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

Abstract

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.

Determinants of binding of oxidized phospholipids on apolipoprotein (a) and lipoprotein (a)

“…The evolution of Lp(a) in apes and monkeys from PLG has occurred concomitantly with losing the lysine binding properties of the KIV 10 LBS. This has occurred through a variety of mechanisms, including loss of KV and/or modifi cations of KIV 10 LBS ( 49,55,56 ). Along with loss of lysine binding, these species also do not have E06 immunoreactivity on Lp(a).…”

“…To generate 8K-IV apo(a)/Lp(a) mice with defective KIV 10 LBS [8K-IV LBS Ϫ apo(a)/Lp(a) mice], the Asp 55 and Asp 57 residues in the KIV 10 LBS apo(a) cDNA construct were replaced by Ala 55 and Ala 57 residues, as previously described ( 35 ). The vector, pRK5 ha8Lysmuta, was digested with Bgl II, polished with Pfu DNA polymerase (Stratagene, La Jolla, CA), and cut with Eco RI.…”

Functional Analysis of the Chimpanzee and Humanapo(a) Promoter Sequences

“…Consequently, it can be considered as a rather low degree of variation, because non-coding regions are usually more subject to variations than coding regions. By comparison, we have reported 1.4% of variation between the coding sequences of the human and chimpanzee apo(a) genes (33).…”

Significant differentiation in the apolipoprotein(a)/lipoprotein(a) trait between chimpanzees from Western and Central Africa

“…Genomic sequences were compared between (sub‐)species using the LPA references sequence for humans (HSA; ENSG00000198670, GRCh38.p3) and PTV (ENSPTRG00000018770; CHIMP2.1.4. ), the corresponding scaffold sequence from PPA (NCBI Reference Sequence: NW_014013999.1), and the published partial sequence for PTT from cloned samples from Gabon (Chenivesse et al, ; Huby et al, ). Positioning of bases and amino acids follows the human sequence with six KIV‐2 copies.…”

“…The KV and the protease domain of PLG are maintained, but the latter seems to have lost plasmin activity (McLean et al, ). The basic domain structure of LPA is shared between humans and chimpanzees (Chenivesse, Huby, Wickins, Chapman, & Thillet, ; Leibundgut et al, ).…”