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R M Lawn

Publications and source records attributed to R M Lawn.

At least 73 records · Page 4Linked to original sources
PubMed1991

Interaction of recombinant apolipoprotein(a) and lipoprotein(a) with macrophages.

Elevated plasma levels of lipoprotein(a), Lp(a), represent a major, inherited risk factor for coronary heart disease, although the mechanism of its action remains unknown. Lp(a) is distinguished from the related LDL particle by the addition of apolipoprotein(a), apo(a). The presence of this large glycoprotein is likely to affect the binding of the particle to the LDL receptor and/or other receptors which may contribute to the atherogenic potential of Lp(a). Here we demonstrate the binding to macrophages of Lp(a) and pure recombinant apo(a) protein, via a specific, high-affinity receptor. This binding could lead to foam cell formation and the localization of Lp(a) to atherosclerotic plaques.

Animals↗
PubMed1990

Apolipoprotein(a) size heterogeneity is related to variable number of repeat sequences in its mRNA.

Plasma apolipoprotein(a) [apo(a)] shows considerable size heterogeneity, existing as discrete glycoprotein isoform variants that range in apparent molecular mass from approximately 400 to 800 kDa. To study the molecular basis of protein size variability, we have isolated liver RNA from individuals with different apo(a) isoforms, and identified apo(a)-specific transcripts using Northern blot analysis. Transcript sizes were shown to be variable (8.0-12 kb) and in all cases were closely correlated with protein masses (590-850 kDa) as determined from immunoblots. Thus, it is almost certain that apo(a) isoform size variation is due to allelic differences in the number of its tandemly repeated sequences of 114 amino acids that resemble kringle four of plasminogen. The high carbohydrate content of apo(a) makes true molecular weight estimations in SDS-PAGE gels difficult. However, a recombinant form of apo(a) containing 17 kringle repeats (calculated molecular mass of 250 kDa) migrates on SDS-PAGE gels only slightly below apoB-100, with an apparent molecular mass of approximately 500 kDa. Since smaller protein isoforms have been observed in the population, this suggests that plasma apo(a) isoforms contain from less than 17 to greater than 30 tandemly repeated kringle units.

Apolipoproteins A↗
PubMed1990

Mammalian cell transient expression of tissue factor for the production of antigen.

We describe a mammalian cell expression system used to rapidly produce microgram quantities of a membrane protein used as an immunogen. A fusion protein expression vector was constructed which contained the signal sequence and 27 amino acids of the Herpes simplex virus glycoprotein D (gD), followed by a factor VIII (fVIII) thrombin cleavage site and the mature tissue factor (TF) sequence. This fusion protein was transiently expressed and then purified using an antibody to gD. The purified fusion protein, gDTF, was incubated with thrombin to remove the gD-fVIII moiety and the resulting rTF served as antigen for the generation of TF-specific antibodies. The antibodies produced were then used for a comparison of the turnover rates of the constitutively and transiently produced fusion protein. In addition, sensitivity to glycosidases indicated that the transiently and constitutively produced recombinant proteins do not contain identical carbohydrate structures.

Amino Acid Sequence↗
PubMed1990

Cellular localization of apolipoprotein D and lecithin:cholesterol acyltransferase mRNA in rhesus monkey tissues by in situ hybridization.

Apolipoprotein D (apoD) and lecithin:cholesterol acyl transferase (LCAT) are found on high density lipoprotein particles (HDLs) and have been postulated to form part of a complex involved in the transport of cholesterol from peripheral tissues to the liver for excretion. We have examined the sites of synthesis of the mRNAs for these two proteins in the rhesus monkey by in situ hybridization. ApoD mRNA-containing cells were widely distributed throughout peripheral tissues in interstitial and connective tissue fibroblasts often associated with blood vessels or capillaries. ApoD mRNA was also found localized in cells associated with peripheral nerves, neuroglial cells, cells in the subarachnoid space on the surface of the brain including the pial cells, perivascular cells, and scattered neurons in the brain. LCAT demonstrated a much more restricted pattern of synthesis and was found to be synthesized by hepatocytes, the basal cell layer of the epidermis, and in brain cell populations distinct from those that synthesize apoD. In the brain LCAT was synthesized by scattered neurons, neuroglial cells, ependymal cells, as well as a discrete cell layer in the cerebellum. ApoD has been shown to possess extensive homology to retinol binding protein, which has a binding pocket for vitamin A. We propose that apoD may also function to bind cholesterol or its derivatives in compartments not in direct contact with the blood. The findings of both apoD and LCAT synthesis in the brain suggest that they play a significant role in lipid transport in the brain.

Animals↗
PubMed1989

Rhesus monkey apolipoprotein(a). Sequence, evolution, and sites of synthesis.

Human lipoprotein(a) is a low density lipoprotein-like lipoprotein whose concentration in plasma is correlated with atherosclerosis. The characteristic protein component of lipoprotein(a) is apolipoprotein(a) (apo(a)) which is disulfide-linked to apolipoprotein B-100. Sequencing of rhesus monkey apo(a) cDNA suggests that this protein, like human apo(a), is highly similar to plasminogen. Sequence data suggests that a plasminogen-like protease activity and kringle 1-, 2-, 3-, and 5-like domains are unnecessary for apo(a) function, but a highly repeated kringle four-like domain is important. Liver is the major site of apo(a) RNA synthesis; reduced amounts of message were also found in testes and brain. Co-expression with apoB-100 and plasminogen in rhesus tissues is not mandatory.

Amino Acid Sequence↗
PubMed1988

The apolipoprotein(a) gene resides on human chromosome 6q26-27, in close proximity to the homologous gene for plasminogen.

Apolipoprotein(a) [apo(a)], the glycoprotein associated with the lipoprotein(a) [Lp(a)] subfraction of plasma lipoproteins, has been shown to exhibit heritable molecular weight isoforms ranging from 400-700 kDa. Increased serum concentrations of Lp(a) correlate positively with the risk of atherosclerosis. Variations in Lp(a) plasma levels among individuals are inherited as a codominant quantitative trait. As part of an effect to define the basis of these variations and further clarify the expression of the protein, we have determined the chromosomal location of the human apo(a) gene. Blot hybridization analysis of DNA from a panel of mouse-human somatic cell hybrids with an apo(a) cDNA probe revealed a complex pattern of bands, all of which segregated with chromosome 6. In situ hybridization yielded a single peak of grain density located on chromosome 6q26-27. Apo(a) cDNA sequences exhibit striking homology to those of the plasma protease plasminogen, and, therefore, we have reexamined the chromosome assignment of the plasminogen gene. We conclude that both the apo(a) and plasminogen genes reside on human chromosome 6q22-27, consistent with a gene duplication mechanism for their evolutionary origin. The results are of significance for the genetic control of apo(a) expression and genetic influences predisposing to atherosclerosis.

Apolipoproteins A↗
PubMed1988

Genetic linkage between lipoprotein(a) phenotype and a DNA polymorphism in the plasminogen gene.

Coronary heart disease risk correlates directly with plasma concentrations of lipoprotein(a) (Lp(a)), a low-density lipoprotein-like particle distinguished by the presence of the glycoprotein apolipoprotein(a) (apo(a)), which is bound to apolipoprotein B-100 (apoB-100) by disulfide bridges. Size isoforms of apo(a) are inherited as Mendelian codominant traits and are associated with variations in the plasma concentration of lipoprotein(a). Plasminogen and apo(a) show striking protein sequence homology, and their genes both map to chromosome 6q26-27. In a large family with early coronary heart disease and high plasma concentrations of Lp(a), we found tight linkage between apo(a) size isoforms and a DNA polymorphism in the plasminogen gene; plasma concentrations of Lp(a) also appeared to be related to genetic variation at the apo(a) locus. We found free recombination between the same phenotype and alleles of the apoB DNA polymorphism. This suggests that apo(a) size isoforms and plasma lipoprotein(a) concentrations are each determined by genetic variation at the apo(a) locus.

Adult↗
PubMed1988

Cloning and mRNA tissue distribution of rabbit cholesteryl ester transfer protein.

The amino acid sequence of rabbit cholesteryl ester transfer protein (CETP) has been obtained from cloned cDNA and genomic sequences. The 496 amino acid rabbit CETP has an overall sequence homology of 81% compared to the 476 amino acid human CETP, with two-thirds of the amino acid substitutions being conservative. Like human CETP, rabbit CETP is extremely hydrophobic, which is consistent with its function in the transfer of neutral lipids. The data implies extensive structural similarity between rabbit and human CETP. Rabbit CETP mRNA is estimated to be 2.2 kilobases in size, 300 nucleotides longer than the corresponding human mRNA, and contains the unusual polyadenylation signal sequence AGTAAA. In rabbit, CETP mRNA is found mainly in the liver, with small amounts also present in adrenal glands and kidney. In contrast to human spleen, rabbit spleen does not have detectable amounts of CETP mRNA. Northern blot analysis of liver poly(A)+ RNAs revealed significant amounts of CETP message in human, rhesus, and rabbit, and undetectable levels in pig, mouse, and rat, in agreement with reported plasma levels of transfer activity.

Amino Acid Sequence↗
PubMed1987

Cloning and expression of human tissue factor cDNA.

Tissue factor is a membrane protein that plays an essential role in the initiation of blood coagulation. When exposed to the circulation, tissue factor interacts with the serine protease factor VII, and the complex triggers fibrin clot formation by activating both factors IX and X of the coagulation cascade. This report describes the cloning and expression of the complementary DNA (cDNA) for human tissue factor. The cDNA encodes a protein of 263 amino acids preceded by a 32 amino acid signal peptide. The predicted protein sequence contains a potential hydrophobic membrane anchoring domain at its carboxy terminus, and bears no significant homology to any other known protein. Tissue factor mRNA of 2400 nucleotides was detected in adipose, adrenal, small intestine and a number of other tissues by Northern blot hybridization analysis. In order to confirm the identity of the cDNA, an expression vector containing the cloned cDNA was used to transfect cultured mammalian cells. These cells produced active tissue factor which was assayed using purified factors VII and X.

Amino Acid Sequence↗
PubMed1987

Human lipoprotein lipase complementary DNA sequence.

Lipoprotein lipase is a key enzyme of lipid metabolism that acts to hydrolyze triglycerides, providing free fatty acids for cells and affecting the maturation of circulating lipoproteins. It has been proposed that the enzyme plays a role in the development of obesity and atherosclerosis. The human enzyme has been difficult to purify and its protein sequence was heretofore undetermined. A complementary DNA for human lipoprotein lipase that codes for a mature protein of 448 amino acids has now been cloned and sequenced. Analysis of the sequence indicates that human lipoprotein lipase, hepatic lipase, and pancreatic lipase are members of a gene family. Two distinct species of lipoprotein lipase messenger RNA that arise from alternative sites of 3'-terminal polyadenylation were detected in several different tissues.

Amino Acid Sequence↗
PubMed1987

Assignment of the human gene for cholesteryl ester transfer protein to chromosome 16q12-16q21.

We have used a cDNA probe for human cholesteryl ester transfer protein (CETP) to determine the chromosomal location for the human gene. Southern blot analysis of DNA from 17 independent mouse-human somatic cell hybrids demonstrated the presence of the gene for human CETP on chromosome 16. Regional mapping of the gene by in situ hybridization was consistent with these results and indicated that the gene resides in the 16q12-21 region of the chromosome. These findings provide an additional polymorphic marker for chromosome 16, as several relatively common restriction fragment length polymorphisms of the gene have previously been reported, and they have significance for studies directed at the identification of genetic factors affecting plasma lipoprotein metabolism and atherosclerosis.

Animals↗
PubMed1987

Partial amino acid sequence of apolipoprotein(a) shows that it is homologous to plasminogen.

Apolipoprotein(a) [apo(a)] is a glycoprotein with Mr approximately equal to 280,000 that is disulfide linked to apolipoprotein B in lipoprotein(a) particles. Elevated plasma levels of lipoprotein(a) are correlated with atherosclerosis. Partial amino acid sequence of apo(a) shows that it has striking homology to plasminogen. Plasminogen is a plasma serine protease zymogen that consists of five homologous and tandemly repeated domains called kringles and a trypsin-like protease domain. The amino-terminal sequence obtained for apo(a) is homologous to the beginning of kringle 4 but not the amino terminus of plasminogen. Apo(a) was subjected to limited proteolysis by trypsin or V8 protease, and fragments generated were isolated and sequenced. Sequences obtained from several of these fragments are highly (77-100%) homologous to plasminogen residues 391-421, which reside within kringle 4. Analysis of these internal apo(a) sequences revealed that apo(a) may contain at least two kringle 4-like domains. A sequence obtained from another tryptic fragment also shows homology to the end of kringle 4 and the beginning of kringle 5. Sequence data obtained from two tryptic fragments show homology with the protease domain of plasminogen. One of these sequences is homologous to the sequences surrounding the activation site of plasminogen. Plasminogen is activated by the cleavage of a specific arginine residue by urokinase and tissue plasminogen activator; however, the corresponding site in apo(a) is a serine that would not be cleaved by tissue plasminogen activator or urokinase. Using a plasmin-specific assay, no proteolytic activity could be demonstrated for lipoprotein(a) particles. These results suggest that apo(a) contains kringle-like domains and an inactive protease domain.

Amino Acid Sequence↗
PubMed1987

Human apolipoprotein D gene: gene sequence, chromosome localization, and homology to the alpha 2u-globulin superfamily.

The exons and bordering intron nucleotides of the human apolipoprotein D (apo D) gene have been sequenced. The protein-coding portion of the gene is divided into five exons which span approximately 12,000 bp. At least one intron interrupts the 5' untranslated region. The gene has been localized to the p14.2----qter region of human chromosome 3. Apo D shares homology with the alpha 2u-globulin superfamily of genes, including approximately 25% amino acid homology with human retinol-binding protein (RBP). Similarity of intron locations in both apo D and RBP suggests that these two genes derived from a common ancestor.

Alpha-Globulins↗
PubMed1987

Detection of hemophilia A carriers using intragenic factor VIII:C DNA polymorphisms.

A DNA polymorphism for an Xbal site in intron 22 of the human factor VIII:C gene extends the utility of DNA methods for carrier detection in families segregating for hemophilia A. While the DNA polymorphism detected by a BclI site in intron 18 of the factor VIII:C gene was informative for 41% of females studied, the BglI/intron 25 polymorphism provided no additional information because of apparent linkage disequilibrium. In contrast, the Xbal intron 22 polymorphism was useful in 53% of women who were uninformative (homozygous) for either the BclI or BglI polymorphisms. Using the BclI/intron 18 and Xbal/intron 22 intragenic polymorphisms, we could provide highly accurate information for 68% of women we studied who were at risk for carriership. The carrier status of the remaining 32% could be determined utilizing the closely linked Taql/St14 DNA polymorphism.

DNA↗
PubMed1986

Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule.

The primary structure of factor VIII consists of 2332 amino acids that exhibit 3 distinct structural domains, including a triplicated region (A domains), a unique region of 909 amino acids (B domain), and a carboxy-terminal duplicated region (C domains), that are arranged in the order A1-A2-B-A3-C1-C2. The B domain (residues 741-1648) of factor VIII is lost when factor VIII is activated by thrombin, which proteolytically processes factor VIII to active subunits of Mr 50,000 (domain A1), 43,000 (domain A2), and 73,000 (domains A3-C1-C2). To determine if the B domain is required for factor VIII coagulant activity, a variant was constructed by using recombinant DNA techniques in which residues 797-1562 were eliminated. This shortened the B domain from 909 to 142 amino acids. This variant factor VIIIdes-797-1652 was expressed in mammalian cells and was found to be functional. The factor VIIIdes-797-1562 protein was purified and shown to be processed by thrombin in the same manner as full-length factor VIII. The factor VIIIdes-797-1562 variant also bound to von Willebrand factor (vWF) immobilized on Sepharose. These results indicate that most of the highly glycosylated B domain of factor VIII is not required for the expression of factor VIII coagulant activity and its interaction with vWF.

Amino Acid Sequence↗
PubMed1986

Identification of a missense mutation in the factor VIII gene of a mild hemophiliac.

DNA probes derived from the cloned factor VIII gene can be used to detect mutations in the factor VIII gene of hemophiliacs. DNA hybridization analysis led to the identification of two contrasting point mutations in the same codon. In a severe hemophiliac with no detectable factor VIII activity, the normal arginine codon (number 2307) is converted to a stop codon, while in a mild hemophiliac with 10 percent of normal activity, this same codon is converted to glutamine.

Amino Acid Sequence↗
PubMed1986

A new polymorphism in the factor VIII gene for prenatal diagnosis of hemophilia A.

A restriction fragment length polymorphism (RFLP) has been found in the gene for clotting factor VIII. Defects in this gene are the cause of hemophilia A. The DNA polymorphism affects an XbaI site in intron 22 of the gene. Two alleles occur in a frequency of 59 and 41 percent of the X chromosomes tested. Furthermore, about 25 percent of females who are homozygous for the previously reported BclI RFLP in the factor VIII gene are heterozygous for the XbaI polymorphism. This new RFLP thus represents a significant addition to available probes for the DNA-based prenatal diagnosis and carrier detection of this disease.

Chromosome Mapping↗