PubMed Health⌕ Search

Biomedical subjects

T Huby

Publications and source records attributed to T Huby.

11 recordsLinked to original sources
PubMed2001

Functional analysis of the chimpanzee and human apo(a) promoter sequences: identification of sequence variations responsible for elevated transcriptional activity in chimpanzee.

Lp(a) concentrations vary considerably among individuals and are primarily determined by the apo(a) gene locus. We have previously shown that mean plasma Lp(a) levels in the chimpanzee are significantly higher than those observed in humans (Doucet, C., Huby, T., Chapman, J., and Thillet, J. (1994) J. Lipid Res 35, 263-270). To evaluate the possibility that this difference may result from a high level of expression of chimpanzee apo(a), we cloned and sequenced 1.4 kilobase (kb) of the 5'-flanking region of the gene and compared promoter activity to that of its human counterpart. Sequence analysis revealed 98% homology between chimpanzee and human apo(a) 5' sequences; among the differences observed, two involved polymorphic sites associated with Lp(a) levels in humans. The TTTTA repeat located 1.3 kb 5' of the apo(a) gene, present in a variable number of copies (n = 5-12) in humans, is uniquely present as four copies in the chimpanzee sequence. The second position concerns the +93 C>T polymorphism that creates an additional ATG start codon in the human apo(a) gene, thereby impairing translation efficiency. In chimpanzee, this position did not appear polymorphic, and a base difference at position +94 precluded the presence of an additional ATG. In transient transfection assays, the chimpanzee apo(a) promoter exhibited a 5-fold elevation in transcriptional activity as compared with its human counterpart. This marked difference in activity was maintained with either 1.4 kb of 5' sequence or the minimal promoter region -98 to +141 of the human and chimpanzee apo(a) genes. Using point mutational analyses, nucleotides present at positions -3, -2, and +8 (relative to the start site of transcription) were found to be essential for the high transcription efficiency of the chimpanzee apo(a) promoter. High transcriptional activity of the chimpanzee apo(a) gene may therefore represent a key factor in the elevated plasma Lp(a) levels characteristic of this non-human primate.

Animals↗
PubMed1998

Molecular cloning of the cDNA encoding the carboxy-terminal domain of chimpanzee apolipoprotein(a): an Asp57 --> Asn mutation in kringle IV-10 is associated with poor fibrin binding.

Insight into the structural features of human lipoprotein(a) [Lp(a)] which underlie its functional implication in fibrinolysis may be gained from comparative studies of apo(a). Indeed, cloning of rhesus monkey apo(a) has shown that a Trp72 --> Arg mutation in the lysine-binding site (LBS) of KIV-10 leads to loss of lysine-binding properties of the rhesus Lp(a) particle. Consequently, comparative studies of apo(a) sequences in different Old World monkey species should further our understanding of the molecular role of Lp(a) in the fibrinolytic process. In contrast to other Old World monkeys, including rhesus monkey, cynomolgus, and baboon, the chimpanzee exhibits an elevated level of Lp(a) and a distinct isoform distribution as compared to humans [Doucet et al. J. Lipid Res. (1994) 35, 263-270]. Clearly then, the chimpanzee is an interesting animal model for study of the structure, function, and potential pathophysiological roles of Lp(a). We have cloned and sequenced the region of chimpanzee apo(a) cDNA spanning KIV-3 to the stop codon. The global organization of this region is similar to that of human apo(a) with the presence of KV, which is absent in rhesus monkey apo(a). Nucleotide sequence comparison indicates a variation of 1.4% between chimpanzee and man and 5.1% between chimpanzee and rhesus monkey. The differences concerned single base changes. An Asp57 --> Asn mutation was detected in KIV-10; this residue is critical to the LBS of KIV-10 in human apo(a). To verify that the Asp57 --> Asn substitution was specific to apo(a), we have also cloned the cDNA-encoding plasminogen, which exhibited an Asp at the corresponding position in kringle IV. Using an in vitro binding assay, we have demonstrated that chimpanzee Lp(a) exhibits poor lysine-specific interaction with both intact and plasmin-degraded fibrin as compared to its human counterpart. We propose that the Asn57 substitution in KIV-10 of chimpanzee apo(a) is responsible for this property. Chimpanzee Lp(a) therefore represents an appropriate particle with which to explore the potential effects of Lp(a) on the fibrinolytic system, such as the inhibition of plasminogen activation or inhibition of t-PA activity.

Amino Acid Substitution↗
PubMed1997

Pathophysiological implication of the structural domains of lipoprotein(a).

Numerous epidemiological studies have shown that lipoprotein(a) (Lp(a)) is an independent risk factor for the premature development of cardiovascular disease. In spite of such evidence, the structural and functional features of this atherogenic, cholesterol-rich particle are not clearly understood. We have demonstrated the presence of two distinct structural domains in apolipoprotein(a) (apo(a)), which are linked by a flexible and accessible region located between kringles 4-4 and 4-5. We have isolated the Lp(a) particle following removal of the N-terminal domain by proteolytic cleavage; the residual particle, containing the C-terminal domain (comprising the region from Kr 4-5 to the protease domain), is linked to apo B-100 by disulphide linkage, and is termed 'mini-Lp(a)'. Mini-Lp(a) exhibited the same binding affinity to fibrin as the corresponding Lp(a). This finding indicated that the kringles responsible for fibrin binding are restricted to Kr 4-5 to Kr 4-10, an observation consistent with the failure of the N-terminal domain to bind to fibrin. N-terminal fragments of apo(a) have been detected in the urine of normal subjects, thereby indicating that part of the catabolism of Lp(a), which is largely indeterminate, could occur via the renal route.

Animals↗
PubMed1996

Expression of a recombinant kringle V of human apolipoprotein(a): antibody characterization and species specificity.

Lipoprotein(a) is a macromolecular complex consisting of a low-density lipoprotein-like particle with an additional glycoprotein, apolipoprotein(a) [apo(a)], linked to apolipoprotein B-100 via a disulfide bond. Apo(a) is highly homologous to plasminogen. We have cloned the sequence corresponding to the kringle V domain of apo(a) from human liver cDNA using an experimental approach involving use of the polymerase chain reaction. The protein product of this clone was expressed in the cytoplasmic compartment of Escherichia coli as a MalE fusion protein. Fusion apo(a) Kr V was isolated from cytoplasmic extracts and purified by amylose-agarose affinity chromatography by eluting with 10 mM maltose. The fusion protein was injected into sheep in order to generate a polyclonal anti-apo(a) Kr V antibody. The antibody raised reacted against both reduced Lp(a) and the C-terminal domain of apo(a), corresponding to a sequence extending from Kr 33 to the C-terminal residue, but did not react with the N-terminal domain containing the repeated Kr IV sequences. The presence of the Kr V sequence was detected in every human apo(a) size isoform tested but only in apo(a) from human and chimpanzee among a panel of apo(a) proteins derived from different animal species.

ATP-Binding Cassette Transporters↗
PubMed1995

Characterization of the N-terminal and C-terminal domains of human apolipoprotein(a): relevance to fibrin binding.

The structural domains of human apolipoprotein(a) (apo(a)) in the lipoprotein(a) (Lp(a)) particle have been recently investigated by limited proteolysis [Huby, T., Doucet, C., Dieplinger, H., Chapman, J., & Thillet, J. (1994) Biochemistry 33, 3335-3341]. We have shown that apo(a) can be cleaved into two structural domains: one was of constant size (170 kDa) and corresponded to the C-terminal (Cter) domain of apo(a). This domain was linked by a disulfide bond to apo B100. By contrast, the N-terminal (Nter) domain, whose size varied according to the digested apo(a) isoform, was not linked to apo B100. We now describe the purification of these apo(a) domains and their interaction with fibrin surfaces in an in vitro binding assay. The Nter domain of apo(a) was purified as a soluble protein in a two-step procedure which involved sequential use of a heparin-Sepharose column and a lysine-Sepharose column. The Cter domain of apo(a), which remained in disulfide linkage with apo B100 of Lp(a), was isolated as a lipoprotein particle by a combination of chromatographic steps on heparin-Sepharose and Q-Sepharose columns. This particle, termed "mini-Lp(a)", appeared homogeneous in nondenaturing polyacrylamide gels and exhibited a particle size (285 A) which was intermediate between that of Lp(a) (300 A) and LDL (265 A). The cleavage site between the respective apo(a) domains was determined by N-terminal sequencing of the purified Cter domain. Such cleavage occurred between residues 3532 and 3533, which are located in the interkringle region between apo(a) kringles 4(4) and 4(5).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Non-enzymatic glycation of lipoprotein(a) in vitro and in vivo.

Nonenzymatic glycation of lipoprotein may contribute to the premature atherogenesis of patients with diabetes mellitus by diverting lipoprotein catabolism from non-atherogenic to atherogenic pathways. It has been demonstrated that the proportion of apolipoprotein B-100 (apo B-100) in glycated form is significantly higher in diabetic patients than in non-diabetic controls, and equally that plasma lipoprotein(a) Lp(a) levels may be increased in diabetic patients. Consequently, we have evaluated the glycation of Lp(a) in vitro and in vivo, by use of a combination of m-aminophenylboronate affinity chromatography and enzyme-linked immunosorbent assay (ELISA) for apo B-100 and Lp(a). In vitro studies were performed on normolipodemic plasma samples containing elevated concentrations of Lp(a). These studies establish that Lp(a) can be glycated in vitro in the presence of high concentrations of glucose, although to a lesser extent than low density lipoprotein (LDL) (15.8% +/- 4.4% and 30.2% +/- 5.4% (P = 0.0001) after a 48 h incubation at 37 degrees C, respectively). We have also shown that apo B-100 was more glycated than apo(a) in the Lp(a) particle. In vivo studies have shown that the percentage of glycated Lp(a) in diabetic patients was significantly higher than in the control population (2.8% +/- 1.07% versus 2.0% +/- 0.43%, P = 0.017). The level of glycated Lp(a) is also positively correlated with that of HbA1c in diabetic patients (r=0.6, P=0.002). Since our results show that Lp(a) is less susceptible to glycation than LDL, we speculate that glycation of LDL may be more relevant to the cardiovascular risk associated with this particle than with Lp(a).

Apolipoprotein B-100↗
PubMed1994

Structural domains of apolipoprotein(a) and its interaction with apolipoprotein B-100 in the lipoprotein(a) particle.

The structural domains of human apolipoprotein(a) [apo(a)] and its interaction with apolipoprotein B-100 (apo B-100) in the lipoprotein(a) [Lp(a)] particle were investigated by limited proteolysis with thermolysin and cathepsin D. We characterized the proteolytic products by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis, followed by immunoblotting using different antibodies. For apo B-100 in Lp(a), the digestion patterns were found to be identical to those previously described [Chen et al. (1989) J. Biol. Chem. 264, 14369-14375; Chen et al. (1991) J. Biol. Chem. 266, 12581-12587] for apo B-100 in LDL. Thus, we compared the digestion patterns of apo B-100 in Lp(a) resolved under reducing and nonreducing migrating conditions. Using an antibody specific for a synthetic peptide of apo B-100 (residues 4004-4021), we confirmed that apo B-100 was linked to apo(a) by its C-terminal end. Various Lp(a)s isolated from several donors, and containing different isoforms, were used to study the structural domains of apo(a). Using the same procedure as for apo B-100, several common features were found for the different isoforms. (1) Apo(a) can be cleaved into two structural domains: one was of constant size (170 kDa) and was linked to apo B-100. Using an antibody specifically directed against kringle V, we demonstrated that this fragment corresponded to the C-terminal part of apo(a). (2) The other domain, whose size varied according to the digested apo(a) isoform, was not linked to apo B-100.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein B-100↗
PubMed1994

Association of elevated lipoprotein(a) levels and coronary heart disease in NIDDM patients. Relationship with apolipoprotein(a) phenotypes.

Non-insulin-dependent diabetes mellitus (NIDDM) is a strong and independent risk factor for coronary heart disease. We assessed the potential relationship between plasma Lp(a) levels, apo(a) phenotypes and coronary heart disease in a population of NIDDM patients. Seventy-one patients with coronary heart disease, who previously have had transmural myocardial infarction, or significant stenosis on coronary angiography, or positive myocardial thallium scintigraphy, or in combination, were compared with 67 patients without coronary heart disease, who tested negatively upon either coronary angiography, myocardial thallium scintigraphy or a maximal exercise test. The prevalence of plasma Lp(a) levels elevated above the threshold for increased cardiovascular risk (> 0.30 g/l) was significantly higher (p = 0.005) in patients with coronary heart disease (33.8%) compared to the control group (13.4%). The relative risk (odds ratio) of coronary heart disease among patients with high Lp(a) concentrations was 3.1 (95% confidence interval, 1.31-7.34; p = 0.01). The overall frequency distribution of apo(a) phenotypes differed significantly between the two groups (p = 0.043). However, the frequency of apo(a) isoforms of low apparent molecular mass (< or = 700 kDa) was of borderline significance (p = 0.067) between patients with or without coronary heart disease (29.6% and 16.4%, respectively). In this Caucasian population of NIDDM patients, elevated Lp(a) levels were associated with coronary heart disease, an association which was partially accounted for by the higher frequency of apo(a) isoforms of small size. In multivariate analyses, elevated levels of Lp(a) were independently associated with coronary heart disease (odds ratio 3.48, p = 0.0233).

Apolipoproteins A↗
PubMed1994

Lipoprotein (a): implication in atherothrombosis.

Substantial experimental evidence now implicates lipoprotein (a) as an independent risk factor for premature cardiovascular disease. Both plasma Lp(a) levels and apo(a) phenotype are strong predictors of risk for ischaemic heart disease. The accumulation of apo(a) in vascular wall tissue and in atherosclerotic plaques and the potential inhibition of fibrinolysis by Lp(a) underlie the enhanced risk of premature cardiovascular disease associated with this cholesterol-rich particle. Recent studies of the capacity of purified Lp(a) isoforms to inhibit fibrinolysis in an in vitro system have revealed that small isoforms of Lp(a) (< or = 500 kDa) are efficient inhibitors of plasminogen activation and bind with high affinity to fibrin. Conversely, large isoforms exert little or no inhibitory effect in this system (> 500 kDa). These data suggest that the potential, high affinity interaction of Lp(a) particles containing small isoforms with fibrin introduces a new, third dimension to the atherothrombotic risk associated with these cholesterol-rich particles.

Apolipoproteins A↗
PubMed1994

Lipoprotein[a] in the chimpanezee: relationship of apo[a] phenotype to elevated plasma Lp[a] levels.

Several studies have documented the presence of Lp[a] in nonhuman primates. However, data are lacking in great apes such as the chimpanzee. We have studied the quantitative distribution of Lp[a], as well as the frequency of apo[a] phenotypes, in a population of chimpanzees living in Gabon. Monoclonal antibody 14A12, directed against human apo[a], failed to recognize chimpanzee Lp[a]. Therefore, Lp[a] was assayed using an ELISA involving two polyclonal antibodies, an anti-human apo[a] and an anti-human apoB-100. Under these conditions, Lp[a] was detected in each of 28 animals. The plasma level of Lp[a] was found to be highly skewed toward elevated values: the mean Lp[a] level was 0.61 mg/ml (SD 0.45) as compared to 0.18 mg/ml (SD 0.16) in a normal Caucasian population (P < 0.0001). Phenotypes for apo[a] were identified by SDS-agarose-gel electrophoresis, followed by immunoblotting and detection by chemiluminescence. Seventeen different isoforms (ranging from 440 to 920 kDa) were found among all the animals as compared to 19 (540 to 960 kDa) in a human population of equivalent number. However, the distribution of apo[a] phenotypes was distinct between these populations. Thus isoforms of low molecular mass occurred with greater frequency in chimpanzee as compared to humans. In both populations, a strong inverse correlation between Lp[a] levels and apo[a] isoform sizes was found in chimpanzees (r = -0.48; P < 0.01) and in man (r = -0.68; P < 0.0002).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗