Disulphide bridges of bovine factor X

Højrup, Peter; Magnusson, Staffan

doi:10.1042/bj2450887

Cited by 44 publications

(21 citation statements)

References 27 publications

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“…There are no cysteines after position 600. It has been determined that in EGF and in the EGF repeat of bovine factor X, disulfide bonds are formed between the cysteine pairs 1 and 3, 2 and 4, and 5 and 6 (17,18). If the same occurs in Bm86, the 576-600 region could form the first two disulfide bonds of an EGF-like region.…”

Section: Resultsmentioning

confidence: 99%

Cloning and expression of a protective antigen from the cattle tick Boophilus microplus.

Rand¹,

Moore²,

Sriskantha³

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

229

151

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Glycoproteins located on the luminal surface of the plasma membrane of tick gut epithelial cells, when used to vaccinate cattle, are capable of stimulating an immune response that protects cattle against subsequent tick infestation. One such tick gut glycoprotein, designated Bm86, has been purified to homogeneity and the amino acid sequences of peptide fragments generated by endoproteinase Lys-C digestion have been determined. We report here the isolation and characterization of a cDNA that encodes Bm86. The nucleotide sequence of the cDNA contains a 1982-base-pair open reading frame and predicts that Bm86 contains 650 amino acids including a 19-amino acid signal sequence and a 23-amino acid hydrophobic region adjacent to the carboxyl terminus. The main feature of the deduced protein sequence is the repeated pattern of 6 cysteine residues, suggesting the presence ofseveral epidermal growth factor-like domains. A fusion protein consisting of 599 amino acids of Bm86 and 651 amino acids of /3-galactosidase was expressed in Escherichia coli as inclusion bodies. Ticks engorging on cattle vaccinated with these inclusion bodies were significantly damaged as a result of the immune response against the cloned antigen.The tick Boophilus microplus is a major ectoparasite of cattle in many parts of the world. A single female cattle tick takes up as much as 1.5 ml of bovine blood, increasing its body weight to =250 mg. It has been estimated that cattle in tropical areas of Australia may become infested with 1000 tick larvae per day, resulting in greatly reduced productivity. In addition, B. microplus is the vector of hematoprotozoal parasites such as Babesia bovis. Chemicals have been used extensively to control ticks and have been partially successful, but this approach suffers from certain drawbacks such as environmental and residue problems, the high incidence of acaricide resistance that has developed in tick populations in the field, the need for frequent administration, and high cost.Recently it was shown that cattle immunized against a Construction and Screening of cDNA Library. RNA was extracted (3) from adult B. microplus (picked from cattle 15 days after infestation), cDNA was synthesized from 4 ,ug of poly(A)+ RNA (4), and cDNA fragments larger than 800 base pairs (bp) were ligated to Agtll (5) to generate a library of 8 x 105 recombinant clones. Oligodeoxynucleotide probes (Table 1) were based on the sequences derived from peptides generated by endoproteinase Lys-C digestion of Bm86 (1).Three nitrocellulose filters were prepared from five 150-mm plates each containing 105 plaques. After prehybridization in 0.6 M sodium pyrophosphate/0.005% heparin (Sigma) at 40°C for 4 hr, hybridization was carried out for 16 hr at 40°C in the same solution with the radioactive oligonucleotide probe. Two of the filters were hybridized with the 63-mer probe while the third was hybridized with a mixture of the 50-, 51-, and 72-mer probes. The filters were washed in 0.3 M NaCl/0.03 M sodium citrate, pH 7.5/0.1% SDS at 45°C a...

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Section: Resultsmentioning

confidence: 99%

Cloning and expression of a protective antigen from the cattle tick Boophilus microplus.

Rand¹,

Moore²,

Sriskantha³

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

229

151

View full text Add to dashboard Cite

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“…Amino acid sequencing identified a heterodimer consisting of the light subunit disulfide linked to a small piece of the heavy subunit starting at Met-296 and presumably extending to Lys-330. The small heavy subunit fragment encompasses Cys-302, which is known to link the FX subunits (38). The end position at Lys-330 is inferred from the prior cleavage known to expose Gly-331 as the NH 2 terminus of the 13-kDa fragment.…”

Section: Fig 6 Effect Of Plasmin-cleaved Fx On Tissue Plasminogen Amentioning

confidence: 99%

Plasmin Converts Factor X from Coagulation Zymogen to Fibrinolysis Cofactor

Pryzdial

Lavigne

Dupuis

et al. 1999

Journal of Biological Chemistry

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Known anticoagulant pathways have been shown to exclusively inhibit blood coagulation cofactors and enzymes. In the current work, we first investigated the possibility of a novel anticoagulant mechanism that functions at the level of zymogen inactivation. Utilizing both clotting and chromogenic assays, the fibrinolysis protease plasmin was found to irreversibly inhibit the pivotal function of factor X (FX) in coagulation. This was due to cleavage at several sites, the location of which were altered by association of FX with procoagulant phospholipid (proPL). The final products were ϳ28 and ϳ47 kDa for proPL-bound and unbound FX, respectively, which did not have analogues when activated FX (FXa) was cleaved instead. We next investigated whether the FX derivatives could interact with the plasmin precursor plasminogen, and we found that plasmin exposed a binding site only on proPL-bound FX. The highest apparent affinity was for the 28-kDa fragment, which was identified as the light subunit disulfide linked to a small fragment of the heavy subunit (Met-296 to ϳLys-330). After cleavage by plasmin, proPL-bound FX furthermore was observed to accelerate plasmin generation by tissue plasminogen activator. Thus, a feedback mechanism localized by proPL is suggested in which plasmin simultaneously inhibits FX clotting function and converts proPL-bound FX into a fibrinolysis cofactor. These data also provide the first evidence for an anticoagulant mechanism aimed directly at the zymogen FX.

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“…Pth, -Cys was identified after its release in the corresponding cycle and eluted just before Pth -Tyr ( H~j r u p and Magnusson, 1987;Marti et al, 1987).…”

Section: Amino Acid Sequence Analysismentioning

confidence: 99%

The multimeric structure and disulfide‐bonding pattern of bovine κ‐casein

Rasmussen¹,

Højrup²,

Petersen

1992

European Journal of Biochemistry

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Bovine Ic-casein was analyzed by SDSIPAGE, MS and amino acid sequence analysis in order to determine its multimeric composition and disulfide-bonding pattern. SDSjPAGE revealed that ICcasein in the native state can range in size from a monomer to a multimeric structure larger than a decamer. Three types of interchain disulfide linkage, Cysl1 -Cysl 1, Cysll -Cys88 and Cys88 -Cys88, were all assigned in multimers purified from [14C]carboxymethylated and untreated bulk milk, as well as a milk sample from a Ic-casein-variant-B homozygote C020. These results indicate that multimerization occurs in a random or at present unpredictable disulfide-bonding pattern regardless of the size of the multimer or the genotype.

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Disulphide bridges of bovine factor X

Cited by 44 publications

References 27 publications

Cloning and expression of a protective antigen from the cattle tick Boophilus microplus.

Cloning and expression of a protective antigen from the cattle tick Boophilus microplus.

Plasmin Converts Factor X from Coagulation Zymogen to Fibrinolysis Cofactor

The multimeric structure and disulfide‐bonding pattern of bovine κ‐casein

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