(Received for publication, July 20, 1995)
From the
In the formation of the lipoprotein(a) (Lp(a)) particle, apolipoprotein(a) (apo(a)) and apolipoprotein B (apoB) are covalently linked via a disulfide bond in both humans and human-apo(a)/apoB transgenic mice. Studies based upon fluorescent labeling of free cysteine residues have suggested that cysteine 3734 of the 4 carboxyl-terminal cysteines of apoB (Cys-3734, Cys-3890, Cys-4190, and Cys-4326) is the most likely candidate to form a disulfide bond with apo(a). However, other recent studies using truncated apoB molecules suggest that Cys-4326, the terminal cysteine of apoB, may be implicated in the binding to apo(a). In order to definitively show which of apoB's carboxyl-terminal cysteines is essential in interacting with apo(a) we have used RecA-assisted restriction enzyme digestion coupled with site-specific mutagenesis to convert Cys-3734 and Cys-4326 to serine within separate 90-kilobase pair apoB P1 phagemid clones. Transgenic mice containing the normal or mutated apoB transgenes were created, and the covalent association of mutated apoB with apo(a) was assessed in mice transgenic for both apoB and apo(a). Analysis by ultracentrifugation and immunoblotting revealed that Cys-4326, but not Cys-3734, was essential in the formation of the covalent bond between apo(a) and apoB in vivo.
Lipoprotein(a) (Lp(a)) ()is formed by the association
of apolipoprotein(a) (apo(a)) with apolipoprotein B (apoB) of low
density lipoprotein (LDL). Although weaker non-covalent interactions
may occur between apoB and apo(a), it is the presence of a disulfide
bond that binds them
covalently(1, 2, 3, 4) . A single
cysteine of apo(a) (Cys-4057) has been identified in forming the in
vitro covalent association of apo(a) with human LDL (5, 6) . The introduction of a point mutation into the
apo(a) molecule to remove Cys-4057 and replace it with either a serine
or glycine was sufficient to prevent the formation of a tight
association between LDL and apo(a). However, the cysteine of apoB
involved in this association has not been unambiguously identified.
Several studies (7, 8, 9) have suggested that Cys-3734 or possibly Cys-4190 of apoB could potentially form a disulfide bridge with apo(a). Many of these studies were based upon the use of fluorescent probes that bind to free cysteine residues of proteins. Fluorescently labeled tryptic peptides of apoB that contain Cys-3734 were absent in the apoB of Lp(a), suggesting that this cysteine was unavailable for binding to the fluorescent probe because of an association with apo(a)(8) . Computer modeling also demonstrated that the peptide region around Cys-3734 was the most energetically favorable to associate with the amino acids surrounding Cys-4057 of apo(a)(8) . Both of these approaches, however, lacked direct experimental evidence for the involvement of Cys-3734 in the binding of apoB to apo(a).
Studies utilizing a truncated form of apoB (apoB-90) expressed in transgenic mice suggested that one of the two cysteines in the deleted terminal 10% of the molecule, Cys-4190 or -4326, could be involved in binding to apo(a)(10) . Another study, using a truncated apoB-94 peptide produced in cell culture, presented evidence not indicated by the fluorescent labeling studies and suggested that Cys-4326, the terminal cysteine of apoB, is involved in disulfide linkage with apo(a) (11) . In both of these studies, deletion of the terminal 6 or 10% of the apoB peptide could not be excluded from inhibiting an association with an alternative cysteine such as Cys-3734. This is important because it has been demonstrated that the conformation of apoB may influence the binding characteristics of apo(a) to apoB on LDL. Individuals with lecithin:cholesterol acyltransferase deficiency have been demonstrated to lack plasma Lp(a)(12) . The LDL of these individuals was found to be deficient in cholesterol, which suggested that interactions between apo(a) and apoB may be impacted upon by the composition and size of LDL mediating conformational changes in apoB. The low binding characteristics of very low density lipoprotein with apo(a) (13) may also be due to conformational changes. Truncated forms of apoB could adopt conformations inhibiting an association with apo(a) in a similar manner.
Apo(a) is not normally expressed in mice, and in human-apo(a) transgenic mice, apo(a) is not covalently linked to murine apoB(14, 15) . However, in mice that contain both transgenes for human apo(a) and apoB, Lp(a) particles, similar to that observed in humans, are present in the plasma of the double transgenic animals(16, 17) . In this study we have used transgenic mice to study the in vivo interactions of apo(a) and apoB. To determine which free cysteine of human apoB is involved in disulfide linkage to apo(a), we have employed site-directed mutagenesis to convert Cys-3734 and Cys-4326 to serines within separate apoB P1-phagemid genomic clones. The P1 clone of the human apoB gene was then used to prepare transgenic mice expressing the mutated protein. In combination with an apo(a)-expressing yeast artificial chromosome transgene(18) , the formation of Lp(a) by the mutant apoB molecules in an in vivo setting was examined.
Mutagenesis of the 1.55-kbp EcoRI fragment containing codon 3734 was carried out in M13, whereas PCR mutagenesis was used for alteration of codon 4326 followed by subcloning of the product to a plasmid for sequencing. The DNA sequences surrounding the mutated sites were determined and compared with that of the published sequence. For both codon 3734 and codon 4326, a single base change was introduced into the apoB sequence converting a thymidine to an adenosine and thus effectively converting a cysteine to a serine in the translated product. No other changes to the apoB gene sequence were detected.
Four Ser-3734 apoB and five Ser-4326 apoB founder mice expressing human apoB as determined by immuno-dot blot assay were created, but only two founders of each mutation type were bred with apo(a) transgenic mice to generate transgenic lines. PCR was used to confirm the presence of the mutated DNA sequences in the genomes of the transgenic mice (Fig. 1). Introduction of the point mutations and conversion of thymidine to adenosine within the apoB DNA sequence introduces TaqI and DdeI restriction enzyme sites for codon 3734 and codon 4326, respectively. No product was obtained from non-transgenic mouse genomic DNA in each case, demonstrating that both primer sets were specific for human DNA. Genomic DNA from apoB transgenic mice gave a 203-bp PCR product from the codon 3734 region and a 331-bp product from the codon 4326 region. However, the product from the Ser-3734 apoB genomic DNA was able to produce the TaqI digestion pattern confirming the presence of the mutated apoB in these mice. Similarly, the PCR product for the codon 4326 region was digested with DdeI in Ser-4326 apoB mice.
Figure 1: PCR amplification of transgenic DNA. Genomic DNA and P1 DNA were amplified by PCR using Taq DNA polymerase. Primers F and G were used to amplify a 203-bp region containing codon 3734. Mutation of codon 3734 introduces a TaqI restriction site. Primers D and E were used to amplify a 331-bp region containing codon 4326. Mutation of this codon introduces a DdeI site. DNA was electrophoresed on 2% Nu-sieve-agarose (FMC) after digesting with TaqI or DdeI. Lane 1, markers; lane 2, non-Tg; lane 3, wt B Tg; lane 4, wt B Tg RE-digested; lane 5, mutated P1; lane 6, mutated P1 RE-digested; lane 7, mutated Tg; lane 8, mutated Tg RE-digested.
The size of human apoB found in plasma from the various transgenic strains was compared by immunoblot analysis (Fig. 2). The human protein found in both of the mutated apoB transgenic lines was identical in size to the human apoB from wild-type (wt) apoB transgenic mouse plasma. Both apoB-48 and apoB-100 bands were present demonstrating normal mRNA editing and synthesis of the peptide.
Figure 2: Human apoB immunoblot of the lipoprotein fraction from transgenic mice. The d < 1.21 g/ml lipoprotein fraction of plasma from mice was collected, electrophoresed, and immunoblotted under reducing conditions as described under ``Experimental Procedures.'' The equivalent of 1 µl of lipoprotein fraction was loaded into each lane, except for wt B in which case 2 µl were loaded.
Figure 3: Human apo(a) immunoblot of plasma fractions from transgenic mice. The d < 1.21 g/ml, lipoprotein-containing (L) or d > 1.21 g/ml, lipoprotein-free (F) fractions of transgenic mouse plasma were collected by ultracentrifugation. The equivalent of 1 µl (for wt B/(a) and Ser-3734 B/(a)) or 2 µl (for (a) and Ser-4326 B/(a)) of plasma fractions was electrophoresed under reducing conditions and immunoblotted using an anti-apo(a) antibody as described under ``Experimental Procedures.''
A significant proportion of apo(a)
was found in the lipoprotein-free fraction of plasma from the wt
apoB/apo(a) mice (Fig. 3). This was a consistent feature of this
line and was attributed to the low concentration of human apoB in these
animals compared with higher expressors. ()To ensure that
adequate levels of apoB were available for maximum binding of apo(a),
the plasma concentration of human apoB was assessed for those mice
presented in Fig. 2and Fig. 3using a human apoB
specific enzyme-linked immunosorbent assay. The wt apoB line contained
14 mg/dl compared with the Ser-3734 apoB and Ser-4326 apoB lines that
contained 59 and 20 mg/dl, respectively. These data demonstrate that
the lack of association between Ser-4326 apoB and apo(a) could not be
attributed to low levels of circulating apoB in these mice.
ApoB contains 25 cysteine residues with at least 16 of these committed to intramolecular disulfide bonds. This leaves 9 potential cysteine residues available for intermolecular interactions(9) . In the present study we chose to replace Cys-3734 and Cys-4326 of human apoB with serine, based upon previous reports suggesting that at least one of these cysteines is a likely candidate for the single disulfide linkage to apo(a). Our results clearly demonstrate that only Cys-4326 is required for the covalent interaction of human apo(a) and human apoB. This is in contrast to a recent study suggesting that 2 molecules of apo(a) are associated per single apoB molecule within the Lp(a) structure(31) . The complete lack of detectable apo(a) within the lipoprotein fraction of the Ser-4326 apoB mice strongly implies that only one molecule of apo(a) is associated covalently with each apoB molecule. Furthermore, the possibility that Cys-3734 is responsible for binding one of the apo(a) molecules also seems unlikely. Non-reduced Lp(a) from Ser-3734 apoB/apo(a) mice demonstrated identical migration compared with Lp(a) from wt apoB/apo(a) mice on SDS-polyacrylamide gels (data not shown), suggesting an equal number of apo(a) molecules bound in each case. However, it is not inconceivable that Lp(a) could require the binding of an initial apo(a) molecule at Cys-4326 to expose alternative binding sites for a second apo(a) molecule.
The large size of the apoB gene has made it difficult to study structural features of this protein. However, with the advent of new technologies such as RARE digestion of DNA, site-specific mutagenesis and manipulation of large genomic clones can be achieved. Here, we have used this approach to demonstrate that Cys-4326 of human apoB, and not Cys-3734, is required for the covalent, disulfide linkage between human apoB and apo(a) in vivo. Furthermore, the transgenic mice developed in these studies may prove useful for the analysis of early events leading to the formation of the disulfide bond. Inhibition of this process could provide new approaches to reducing plasma levels of Lp(a)