Monkey Apo Research Articles

Elements in the C terminus of apolipoprotein [a] responsible for the binding to the tenth type III module of human fibronectin

In previous studies, we showed that the C-terminal domain, F2, but not the N-terminal domain, F1, is responsible for the binding of apolipoprotein [a] (apo[a]) to human fibronectin (Fn). To pursue those observations, we prepared, by both elastase digestion and recombinant technology, subsets of F2 of a different length containing either kringle (K) V or the protease domain (PD). We also studied rhesus monkey apo[a], which is known to contain PD but not KV. In the case of Fn, we used both an intact product and its tenth type III module (10FN-III) expressed in Escherichia coli. The binding studies carried out on microtiter plates showed that the affinity of F2 for immobilized 10FN-III was approximately 6-fold higher than that for Fn (dissociation constants = 1.75 +/- 0.31 nM and 10.25 +/- 1.62 nM, respectively). The binding was also exhibited by rhesus apo[a] and by an F2 subset containing the PD linked to an upstream microdomain comprising KIV-8 to KIV-10 and KV, inactive by itself. Competition experiments on microtiter plates showed that both Fn and 10FN-III, when in solution, are incompetent to bind F2. Together, our results indicate that F2 binds to immobilized 10FN-III more efficiently than whole Fn and that the binding can be sustained by truncated forms of F2 that contain the catalytically inactive PD linked to an upstream four K microdomain.

Baboon Lipoprotein(a) Binds Very Weakly to Lysine−Agarose and Fibrin Despite the Presence of a Strong Lysine-Binding Site in Apolipoprotein(a) Kringle IV Type 10

Human apolipoprotein(a) kringle IV type 10 [apo(a) KIV(10)] contains a strong lysine-binding site (LBS) that mediates the interaction of Lp(a) with biological substrates such as fibrin. Mutations in the KIV(10) LBS have been reported in both the rhesus monkey and chimpanzee, and have been proposed to explain the lack of ability of the corresponding Lp(a) species to bind to lysine and fibrin. To further the comparative analyses with other primate species, we sequenced a segment of baboon liver apo(a) cDNA spanning KIV(9) through the protease domain. Like rhesus monkey apo(a), baboon apo(a) lacks a kringle V (KV)-like domain. Interestingly, we found that the baboon apo(a) KIV(10) sequence contains all of the canonical LBS residues. We sequenced the apo(a) KIV(10) sequence from an additional 10 unrelated baboons; 17 of 20 alleles encoded Trp at position 70, whereas only two alleles encoded Arg at this position and thus a defective LBS. Despite the apparent presence of a functional KIV(10) LBS in most of the baboons, none of the Lp(a) in the plasma of the corresponding baboons bound specifically to lysine-Sepharose (agarose) even upon partial purification. Moreover, baboon Lp(a) bound very poorly to plasmin-modified fibrinogen. Expression of baboon and human KIV(10) in bacteria allowed us to verify that these domains bind comparably to lysine and lysine analogues. We conclude that presentation of KIV(10) in the context of apo(a) lacking KV may interfere with the ability of KIV(10) to bind to substrates such as fibrin; this paradigm may also be present in other non-human primates.

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.

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.

Species-specific polymorphism in the promoter of the apolipoprotein A-I gene: restoration of human transcriptional efficiency by substitution at positions −189, −144 and −48 bp

Previous studies indicate that species-specific differences in apolipoprotein A-I (apo A-I) expression could be largely explained by cis-acting factors located within or near the 5′ flanking region ( −231 to +223 bp, where +1 is the start site of transcription). In the present studies, we have localized 7 sites within the (−231 to −15 bp) region of the African green monkey apo A-I gene that differ from the human apo A-I gene 5′ flanking region. To identify which of the 7 polymorphic sites were essential for the species-specific differences in apo A-I gene expression, mutated promoter constructs were transfected into HepG2 cells and reporter gene expression was measured. Each of the 7 sites within a defined 5′ flanking region of the human gene was individually mutated to the African green nucleotide sequence found at that position. Three of the sites (−189, −144 and −48) were found to raise the human apo A-I promoter activity to approx. 60–65% of the African green promoter. While double mutations (−144/ −48 by and −89/ −144 bp), restored the human apo A-I promoter activity to 100% of that found with the African green monkey promoter. Additional studies revealed similar DNA: protein interactions with DNA probes from either human or African green monkey and HepG2 cell nuclear extract. In conclusion, these studies demonstrate that double and triple nucleotide substitutions within the human apo A-I promoter are sufficient to restore gene expression in HepG2 cells to levels seen with the African green monkey promoter. These data suggest that sites −189, −144 and −48 bp are involved in significantly altering the binding affinity of a nuclear factor determining the species-specific level of apo A-I gene transcription.

Nucleotide sequence of the cynomolgus monkey apolipoprotein A-I gene and corresponding flanking regions

The cynomolgus monkey ( Macaca fascicularis) apolipoprotein A–I (apo A–I) gene and corresponding flanking regions have been isolated from a cynomolgus monkey genomic library and completely sequenced. Comparison with the human sequence shows a > 90% homology with the monkey sequence, overall. The monkey apo A–I structural gene (1856 bp) is representative of the general apolipoprotein gene structure and consists of four exons and three introns. Minor differences exist between the monkey and human structural gene sequence but do not result in significant changes. Sequences preceding the putative monkey apo A–I promoter are virtually identical to the human apo A–I enhancer region with an exact match to the three enhancer DNA binding sites.

Plasma lipoprotein(a) concentration is controlled by apolipoprotein(a) (apo(a)) protein size and the abundance of hepatic apo(a) mRNA in a cynomolgus monkey model

The cynomolgus macaque was used as a model to study lipoprotein(a) (Lp(a)). Antibodies to Lp(a) were used in Ouchterlony and Western blot analysis to show that cynomolgus monkey and human Lp(a) were similar immunochemically. Monkey Lp(a) levels were measured by a quantitative sandwich enzyme-linked immunosorbent assay in 117 animals, and Lp(a) varied in concentration from 1 to 64 mg/dl. Individual monkeys had apo(a) glycoprotein sizes as either single- or double-band phenotypes that ranged from 400 to 750 kDa. Monkey apo(a) transcript lengths varied from 8.5 to 13.6 kilobases. The Lp(a) concentration, apo(a) glycoprotein size, and apo(a) transcript length distributions were similar to those in humans. In the monkeys, there was a very high correlation between apo(a) transcript size and apo(a) protein size (R = 0.93, p = 0.0001). This variation in apo(a) transcript and protein size was shown to be due to the number of kringle IV repeats in apo(a) mRNA and DNA. Monkey plasma Lp(a) concentrations correlated inversely with apo(a) glycoprotein size (R = 0.43, p = 0.0016) and directly with hepatic apo(a) mRNA abundance (R = 0.54, p = 0.004). Apo(a) transcript lengths did not correlate with hepatic apo(a) mRNA levels. This suggests that apo(a) size and mRNA levels have major independent effects on plasma Lp(a) concentration. In multivariate analysis, they account for up to 58% of the variability in Lp(a) concentration. In summary, these data provide insight into the regulation of Lp(a) levels and suggest that the cynomolgus monkey is a suitable model in which to study the role of Lp(a) in the pathogenesis of atherosclerosis.

Purification, cloning and nucleotide sequence determination of cynomolgus monkey apolipoprotein C-11: comparison to the human sequence

We have purified apolipoprotein C-II (apo C-II) from cynomolgus monkey plasma, prepared antibody against it and used the antibody to isolate a cDNA containing the complete coding sequence for cynomolgus monkey apo C-II. Sequence analysis indicated that the monkey apo C-II cDNA was 200 bp longer than the human and the difference in size was all in the 5 degrees untranslated region of mRNA. This was confirmed by Northern analysis of human and monkey RNA. There was an open reading frame in the monkey apo C-II cDNA sequence encoding a preprotein of 101 amino acids - identical in size to the human protein. The carboxyl terminal 44 amino acids of the protein were 100% homologous to the human apo C-II amino acid sequence indicating evolutionary conservation of both structure and function. However, the amino terminal 35 amino acids of the protein were only 75% homologous and the amino terminal 19 amino acids were only 58% homologous to the human sequence. The amino acid sequence derived from the nucleotide sequence predicts a more basic protein than the human apo C-II and this is confirmed by isoelectric focusing and immunoblotting.

Apolipoprotein A-I metabolism in cynomolgus monkey. Identification and characterization of beta-migrating pools.

Fresh plasma from control (C) and hypercholesterolemic (HC) cynomolgus monkeys was analyzed by agarose electrophoresis-immunoblotting with antibody to cynomolgus monkey apolipoprotein (apo) A-I. Two bands were evident on the autoradiogram: an alpha-migrating band (high density lipoprotein) and a beta-migrating band that comigrated exactly with cynomolgus monkey low density lipoprotein (LDL). The presence of beta-migrating apo A-I in the plasma of these monkeys was confirmed by Geon-Pevikon preparative electrophoresis, crossed immunoelectrophoresis, and isotope dilution studies in which radiolabeled apo A-I was found to equilibrate also with alpha- and beta-migrating pools of apo A-I in the plasma. Subfractionation of C and HC plasma by agarose column chromatography (Bio-Gel A-0.5M and A-15M) followed by agarose electrophoresis-immunoblotting indicated that the beta-migrating apo A-I in C was relatively homogeneous and eluted with proteins of Mr approximately 50 kD [apo A-I(50 kD)], whereas two beta-migrating fractions were identified in HC, one that eluted with the 50-kD proteins, and the other that eluted in the LDL Mr range [apo A-I(LDL)]. The apo A-I(LDL) was precipitated by antibody to cynomolgus monkey apo B. The apo A-I(50 kD) accounted for 5 +/- 1% (mean +/- SD) of the plasma apo A-I in C plasma, and 15 +/- 7% in HC plasma. No apo A-I(LDL) was detected in C plasma, but that fraction accounted for 9 +/- 7% of the apo A-I in HC plasma. These data establish the presence of multiple pools of apo A-I in the cynomolgus monkey, which must be taken into consideration in any comprehensive model of apo A-I metabolism in this species.

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.

The baboon apolipoprotein E gene: Structure, expression, and linkage with the gene for apolipoprotein C-I

To develop the baboon model for molecular genetic studies of atherosclerosis, we have cloned and sequenced the baboon apolipoprotein E (apo E) gene. The baboon apo E gene encodes the E4 isoform with respect to specific amino acid positions, suggesting that the common ϵ3 allele is not the primal human allele. Rather than accumulating predominantly synonymous nucleotide changes, 50% of substitutions in human and baboon apo E gene coding regions cause amino acid substitutions. However, comparisons of these apo E proteins show conservation of amphipathic helices required for apo E-lipid interactions. The human and baboon apo E genes have diverged less extensively than those from rat and mouse, providing further evidence for a slowing of molecular evolution in primate species. The baboon and rhesus monkey apo E genes (intron 2) contain two Alu repeats that are absent in the human gene, indicating insertion after the divergence of human and cercopithecine lineages, but before the baboon/rhesus divergence. S1 nuclease studies show that transcription of the baboon apo E gene starts at two different positions, one of which corresponds to the human gene start site. To examine linkage of apolipoprotein genes in the baboon genome, we have used a human cDNA probe to detect apo C-I gene sequences approximately 4 kb from the 3′ end of the baboon apo E gene.

Asymmetry of apolipoprotein A-I in solution as assessed from ultracentrifugal, viscometric, and fluorescence polarization studies

The solution properties of apoliporprotein A-I (apo A-I) isolated from human and rhesus monkey serum highdensity lipoproteins were examined by the techniques of fluorescence polarization, sedimentaion velocity, sedimentation equilibrium, and viscometry as a function of protein concentration. The ultracentrifugal studies confirmed previous reports that human and rhesus monkey apo A-I undergo a concentration-dependent, reversible self-association in aqueous solutions. The sedimentaion velocity data, extrapolated to infinite dilution, indicated that the apo A-I monomer is asymmetric, with an axial ratio of about 6. The reduced viscosity of apo A-I solutions was also markedly affected by protein concentration; within the range of concentrations studied, the values for human apo A-I ranged from 11.19 to 21.24 mL/g and for rhesus apo A-I, from 8.26 to 16.83 mL/g. The intrinsic viscosity, 6.58 mL/g, of the apo A-I monomer was significantly higher than that of a typical globular protein (3-4 mL/g); the axial ratio, 5.5, was in agreement with that obtained from the ultracentrifugation analyses. In the fluorescence polarization studies, human and rhesus apo A-I were dansylated to the extent of 1.0 to 2.5 mol of dansyl group/mol of apo A-I. This chemical modification was attended by no appreciable change in the secondary structure of apomore » A-I, as assessed by circular dichroism spectra. The apparent sedimentation coefficient, molecular weight, and rotational relaxation time of each of the dansylated proteins indicated that both human and rhesus monkey apo A-I self-associated to a lesser extent than the unmodified apo A-I. At protein concentrations ranging from 0.002 to 3.0 g/L, the fluorescence polarization values of dansylated apo A-I remained essentially constant (0.185 to 0.192), although rotational relaxation times indicated self-association at the higher concentrations.« less