirmed by immunoprecipitation-Western blot analysis. We found that Adam7 and Itm2b are located in detergent-resistant regions known to be highly correlated with membrane lipid rafts. We further found that the association of Adam7 with Itm2b is remarkably promoted during sperm capacitation owing to a conformational change of Adam7 that occurs in concert with the capacitation process. Thus, our results suggest that Adam7 functions in fertilization through the formation of a chaperone complex and enhanced association with Itm2b during capacitation in sperm.     

Expression and relationship of male reproductive ADAMs in mouse

A number of a disintegrin and metalloprotease (ADAM) family members are expressed in mammalian male reproductive organs such as testis and epididymis. These reproductive ADAMs are divided phylogenically into three major groups: ADAMs 1, 4, 6, 20, 21, 24, 25, 26, 29, 30, and 34 (the first group); ADAMs 2, 3, 5, 27, and 32 (the second group); and ADAMs 7 and 28 (the third group). Previous mouse knockout studies indicate that ADAM1, ADAM2, and ADAM3 have intricate expressional relationships, playing critical roles in fertilization. In the present study, we analyzed processing, biochemical characteristics, localization, and expressional relationship of the previously-unexplored, second-group ADAMs (ADAM5, ADAM27, and ADAM32). We found that all of the three ADAMs are made as precursors in the testis and processed during epididymal maturation, and that ADAM5 and ADAM32, but not ADAM27, are located on the sperm surface. Using sperm from Adam2(-/-) and Adam3(-/-) mice, we found that, among the three ADAMs, the level of ADAM5 is modestly and severely reduced in Adam3 and Adam2 knockout sperm, respectively. Further, we analyzed ADAM7, an epididymis-derived sperm surface ADAM from the separate phylogenetic group, in the knockout sperm. We found that the level of ADAM7 is also significantly reduced in both Adam2 and Adam3-null sperm. Taken together, our results suggest a novel expressional relationship of ADAM5 and ADAM7 with ADAM2 and ADAM3, which play critical roles in fertilization.     

Human NOTCH2 Is Resistant to Ligand-independent Activation by Metalloprotease Adam17

Cell surface receptors of the NOTCH family of proteins are activated by ligand induced intramembrane proteolysis. Unfolding of the extracellular negative regulatory region (NRR), enabling successive proteolysis by the enzymes Adam10 and γ-secretase, is rate-limiting in NOTCH activation. Mutations in the NOTCH1 NRR are associated with ligand-independent activation and frequently found in human T-cell malignancies. In mammals four NOTCH receptors and five Delta/Jagged ligands exist, but mutations in the NRR are only rarely reported for receptors other than NOTCH1. Using biochemical and functional assays, we compared the molecular mechanisms of ligand-independent signaling in NOTCH1 and the highly related NOTCH2 receptor. Both murine Notch1 and Notch2 require the metalloprotease protease Adam17, but not Adam10 during ligand-independent activation. Interestingly, the human NOTCH2 receptor is resistant to ligand-independent activation compared with its human homologs or murine orthologs. Taken together, our data reveal subtle but functionally important differences for the NRR among NOTCH paralogs and homologs.     

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.     

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.     

Human ADAM33: protein maturation and localization

ADAM33 (a disintegrin and metalloprotease) was recently found to be a novel asthma susceptibility gene. Domain-specific antibodies were used to study its expression and processing. When the pro-domain and catalytic domain were expressed by a stable-transfected cell line, the pro-domain was removed by cleavage within a putative furin cleavage site. The catalytic domain was active in an alpha(2)-macroglobulin complex formation assay and mutation of the catalytic site glutamic acid (E346A) eliminated activity. In transient transfections using the full-length protein, a pro-form and mature form were detectable and alternate glycosylation was demonstrated at sites within the catalytic domain. ADAM33 was detected on the cell surface, with the majority of protein detected intracellularly. The E346A mutation had no significant effect on protein processing. Endogenous ADAM33 was detected in bronchus tissue, bronchial smooth muscle cells, and MRC-5 fibroblasts, consistent with a role in the pathophysiology of asthma.     

ADAMDEC1 Is a Metzincin Metalloprotease with Dampened Proteolytic Activity

ADAMDEC1 (Decysin-1) is a putative ADAM (a disintegrin and metalloprotease)-like metalloprotease with an unknown physiological role, selectively expressed in mature dendritic cells and macrophages. When compared with other members of the ADAM family, ADAMDEC1 displays some unusual features. It lacks the auxiliary cysteine-rich, EGF, and transmembrane domains, as well as the cytoplasmic tail. The active site of ADAMDEC1 is unique by being the only mammalian ADAM protease with a non-histidine zinc ligand, having an aspartic acid residue instead. Here we demonstrate that ADAMDEC1, despite these unique features, functions as an active metalloprotease. Thus, ADAMDEC1 is secreted as a mature, glycosylated, and proteolytically active metalloprotease, capable of cleaving macromolecular substrates. In the recombinant form, three of the four potential N-linked glycosylation sites are modified by carbohydrate attachment. Substitution of basic residues at the predicted proprotein convertase cleavage site blocks proprotein processing, revealing both specific ADAMDEC1-dependent and specific ADAMDEC1-independent cleavage of the prodomain. The pro-form of ADAMDEC1 does not have proteolytic activity, demonstrating that the prodomain of ADAMDEC1, like in other members of the ADAM family, confers catalytic latency. Interestingly, the proteolytic activity of mature ADAMDEC1 can be significantly enhanced when a canonical ADAM active site with three zinc-coordinating histidine residues is introduced.     

Binding of ADAM12, a marker of skeletal muscle regeneration, to the muscle-specific actin-binding protein, alpha -actinin-2, is required for myoblast fusion

ADAM12 belongs to the transmembrane metalloprotease ADAM ("a disintegrin and metalloprotease") family. ADAM12 has been implicated in muscle cell differentiation and fusion, but its precise function remains unknown. Here, we show that ADAM12 is dramatically up-regulated in regenerated, newly formed fibers in vivo. In C2C12 cells, ADAM12 is expressed at low levels in undifferentiated myoblasts and is transiently up-regulated at the onset of differentiation when myoblasts fuse into multinucleated myotubes, whereas other ADAMs, such as ADAMs 9, 10, 15, 17, and 19, are expressed at all stages of differentiation. Using the yeast two-hybrid screen, we found that the muscle-specific alpha-actinin-2 strongly binds to the cytoplasmic tail of ADAM12. In vitro binding assays with GST fusion proteins confirmed the specific interaction. The major binding site for alpha-actinin-2 was mapped to a short sequence in the membrane-proximal region of ADAM12 cytoplasmic tail; a second binding site was identified in the membrane-distal region. Co-immunoprecipitation experiments confirm the in vivo association of ADAM12 cytoplasmic domain with alpha-actinin-2. Overexpression of the entire cytosolic ADAM12 tail acted in a dominant negative fashion by inhibiting fusion of C2C12 cells, whereas expression of a cytosolic ADAM12 lacking the major alpha-actinin-2 binding site had no effect on cell fusion. Our results suggest that interaction of ADAM12 with alpha-actinin-2 is important for ADAM12 function.     

A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice

Staphylococcus aureus is a major cause of human disease, responsible for half a million infections and approximately 20,000 deaths per year in the United States alone. This pathogen secretes α-hemolysin, a pore-forming cytotoxin that contributes to the pathogenesis of pneumonia. α-hemolysin injures epithelial cells in vitro by interacting with its receptor, the zinc-dependent metalloprotease ADAM10 (ref. 6). We show here that mice harboring a conditional disruption of the Adam10 gene in lung epithelium are resistant to lethal pneumonia. Investigation of the molecular mechanism of toxin-receptor function revealed that α-hemolysin upregulates ADAM10 metalloprotease activity in alveolar epithelial cells, resulting in cleavage of the adherens junction protein E-cadherin. Cleavage is associated with disruption of epithelial barrier function, contributing to the pathogenesis of lethal acute lung injury. A metalloprotease inhibitor of ADAM10 prevents E-cadherin cleavage in response to Hla; similarly, toxin-dependent E-cadherin proteolysis and barrier disruption is attenuated in ADAM10-knockout mice. Together, these data attest to the function of ADAM10 as the cellular receptor for α-hemolysin. The observation that α-hemolysin can usurp the metalloprotease activity of its receptor reveals a previously unknown mechanism of pore-forming cytotoxin action in which pathologic insults are not solely the result of irreversible membrane injury and defines ADAM10 inhibition as a strategy to attenuate α-hemolysin-induced disease.     

Selective modulation of integrin-mediated cell migration by distinct ADAM family members

A disintegrin and a metalloprotease (ADAM) family members have been implicated in many biological processes. Although it is recognized that recombinant ADAM disintegrin domains can interact with integrins, little is known about ADAM-integrin interactions in cellular context. Here, we tested whether ADAMs can selectively regulate integrin-mediated cell migration. ADAMs were expressed in Chinese hamster ovary cells that express defined integrins (alpha4beta1, alpha5beta1, or both), and cell migration on full-length fibronectin or on its alpha4beta1 or alpha5beta1 binding fragments was studied. We found that ADAMs inhibit integrin-mediated cell migration in patterns dictated by the integrin binding profiles of their isolated disintegrin domains. ADAM12 inhibited cell migration mediated by the alpha4beta1 but not the alpha5beta1 integrin. ADAM17 had the reciprocal effect; it inhibited alpha5beta1- but not alpha4beta1-mediated cell migration. ADAM19 and ADAM33 inhibited migration mediated by both alpha4beta1 and alpha5beta1 integrins. A point mutation in the ADAM12 disintegrin loop partially reduced the inhibitory effect of ADAM12 on cell migration on the alpha4beta1 binding fragment of fibronectin, whereas mutations that block metalloprotease activity had no effect. Our results indicate that distinct ADAMs can modulate cell migration mediated by specific integrins in a pattern dictated, at least in part, by their disintegrin domains.     

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.     

Shedding of the transferrin receptor is mediated constitutively by an integral membrane metalloprotease sensitive to tumor necrosis factor alpha protease inhibitor-2

The transferrin receptor (TfR) is a transmembrane protein that mediates cellular uptake of iron. Although the serum concentration of the soluble TfR (sTfR) is altered in several diseases and used for diagnostic purposes, the identity and regulation of the shedding protease is unknown. In this study we quantified sTfR release from microsomal membranes and leukocytic cell lines in the presence of numerous protease inhibitors and cell activating compounds. We show that sTfR release is mediated by an integral membrane metalloprotease and can be inhibited by matrix metalloproteinase inhibitor 2 and tumor necrosis factor alpha protease inhibitor-2 (TAPI-2). Cleavage is also inhibited by a specific furin inhibitor, indicating that the protease is activated by a furin-like proprotein convertase. Whereas stimulation of the cells by the ectodomain shedding activator phorbol 12-N-myristate 13-acetate did not alter sTfR release significantly, the phosphatase inhibitor pervanadate led to an increase of TfR shedding in several leukocytic cell lines. Our results suggest that TfR shedding is constitutively mediated by a member of the metalloprotease family known as ADAM (for a disintegrin and metalloprotease).