皖南尖吻蝮蛇毒中类去整合蛋白/富含半胱氨酸两结构域cDNA的克隆和表达

为了探讨出血毒金属蛋白酶结构功能关系 ,通过 RT- PCR方法 ,从皖南尖吻蝮蛇( Agkistrodon acutus)毒腺总 RNA中扩增得到编码 P- 型出血毒金属蛋白酶的完整类去整合蛋白和富含半胱氨酸两个结构域 c DNA( AA/DC) .它全长 964bp的 c DNA,开放阅读框架编码 2 1 6个氨基酸残基 ,序列比较分析表明它同来自 Bothrops jararaca的 jararhagin- C、来自 Crotalus atrox的 catrocollastatin- C有很高的同源性 .在类去整合蛋白结构域中 ,Ser- Glu- Cys- Asp( SECD)代替了去整合蛋白中相应部位的 Arg- Gly- Asp( RGD)三肽序列 .将编码区基因克隆入 p GEX- 2 T载体中 ,转化大肠杆菌 TG- 1 ,用 IPTG诱导表达 ,表达产物具有抑制胶原诱导的血小板凝集活性 ,但不抑制ADP诱导的血小板凝集 .该研究为进一步阐述蛇毒金属蛋白酶结构功能关系和药物开发奠定了基础 .     

两个菜花烙铁头蛇毒金属蛋白酶去整合素基因的克隆与序列分析(英文)

从菜花烙铁头蛇的毒腺中 ,利用RT PCR进行体外扩增 ,克隆到 2个金属蛋白酶 去整合素基因 ,命名为TJM 1、TJM 2 .TJM 1cDNA全长为 15 2 8bp ,编码 4 81个氨基酸 ;TJM 2cDNA全长为 15 78bp ,编码 4 84个氨基酸 .TJM 1和TJM 2都属于Ⅱ型蛇毒金属蛋白酶 ,由信号肽、前肽、金属蛋白酶、间隔肽和去整合素 5部分组成 .Ⅱ型蛇毒金属蛋白酶氨基酸序列的比较及进化分析显示 ,它可进一步分为两类 ,一类包括大多数Ⅱ型蛇毒金属蛋白酶 (其中含有TJM 1) ,而TJM 2和agkistin则组成了另一类 .并且TJM 2和agkistin的第 4 0 7位和第 4 2 6位残基都是半胱氨酸 ,而在其它Ⅱ型金属蛋白酶的相应位置 ,4 0 7位是丝氨酸 ,4 2 6位则缺失 .TJM 2和agkistin均有可与整合素α2 Ⅰ区域特异性结合的片段Q NRKRHDNAQ(残基 2 76～ 2 84 ) ,这个片段在其它Ⅱ型金属蛋白酶中并没有发现 .因此推断 ,TJM 2和agkistin可能属于一类新型的Ⅱ型蛇毒金属蛋白酶 .     

Structural and functional significance of disulfide bonds in saxatilin,a 7.7 kDa disintegrin

Saxatilin is a 7.7 kDa disintegrin that belongs to a family of homologous protein found in several snake venoms. Six disulfide bond locations of the disintegrin were determined by enzymatic cleavage and matrix-assisted-laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF). Functional implications of the disulfide bonds related to the biological activity of saxatilin were investigated with recombinant protein species produced by site-directed mutagenesis of saxatilin. Several lines of experimental evidence indicated that three disulfide bonds, Cys21-Cys35, Cys29-Cys59, and Cys47-Cys67, of the disintegrin are closely associated with its biological function such as its ability to block the binding of integrin GPIIb-IIIa and alpha(v)beta(3) with fibrinogen and extracellular matrix. Those disulfide linkages were also revealed to be important for maintaining the functional structure of the protein molecule. On the other hand, the disulfide bridges of Cys6-Cys15 and Cys8-Cys16 do not appear to be critical for the molecular structure and function of saxatilin.     

Molecular cloning, functional expression, and molecular modeling of bothrostatin, a new highly active disintegrin from Bothrops jararaca venom

Disintegrins are among the most potent antagonists of several integrins. A cDNA encoding a novel disintegrin, bothrostatin, was cloned from a Bothrops jararaca cDNA library. The precursor of bothrostatin contains a pro, a metalloproteinase, and an RGD-disintegrin domain. The disintegrin domain expressed in Escherichia coli showed high inhibitory activity on collagen-induced platelet aggregation (IC(50) of 12nM), and thus it can be used as a useful tool for studies of integrin-ligand interaction. Furthermore, we used the comparative modeling approach to obtain a model of the 3D structure of bothrostatin. Our results suggest that bothrostatin adopts a globular, closed structure in solution. The RGD motif is exposed to the solution by the loop formed by residues 45-59 and is very close to the C-terminal domain forming a finger-like structure. The proximity of the RGD loop and the C-terminal residues, which is maintained by the Cys47-Cys66 bond, suggests that the C-terminal residues are involved in the ability of bothrostatin to interact with its ligands.     

Echistatin disulfide bridges: selective reduction and linkage assignment.

Echistatin is the smallest member of the disintegrin family of snake venom proteins, containing four disulfides in a peptide chain of 49 residues. Partial assignment of disulfides has been made previously by NMR and chemical approaches. A full assignment was made by a newly developed chemical approach, using partial reduction with tris-(2-carboxyethyl)-phosphine at acid pH. Reduction proceeded in a stepwise manner at pH 3, and the intermediates were isolated by high performance liquid chromatography. Alkylation of free thiols, followed by sequencer analysis, enabled all four bridges to be identified: (1) at 20 degrees C a single bridge linking Cys 2-Cys 11 was broken, giving a relatively stable intermediate; (2) with further treatment at 41 degrees C the bridges Cys 7-Cys 32 and Cys 8-Cys 37 became accessible to the reagent and were reduced at approx. equal rates; (3) the two bicyclic peptides produced in this manner were less stable and could be reduced at 20 degrees C to a peptide that retains a single bridge linking Cys 20-Cys 39; and (4) the monocyclic peptide can be reduced to the linear molecule at 20 degrees C. Some disulfide exchange occurred during alkylation of the bicyclic intermediates, but results unambiguously show the pattern to be [2-11; 7-32; 8-37; 20-39]. A comparison is made with kistrin, a longer disintegrin whose disulfide structure has been proposed from NMR analysis.     

The disulfide bridge pattern of snake venom disintegrins, flavoridin and echistatin.

Flavoridin and echistatin, isolated from the venom of Trimeresurus flavoviridis and Echis carinatus, respectively, belong to the disintegrin family of integrin beta 1 and beta 3 inhibitors of low molecular weight RGD-containing, cysteine-rich peptides. Since disulfide bonds are critical for expression of biological activity, we sought to determine their location in these two proteins. In flavoridin, direct evidence for the existence of linkage between Cys4-Cys19 and between Cys45 and Cys64 was obtained by analysis of proteolytic products, and indirect evidence suggests links between Cys6-Cys14 and Cys13-Cys36. In echistatin, links between Cys8-Cys37 and Cys20-Cys39 were identified by direct chemical analysis.     

Inhibitory antibodies against endopeptidase activity of human adamalysin 19.

Human adamalysin 19 (hADAM19)/meltrin beta is a member of the ADAM (a disintegrin and metalloproteinase) family and an active metalloproteinase. It is a new metalloproteinase and disintegrin dendritic cell antigen marker. Adamalysin 19 gene was expressed in normal and transformed tissues and cells such as placenta, brain, heart, leukocytes, and colorectal adenocarcinoma SW480. To develop specific tools to investigate the functions of hADAM19, peptide antigens were rationally selected and specific polyclonal antibodies (pAbs) were developed to modulate hADAM19 activity. Anti-metalloproteinase and anti-disintegrin domain IgG molecules inhibited the alpha-2-macroglobulin cleavage by hADAM19; however, their pre-immune and anti-pro-domain IgG molecules did not. Since anti-disintegrin IgG also neutralized the proteolytic activity, the disintegrin domain may affect the hADAM19 protein folding and/or substrate binding. These pAbs may be used to specifically localize the hADAM19 protein in tissues and cells and elucidate its biological and pathological functions such as processing pro-growth factors.     

Native cysteine residues are dispensable for the structure and function of all five yeast mitotic septins

Budding yeast septins assemble into hetero‐octamers and filaments required for cytokinesis. Solvent‐exposed cysteine (Cys) residues provide sites for attaching substituents useful in assessing assembly kinetics and protein interactions. To introduce Cys at defined locations, site‐directed mutagenesis was used, first, to replace the native Cys residues in Cdc3 (C124 C253 C279), Cdc10 (C266), Cdc11 (C43 C137 C138), Cdc12 (C40 C278), and Shs1 (C29 C148) with Ala, Ser, Val, or Phe. When plasmid‐expressed, each Cys‐less septin mutant rescued the cytokinesis defects caused by absence of the corresponding chromosomal gene. When integrated and expressed from its endogenous promoter, the same mutants were fully functional, except Cys‐less Cdc12 mutants (which were viable, but exhibited slow growth and aberrant morphology) and Cdc3(C124V C253V C279V) (which was inviable). No adverse phenotypes were observed when certain pairs of Cys‐less septins were co‐expressed as the sole source of these proteins. Cells grew less well when three Cys‐less septins were co‐expressed, suggesting some reduction in fitness. Nonetheless, cells chromosomally expressing Cys‐less Cdc10, Cdc11, and Cdc12, and expressing Cys‐less Cdc3 from a plasmid, grew well at 30°C. Moreover, recombinant Cys‐less septins—or where one of the Cys‐less septins contained a single Cys introduced at a new site—displayed assembly properties in vitro indistinguishable from wild‐type. Proteins 2013; 81:1964–1979. © 2013 Wiley Periodicals, Inc.     

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.     

Inhibition of the tumor necrosis factor-alpha-converting enzyme by its pro domain

Tumor necrosis factor-alpha-converting enzyme (TACE) is a disintegrin metalloproteinase that processes tumor necrosis factor and a host of other ectodomains. TACE is biosynthesized as a zymogen, and activation requires the removal of an inhibitory pro domain. Little is known about how the pro domain exerts inhibition for this class of enzymes. To study the inhibitory properties of the pro domain of TACE, we have expressed it in isolation from the rest of the protease. Here we show that the TACE pro domain (TACE Pro) is a stably folded protein that is able to inhibit this enzyme. TACE Pro inhibited the catalytic domain of TACE with an IC(50) of 70 nm. In contrast, this inhibitory potency decreased over 30-fold against a TACE form containing the catalytic plus disintegrin/cysteine-rich domains (IC(50) greater that 2 microm). The disintegrin/cysteine-rich region in isolation also decreases the interaction of TACE Pro with the catalytic domain. Surprisingly, we found that the cysteine switch motif located in TACE Pro was not essential for inhibition of the enzymatic activity of TACE; the pro domain variant C184A showed the same inhibitory potency against both TACE forms as wild type TACE Pro. X-ray absorption spectroscopy experiments indicate that binding of TACE Pro to the catalytic domain does include ligation of the catalytic zinc ion via the sulfur atom of its conserved Cys(184) residue. Moreover, the binding of TACE Pro to the catalytic zinc ion partially oxidizes the catalytic zinc ion of the enzyme. Despite this, the nature of the interaction between the pro and catalytic domains of TACE is not consistent with a simple competitive model of inhibition based on cysteine switch ligation of the zinc ion within the active site of TACE.     

Characteristics of two genes encoding proteins with an ADAM-type metalloprotease domain, which are induced during the molting periods in Bombyx mori

By microarray analyses, we identified two genes (BmADAMTS-1 and BmADAMTS-like) encoding a protein, which are induced during the pupal ecdysis in the wing discs of Bombyx mori; these genes are homologous to ADAMTS family members (a disintegrin and metalloproteinase domain, with thrombospondin type-1 repeats). A complete metal-binding motif of the ADAM-type metalloprotease domain (HEXXHXXGXXHD) was contained in both amino acid sequences. However, thrombospondin type 1 (TSP-1) repeats were observed only in BmADAMTS-1. The BmADAMTS-1 gene was expressed in the hemocyte and midgut of the larvae at day 2 of wandering stage (W2), and strongly induced during the pupal ecdysis in the hemolymph. The BmADAMTS-like gene was expressed in the epithelial tissues of the larvae at W2, and had expression peaks slightly later than the BmADAMTS-1 gene. Our results indicate that BmADAMTS-1 and BmADAMTS-like may cleave the extracellular matrix (ECM) in the degenerating and remodeling tissues during the molting periods.     

Molecular cloning and mapping of a novel ADAM gene (ADAM29) to human chromosome 4

Members of the ADAM family (type I integral membrane protein with a disintegrin and metalloprotease domain) have been implicated in many important biological processes involving cell-cell and cell-matrix interactions, such as fertilization and myoblast fusion. We report here the cDNA sequence of a novel human ADAM gene (ADAM29) that contains a putative fusion peptide. Northern blot analysis revealed that the mRNA of ADAM29 is highly expressed in the testis. By radiation hybrid panel mapping, the ADAM29 gene was assigned to human chromosome 4q34.2-qter.     

Evidence for disulfide involvement in the regulation of intramolecular autolytic processing by human adamalysin19/ADAM19

Human adamalysin 19 (a disintegrin and metalloproteinase 19, hADAM19) is activated by furin-mediated cleavage of the prodomain followed by an autolytic processing within the cysteine-rich domain at Glu586-Ser587, which occurs intramolecularly, producing an NH2 terminal fragment (N-fragment) associated with its COOH-terminal fragment (C-fragment), most likely through disulfide bonds. When stable Madin-Darby canine kidney (MDCK) transfectants overexpressing soluble hADAM19 were treated with dithiothreitol (DTT) or with media at pH 6.5, 7.5, or 8.5, the secretion and folding of the enzyme were not affected. Autolytic processing was blocked by DTT and pH 6.5 media, which favor disulfide reduction, but was increased by pH 8.5 media, which promotes disulfide formation. Cys605, Cys633, Cys639, and Cys643 of the C-fragment appear to be partially responsible for the covalent association between the C-fragment and the N-fragment. A new autolytic processing site at Lys543-Val544 was identified in soluble mutants when these cysteine residues were individually mutated to serine residues. Shed fragments were also detectable in the media from MDCK cells stably expressing the full-length Cys633Ser mutant. Ilomastat/GM6001 inhibited hADAM19 with an IC50 of 447 nM, but scarcely affected the shedding process. The cysteine-rich domain likely forms disulfide bonds to regulate the autolytic processing and shedding of hADAM19.     

Loss of ectodomain shedding due to mutations in the metalloprotease and cysteine-rich/disintegrin domains of the tumor necrosis factor-alpha converting enzyme (TACE)

Tumor necrosis factor-alpha converting enzyme (TACE), a multidomain protease essential for development and disease, releases the ectodomains from many transmembrane proteins in a regulated fashion. To understand the mechanism underlying the regulation of TACE activity, we sought to identify the cause of ectodomain shedding deficiencies in two mutated CHO sublines designated M1 and M2. Transfection of expression vectors for human and mouse TACE restored ectodomain shedding of TNF-alpha and TGF-alpha, suggesting that defects in the TACE gene contribute to the phenotype of M1 and M2 cells. The overall levels of endogenous TACE forms in M1 cells were significantly lower than those found in their parental cells, whereas only TACE zymogen, but not its mature form, was detectable in M2 cells. Molecular analyses suggested that M1 cells contained only one expressible TACE allele encoding an M435I point mutation in the catalytic center of the protease, and M2 cells produced two TACE variants with distinct point mutations in the catalytic domain (C225Y) and the cysteinerich/disintegrin domain (C600Y). Overexpression of the C225Y and C600Y TACE by transient transfection largely compensated for maturation defects in the variants but failed to restore TNF-alpha and TGF-alpha release in the shedding-defective CHO cell lines and fibroblasts derived from TACE-null mouse embryo. Further mutagenesis and functional analyses demonstrated that Cys(600) was absolutely essential for ectodomain shedding, suggesting that Cys(600), similar to Cys(225), participates in disulfide bonding, which is critical for both the processing and catalysis of TACE.     

Assignment of all four disulfide Bridges in echistatin

Echistatin is a 49-amino-acid protein fromEchis carinatus venom. It contains four disulfide bonds. Since the disulfide bonding is critical for biological activity, it is very important to assign the disulfide linkage in this protein. Echistatin was incubated in 250 mM oxalic acid at 100°C for 4 hr under nitrogen. Under these conditions, many overlapping disulfide-containing peptides were identified by ionspray mass spectrometry. Ionspray MS/MS data indicate that the four disulfide bonds are Cys 2–Cys 11, Cys 7–Cys 32, Cys 8–Cys 37, and Cys 20–Cys 39. To our knowledge, this is the first time all four disulfide bonds in echistatin have been assigned in one experiment without disulfide bond exchange. This approach, which combines oxalic acid hydrolysis and ionspray MS/MS, may be very useful for assigning disulfide bridges in other proteins from the disintegrin family.     

The lymphocyte metalloprotease MDC-L (ADAM 28) is a ligand for the integrin alpha4beta1.

The interaction of lymphocytes with other cells is critical for normal immune surveillance and response. MDC-L (ADAM 28), a member of the ADAM (a disintegrin and metalloprotease) protein family, is expressed on the surface of human lymphocytes. ADAMs possess a disintegrin-like domain similar in sequence to small non-enzymatic snake venom peptides that act as integrin antagonists. We report here that the disintegrin domain of MDC-L is recognized by the leukocyte integrin alpha(4)beta(1). Recombinant Fc fusion proteins possessing the disintegrin domain of MDC-L supported adhesion of the T-lymphoma cell line, Jurkat, in a concentration- and divalent cation-dependent manner. Adhesion of Jurkat cells to the disintegrin domain of MDC-L was inhibited by an anti-MDC-L monoclonal antibody (mAb), Dis1-1. The epitope for mAb Dis1-1 was localized within 59 residues of the disintegrin domain. Recombinant expression of this 59-residue fragment of the disintegrin domain also supported cell adhesion. Adhesion of Jurkat cells to the MDC-L disintegrin domain was specifically inhibited by anti-alpha(4) and anti-beta(1) function-blocking mAbs. Furthermore, adhesion of various cell lines to MDC-L correlated with expression of the integrin alpha(4)-subunit. Transfected K562 cells expressing alpha(4)beta(1) adhered to the disintegrin domain in contrast to non-transfected K562 cells. We further investigated the binding of recombinant MDC-L disintegrin domain (rDis-Fc) in solution. The rDis-Fc was found to bind to Jurkat cells in solution in a concentration-dependent and saturable manner. Both adhesion and solution binding of rDis-Fc was inhibited by the alpha(4)beta(1) ligand mimetic CS-1 peptide. Additionally, recognition of the MDC-L disintegrin domain required "activation" of lymphocyte beta(1) integrins. The interaction of MDC-L with alpha(4)beta(1) may potentially regulate metalloprotease function by targeting or sequestering the active protease on the cell surface. These results suggest a potential role for the lymphocyte ADAM, MDC-L, in the interaction of lymphocytes with alpha(4)beta(1)-expressing leukocytes.     

ADAM19 autolysis is activated by LPS and promotes non-classical secretion of cysteine-rich protein 2

ADAM family proteins are type I transmembrane, zinc-dependent metalloproteases. This family has multiple conserved domains, including a signal peptide, a pro-domain, a metalloprotease domain, a disintegrin (DI) domain, a cysteine-rich (Cys) domain, an EGF-like domain, a transmembrane domain, and a cytoplasmic domain. The Cys and DI domains may play active roles in regulating proteolytic activity or substrate specificity. ADAM19 has an autolytic processing activity within its Cys domain, and the processing is necessary for its proteolytic activity. To identify a new physiological function of ADAM19, we screened for associating proteins by using the extracellular domain of ADAM19 in a yeast two-hybrid system. Cysteine-rich protein 2 (CRIP2) showed an association with ADAM19 through its DI and Cys domains. Sequence analysis revealed that CRIP2 is a secretable protein without a classical signal. CRIP2 secretion was increased by overexpression of ADAM19 and decreased by suppression of ADAM19 expression. Moreover, CRIP2 secretion increased in parallel with the autolytic processing of ADAM19 stimulated by lipopolysaccharide. These findings suggest that ADAM19 autolysis is activated by lipopolysaccharide and that ADAM19 promotes the secretion of CRIP2.     

Male Germ Cell-Expressed Mouse Gene TAZ83 Encodes a Putative, Cysteine-rich Transmembrane Protein (cyritestin) Sharing Homologies with Snake Toxins and Sperm-Egg Fusion Proteins

Data obtained by cloning of a mouse cDNA ( TAZ83 ) are presented. Its corresponding gene is expressed in meiotic and haploid testicular germ cells. The gene encodes a putative, cysteine-rich transmembrane protein with a deduced molecular weight of 90 kilodaltons and an isoelectric point of 5.4. Cysteine patterns within the predicted amino acid sequence of the TAZ83 gene product ( cyritestin , cysteine-rich, testicular) are highly conserved when compared to various snake toxins of the disintegrin metalloproteinase type. The cysteine pattern conservation between cyritestin and a guinea pig sperm-egg fusion protein suggests that TAZ83 codes for a mouse protein with comparable properties or function.     

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.