Expression levels of A disintegrin and metalloproteases (ADAMs), and Th17-related cytokines and their association with Helicobacter pylori infection in patients with gastroduodenal diseases.

Expression levels of A disintegrin and metalloproteases (ADAMs) (10 and 17) and Th17-related cytokines [interleukin (IL) 17A, IL-17F, IL-33, IL-23, IL-23R] were investigated by quantitative real time polymerase chain reaction in gastric biopsies of patients with different gastroduodenal pathologies in the presence and absence of Helicobacter pylori infection. Patients with gastric cancer (GC) (n = 70, intestinal-type 38 and diffuse type 32), peptic ulcer disease [n = 50, duodenal ulcer (DU) 16 and gastric ulcer (GU) 34] and functional dyspepsia (n = 120) were included in the study. Further, the expression levels of ADAMs and Th17 cytokines were correlated with H. pylori cytotoxin-associated genes pathogenicity island (cagPAI) status. Expression levels of ADAMs (10 and 17) and Th17-related cytokines (IL-17A, IL-23, IL-23R) were significantly higher in H. pylori-positive than in H. pylori-negative gastric biopsies. Significant increase in ADAM17 and Th17 cytokines (IL-17A and IL-23) expressions was observed in patients with GU and intestinal-type GC in the presence of H. pylori infection and in strains harbouring intact cagPAI. Expression levels of IL-17A, IL-23 and ADAM17 were strongly correlated with GU and intestinal-type GC and weakly with DU and diffuse-type GC in the presence of H. pylori infection. Higher expression levels of ADAM17 and Th17 cytokines (IL-17A and IL-23), and their strong correlation with GU and intestinal-type GC patients in the presence of H. pylori and its intact cagPAI status, suggest a possible role of strain specificity in the pathogenesis of these diseases.

Synergistic antitumoral effect of IL-12 gene cotransfected with antiangiogenic genes for angiostatin, endostatin, and saxatilin.

Previously, it was reported that the cotransfection of angiostatin K1-3, endostatin, and saxatilin genes using cationic liposomes significantly inhibited tumor progression. IL-12 is a well-known immune modulator that promotes Th1-type antitumor immune responses and also induces antiangiogenic effects. In this study, we have examined the antitumoral function of the IL-12 gene cotransfected with antiangiogenic genes for angiostatin K1-3, endostatin, and saxatilin by O,O'-dimyristyl-N-lysyl glutamate (DMKE) cationic liposomes in a mouse tumor model. According to our results, the administration of the IL-12 gene or the genes for angiostatin K1-3, endostatin, and saxatilin exhibited effective inhibition of B16BL6 melanoma growth in mice. In particular, intravenous administration of the IL-12 gene along with intratumoral administration of the three antiangiogenic genes synergistically inhibited the B16BL6 tumor growth. These results suggest that systemically expressed IL-12 enhances antitumoral efficacy of locally expressed antiangiogenic proteins.

"Insularin, a disintegrin from Bothrops insularis venom: inhibition of platelet aggregation and endothelial cell adhesion by the native and recombinant GST-insularin proteins".

Insularin (INS) was obtained from Bothrops insularis venom by reversed-phase high-performance liquid chromatography using a C(18) column and characterized as a disintegrin by peptide mass fingerprint and inhibition of ADP-induced platelet aggregation. A cDNA coding for P-II a metalloproteinase/disintegrin was cloned from a cDNA library from B. insularis venom glands. The deduced protein sequence possesses 73 amino acid residues, including the N-terminal, internal peptides of native insularin, the ARGDNP-sequence and 12 cysteines in a conserved alignment. This cDNA fragment was subcloned in the pGEX-4T-1 vector and expressed in a prokaryotic expression system as a fusion protein with glutathione S-transferase (GST-INS). Both native and recombinant insularin inhibited ADP-induced platelet aggregation and endothelial cells (HUVEC) adhesion with similar activities indicating that GST-INS folded correctly and preserved the integrin-binding loop. Insularin may be a tool in studies that involve platelets and endothelial cell adhesion dependent on alphaIIbeta3 and alphavbeta3 integrins.

Expression and functional characterization of a recombinant targeted toxin with an uPA cleavable linker in Pichia pastoris.

A recombinant targeted toxin (Disintegrin-Conj-Mel) was developed that contained a disintegrin connected to cytotoxic melittin by a urokinase plasminogen activator (uPA)-cleavable linker. This recombinant targeted toxin was designed to target tumor cells expressing integrin αvß3. The fusion gene was expressed under the control of the promoter AOX1 in Pichia pastoris. Electrophoresis by SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain, demonstrated that an approximately 13 kDa fusion protein was secreted into the culture medium. The molecular weight was that calculated from the predicted amino acid sequence. After optimizing the growth and expression conditions of the transformant strain, about 160 mg/L of the recombinant protein was achieved. The recombinant protein was purified to more than 95% purity by SP Sepharose ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The hemolysis bioactivity test revealed that the fusion had no hemolytic activity or cytotoxicity against uPA non-expressing 293 cells, but exerted dose-dependent inhibition on uPA-expressing A549 cell proliferation.

Recombinant expression in human cells of active integrin alpha 1 beta 1-blocking RTS-disintegrin jerdostatin.

Jerdostatin, an RTS short disintegrin cloned from Protobothrops jerdonii and recombinantly produced in Escherichia coli, is a potent and specific antagonist of the alpha(1)beta(1) integrin. Jerdostatin selectively blocked the adhesion of alpha(1)beta(1)-K562 cell to collagens I and IV in vitro and angiogenesis in vivo. Here we report the recombinant production of jerdostatin in a mammalian cell system, a prerequisite for developing a conditional transgenic mouse to investigate the effect of systemic expression of jerdostatin on tumor development. For proper export of jerdostatin, a secretion leader sequence was engineered at the protein's N-terminus. A FLAG epitope was also included at the N-terminus of the mature disintegrin to facilitate its isolation and characterization of recombinant jerdostatin (rJerd). This pRc-CMV/FLAG-rJerd construct was transiently expressed in HEK-293 cells and was efficiently secreted into the culture medium. rJerd bound to recombinant soluble alpha(1)beta(1) integrin in a saturable and cation-independent manner. Soluble rJerd also inhibited the binding of alpha(1)beta(1) integrin to the CB3 fragment of collagen IV in a dose-dependent manner (IC(50) 570 nM). Mammalian cell-expressed jerdostatin disrupted the adhesion of RuGli cells to collagen IV. Our results highlight pRc-CMV/FLAG-rJerd as a suitable construct for expressing soluble active alpha(1)beta(1)-blocking jerdostatin in a mammalian cell system.

Expression, purification and characterization of recombinant Jerdonitin, a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains.

Jerdonitin is a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains. In this study, we established a high-level expression system in Pichia pastoris and developed a purification strategy for the recombinant Jerdonitin. This recombinant Jerdonitin degraded fibrinogen at a level of activity comparable with its wild type. The effects of recombinant Jerdonitin on inhibiting ADP-induced human platelet aggregation were in a dose-dependent manner with an IC(50) of 248nM. In addition, we reported here that Jerdonitin can significantly inhibit the growth of several cell lines, including human liver cancer cells (Bel7402), human leukemia cells (K562) and human gastric carcinoma cells (BGC823). This study offers recombinant Jerdonitin that will be valuable for further functional and structural studies of Jerdonitin.

Role of ADAM-9 disintegrin-cysteine-rich domains in human keratinocyte migration.

ADAM-9 belongs to a family of transmembrane, disintegrin-containing metalloproteinases involved in protein ectodomain shedding and cell-cell and cell-matrix interactions. The aim of this study was to analyze the expression of ADAM-9 in skin and to assess the role of this proteolytic/adhesive protein in skin physiology. In normal skin, ADAM-9 expression was detected in both the epidermis and dermis and in vitro in keratinocytes and fibroblasts. Here we report that ADAM-9 functions as a cell adhesion molecule via its disintegrin-cysteine-rich domain. Using solid phase binding assays and antibody inhibition experiments, we demonstrated that the recombinant disintegrin-cysteine-rich domain of ADAM-9 specifically interacts with the beta1 integrin subunit on keratinocytes. This was corroborated by co-immunoprecipitation. In addition, engagement of integrin receptors by the disintegrin-cysteine-rich domain resulted in ERK phosphorylation and increased MMP-9 synthesis. Treatment with the ERK inhibitor PD98059 inhibited MMP-9 induction. Furthermore, the presence of the soluble disintegrin-cysteine-rich domain did not interfere with cell migration on different substrates. However, keratinocytes adhering to the immobilized disintegrin-cysteine-rich domain showed increased motility, which was partially due to the induction of MMP-9 secretion. In summary, our results indicate that the ADAM-9 adhesive domain plays a role in regulating the motility of cells by interaction with beta1 integrins and modulates MMP synthesis.

Expression of a disintegrin and metalloprotease (ADAM and ADAMTS) enzymes in human non-small-cell lung carcinomas (NSCLC).

A Disintegrin and Metalloprotease (ADAM) are transmembrane proteases displaying multiple functions. ADAM with ThromboSpondin-like motifs (ADAMTS) are secreted proteases characterised by thrombospondin (TS) motifs in their C-terminal domain. The aim of this work was to evaluate the expression pattern of ADAMs and ADAMTS in non small cell lung carcinomas (NSCLC) and to investigate the possible correlation between their expression and cancer progression. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunohistochemical analyses were performed on NSCLC samples and corresponding nondiseased tissue fragments. Among the ADAMs evaluated (ADAM-8, -9, -10, -12, -15, -17, ADAMTS-1, TS-2 and TS-12), a modulation of ADAM-12 and ADAMTS-1 mRNA expression was observed. Amounts of ADAM-12 mRNA transcripts were increased in tumour tissues as compared to the corresponding controls. In sharp contrast, ADAMTS-1 mRNA levels were significantly lower in tumour tissues when compared to corresponding nondiseased lung. These results were corroborated at the protein level by Western blot and immunohistochemistry. A positive correlation was observed between the mRNA levels of ADAM-12 and those of two vascular endothelial growth factor (VEGF)-A isoforms (VEGF-A(165) and VEGF-A(121)). Taken together, these results providing evidence for an overexpression of ADAM-12 and a lower expression of ADAMTS-1 in non-small-cell lung cancer suggest that these proteases play different functions in cancer progression.

A secreted form of ADAM9 promotes carcinoma invasion through tumor-stromal interactions.

Tumor cell invasion is a process regulated by integrins, matrix-degrading enzymes, and interactions with host tissue stromal cells. The ADAM family of proteins plays an important role in modulating various cellular responses. Here, we show that an alternatively spliced variant of ADAM9 is secreted by hepatic stellate cells and promotes carcinoma invasion. ADAM9-S induced a highly invasive phenotype in several human tumor cell lines in Matrigel assays, and the protease activity of ADAM9-S was required for invasion. ADAM9-S binds directly to alpha6beta4 and alpha2beta1 integrins on the surface of colon carcinoma cells through the disintegrin domain. ADAM9-S was also able to cleave laminin and promote invasion. Analysis of human liver metastases revealed that ADAM9 is expressed by stromal liver myofibroblasts, particularly those that are localized within the tumor stroma at the invasive front. These results emphasize the importance of tumor-stromal interactions in invasion and suggest that ADAM9-S can be an important determinant in the ability of cancer cells to invade and colonize the liver.

Altered patterns of gene expression specific to thoracic aortic aneurysms: microarray analysis of surgically resected specimens.

Changes in the expression levels of several genes have been described in aortic aneurysm specimens, however, the spectrum of diverse molecular alterations remains to be elucidated. We attempted to identify key molecules that modulate the pathogenesis of aortic aneurysm, using a complimentary DNA microarray carrying approximately 13,000 human genes. Segments of thoracic aortic aneurysms (TAA) and adjacent normal thoracic aortic tissues without aneurysmal changes (NTA) were obtained from 20 patients undergoing graft surgery. RNA obtained from five pairs of TAA and NTA samples was compared to determine aneurysm-specific alterations using microarray. Further, the expression levels of several genes of interest were verified in the remaining specimens by real-time reverse transcription-polymerase chain reaction (RT-PCR). In microarray assays, several types of the matrix metalloproteinases were upregulated as reported previously. Also, 220 genes suggested to be involved in protein degradation, inflammation, apoptosis, stress response, intracellular signaling, and other processes were significantly upregulated. Many of these genes have not been previously implicated in cardiovascular disease. The real time RT-PCR independently confirmed that the expression levels of MMP-2, MMP-9, ADAMTS-1, and caspase 4 were consistently increased in TAA. The results indicate that many genes are involved in a complicated manner in the pathogenesis of TAA. Investigation of these genes will help clarify the pathogenesis of this disease, and may lead to the discovery of novel therapeutic targets.

Dynamic induction of ADAMTS1 gene in the early phase of acute myocardial infarction.

Extracellular matrix (ECM)-degrading enzymes such as matrix metalloproteases (MMPs) play an essential role in the repair of infarcted tissue, which affects ventricular remodeling after myocardial infarction. ADAMTS1 (A disintegrin and metalloprotease with thrombospondin motifs), a newly discovered metalloprotease, was originally cloned from a cancer cell line, but little is known about its contribution to disease. To test the hypothesis that ADAMTS1 appears in infarcted myocardial tissue, we examined ADAMTS1 mRNA expression in a rat myocardial infarction model by Northern blotting, real-time RT-PCR and in situ hybridization. Normal endothelium expressed little ADAMTS1 mRNA, while normal myocardium expressed no detectable ADAMTS1 mRNA. Up-regulation of ADAMTS1 was demonstrated by Northern blot analysis and real-time RT-PCR at 3 h after coronary artery ligation. In situ hybridization revealed strong ADAMTS1 mRNA signals in the endothelium and myocardium in the infarcted heart, mainly in the infarct zone, at 3 h after myocardial infarction. The rapid and transient up-regulation of the ADAMTS1 gene in the ischemic heart was distinct from the regulatory patterns of other MMPs. Our study demonstrated that the ADAMTS1 gene is a new early immediate gene expressed in the ischemic endothelium and myocardium.

Aberrant alternative exon use and increased copy number of human metalloprotease-disintegrin ADAM15 gene in breast cancer cells.

ADAM genes have been associated with cancer, with ADAM expression, genomic rearrangements, and, by implication of ADAM proteins in the altered behavior found in tumor cells. In the present study, increased copy number of the ADAM15 gene in human breast cancer cell lines was demonstrated by fluorescence in situ hybridization. This was not reflected in mRNA levels, however. Instead, the use of alternative ADAM15 exons appeared erratic, leading to aberrant combinations of ADAM15 mRNA isoforms in the cancer cells. Clustering analysis indicated that these isoform patterns were nonrandom, suggesting a failure in the regulation mechanism or mechanisms of the alternative exon usage. Altered regulation of alternative exon usage may provide a useful target for cancer diagnostics development. ADAM15 would be particularly appropriate for breast cancer diagnostics because the various combinations of its three alternatively used exons can be readily examined with a simple, straightforward PCR protocol.

ADAM gene expression and regulation during human osteoclast formation.

In this study, we identified the expression and the regulation of ADAM members (a disintegrin and metalloprotease) at both gene and protein levels during human osteoclast differentiation and activity. Human peripheral blood monocytes (HPBMC) treated with M-CSF and RANKL were used as an in vitro fusion model. In parallel, we used human osteoclastoma (OCL) tumor as a source of mature osteoclasts, and human osteoblastic cells as a control representing nonfusing and non-resorbing bone cells. RT-PCR using ADAM-specific primers enabled us to identify the expression of ADAM 8, 9, 10, 15, 17, and 28 in both osteoclasts and osteoblasts. Using primers specific for each ADAM 12 isoform (L and S), we observed a strong signal for both forms (ADAM 12L and ADAM 12S) in osteoblastic cells, while only ADAM 12S was detectable in HPBMC-derived osteoclasts and osteoclastoma. Gene regulation was studied using real-time PCR analysis performed during HPBMC differentiation; this showed a progressive increase of ADAM 12 mRNA level from day 1 to 8 of the culture, while at around day 9, ADAM 12 mRNA level decreased 2-fold. We also showed that ADAM 8, ADAM 17, and ADAM 28 decreased according to the stage of HPBMC differentiation or fusion. ADAM 10 was unaltered during cell fusion. However, confocal immunolocalization showed that ADAM 10 protein re-localized from the nuclei and cytoplasm to the plasma membrane during culture and to the ruffled border in resorbing cells. The same re-localization process was observed using an ADAM 12S-specific antibody during HPBMC differentiation. Between days 12 and 14, ADAM 12 co-localized with the F-actin ring, and at day 15, a strong signal was also present in ruffled border or sealing zone area of osteoclasts. Our results describe the expression and regulation of various ADAMs in human bone cells and the selective expression of ADAM 12L in osteoblasts. Our gene regulation and protein localization studies suggest a function for ADAM 10 and ADAM 12S in the formation of osteoclasts from HPBMC and resorption activity.

Overexpression of ADAM9 in non-small cell lung cancer correlates with brain metastasis.

The "a disintegrin and metalloprotease" (ADAM) family contributes to regulation of the cell-cell and cell-matrix interactions that are critical determinants of malignancy. To determine the relationship between metastasis and ADAM proteins, we compared the mRNA levels of ADAM9, -10, -12, -15, and -17 in sublines of an EBC-1 lung cancer cell line that were highly metastatic to either brain or bone. ADAM9 mRNA levels were significantly higher in highly brain-metastatic sublines than in the parent or highly bone-metastatic sublines. To elucidate the role of ADAM9 in brain metastasis, we stably transfected A549 and EBC-1 cells with a full-length ADAM9 expression vector. Compared with mock-transfectants, ADAM9 overexpression resulted in increased invasive capacity in response to nerve growth factor, increased adhesion to brain tissue, and increased expression of integrin alpha 3 and beta 1 subunits. Administration of the anti-beta 1 monoclonal antibody attenuated this increase in invasive and adhesive activity. Intravenous administration of ADAM9-overexpressing A549 cells to mice resulted in micrometastatic foci in the brain and multiple metastatic colonies in the lungs. In contrast, administration of parent and mock-transfected A549 cells to mice resulted in lung tumors without brain metastasis. These results suggest that ADAM9 overexpression enhances cell adhesion and invasion of non-small cell lung cancer cells via modulation of other adhesion molecules and changes in sensitivity to growth factors, thereby promoting metastatic capacity to the brain.

Expression of ADAMs (a disintegrin and metalloproteases) and TIMP-3 (tissue inhibitor of metalloproteinase-3) in human prostatic adenocarcinomas.

A disintegrin and metalloproteases (ADAMs) are proteins that contain both a disintegrin and metalloprotease domain and have potential implications for the metastasis of human cancer cells via cell adhesion and protease activities. In this study, we analyzed the expression levels of ADAM-9, ADAM-10 and ADAM-17 (TNF-alpha converting enzyme, TACE), and TIMP-3 (tissue inhibitor of metalloproteinase-3) in human prostatic tumor cell lines as well as in clinical patient materials (BPH and tumor tissue samples). Human prostatic tumor cell lines (MDA PCa 2b, LNCaP-C33, -C51, -C81, -Pro5, -Ln3, -C4-2, PC3, and DU145) showed varied levels of expression for ADAM-9, -10 and -17 mRNA. A strong expression of ADAM-17/TACE was further revealed by Western blot analysis in prostatic tumor cell lines. In the case of clinical material, all the tumor samples (8/8) revealed the expression of ADAM-9, -10, and -17 compared to the specimens of benign prostatic hyperplasia (BPH) where 80% of (8/10) samples showed the expression of ADAM-9, 86% (6/7) of ADAM-10, and 30% (3/10) of ADAM-17/TACE. Interestingly, expression of a potent inhibitor of ADAM-17 (TIMP-3) was not detected in any prostatic tumor cell lines (0/9), while TIMP-3 expression was detected in 82% (9/11) of BPH samples. Androgen-sensitive LNCaP-C33 cells exhibited differences in ADAMs regulation by 5alpha-dihydrotestosterone (DHT), while such differences were not detectable in androgen-independent LNCaP-C81 cells. These results suggest that an inverse expression pattern of ADAM-17/TACE and TIMP-3, and the regulation of ADAMs with DHT might play an important role in the pathogenesis of prostate cancer.

BaG, a new dimeric metalloproteinase/disintegrin from the Bothrops alternatus snake venom that interacts with alpha5beta1 integrin.

The alpha(5)beta(1) integrin is one of the major fibronectin receptors which plays an essential role in the adhesion of normal and tumor cells to extracellular matrix. Here, we describe the isolation and characterization of a novel dimeric metalloproteinase/disintegrin, which is an inhibitor of fibronectin binding to the alpha(5)beta(1) integrin. This protein (BaG) was isolated from the venom of the South American snake Bothrops alternatus by gelatin-Sepharose affinity and anion exchange chromatography. The molecular mass of BaG was approximately 130 kDa under non-reducing conditions and 55 kDa under reducing conditions by SDS-PAGE. BaG shows proteolytic activity on casein that was inhibited by EDTA. 1,10-phenanthroline-treated BaG (BaG-I) inhibits ADP-induced platelet aggregation with an IC(50) of 190 nM. BaG-I inhibits fibronectin-mediated K562 cell adhesion with an IC(50) of 3.75 microM. K562 cells bind to BaG-I probably through interaction with alpha(5)beta(1) integrin, since anti-alpha(5)beta(1) antibodies inhibited K562 cell adhesion to BaG-I. In addition, BaG-I induces the detachment of K562 cells that were bound to fibronectin. In summary, we have purified a novel, dimeric snake venom metalloproteinase/disintegrin that binds to the alpha(5)beta(1) integrin.

Metallothionein-1 messenger RNA transcription in steroid-secreting cells of the rat ovary during the periovulatory period.

An increase in metallothionein 1 (MT-1) mRNA was detected in the ovaries of immature Wistar rats that were primed with s.c. injection of 10 IU eCG followed 48 h later by 10 IU hCG s.c. to initiate the ovulatory process. Ovarian RNA was extracted at 0, 2, 4, 8, 12, 24, 72, 144, and 288 h after the primed animals were injected with hCG. These extracts were used for reverse transcription polymerase chain reaction (RT-PCR) differential display and Northern analyses that yielded complementary gene fragments for MT-1. Expression of MT-1 mRNA increased significantly by 24 h after hCG treatment and reached a peak at 144 h after hCG. In contrast, a disintegrin and metalloproteinase with thrombospondin motifs and a tissue inhibitor of metalloproteinase 1, which were also detected by the RT-PCR differential display procedure, reached a peak at 12 h after hCG and returned to control levels in the ovaries by 72 h after hCG. In situ hybridization indicated that most of the MT-1 mRNA was expressed in the vicinity of the theca interna of preovulatory follicles and in the lutein granulosa of postovulatory follicles. Thus, MT-1 mRNA expression is primarily in the vicinity of steroid-secreting areas of the ovary. The substantial increase in MT-1 mRNA expression might be important in protecting the ovarian tissues from oxidative stress generated by ovarian inflammatory events during the ovulatory process and luteinization.

Tumor necrosis factor alpha induces a metalloprotease-disintegrin, ADAM8 (CD 156): implications for neuron-glia interactions during neurodegeneration.

ADAM proteases, defined by extracellular disintegrin and metalloprotease domains, are involved in protein processing and cell-cell interactions. Using wobbler (WR) mutant mice, we investigated the role of ADAMs in neurodegeneration and reactive glia activation in the CNS. We found that ADAM8 (CD 156), a suspected leukocyte adhesion molecule, is expressed in the CNS and highly induced in affected CNS areas of WR mice, in brainstem and spinal cord. ADAM8 mRNA and protein are found at low levels throughout the normal mouse CNS, in neurons and oligodendrocytes. In the WR CNS regions in which neurodegeneration occurs, ADAM8 is induced in neurons, reactive astrocytes, and activated microglia. Similarly, the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) is upregulated and shows the same cellular distribution. In primary astrocytes from wild-type and WR mice, in primary cerebellar neurons, and in mouse motoneuron-like NSC19 cells, ADAM8 expression was induced up to 15-fold by mouse TNF-alpha, in a dose-dependent manner. In both cell types, ADAM8 was also induced by human TNF-alpha, indicating that TNF receptor type I (p55) is involved. Induction of ADAM8 mRNA was suppressed by treatment with an interferon-regulating factor 1 (IRF-1) antisense oligonucleotide. We conclude that IRF-1-mediated induction of ADAM8 by TNF-alpha is a signaling pathway relevant for neurodegenerative disorders with glia activation, proposing a role for ADAM8 in cell adhesion during neurodegeneration.

Jararhagin ECD-containing disintegrin domain: expression in escherichia coli and inhibition of the platelet-collagen interaction.

Jararhagin, a hemorrhagin from Bothrops jararaca venom, is a soluble snake venom component comprising metalloproteinase and disintegrin cysteine-rich domains and, therefore, is structurally closely related to the membrane-bound A Disintegrin And Metalloproteinase (ADAMs) protein family. Its hemorrhagic activity is associated with the effects of both metalloproteinase and disintegrin domains; the metalloproteinase enzymatically damages the endothelium and the disintegrin domain inhibits platelet-collagen interactions. The expression of whole jararhagin or its disintegrin domain has never been attempted before. The aim of this study was to investigate whether we could express the disintegrin domain of jararhagin and to verify whether this domain displays an inhibitory effect on the platelet-collagen interaction. Therefore, the cDNA fragment coding for the disintegrin plus cysteine-rich domains of jararhagin was cloned into the pET32a vector, used to transform the Escherichia coli AD494(DE3)pLysS strain. The thioredoxin-disintegrin fusion protein was recovered from the soluble extract of the cells, yielding up to 50 mg/liter culture. The fusion protein was isolated using polyhistidine binding resin which resulted in a main band of 45 kDa recognized by anti-native jararhagin antibodies. Antibodies raised in rabbits against the fusion protein had high enzyme-linked immunosorbent assay titers against native jararhagin and detected a band of 52 kDa on Western blots of whole B. jararaca venom demonstrating that these antibodies recognize the parent jararhagin molecule. Treatment of the fusion protein with enterokinase, followed by further capture of the enzyme, resulted in a band of 30 kDa, the expected size for jararhagin-C. Further purification of the cleaved disintegrin using FPLC Mono-Q columns resulted in one fraction capable of efficiently inhibiting collagen-induced platelet aggregation in a dose-dependent manner (IC(50) of 8.5 microg/ml).

Metalloproteinase-like, disintegrin-like, cysteine-rich proteins MDC2 and MDC3: novel human cellular disintegrins highly expressed in the brain.

Cellular disintegrins are a family of membrane-anchored proteins structurally related to snake venom disintegrins, and are potential regulators of cell-cell and cell-matrix interactions. The members of this protein family are also called ADAMs (a disintegrin and metalloproteinase) or MDC proteins (metalloproteinase-like disintegrin-like cysteine-rich), because they all contain disintegrin-like and metalloproteinase-like domains. In this paper, we report the cloning and sequence analysis of two novel additional members of this family, which we have termed MDC2 and MDC3. The deduced amino acid sequences reveal that the two proteins possess typical cellular disintegrin structures [that is, pro-, metalloproteinase-like, disintegrin-like, cysteine-rich, epidermal growth factor-like, transmembrane, and cytoplasmic domains] and exhibit high sequence similarity with human MDC/ADAM11 protein [Katagiri, Harada, Emi and Nakamura (1995) Cytogenet. Cell Genet. 68, 39-44]. A zinc-binding motif, which is critical for proteinase activity, is disrupted in the metalloproteinase-like domain of MDC2 and MDC3, as well as MDC/ADAM11. In the disintegrin-like domain of snake venom short disintegrins, the RDG-containing loops are critical for integrin binding. These three MDCs do not contain the RDG sequences, but the corresponding loops in these proteins are similar to each other. Northern blot analysis revealed that the mRNAs of MDC2, MDC3 and MDC/ADAM11 are highly expressed in the brain. These findings suggest that these proteins may function as integrin ligands in the brain.