Primary structure of two-chain botrocetin, a von Willebrand factor modulator purified from the venom of Bothrops jararaca.

Usami, Yoshiko; Fujimura, Yoshihiro; Suzuki, Masami; Ozeki, Yasuhiro; Nishio, Kenji; Fukui, Hiromu; Titani, Koiti

doi:10.1073/pnas.90.3.928

Cited by 110 publications

(67 citation statements)

References 32 publications

(27 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The two chains of bothrojaracin present a high degree of identity (47 %). They are similar to botrocetin [8], IX/X-bp [5] and alboaggregin-B [lo] (Fig. 3), with identities of 80, 57 and 54% for the A chains, and 66, 54 and 56% for the B chains, respectively.…”

Section: Resultsmentioning

confidence: 90%

Molecular Cloning and Expression of Bothrojaracin, A Potent Thrombin Inhibitor from Snake Venom

Arocas

Castro

Zingalf

et al. 1997

European Journal of Biochemistry

View full text Add to dashboard Cite

Bothrojaracin is a potent and selective thrombin inhibitor that has been isolated from the venom of Botlzrops jaruruca. It does not interact with the catalytic site of the enzyme but binds to both anionbinding exosites 1 and 2 resulting in a potent inhibition of thrombin activity towards fibrinogen and platelets [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C. & Bon, C. (1993) Biochemistry 32, 10794-I0 8021. Bothrojaracin is a 27-kDa protein composed of two disulfide-linked polypeptide chains, A and B, of 15 kDa and 13 kDa, respectively. The sequences of A and B chains determined by molecular cloning exhibit a high degree of identity with other snake venom lectin-like proteins. In contrast to other ligands that interact with thrombin exosite 1, the amino acid sequence of bothrojaracin does not contain an acidic sequence similar to the C-terminal tail of hirudin. Expression of functional bothrojaracin was achieved in COS cells upon transfection with two pcDNA3 vectors containing the complete cDNAs. Recombinant bothrojaracin, which was secreted into the medium, was able to bind to and inhibit thrombin. When expressed alone, the B chain formed inactive dimers that were secreted into the culture medium. In contrast, no bothrojaracin-related protein was detected in conditioned media from cells transfected with the A chain.Keywords: thrombin inhibitor; snake-venom protein; C-type lectin; molecular cloning and sequencing; expression of recombinant heterodimeric protein.Snake venoms are a rich source of enzymes and inhibitors involved in blood coagulation or platelet activation [I]. Several of these venom proteins, with molecular masses of approximately 30 kDa, consist of two disulfide-linked similar subunits containing a C-type lectin motif 12, 31 and exhibit a high degree of similarity : factor IX/factor X-binding protein (IX/X-bp) interacts with coagulation factors TX and X in a calcium-dependent manner [4-61; botrocetin binds von Willebrand factor, inducing its binding to glycoprotein Ib [7, 81; and alboaggregin-B [9, 101, echicetin [11, 121, agkicetin 1131 andjararaca glycoprotein-Ib-binding protein [ 141 bind platelet glycoprotein Ib. However, most of these proteins do not bind to carbohydrates, and thus cannot be considered as true C-type lectins. We have identified and purified previously bothrojaracin, a protein from the venom of Bothvops ,juraruca, which belongs to the same protein family, as shown by the N-terminal sequences of its two chains 1251. Bothrojaracin is a 27-kDa protein composed of two disulfide-linked polypeptide chains of 13 kDa and 15 kDa and presents several isoforms. Bothrojaracin binds both anion-binding exosites 1 and 2 of thrombin, resulting in potent inhibition of thrombin activity on fibrinogen and platelets, but it does not mask the thrombin catalytic site [lS, 161, in In the present report, we describe the molecular cloning and the expression of bothrojaracin in COS cells. The entire protein sequence of the two chains of bothrojaracin, deduced from the cDNA cloner, confirmed their...

show abstract

Section: Resultsmentioning

confidence: 90%

Molecular Cloning and Expression of Bothrojaracin, A Potent Thrombin Inhibitor from Snake Venom

Arocas

Castro

Zingalf

et al. 1997

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…The Nowa CTLD belongs to the intron-positive subfamiliy of CTLDs, which possess six Cys-residues (Drickamer, 1989), with disulfide bonds between Cys(1)-Cys(2), Cys(3)-Cys(6), and Cys(4)-Cys(6) demonstrated in many of these CTLDs (Llera et al, 2001;Usami et al, 1993). The CTLD of Nowa lacks the residues with carbonyl side chains implicated in Ca 2+ -dependent sugar binding (Weis et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

Nowa, a novel protein with minicollagen Cys-rich domains, is involved in nematocyst formation inHydra

Engel

Oezbek

Engel

et al. 2002

Journal of Cell Science

View full text Add to dashboard Cite

In addition, the nucleotide sequence for the Nowa cDNA was shown with an incorrect GenBank accession number. The correct GenBank accession number is AF539862. AUTHOR CORRECTION IntroductionPattern formation within single cells has been recently recognized as a fundamental but not well understood aspect of cell biology (Shulman and St Johnston, 1999). A particularly fascinating example is the morphogenesis of the nematocyst, a complex structure that develops inside a giant secretory vesicle in the cnidarian nematocyte (Slautterback and Fawcett, 1959;Holstein, 1981). Upon stimulation of the nematocyte, the nematocyst discharges explosively, a process that takes less than 3 milliseconds (Holstein and Tardent, 1984).The basic structure of the nematocyst consists of a capsule with a double-layered wall, a matrix with an inverted tubule bearing spines, and an operculum. Based on this structure, a wide diversity of morphological types of nematocysts (Fig. 1A) has evolved that serve different functions such as capture of prey and defense (Mariscal, 1974;.Nematocyst morphogenesis can be subdivided into five stages (Holstein, 1981). (1) An early growth phase during which the capsule primordium forms and grows by addition of new vesicles to the vesicle harboring the capsule. (2) A late growth phase during which a tubule forms outside the capsule by addition of more vesicles; capsule and tubule wall form a continuous structure. (3) Invagination of the long external tubule into the capsule. (4) An early maturation phase leading to the formation of spines by condensation of the protein spinalin (Koch et al., 1998) inside the invaginated tubule. (5) A final late maturation step during which poly-γ-glutamate is synthesized in the matrix of the capsule. This generates an osmotic pressure of 150 bar that drives discharge (Weber, 1990;Szczepanek et al., 2002).The extremely high pressure in mature capsules requires high tensile strength of the wall. This tensile strength is mediated by minicollagens, a family of very short collagens that form the capsule's inner wall (Kurz et al., 1991;Holstein et al., 1994). In a previous paper (Engel et al., 2001), we have shown that wall maturation involves polymerization of minicollagens to an insoluble polymer. This polymerization is mediated by disulfides in the minicollagen cysteine-rich domains (MCCR domains) that undergo a switch from intrachain to inter-chain disulfide bonds in the late maturation phase. The inner wall layer, formed by minicollagens, is covered by an outer wall layer, which is more electron-dense than the inner wall in EM sections of nematocysts (Holstein, 1981;Watson and Mariscal, 1984) and appeared as a layer of globular material in field emission scanning electron microscopy (Holstein et al., 1994). The molecular nature of this outer wall was previously unknown.In this study we used the monoclonal antibody H22 (mAb H22) (Kurz et al., 1991), which stained the outer wall of Hydra nematocysts throughout morphogenesis and in mature readyto-discharge nematocysts in the tent...

show abstract

“…However, TSL possesses an additional cysteine residue at position 2. In addition, TSL showed the following percentages of identity with other heterodimeric snake venom C-type-lectin-like proteins : the α and β subunits of botrocetin [23], 39.4 % and 38.2 % respectively ; the β and α subunits of echicetin [5], 37.8 % and 36.3 % respectively ; the B and A chains of habu IX\X-binding protein [3], 33.6 % and 33.3 % respectively. From the known patterns of disulphide bridges in three of the C-type-lectin-like proteins from snake venoms (e.g.…”

Section: Sequence Similarity Between Tsl and Other C-type Crdsmentioning

confidence: 99%

“…From the known patterns of disulphide bridges in three of the C-type-lectin-like proteins from snake venoms (e.g. factor IX\X-binding protein from Trimeresurus fla o iridis [24], botrocetin from Bothrops jararaca [23], and the homodimeric rattlesnake lectin from C. atrox [1]), the location of the disulphide bridges in the TSL protein could be deduced. Four intrachain disulphide bridges linked Cys$ to Cys"%, Cys$" to Cloning of a novel snake venom lectin Cys"$", Cys$) to Cys"$$, and Cys"!'…”

Section: Sequence Similarity Between Tsl and Other C-type Crdsmentioning

confidence: 99%

Cloning of a galactose-binding lectin from the venom of Trimeresurus stejnegeri

Xia

et al. 1999

Biochemical Journal

View full text Add to dashboard Cite

A galactose-binding lectin isolated from the venom of Trimeresurus stejnegeri is a homodimer C-type lectin. The cloned cDNA encoding the monomer of Trimeresurus stejnegeri lectin (TSL) was sequenced and found to contain a 5h-end non-coding region, a sequence which encodes 135 amino acids, including a typical 23 amino acid signal peptide followed by the mature protein sequence, a 3h-end non-coding region, a polyadenylation signal, and a poly(A) region. To completely characterize the deduced amino acid sequence, on-line HPLC-MS and tandem MS were used to analyse the intact monomer and its proteolytic peptides. A modified peptide fragment was also putatively identified by HPLC-MS analysis. The deduced amino acid

show abstract

Primary structure of two-chain botrocetin, a von Willebrand factor modulator purified from the venom of Bothrops jararaca.

Cited by 110 publications

References 32 publications

Molecular Cloning and Expression of Bothrojaracin, A Potent Thrombin Inhibitor from Snake Venom

Molecular Cloning and Expression of Bothrojaracin, A Potent Thrombin Inhibitor from Snake Venom

Nowa, a novel protein with minicollagen Cys-rich domains, is involved in nematocyst formation inHydra

Cloning of a galactose-binding lectin from the venom of Trimeresurus stejnegeri

Contact Info

Product

Resources

About