RsaI polymorphism in von Willebrand factor (vWF) at codon 789.
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Base Sequence↗
Biomedical subjects
S T Lord
Publications and source records attributed to S T Lord.
Analysis of fibrinogen A alpha-fusion proteins. Mutants which inhibit thrombin equivalently are not equally good substrates.
We have examined the interaction of thrombin with fibrinogen A alpha chain residues 7-16. Using genetically engineered constructions, we have synthesized in Escherichia coli a fibrinogen A alpha 1-50 fusion protein and seven mutant proteins with single amino acid substitutions. These are: Asp7----Ala, Phe8----Tyr, Glu11----Ala, Gly12----Val, Gly13----Val, Gly14----Val, and Arg16----Leu. Competitive immunoassay of cell lysates showed that all the mutations but one, Arg16----Leu, altered the structure of the protein such that cross-reactivity with the A alpha-specific monoclonal antibody, Y18, was significantly reduced. The fusion proteins were purified and analyzed as thrombin inhibitors and substrates. All the fusion proteins are competitive inhibitors of the amidolytic hydrolysis of Spectrozyme TH, a thrombin-specific chromogenic substrate, with inhibition constants corresponding to that for fibrinogen. We conclude that these 7 amino acid substitutions do not alter thrombin binding to the fusion proteins. The fusion proteins were tested as substrates by monitoring thrombin-dependent peptide release. The natural sequence and three mutants, Asp7----Ala, Glu11----Ala, and Gly14----Val, are good substrates. The other mutants are either poor substrates or are not cleaved by thrombin within A alpha 1-50. These results indicate that residues between Asp7 and Arg16 are critical to efficient peptide hydrolysis, whereas residues outside this region are critical to thrombin binding.
Amino Acid Sequence↗
Analysis of fibrinogen alpha residues 27-50: interactions with thrombin.
In order to further understand the unusual specificity of thrombin activity, we have examined the interactions of thrombin with fibrinogen alpha-chain residues 27-50 by two complementary approaches. Using genetically engineered constructions, we have synthesized a fusion protein containing fibrinogen A alpha-residues 1-50 and seven variants of this protein with substitutions in alpha-residues 33-41. These fusion proteins were purified and analysed as thrombin inhibitors and substrates. Substitution of Phe35----Leu of Asp40----Gly substantially increased Ki, but all the fusion proteins were equally good substrates when assayed at concentrations equivalent to Ki. These results indicate that Phe35 and Asp40 are important for thrombin binding, but not catalysis. We have also synthesized a peptide analogous to fibrinogen alpha 27-50. This peptide was a competitive inhibitor of thrombin-catalysed cleavage of fibrinogen, but was not an inhibitor of hydrolysis of small chromogenic substrates. These data further indicate that residues alpha 27-50 are important in the thrombin-fibrinogen interaction.
Amino Acid Sequence↗
The utility of a HindIII polymorphism of factor VIII examined by rapid DNA analysis.
A previously described HindIII restriction fragment length polymorphism (RFLP) of factor VIII (FVIII) has its polymorphic site in the unsequenced nineteenth intron. We have located the polymorphic site, as well as an invariant site, by amplifying and sequencing IVS 19 using the polymerase chain reaction (PCR). The oligonucleotide primers were synthesized from known FVIII sequence on either side of the 19-20 splice junction. The amplified product was cloned into a plasmid and sequenced by the dideoxy chain termination method. The polymorphic HindIII site was 103 bp and the invariant site 184 bp from the 3' end of the nineteenth exon. The frequency of the polymorphism was determined in 457 subjects (643 chromosomes) of seven ethnic groups on whom frequency of the BclI RFLP of IVS 18 was also assessed. The HindIII site is highly polymorphic in all groups, approximately 0.25:0.75, the expected heterozygosity averaging 37.6%, and the observed number of heterozygotes did not differ significantly from expectation. The (+):(-) allelic ratio is similar in all groups, except African-Americans in whom it is reversed. Strong allelic association (linkage disequilibrium) is present between the HindIII polymorphism of IVS 19 and the BclI polymorphism of IVS 18.
Alleles↗
High-resolution NMR studies of fibrinogen-like peptides in solution: interaction of thrombin with residues 1-23 of the A alpha chain of human fibrinogen.
The interaction of the following human fibrinogen-like peptides with bovine thrombin was studied by use of one- and two-dimensional NMR techniques in aqueous solution: Ala(1)-Asp-Ser-Gly-Glu-Gly-Asp-Phe(8)-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16 )- Gly(17)-Pro-Arg(19)-Val(20)-Val-Glu-Arg (F10), residues 1-16 of F10 (fibrinopeptide A), residues 17-23 of F10 (F12), residues 1-20 of F10 (F13), residues 6-20 of F10 with Arg(16) replaced by a Gly residue (F14), and residues 6-19 of F10 with Arg(16) replaced by a Leu residue (F15). At pH 5.3 and 25 degrees C, the Arg(16)-Gly(17) peptide bonds of both peptides F10 and F13 were cleaved instantaneously in the presence of 0.6 mM thrombin, whereas the cleavage of the Arg(19)-Val(20) peptide bonds in peptides F12, F13, and F14 took over 1 h for completion. On the basis of observations of line broadening, fibrinopeptide A was found to bind to thrombin. While resonances from residues Ala(1)-Glu(5) were little affected, binding of fibrinopeptide A to thrombin caused significant line broadening of NH and side-chain proton resonances within residues Asp(7)-Arg(16). There is a chain reversal within residues Asp(7)-Arg(16) such that Phe(8) is brought close to the Arg(16)-Gly(17) peptide bond in the thrombin-peptide complex, as indicated by transferred NOEs between the aromatic ring protons of Phe(8) and the C alpha H protons of Gly(14) and the C gamma H protons of Val(15). A similar chain reversal was obtained in the isolated peptide F10 at a subzero temperature of -8 degrees C. The titration behavior of Asp(7) in peptide F13 does not deviate from that of the reference peptide, N-acetyl-Asp-NHMe at both 25 and -8 degrees C, indicating that no strong interaction exists between Asp(7) and Arg(16) or Arg(19). Peptides with Arg(16) replaced by Gly and Leu, respectively, i.e., F14 and F15, were also found to bind to thrombin but with a different conformation, as indicated by the absence of the long-range NOEs observed with fibrinopeptide A. Residues Asp(7)-Arg(16) constitute an essential structural element in the interaction of thrombin with fibrinogen.
Amino Acid Sequence↗
The Malmö polymorphism of factor IX: establishing the genotypes by rapid analysis of DNA.
A DNA polymorphism in the coding region of coagulation factor IX--potentially valuable for carrier detection, prenatal diagnosis, and population studies--was described in 1985. It had been discovered with monoclonal antibodies that distinguish between threonine and alanine as the 148th residue of the peptide. Its use as a diagnostic tool has been limited because threonine-containing factor IX (Malmö A) is dominant to alanine-containing factor IX (Malmö B) in immunoassays of plasma; therefore, detection of Malmö heterozygotes is not possible in all instances. A DNA method for recognizing all heterozygotes has been developed, but it also has limitations. We report the development of another DNA procedure based on amplification of the relevant DNA with the polymerase chain reaction (PCR). This method is quick, avoids the use of isotopes and x-ray film, and specifically identifies all the Malmö genotypes: hemizygotes, homozygotes, and heterozygotes. The procedure can be performed satisfactorily on small samples of blood (less than 1 mL) as suggested by Kogan et al (N Engl J Med 317:985, 1987). The method described is applicable to any genetic polymorphism that overlaps a restriction enzyme recognition site.
Base Sequence↗
Expression of a fibrinogen fusion peptide in Escherichia coli: a model thrombin substrate for structure/function analysis.
The initial event in fibrin clot formation is the thrombin-catalyzed cleavage of the A alpha chain of human fibrinogen. Most of the information required for thrombin recognition and cleavage of the A alpha chain lies in the amino terminal 51 residue CNBr fragment. By selective modification of residues in this region, we probed the features that participate in thrombin interactions. We constructed a vector which expressed a tripartite protein (tribrid) consisting of amino acids 1 to 50 of the A alpha chain followed by 60 amino acids of chicken collagen and the beta-galactosidase protein from Escherichia coli. Cell lysates run on NaDodSO4-polyacrylamide gels contained the predicted band of molecular weight (mol wt) 125,000. The tribrid reacted with a monoclonal antibody, Mab-Y18, which recognizes the amino terminus of the A alpha chain. When cell lysates were incubated with thrombin, FPA was released. By including one heterogeneous oligonucleotide in the construction, we generated plasmids that encoded three specific amino acid substitutions. Surprisingly, changing Gly14 to Val did not alter thrombin cleavage, although recognition by Mab-Y18 was lost. Substitution of lie for Arg23 did not alter either thrombin cleavage or monoclonal recognition. Substitution of Leu for Arg 16 altered thrombin cleavage; unexpectedly, recognition by Mab-Y18 was not changed.
Amino Acid Sequence↗
Expression in Escherichia coli of the human fibrinogen B beta chain and its cleavage by thrombin.
The human fibrinogen B beta chain was expressed in Escherichia coli to study the functions of fibrinogen associated with this subunit. Recombinant B beta chains were expressed at 100 ng/mL in an IPTG-dependent manner. A first cistron sequence, inserted into the expression vector 5' to the B beta chain cDNA, was required to express the protein. Recombinant B beta chains were expressed within five minutes after induction with IPTG and were soluble in physiologic buffers. The recombinant B beta chains migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a rate identical to B beta chains from fibrinogen treated with N-glycanase. Recombinant B beta chains were cleaved by thrombin, as demonstrated by the loss of cross-reactivity with a monoclonal antibody (MoAb) specific for the undigested B beta 1-42 fragment. The levels of expression of the B beta chain were much lower than those reported previously for the gamma chain of fibrinogen expressed in a similar vector in E coli. However, these levels are sufficient to allow further characterization of this fibrinogen subunit.
Base Sequence↗
High-level expression of a functional human fibrinogen gamma chain in Escherichia coli.
Human fibrinogen gamma chain has been expressed intact at high levels in Escherichia coli. The construction of the expression plasmid, p253, is described. Synthesis of the recombinant protein is isopropyl-beta-D-thiogalactopyranoside-dependent and is driven by the tac promoter. Western analysis of E. coli lysates demonstrates a novel protein of approx. 45 kDa which cross-reacts with antisera to human fibrinogen gamma chains. The protein is not soluble in common E. coli lysis buffers and becomes soluble in 6 M guanidine.HCl or 6 M urea. Initial insolubility is due to interchain disulfide bond formation and to noncovalent interactions. Induced cells examined by phase-contrast microscopy contain dense inclusion bodies. A known function of the gamma chains of human fibrinogen is the clumping of Staphylococcus aureus Newman D2C cells [Hawiger et al., Biochemistry (1982) 1407-1413]. We demonstrate that suspensions of recombinant gamma chains retain the ability to clump cells from this strain of S. aureus.
Bacterial Proteins↗
High-resolution NMR studies of fibrinogen-like peptides in solution: resonance assignments and conformational analysis of residues 1-23 of the A alpha chain of human fibrinogen.
The proton resonances of the following synthetic linear human fibrinogen-like peptides were completely assigned with two-dimensional NMR techniques in solution: Ala(1)-Asp-Ser-Gly-Glu-Gly-Asp(7)-Phe-Leu-Ala-Glu-Gly(12)-Gly(13)-Gly(14)- Val(15)-Arg(16)-Gly-Pro-Arg-Val-Val-Glu-Arg (F10), Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly(13)-Gly(14)-Val-Arg (F11), and Gly-Pro-Arg-Val-Val-Glu-Arg (F12). No predominant structure was found in the chain segment from Ala(1) to Gly(6) for F10 in both H2O and dimethyl sulfoxide. The previous suggestion that there is a hairpin loop involving residues Gly(12) to Val(15) in the A alpha chain of human fibrinogen is supported by the slow backbone NH exchange rates of Gly(14) and Val(15), by an unusually small NH chemical shift of Val(15), and by strong sequential NOE's involving this region in F10. This local chain fold within residues Asp(7) to Val(20) may place the distant Phe residue near the Arg(16)-Gly(17) peptide bond which is cleaved by thrombin.
Amino Acid Sequence↗
The Malmö polymorphism of coagulation factor IX, an immunologic polymorphism due to dimorphism of residue 148 that is in linkage disequilibrium with two other F.IX polymorphisms.
A mouse monoclonal antibody (MAB 9.9) to coagulation factor IX (F.IX) detects a polymorphism in the plasma of normal people. Its epitope has been narrowed down to less than 6 amino acids in the activation peptide of the X-linked F.IX protein. The activation peptide contains a dimorphism--Thr:Ala--at position 148 of the protein. Using synthetic oligonucleotides, we have demonstrated that (1) the F.IX which reacts with 9.9 has Thr at position 148 and (2) that which does not has Ala. Positive reactors (148thr) are designated Malmö A, and negative reactors (148ala) are designated Malmö B. The plasma levels of AA women are indistinguishable from those of A men, and both B men and BB women are null against MAB 9.9. The plasma level of Malmö A in AB women is approximately half that of AA women, and "lyonization" is clearly operating in the heterozygotes. The dimorphism is in strong linkage disequilibrium with two other intragenic RFLPs, TaqI and XmnI. Furthermore, intragenic crossing-over--including double crossing-over--appears to have occurred between the three sites. Seven of the eight possible haplotypes have been identified, five in men and two others in women. The immunoassay that identifies approximately 50% of the AB women in the pool of Malmö A females with 95% confidence identifies men unambiguously as A or B. The assay would be very useful for population-genetic studies of the Malmö epitope if the studies were limited to men.
Antibodies, Monoclonal↗
Hemophilia B Durham: a mutation in the first EGF-like domain of factor IX that is characterized by polymerase chain reaction.
Two men with factor IX (FIX)antigen-positive (CRM+) hemophilia B were selected for study because of their abnormal expression of an immunologically defined epitope previously localized to the EGF-like domains of the molecule. Exons IV and V (coding for the first and second EGF-like domains) of FIX were amplified 10(7) times from the patients' genomic DNA by using polymerase chain reaction (PCR) technology and sequenced. Both patients had identical mutations which resulted in the highly conserved Gly 60 residue being changed to Ser. PCR-amplified exon IV from six normal males had the previously defined canonic sequence. The correlation between the mutation and defective epitope expression in the two patients suggests that a change in the tertiary structure of the EGF-like domain is likely to cause the mild hemophilia B.
Adult↗
Population genetics of coagulant factor IX: frequencies of two DNA polymorphisms in five ethnic groups.
Two frequently used restriction-enzyme polymorphisms (RFLPs) of coagulant F.IX, TaqI and XmnI, have been examined in five ethnic groups: white Americans, black Americans, East Indians, Chinese, and Malays. There is a distinct "cline" in the frequencies of both polymorphisms, from white Americans to Malays. The rarer type 2 alleles of both polymorphisms, in which middle recognition sites are present--and which in our sample reach their highest frequencies in white Americans--are marginally higher in four groups of Europeans previously reported by others. The frequencies of the rarer alleles are significantly higher in Europeans than in black Americans and East Indians, and these alleles are essentially absent in Chinese and Malays. The frequency of heterozygosity diminishes in the same order, being zero in Malays for both polymorphisms. The polymorphisms are in strong linkage disequilibrium, and in all groups the type 1 allele for TaqI is disproportionately accompanied by the type 1 allele for XmnI. The paucity of type 2 alleles and the low rate of heterozygosity in four non-European groups suggest that the polymorphisms will be of little diagnostic value south of Gibraltar and east of Suez. This prediction is confirmed by the observed haplotype frequencies in the black American and the Oriental groups.
Alleles↗
In vitro methylation doubles the efficiency of oligonucleotide-directed mutagenesis.
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Methylation↗
Expression of a cloned human fibrinogen cDNA in Escherichia coli: synthesis of an A alpha polypeptide.
The construction of a plasmid, p166.9, for the controlled synthesis of the A alpha-chain of human fibrinogen in Escherichia coli is described. The plasmid combines the tac promoter, constructed for controlled high-level peptide expression, with the promoter and signal peptide codons from an E. coli plasmid beta-lactamase gene and a cDNA of the A alpha-chain of human fibrinogen. The tac promoter is repressed in lacIQ strains of E. coli and induced by isopropylthio-beta-D-galactoside (IPTG). Protein blot analysis of lysates of cells carrying p166.9 demonstrates the IPTG-dependent synthesis of polypeptides which cross react with antisera to the A alpha-chain of human fibrinogen. The largest and predominant species corresponds to an apparent molecular weight of 63,000. When the cell growth media or cell lysates are treated with thrombin, the enzyme which normally releases fibrinopeptide A (FPA) from the A alpha-chain, FPA-like peptides are detectable by radioimmunoassay with antiserum prepared against human FPA. Thrombin-treated cell growth media prepared 4 hr after IPTG induction contained 340 ng/ml of FPA-like material; using a mass ratio of 40 for FPA to A alpha, this indicates that the A alpha-peptide concentration in the culture media is 13 micrograms/ml.
Cloning, Molecular↗
A transcriptionally active monkey genomic segment homologous to the regulatory region of simian virus 40 is associated with DNase I-hypersensitive sites.
Segments of monkey genomic DNA that are homologous to the control region around the origin of replication of simian virus 40 were previously cloned and characterized (Queen et al., Mol. Cell Biol. 1:1061-1068, 1981). We describe here two DNase I-hypersensitive sites that map in the region of monkey chromatin around one such ori-like segment. One of these sites lies within the simian virus 40 homologous segment which is also a site from which transcription initiates bidirectionally (J. Saffer and M. Singer, submitted for publication).
Animals↗
Partial mRNA sequences for human A alpha, B beta, and gamma fibrinogen chains: evolutionary and functional implications.
Using rat cDNA and genomic probes to screen a human liver cDNA library, we have isolated clone of 2,274, 855, and 736 base pairs (bp) coding for the A alpha, B beta and gamma chains of human fibrinogen. Sequence analysis reveals a hitherto unrecognized extension of 15 amino acids at the carboxyl terminus of the A alpha chain, the terminal residue of which is proline. This brings the known length of the human A alpha chain to 625 amino acids. The 13-amino-acid repeated region in the midportion of the A alpha chain clearly has arisen through an 8-fold duplication of a 39-bp genetic element, which itself appears to have been constructed from smaller 6-bp repeating units. Greater than 50% sequence homology between B beta- and gamma-chain coding regions confirms postulates that these genes have arisen by duplication and subsequent divergence of an ancestral gene. A comparison of human and rat gamma-chain cDNAs shows more than 88% sequence homology over the carboxyl-terminal 162 amino acids, implying strong selective pressures on these portions of the gamma-chain gene.
Adult↗
The labeling with 8-azido-cyclic adenosine monophosphate of proteins in vesicles of sarcoplasmic reticulum from rabbit skeletal muscle.
Photoinduced labeling with 8-azido-cyclic AMP of proteins in vesicles from sarcoplasmic reticulum rabbit skeletal muscle has been examined. At concentrations of 0.1 microM or less, specific labeling of three or four bands, representing trace components in the SDS gels, was seen. This labeling was prevented by cyclic AMP. Some of the bands correspond approximately in apparent molecular weight to subunits of cyclic AMP-dependent protein kinases reported in other systems. Attempts to use this reagent as a probe for protein position in the membrane have been unsuccessful since 8-azido-cyclic AMP passes into and through the membrane. The reagent also appears to partition with a linear concentration dependence into the vesicular membrane itself, presumably into the lipid portion.
Affinity Labels↗