Cloning and chromosomal mapping of mouse ADAM11, ADAM22 and ADAM23.

A cellular disintegrin, also called MDC and ADAM is a recently discovered gene family that encodes protein with disintegrin-like and metalloprotease-like domains. We have reported the identification of human cDNAs encoding novel ADAM family proteins that we named MDC2 and MDC3 because of their structural similarity to the MDC (Sagane, K. et al., 1998. Biochem. J. 334, 93-98). The Human Gene Nomenclature Committee assigned the gene symbols ADAM11 for the MDC, ADAM22 for the MDC2 and ADAM23 for the MDC3. Here we report the isolation of three novel murine cDNAs encoding the proteins closely related to the human ADAM11, ADAM22 and ADAM23. Their chromosomal locations in the mouse were identified by interspecies backcross mapping. The loci of these murine ADAM genes were in good accordance with the location of each human ortholog, ADAM11, ADAM22 and ADAM23. These findings suggest that three murine cDNAs that we have isolated are the murine ADAM11, ADAM22 and ADAM23 cDNAs. Northern blot analysis shows that all of these three murine ADAMs were highly expressed in the mouse brain. The structures of these ADAM proteins strongly suggest that they could function as integrin receptors. The implications of the cellular disintegrins in neural development are discussed.     

LGI1 and LGI4 bind to ADAM22, ADAM23 and ADAM11

The transmembrane protein ADAM22 is expressed at high levels in the brain. From its molecular structure, ADAM22 is thought to be an adhesion molecule or a receptor because it has functional disintegrin-like and cysteine-rich sequences in its ectodomain. The phenotypic analysis of ADAM22-deficient mice has indicated the important roles played by ADAM22 in proper neuronal function and peripheral nerve development, however, the precise molecular function of ADAM22 is still unknown. To understand the function of ADAM22 on a molecular basis, we identified ADAM22 binding proteins by using immunoprecipitation and mass spectrometric analysis. This analysis revealed that Leucine-rich glioma inactivated 1 (LGI1) is the most potent ADAM22 binding protein in mouse brain. By our quantitative cell-ELISA system, we demonstrated the specific binding of LGI1 with ADAM22. Furthermore, we showed that LGI4, a putative ADAM22 ligand, also bound to ADAM22. Characterization of the binding specificity of LGI1 and LGI4 suggested that ADAM22 is not a sole receptor, because ADAM11 and ADAM23 had a significant binding ability to LGI1 or LGI4. Therefore, LGI-ADAM system seems to be regulated not only by the affinity but also by the cell-type-specific expression of each protein. Our findings provide new clues to understand the functions of LGI1 and LGI4 as an ADAMs ligand.     

Putative function of ADAM9, ADAM10, and ADAM17 as APP alpha-secretase

The putative alpha-secretase cleaves the amyloid precursor protein (APP) of Alzheimer's disease in the middle of the amyloid beta peptide (Abeta) domain. It is generally thought that the alpha-secretase pathway mitigates Abeta formation in the normal brain. Several studies have suggested that ADAM9, ADAM10, and ADAM17 are candidate alpha-secretases belonging to the ADAM (a disintegrin and metalloprotease) family, which are membrane-anchored cell surface proteins. In this comparative study of ADAM9, ADAM10, and ADAM17, we examined the physiological role of ADAMs by expressing these ADAMs in COS-7 cells, and both "constitutive" and "regulated" alpha-secretase activities of these ADAMs were determined. We tried to suppress the expression of these ADAMs in human glioblastoma A172 cells, which contain large amounts of endogenous alpha-secretase, by lipofection of the double-stranded RNA (dsRNA) encoding each of these ADAMs. The results indicate that ADAM9, ADAM10, and ADAM17 catalyze alpha-secretory cleavage and therefore act as alpha-secretases in A172 cells. This is the first report that to suggest the endogenous alpha-secretase is composed of several ADAM enzymes.     

Fluorescent substrates for the proteinases ADAM17, ADAM10, ADAM8, and ADAM12 useful for high-throughput inhibitor screening

In this paper we describe novel fluorescent substrates for the human ADAM family members ADAM17, ADAM10, ADAM8, and ADAM12 that have good specificity constants and are useful for high-throughput screening of inhibitors. The fluorescence resonance energy transfer substrates contain a 4-(4-dimethylaminophenylazo)benzoyl and 5-carboxyfluorescein (Dabcyl/Fam) pair and are based on known cleavage sequences in precursor tumor necrosis factor-alpha (TNF-alpha) and CD23. The precursor TNF-alpha-based substrate, Dabcyl-Leu-Ala-Gln-Ala-Homophe-Arg-Ser-Lys(Fam)-NH2, is a good substrate for all the ADAMs tested, including ADAM12 for which there is no reported fluorescent substrate. The CD23-based substrate, Dabcyl-His-Gly-Asp-Gln-Met-Ala-Gln-Lys-Ser-Lys(Fam)-NH2, is more selective, being hydrolyzed efficiently only by ADAM8 and ADAM10. The substrates were used to obtain inhibition constants for four inhibitors that are commonly used in shedding assays: TMI-1, GM6001, GW9471, and TAPI-2. The Wyeth Aerst compound, TMI-1, is a potent inhibitor against all of the ADAMs tested and is slow binding against ADAM17.     

The ADAM family

Notch signaling is integral to a large number of developmental and homeostasis events, and either gain or loss of Notch signaling results in a wide range of defects. Notch must be processed by several proteases, including a member of the ADAM (a disintegrin and metalloprotease) family to mediate downstream signaling. Until recently, interactions of Notch with specific ADAMs in different contexts were unclear. ADAM10 is now known to be specifically essential for development and homeostasis of mouse epidermis and cardiovascular structures, and ADAM17 may not be able to fully replace ADAM10 in these contexts. However, Notch from T-cell acute lymphoblastic leukemia (T-ALL) patients can be cleaved by both ADAMs 10 and 17. Studies have revealed that ADAM10 is necessary for Notch processing when Notch is activated by a ligand, while ADAM17 is the major protease for processing Notch that is activated independently of ligand in both flies and mammals.     

ADAM function in embryogenesis

Cleavage of proteins inserted into the plasma membrane (shedding) is an essential process controlling many biological functions including cell signaling, cell adhesion and migration as well as proliferation and differentiation. ADAM surface metalloproteases have been shown to play an essential role in these processes. Gene inactivation during embryonic development have provided evidence of the central role of ADAM proteins in nematodes, flies, frogs, birds and mammals. The relative contribution of four subfamilies of ADAM proteins to developmental processes is the focus of this review.     

A disintegrin and metalloproteinase (ADAM)-mediated ectodomain shedding of ADAM10

A disintegrin and metalloproteinase (ADAM) 10 is a type I transmembrane glycoprotein responsible for the ectodomain shedding of a range of proteins including the amyloid precursor protein implicated in Alzheimer's disease. In this study we demonstrate that ADAM10 itself is subject to shedding by one or more ADAMs. Expression of epitope-tagged wild-type ADAM10 in SH-SY5Y cells enabled the detection of a soluble ectodomain in conditioned medium. Shedding of the ADAM10 ectodomain was inhibited by a known ADAM inhibitor with a reciprocal accumulation of the full-length mature protein in both cell lysates and extracellular membrane vesicles. Shedding was also stimulated by phorbol ester treatment of cells. A glycosylphosphatidylinositol-anchored form of ADAM10 lacking the cytosolic, transmembrane and α-helical juxtamembrane regions of the wild-type protein was shed in a similar manner. Furthermore, a truncated soluble ADAM10 construct, although correctly post-translationally processed and catalytically active against a synthetic peptide substrate, was incapable of shedding cell-associated amyloid precursor protein. Finally, we show that ADAM9 is, at least in part, responsible for the ectodomain shedding of ADAM10. In conclusion, this is a new mechanism by which levels of ADAM10 are regulated and may have implications in a range of human diseases including Alzheimer's disease.     

Possible function of the ADAM1a/ADAM2 Fertilin complex in the appearance of ADAM3 on the sperm surface

In mouse, two different isoforms of ADAM1 (fertilin alpha), ADAM1a and ADAM1b, are produced in the testis. ADAM1a is localized within the endoplasmic reticulum of testicular germ cells, whereas epididymal sperm contain only ADAM1b on the plasma membrane. In this study, we show that the loss of ADAM1a results in the male infertility because of the severely impaired ability of sperm to migrate from the uterus into the oviduct through the uterotubal junction. However, epididymal sperm of ADAM1a-deficient mice were capable of fertilizing cumulus-intact, zona pellucida-intact eggs in vitro despite the delayed dispersal of cumulus cells and the reduced adhesion/binding to the zona pellucida. Among testis (sperm)-specific proteins examined, only the level of ADAM3 (cyritestin) was strongly reduced in ADAM1a-deficient mouse sperm. Moreover, the appearance of ADAM3 on the sperm surface was dependent on the formation of a fertilin protein complex between ADAM1a and ADAM2 (fertilin beta) in testicular germ cells, although no direct interaction between the fertilin complex and ADAM3 was found. These results suggest that ADAM1a/ADAM2 fertilin may be implicated in the selective transport of specific sperm proteins including ADAM3 from the endoplasmic reticulum of testicular germ cells onto the cell surface. These proteins then can participate in sperm migration into the oviduct, the dispersal of cumulus cells, and sperm binding to the zona pellucida.     

Catalytic properties of ADAM19

ADAMs are membrane-anchored glycoproteins with functions in fertilization, heart development, neurogenesis, and protein ectodomain shedding. Here we report an evaluation of the catalytic activity of recombinantly expressed soluble forms of ADAM19, a protein that is essential for cardiovascular morphogenesis. Proteolytic activity of soluble forms of ADAM19 was first demonstrated by their autocatalytic removal of a purification tag (Myc-His) and their ability to cleave myelin basic protein and the insulin B chain. The metalloprotease activity of ADAM19 is sensitive to the hydroxamic acid-type metalloprotease inhibitor BB94 (batimastat) but not to tissue inhibitors of metalloproteases (TIMPs) 1-3. Moreover, ADAM19 cleaves peptides corresponding to the known cleavage sites of tumor necrosis factor-alpha (TNF-alpha), TNF-related activation-induced cytokine (TRANCE, also referred to as osteoprotegerin ligand), and kit ligand-1 (KL-1) in vitro. Although ADAM19 is not required for shedding of TNFalpha and TRANCE in mouse embryonic fibroblasts, its overexpression in COS-7 cells results in strongly increased TRANCE shedding. This suggests a potential role for ADAM19 in shedding TRANCE in cells where both molecules are highly expressed, such as in osteoblasts. Interestingly, our results also indicate that ADAM19 can function as a negative regulator of KL-1 shedding in both COS-7 cells and mouse embryonic fibroblasts, instead of acting directly on KL-1. The identification of potential in vitro substrates offers the basis for further functional studies of ADAM19 in cells and in mice.     

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.     

Identification of ADAM10 as a major TNF sheddase in ADAM17-deficient fibroblasts

ADAM17 (a disintegrin and metalloprotease)-deficient murine fibroblasts stably transfected with proTNF cDNA release significant amounts of biologically active soluble TNF. The enzyme responsible for this activity is a membrane protein that hydrolyzes the peptide bond Ala⁷⁶:Val⁷⁷ within proTNF. Its activity is inhibited by 1,10-phenantroline and GM6001, insusceptible to TIMP-2 (tissue inhibitor of metalloproteinases-2), and stimulated by ionomycin. These characteristics match ADAM10. The moderate silencing of ADAM10 by shRNA resulted in a significant inhibition of TNF shedding. There was no correlation between the level of ADAM10 expression and the presence of active ADAM17. Our results indicate that ADAM10 may function as the TNF sheddase in cells which lack ADAM17 activity.     

The enzymatic activity of ADAM8 and ADAM9 is not regulated by TIMPs

Prostaglandin E2 (PGE2)-dependent effects on various cell responses are regulated by respective PGE2 receptors (EP1, EP2, EP3, EP4) expressing in target cells. Alveolar type II cell (a main progenitor cell of lung adenocarcinoma) expressed only EP4, while human lung adenocarcinoma cells (A549) expressed EP3 as well as EP4. An antagonistic effect of EP3 against EP4 through the modulation of cyclic AMP level is required for PGE2-mediated activation of Ras signal pathway in A549 cells. These results suggest that the expression of EP3 may be a critical factor for the PGE2-mediated activation of Ras signal pathway in A549 cells.     

Shedding light on ADAM metalloproteinases

ADAM metalloproteinase disintegrins have emerged as the major proteinase family that mediates ectodomain shedding, the proteolytic release of extracellular domains from their membrane-bound precursors. Recent gene-manipulation studies have established the role of ADAM-mediated shedding in mammalian physiology and, in addition, raised the issue of functional redundancy among ADAM sheddases. ADAM sheddases activate, for example, growth factors and cytokines, thus regulating signalling pathways that are important in development and pathological processes such as cancer. The recent studies have also begun to elucidate the substrate specificity and the mechanisms that control ADAM-mediated shedding events that regulate, for example, growth-factor and Notch signalling, and the processing of the amyloid precursor protein.     

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.