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J. Biol. Chem., Vol. 279, Issue 4, 2679-2688, January 23, 2004

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Quantitative Evaluation of the Contribution of Weak Lysine-binding Sites Present within Apolipoprotein(a) Kringle IV Types 6–8 to Lipoprotein(a) Assembly^*

Lev Becker, P. Michael Cook, Theodore G. Wright, and Marlys L. Koschinsky

From the Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada

Received for publication, August 25, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
During lipoprotein(a) (Lp(a)) assembly, non-covalent interactions between apolipoprotein(a) (apo(a)) and low density lipoprotein precede specific disulfide bond formation. Studies have shown that the non-covalent step involves an interaction between the weak lysine-binding sites (WLBS) present within each of apo(a) kringle IV types 6, 7, and 8 (KIV_6–8), and two lysine residues (Lys⁶⁸⁰ and Lys⁶⁹⁰) within the NH₂ terminus of the apolipoprotein B-100 (apoB) component of low density lipoprotein. In the present study, we introduced single point mutations (E56G) into each of the WLBS present in apo(a) KIV_6–8 and expressed these mutations in the context of a 17-kringle (17K) recombinant apo(a) variant. Single mutations that disrupt the WLBS in KIV₆, KIV₇, and KIV₈, as well as mutants that disrupt the WLBS in both KIV₆ and KIV₇, or both KIV₇ and KIV₈, were assessed for their ability to form non-covalent and covalent Lp(a) complexes. Our results demonstrate that both apo(a) KIV₇ and KIV₈, but not KIV₆, are required for maximally efficient non-covalent and covalent Lp(a) assembly. Single mutations in the WLBS of KIV₇ or KIV₈ resulted in a 3-fold decrease in the affinity of 17K recombinant apo(a) for apoB, and a 20% reduction in the rate of covalent Lp(a) formation. Tandem mutations in the WLBS in both KIV₇ and KIV₈ resulted in a 13-fold reduction in the binding affinity between apo(a) and apoB, and a 75% reduction in the rate of the covalent step of Lp(a) formation. We also showed that KIV₇ and KIV₈ specifically bind with high affinity to apoB-derived peptides containing Lys⁶⁹⁰ or Lys⁶⁸⁰, respectively. Taken together, our data demonstrate that specific interactions between apo(a) KIV₇ and KIV₈ and Lys⁶⁸⁰ and Lys⁶⁹⁰ in apoB mediate a high affinity non-covalent interaction between apo(a) and low density lipoprotein, which dictates the efficiency of covalent Lp(a) formation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Lipoprotein(a) (Lp(a))¹ contains a low density lipoprotein (LDL)-like moiety that is attached to the glycoprotein apolipoprotein(a) (apo(a)) by a single disulfide bond (1, 2). The LDL moiety of Lp(a) is similar to plasma-derived LDL both in lipid composition as well as in the presence of apolipoprotein B-100 (apoB). As such, the apo(a) component likely confers the unique structural and functional properties that have been attributed to Lp(a). Apo(a) contains tandem repeats of a sequence that closely resembles plasminogen kringle IV, followed by sequences that are highly homologous to the kringle V and protease domains of plasminogen (3). Based on amino acid sequence, apo(a) contains 10 distinct subclasses of plasminogen kringle IV-like domains (3); the kringle IV type 2 domain (KIV₂) is present in a variable number of identically repeated copies, which is the molecular basis for the observed isoform size heterogeneity of Lp(a) (4, 5). An unpaired cysteine residue in apo(a) KIV₉ (Cys⁶⁷) is involved in disulfide linkage with apoB to form covalent Lp(a) particles (1, 2).

Numerous epidemiological studies (both case-control and prospective designs) have identified elevated plasma Lp(a) concentrations as a risk factor for the development of atherosclerotic disorders including coronary heart disease (reviewed in Refs. 6 and 7). Conventional therapies such as lifestyle changes and statin therapy, which are highly effective in lowering plasma LDL concentrations, have been relatively unsuccessful in reducing Lp(a) concentrations (7). As such, alternative strategies for lowering plasma Lp(a) levels have been suggested, including inhibition of the assembly of Lp(a) particles (8–10). It is generally accepted that Lp(a) assembly is a two-step process in which initial non-covalent interactions between apo(a) and apoB precede disulfide bond formation (10, 11), which occurs between cysteine residues located within the carboxyl-terminal ends of the respective proteins (1, 2, 12, 13). Although previous data indicate that the efficiency of the covalent step of Lp(a) assembly is dictated by the affinity of the non-covalent interaction between apo(a) and apoB (11, 14), we have recently demonstrated that the conformation of apo(a) is also an important determinant of the rate of covalent Lp(a) particle formation (15). Apo(a) adopts a closed conformation that inhibits covalent Lp(a) assembly, possibly by restricting access to the free sulfhydryl group in apo(a) KIV₉ (15). In this regard, we have previously shown that the addition of the lysine analog -aminocaproic acid (-ACA) elicits a conformational change in apo(a) to a more open structure, which results in a 6-fold enhancement in its ability to form covalent Lp(a) particles.

Sequences have been identified within both the NH₂ and COOH termini of apoB that mediate its non-covalent interaction with apo(a) (9, 16, 17). Previous data from our group underscore a role for sequences within the NH₂-terminal 18% of apoB (apoB-18) in mediating its lysine-dependent, non-covalent association with apo(a) (16). More recently, using a combination of citraconic anhydride modification and site-directed mutagenesis, we have identified two lysine residues (Lys⁶⁸⁰ and Lys⁶⁹⁰) within apoB-18 that are required for non-covalent binding to apo(a) (18); mutation of Lys⁶⁸⁰ was found to completely abolish non-covalent binding to apo(a), whereas mutation of Lys⁶⁹⁰ significantly diminished the affinity of this interaction.

With respect to sequence requirements in apo(a) that mediate its non-covalent interaction with apoB, several groups have shown that the weak lysine-binding sites (WLBS) present within apo(a) KIV_5–8 are likely involved in mediating this interaction (11, 14, 19–21). Studies performed by several investigators have underscored the importance of KIV₆ (10, 14, 20, 21) and possibly KIV₇ (10, 14, 21) in this process. Previous data from our group also suggest that the WLBS in apo(a) KIV₈ plays a major role in non-covalent binding to LDL (14); all studies to date have demonstrated no role for apo(a) KIV₅. In the above studies, domains in apo(a) involved in non-covalent binding to apoB were identified by sequential truncation of full-length apo(a), followed by assessment of binding to immobilized LDL. Using this approach, however, neither the individual contribution of each WLBS to the non-covalent interaction with apoB in the context of full-length apo(a) nor the quantitative relationship between apo(a)/LDL non-covalent interactions and covalent Lp(a) assembly can be assessed. Indeed, based on our recent studies, the truncated derivatives previously used would lack the closed conformation that is adopted by full-length apo(a).²

In the present study, we generated mutations in the WLBS of apo(a) KIV₆, KIV₇, and KIV₈, as well as tandem WLBS mutants in both KIV₆ and KIV₇, or KIV₇ and KIV₈; all of these mutations were made in the context of a 17-kringle-containing apo(a) variant (17K; Ref. 22), which corresponds to a physiologically relevant apo(a) isoform (4, 5). The apo(a) variants were assessed for their ability to associate with LDL in solution and for the efficiency with which they form covalent Lp(a) particles. We further determined whether lysine residues (Lys⁶⁸⁰ and Lys⁶⁹⁰) within apoB-18 are important for binding to the WLBS in apo(a) by measuring solution phase interactions between bacterially expressed apo(a) KIV₇ and KIV_8, and synthetic peptides containing apoB Lys⁶⁸⁰ and Lys⁶⁹⁰. Taken together, our data identify a critical role for the WLBS in both apo(a) KIV₇ and KIV₈ in mediating non-covalent association with the Lys⁶⁹⁰ and Lys⁶⁸⁰ residues in apoB, respectively, and demonstrate that these interactions are required for the covalent step of Lp(a) formation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Construction and Expression of Recombinant Apo(a) Variants in Mammalian Cells—The plasmid encoding the 6K r-apo(a) derivative (pRK5ha6 plasmid) (11) was used as a PCR template in the construction of an expression plasmid corresponding to the 17K_KIV6E56G variant. The mutation was generated by PCR using two sets of primer pairs (1 and 2, and 3 and 4; see Table I). Fragments containing the mutation were then inserted, using a number of steps, into the 17K r-apo(a) expression plasmid pRK5ha17 (22), to give the final expression plasmid designated pRK5ha17_KIV6E56G. The pRK5ha6 plasmid was also used as a PCR template in the construction of an expression plasmid encoding the 17K_KIV7E56G variant. Fragments generated by PCR using primer pair 5 and 6 and pair 7 and 4 were used (see Table I) to create the mutation. Fragments containing the mutation were cloned into pRK5ha17 to generate the expression plasmid designated pRK5ha17_KIV7E56G.

Human embryonic kidney (293) cells stably expressing each of the r-apo(a) derivatives were generated as previously described (22). Briefly, these cells were transfected by the calcium phosphate co-precipitation method using 10 µg of each r-apo(a) plasmid and 1 µg of a plasmid encoding the neomycin resistance gene per 100-mm plate. Stable transformants were selected by culturing the cells in the presence of 0.8 mg/ml G418 (Invitrogen) until foci developed. At this time, the foci were picked and screened for apo(a) expression by Western blot analysis.

Construction and Expression of Apo(a) KIV₇ and KIV₈ in Escherichia coli—Sequences encoding apo(a) KIV₇ were cloned into the bacterial expression vector pET16b (Stratagene) as previously described (23). For the construction of KIV₈ in pET16b, a similar strategy was employed as for KIV₇. Briefly, sequences corresponding to the KIV₈ domain were amplified from the pRK5-SK₈ vector.³ using primers 10 and 11 (Table I) and were cloned into the NdeI site in pET16b. For both KIV₇ (23) and KIV₈, the expression plasmids contain sequences corresponding to the respective kringle domains, as well as the entire NH₂- and COOH-terminal interkringle sequences (3).

Purification of LDL—LDL was purified from the plasma of a normolipidemic volunteer as previously described (18). The concentration of purified LDL was determined using a modified Bradford assay, and the lipoprotein was stored at 4 °C in the presence of 1 mM phenylmethylsulfonyl fluoride and 1 mM EDTA for a maximum of 3 days prior to use.

Purification of Recombinant Apo(a) Variants—All r-apo(a) variants used in this study, with the exception of KIV₇ and KIV₈, were purified from the conditioned medium of stably expressing 293 cell lines using lysine-Sepharose chromatography as previously described (24). Protein concentrations for all r-apo(a) derivatives were obtained by absorbance measurements at 280 nm (corrected for Rayleigh scattering) using previously determined extinction coefficients (25). Protein integrity and purity were assessed by SDS-PAGE under non-reducing and reducing conditions followed by staining with Coomassie Blue.

Single kringle constructs encoding KIV₇ or KIV₈ were used to transform BL21 (DE3) cells (Novagen); LB containing ampicillin was inoculated with an overnight culture of transformed BL21 (DE3) cells and grown to an A₆₀₀ of 0.8. Protein expression was induced by the addition of isopropyl-1-thio--D-galactopyranoside to a final concentration of 1mM, and cultures were grown for an additional 3 h at 37 °C. Cells were harvested by centrifugation, and the cell pellets were lysed by sonication. Following centrifugation, the insoluble pellet was then solubilized by the addition of a buffer containing 6 M guanidine HCl. The purification of the single kringles was performed essentially as we have previously described for KIV_7, using Ni²⁺-Sepharose chromatography and gel filtration (23), with a few minor modifications. Following gel filtration, proteins were dialyzed against 20 mM Tris-HCl pH 7.9, 1 mM EDTA for 2 h, and were then dialyzed against double-distilled H₂O. Purified proteins were concentrated by lyophilization; before use, protein pellets were dissolved in 20 mM Tris-HCl, pH 7.9, and protein concentrations were determined by measurement of absorbance at 280 nm (corrected for Rayleigh scattering), using calculated extinction coefficients (E_0.1% = 2.12 and 2.24 for KIV₇ and KIV₈, respectively) (25). Protein integrity and purity were assessed by SDS-PAGE under non-reducing and reducing conditions, followed by staining with Coomassie Blue.

Analytical Ultracentrifugation of Apo(a)—Sedimentation velocity experiments on the 17K and 17K variants were performed using a Beckman XL-I analytical ultracentrifuge as previously described (15).

Modification of LDL 5'-Iodoacetamidofluorescein—LDL was labeled with 5'-iodoacetamidofluorescein as previously described (15). The concentration of fluorescein-labeled LDL (flu-LDL) was determined using a modified Bradford assay, and the modified lipoprotein was stored at 4 °C for no longer than 3 days prior to use.

Binding of Recombinant Apo(a) to Flu-LDL—Apo(a) binding to flu-LDL was performed as previously described (15). Briefly, fluorescein fluorescence measurements of flu-LDL were performed using an LS50B Luminescence Spectrometer (PerkinElmer Life Sciences). Flu-LDL (50 nM) was titrated with 17K, 17K_KIV6E56G, 17K_KIV7E56G, 17K_KIV8E56G, 17K_KIV6/7E56G, and 17K_KIV7/8E56G. Titrations were performed in HEPES-buffered saline (20 mM HEPES, pH 7.4, 150 mM NaCl) containing 0.01% Tween 20 in a quartz cuvette, which had been conditioned with this buffer prior to use. Fluorescein was excited at a wavelength of 495 nm and a slit width of 2.5 nm, whereas fluorescein emission was detected at a wavelength of 530 nm and a slit width of 5 nm, with a 510-nm cut-off filter placed in the emission beam. Ligand solutions, containing 50 nM flu-LDL to eliminate dilution effects, were added in a stepwise manner until saturation of the fluorescence change was attained. To determine the K_D for the interaction between apo(a) and flu-LDL, titration curves were subjected to non-linear regression analysis using the following quadratic equation (Equation 1).

(Eq. 1)

I is the measured fluorescence change, dI is the difference between the fluorescence coefficient for LDL in the free and apo(a)-bound states, K_D is the dissociation constant, and [A]₀ and [B]₀ are the total concentrations of apo(a) and flu-LDL, respectively.

Transient Transfection and Metabolic Labeling of 17K and Recombinant Apo(a) Variants with [³⁵S]Cysteine—Plamids encoding 17K, 17K_KIV6E56G, 17K_KIV7E56G, 17K_KIV8E56G, 17K_KIV6/7E56G, and 17K_KIV7/8E56G were transiently transfected into 293 cells and metabolically labeled with [³⁵S]cysteine as previously described (15). Labeled conditioned medium was harvested and stored at –70 °C prior to use.

Covalent Lp(a) Assembly Assays—In vitro covalent Lp(a) assembly assays were performed essentially as previously described (15). Briefly, purified native LDL (50 nM) was incubated with conditioned medium containing 2 nM 17K or each of the 17K variants at 37 °C in a total volume of 300 µl. At selected time points (0–8 h), a 30-µl aliquot was removed, added to an equal volume of 2x Laemmli sample buffer (26) in the absence of a reducing agent, and heated at 95 °C for 5 min. Samples were then subjected to SDS-PAGE on 4% polyacrylamide gels. Following electrophoresis, gels were placed in fixing solution (25% methanol, 12.5% acetic acid in double-distilled H₂O) for 20 min, followed by incubation in Amplify solution (Amersham Life Sciences) for 30 min. The gels were then dried and exposed to a PhosphorImager screen (Bio-Rad) for 16 h. The screen was developed using a Bio-Rad Molecular Imager FX, and the bands were quantified using Quantity One 4.0.1 densitometry software. The extent of covalent Lp(a) particle formation was quantified according to the following formula: %Lp(a) = [Lp(a)]/([Lp(a)] + [apo(a)]) x 100. The data were fit according to an exponential rise to maximum formula given in Equation 2, which was derived from first principles assuming that covalent Lp(a) formation occurs as a two-step process.

(Eq. 2)

%Lp(a) is the amount of Lp(a) formed at time t, t is the time of reaction, and a is a constant is described by the following relationship.

(Eq. 3)

[B]₀ is the total concentration of LDL, K_D is the dissociation constant for the interaction between apo(a) and LDL, and k is the rate constant for disulfide bond formation.

ApoB Peptide Synthesis and Purification—ApoB peptides corresponding to amino acids 675–689 (apoB675–689) and 685–699 (apoB685–699) in the primary sequence of apoB were synthesized by the Alberta Peptide Institute (Edmonton, Alberta, Canada). Both of the peptides were purified using high performance liquid chromatography, lyophilized, and stored at –20 °C. Peptides were reconstituted in 20 mM Tris base, and the solution was adjusted to pH 7.9 by the dropwise addition of 1 M HCl. Reconstituted peptides were stored at –70 °C prior to use.

Measurement of the Binding of -ACA and ApoB Peptides to KIV₇ and KIV₈ Using Intrinsic Fluorescence—Intrinsic fluorescence experiments were conducted using an LS50B Luminescence Spectrometer (PerkinElmer Life Sciences). Apo(a) KIV₇ and KIV₈ were each titrated with -ACA, apoB675–689, or apoB685–699. Titrations were performed in 20 mM Tris-HCl, pH 7.9, containing 0.01% Tween 20 in a quartz cuvette, which had been conditioned with this buffer prior to use. Tryptophan residues were excited at a wavelength of 280 nm and a slit width of 2.5 nm, whereas tryptophan emission was detected at a wavelength of 340 nm and a slit width of 5 nm with a 290-nm cut-off filter placed in the emission beam. Ligand solutions, containing equimolar concentrations of KIV₇ or KIV₈ to eliminate dilution effects, were added in a stepwise manner until saturation of the fluorescence change was attained. To determine K_D values for the interactions, titration curves were subjected to non-linear regression analysis using hyperbolic equations for binding to -ACA, and quadratic equations (see Equation 1) for binding to the apoB-derived peptides. For titrations with -ACA, a final concentration of 2 µM KIV₇ or KIV₈ was used, whereas titrations with the apoB-derived peptides were performed using 200 nM KIV₇ or KIV₈. Because apoB685–699 contains a tryptophan residue, an additional control experiment was performed in which identical concentrations of the peptide to those used in the titrations of KIV₇ and KIV₈ were added to buffer. The fluorescence values obtained from this titration were subjected to linear regression; the slope from this fit (i₀) was used to correct for the direct contribution of apoB685–699 to the fluorescence signal obtained from the titrations of KIV₇ and KIV₈ with this peptide. The formula used to obtain K_D values for the interactions between apoB685–699 and KIV₇ or KIV₈ is as follows.

(Eq. 4)

I is the fluorescence change, i₀ is the fluorescence coefficient for free apoB685–699, [B]₀ and [A]₀ are the total concentrations of apoB685–699 and KIV₇ or KIV₈, respectively, dI is the difference between the fluorescence coefficients for KIV₇ or KIV₈ in the free and peptide-bound forms, and K_D is the binding constant for the interaction between KIV₇ or KIV₈ and apoB685–699.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Expression and Purification of Recombinant Apo(a) Variants—17K r-apo(a) as well as variants containing mutations in the WLBS in KIV_6–8 (Fig. 1A) were stably expressed in 293 cells and purified using lysine-Sepharose affinity chromatography. Protein integrity and purity were verified by SDS-PAGE followed by staining with Coomassie Blue (Fig. 1B). Apo(a) KIV₇ (23) and KIV₈ (Fig. 1A) were expressed in E. coli, refolded, and purified to homogeneity as determined by SDS-PAGE under reducing and non-reducing conditions followed by staining with Coomassie Blue (Fig. 1C).

View larger version (32K): [in this window] [in a new window]   FIG. 1. Recombinant apo(a) variants used in this study. Panel A, recombinant apo(a) variants corresponding to 17K, 17KKIV6E56G, 17KKIV8E56G, 17KKIV7E56G, 17KKIV6/7E56G, and 17KKIV7/8E56G were constructed in the pRK5 vector and expressed in human embryonic kidney (293) cells as outlined under "Experimental Procedures." Each mutant apo(a) variant contains an amino acid substitution at position 56 (denoted by an asterisk) within one or more of the WLBS, where position 1 corresponds to the first cysteine residue in the kringle. Sequences corresponding to either KIV7 or KIV8 were assembled in the pET16b vector and expressed in E. coli, as described under "Experimental Procedures." Panel B, 17K, 17KKIV6E56G, 17KKIV7E56G, 17KKIV8E56G, 17KKIV6/7-E56G, and 17KKIV7/8E56G r-apo(a) derivatives were purified using lysine-Sepharose chromatography (24). To assess protein integrity and purity, purified r-apo(a) derivatives (5 µg) corresponding to 17K (1), 17KKIV6E56G (2), 17KKIV7E56G (3), 17KKIV8E56G (4), 17KKIV6/7E56G (5), and 17KKIV7/8E56G (6) were subjected to SDS-PAGE under non-reducing (NR) and reducing (R) and stained with Coomassie Blue. Panel C, apo(a) KIV8 was purified using a similar procedure to that previously described for apo(a) KIV7 (23). To assess protein integrity and purity, purified apo(a) KIV8 (10 µg) was subjected to SDS-PAGE under non-reducing (NR) and reducing (R) and stained with Coomassie Blue.

View larger version (10K): [in this window] [in a new window]   FIG. 2. Effect of -ACA on the sedimentation coefficient of 17KKIV6/7-E56G r-apo(a). Sedimentation coefficients for 17KKIV6/7E56G in the presence of -ACA (0, 5, 10, 25, 50, and 100 mM) were determined using analytical ultracentrifugation. The line represents regression of the data to a hyperbolic equation.

View larger version (14K): [in this window] [in a new window]   FIG. 3. Non-covalent binding between 17K recombinant apo(a) and 17KKIV7/8E56G, and flu-LDL. Flu-LDL was titrated with either 17K r-apo(a) (•) or 17KKIV7/8E56G () until the fluorescence approached saturation. The fluorescence changes recorded during the titrations were modeled according to Equation 1 (solid lines) to obtain the affinity for the non-covalent interaction between the r-apo(a) species and flu-LDL (KD).

View larger version (28K): [in this window] [in a new window]   FIG. 4. The effect of mutation of the weak lysine-binding sites in KIV7 and KIV8 in 17K on the covalent step of Lp(a) assembly. In vitro covalent Lp(a) assembly assays were used to study the kinetics of covalent Lp(a) particle formation over an 8-h time course. Panel A, Lp(a) was separated from free apo(a) by SDS-PAGE on a 4% gel and visualized by autoradiography. The percentage of Lp(a) formed was determined using densitometry to measure the amount of Lp(a) and free apo(a) at a given time (t). A representative autoradiograph for 17KKIV7/8-E56G and 17K is shown. Panel B, the percentage of Lp(a) formed using metabolically labeled r-apo(a) (17K (•), 17KKIV6E56G (), 17KKIV7E56G (), 17KKIV8E56G (), 17KKIV6/7E56G, (), and 17KKIV7/8E56G ()) was modeled with respect to t using Equation 2 (solid lines) to obtain a constant (a) that represents the efficiency of covalent Lp(a) assembly.

View larger version (10K): [in this window] [in a new window]   FIG. 5. Relationship between the non-covalent and covalent steps of Lp(a) assembly. The KD values obtained for the interaction between flu-LDL and 17K, 17KKIV6E56G, 17KKIV7E56G, 17KKIV8E56G, 17KKIV6/7 E56G, and 17KKIV7/8E56G (KD) were regressed as a function of the efficiency with which these recombinant variants form covalent Lp(a) particles (corresponding to the term a) using Equation 3 (line). The fit obtained was used to calculate the rate constant for disulfide bond formation (k) for all of the 17K r-apo(a) species used.

Initially, we examined the ability of bacterially expressed apo(a) KIV₇ and KIV₈ to bind to -ACA using intrinsic fluorescence as previously described (23). Titration of KIV₇ with -ACA resulted in an increase in fluorescence, whereas titration of KIV₈ yielded a decrease in fluorescence (Fig. 6). The observed changes in fluorescence (I) were modeled with respect to the concentration of -ACA according to a simple hyperbolic relationship to obtain affinities (K_D) for the interaction between apo(a) KIV₇ or KIV₈ and -ACA. The results indicated that apo(a) KIV₇ and KIV₈ bound -ACA with comparable affinities (K_D = 217 µM, K_D = 800 µM, respectively); the value obtained for KIV₇ is comparable with what we have previously reported (23). The affinity of the respective kringles containing the WLBS for -ACA is substantially less than the affinity of KIV₁₀, containing a strong LBS, for this ligand (33 µM; Ref. 23). We used intrinsic fluorescence to investigate the ability of apo(a) KIV₇ and KIV₈ to bind apoB675–689 and apoB685–699. Titration of KIV₈ with apoB675–689 yielded a decrease in fluorescence that was subsequently modeled to obtain a K_D of 167 ± 46 nM (Fig. 7A). This corresponds to a 4000-fold increase in affinity of KIV₈ for apoB675–689 compared with -ACA. Interestingly, titration of KIV₇ with apoB675–689 did not elicit a change in intrinsic fluorescence (Fig. 7A), suggesting that KIV₇ does not bind to apoB675–689. In contrast, however, titration of KIV₇ with apoB685–699 elicited a non-linear increase in intrinsic fluorescence (Fig. 7B). Subsequent modeling of the fluorescence change (I) with respect to the concentration of the apoB685–699 peptide according to Equation 4 yielded a K_D of 1.2 ± 0.18 µM, indicating that KIV₇ interacts with this peptide with high affinity compared with -ACA. Titration of KIV₈ with apoB685–699 did not elicit a detectable change in fluorescence (Fig. 7B), suggesting that this peptide does not interact with KIV₈.

View larger version (13K): [in this window] [in a new window]   FIG. 6. Measurement of the affinity of apo(a) KIV7 and KIV8 for -ACA using intrinsic fluorescence. Apo(a) KIV7 or KIV8 (2 µM) were titrated with -ACA, and the resultant fluorescence changes in intrinsic fluorescence were measured. The lines represent the fit obtained when the fluorescence changes for KIV7 () or KIV8 (•) as a function of -ACA concentration were regressed to a hyperbolic equation to yield the affinities for the interactions between KIV7 or KIV8 and -ACA (KD).

View larger version (10K): [in this window] [in a new window]   FIG. 7. Measurement of the affinity of apo(a) KIV7 and KIV8 for apoB675–689 and apoB685–699 using intrinsic fluorescence. Panel A, apo(a) KIV8 (•) (200 nM) or KIV7 () were titrated with apoB675–689 and the tryptophan fluorescence changes were recorded. The line represents the fit obtained when the data were modeled according to the quadratic relationship described by Equation 1, yielding a dissociation constant (KD) for the interaction between KIV8 and apoB675–689. Panel B, apo(a) KIV7 (•) or KIV8 () were titrated with apoB685–699 and the resultant fluorescence changes were fit with respect to the peptide concentration according to Equation 4, yielding a KD for the interaction between KIV7 and apoB685–699.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have recently shown that the conformational status of apo(a) is an important determinant of the efficiency covalent of Lp(a) assembly (15). Apo(a) adopts a closed conformation that can be converted to an open form by the addition of the lysine analog -ACA. Importantly, the conformational status of apo(a) does not alter its affinity for LDL (K_D), but rather influences the rate constant for disulfide bond formation (k), possibly by restricting access of LDL to the free cysteine in apo(a) KIV₉. We have also recently demonstrated that the closed conformation of apo(a) is maintained by an intramolecular interaction between the strong lysine-binding site in KIV₁₀ (located in the COOH-terminal half of the molecule) and sequences within the amino-terminal half of the molecule.² As such, the closed conformation of apo(a) is disrupted when truncated apo(a) variants are used. This is important in that the studies used to identify domains in apo(a) that are required for Lp(a) assembly have been conducted using truncated apo(a) variants (11, 14, 20, 21). In light of our recent discovery of the role of the conformational status of apo(a), a quantitative re-evaluation of the role of the respective WLBS is required. As we have previously shown, although the conformational status of apo(a) does not directly affect the measurement of non-covalent binding between apo(a) and LDL, it complicates measurements of the covalent step of assembly (15), which are necessary to assess the productivity of the non-covalent step. Our original postulate was that the affinity of the non-covalent step dictated the efficiency of the covalent assembly. However, we have observed that the KIV₉-P r-apo(a) variant binds to LDL with high affinity in solution (K_D = 48 nM),² but forms covalent Lp(a) particles with very low efficiency (11). In the context of this observation, the present study is significant in that it implies a role for the non-covalent step in orienting the apo(a) and apoB molecules and suggests a key role for the WLBS in KIV_7–8 in this respect. On this basis, our studies provide the first evidence that the role of the WLBS in apo(a) is not simply to anchor apo(a) to LDL, but rather to localize apo(a) in the correct orientation to ensure efficient disulfide bond formation.

Elucidation of the molecular structures of apo(a) KIV₆ by NMR (27) and apo(a) KIV₇ by x-ray crystallography (28) has provided a rationale for the use of site-directed mutagenesis to disrupt the lysine-binding properties of the WLBS as opposed to an approach that involves deleting entire WLBS-containing kringle domains. The lysine-binding pockets in each of apo(a) KIV_6–8 contains three centers that coordinate -ACA: a cationic center (Arg³⁵ and Arg⁶⁹), which coordinates the carboxylate group; an anionic center (Arg⁵⁴ and Glu⁵⁶), which coordinates the amino group; and a hydrophobic trough (Trp⁶⁰, Tyr⁶², and Trp⁷⁰), which stabilizes the methylene groups (28). Together, these three centers account for the interaction of the WLBS with free lysine, lysine analogs, and lysyl residues on physiological binding partners such as apoB. Using site-directed mutagenesis, we introduced E56G point mutations into 17K r-apo(a) to generate mutant variants with diminished lysine-binding properties in KIV₆ (17K_KIV6E56G), KIV₇ (17K_KIV7E56G), KIV₈ (17K_KIV8E56G), KIV₆ and KIV₇ (17K_KIV6/7E56G), and KIV₇ and KIV₈ (17K_KIV7/8E56G). Importantly, none of the point mutations introduced affected the s_20,w of 17K r-apo(a) in the analytical ultracentrifuge, indicating that the mutations do not alter the conformational status of 17K r-apo(a) (Table II).

Because the mutant r-apo(a) variants were expressed in the context of the 17K r-apo(a), we were able to assess the role of each of the WLBS in both the non-covalent and covalent steps of Lp(a) assembly. The data clearly indicate that the WLBS in KIV₇ and KIV₈, but not KIV₆, contribute to the initial non-covalent association between apo(a) and LDL (Fig. 3B, Table III). Mutation of either KIV₇ or KIV₈ resulted in a 4-fold reduction in the affinity of apo(a) for LDL, whereas tandem mutation of both WLBS led to a 13-fold decline in the affinity. We further demonstrated that the WLBS in KIV₇ and KIV₈, but not KIV₆, are required for covalent Lp(a) particle formation. Importantly, the efficiency of covalent Lp(a) particle formation (a value) for the wild-type and mutant 17K r-apo(a) variants exhibited an inverse relationship with the affinity for LDL (K_D) obtained in the non-covalent assays (Fig. 5). This relationship agrees with the model that we have derived from first principles based on the assumption that Lp(a) assembly is a two-step process (see Equations 2 and 3). Our observation that the data obtained in both the non-covalent and covalent assays follow this relationship implies that the K_D values obtained in the non-covalent assay describe productive binding interactions between apo(a) and LDL (i.e. interactions that result in disulfide bond formation). Furthermore, the relationship between K_D and a that is predicted by Equation 3 demonstrates clearly that mutation of the WLBS in apo(a) KIV₇ and KIV₈ alters the efficiency of Lp(a) assembly at the level of K_D and not k. This result is consistent with data obtained from analytical ultracentrifugation, which shows that mutation of the WLBS in apo(a) does not alter the s_20,w of 17K r-apo(a). In addition, the k value (0.0613 h^–1) obtained from the fit of K_D versus a is in excellent agreement with the k value (0.0497 h^–1) previously measured for 17K r-apo(a) (15).

Although the involvement of the WLBS in the non-covalent interaction between apo(a) and LDL has been demonstrated by numerous groups (14, 19–21), the data from the present study represent the first quantitative assessment of the role of each of the WLBS present in apo(a) KIV_6–8 to both the non-covalent and covalent steps of Lp(a) assembly. Our findings are not in agreement with previous studies, which suggested that apo(a) KIV₆ mediates the non-covalent interaction with LDL (14, 20, 21). It is important to note, however, that in all of these studies roles for apo(a) KIV₆-KIV₈ in apo(a)-LDL interactions were identified using truncated variants in which entire kringle domains containing the WLBS were removed.

Our present observation that two WLBS present in each of apo(a) KIV₇ and KIV₈ mediate non-covalent Lp(a) assembly is in good agreement with our previous report suggesting that two lysine residues (Lys⁶⁸⁰ and Lys⁶⁹⁰) within the NH₂-terminal 18% of apoB (apoB-18) are required for binding to WLBS present within apo(a) KIV_5–8 (18). Studies from another group, however, suggest that lysine residue(s) within the carboxyl-terminal end of apoB mediate non-covalent binding to apo(a). In this context, a recent study demonstrated that a lysine-rich, apoB-derived peptide corresponding to amino acids 4372–4392 (apoB4372–4392) binds apo(a) non-covalently and inhibits covalent Lp(a) particle formation with a higher potency than -ACA (9). Taken together, these studies suggest that several regions of apoB may be involved in non-covalent association with apo(a). Evaluation of the relative contribution of these sequences should be addressed in future studies; the current study is an extension of our previous work and thus focuses on the lysine-dependent non-covalent interaction between apo(a) KIV_6–8 and sequences within the amino-terminal 18% of apoB (16, 18).

To investigate whether the WLBS in KIV₇ and KIV₈ are specific for Lys⁶⁸⁰ and Lys⁶⁹⁰ in LDL, we measured the affinity of these kringles for -ACA, as well as peptides spanning Lys⁶⁸⁰ (apoB675–689) or Lys⁶⁹⁰ (apoB685–699). Both KIV₇ and KIV₈ bound -ACA with comparable affinities (Fig. 6; K_D = 217 and 800 µM, respectively) that are in keeping with previously reported values for the interaction between -ACA and the WLBS in apo(a) (15, 23, 27). Analysis of the primary sequence alignment of apo(a) KIV₇ and KIV₈ reveals that both of these kringle domains contain all of the crucial amino acid residues that form the lysine-binding pocket, an observation that explains their similar affinity for -ACA (3). Interestingly, KIV₈ bound apoB675–689 with a 4000-fold higher affinity (Fig. 7A; K_D = 167 nM) than -ACA, suggesting that flanking sequences surrounding Lys⁶⁸⁰ are required for high affinity binding. Moreover, KIV₇ did not interact with apoB675–689, suggesting that sequences flanking Lys⁶⁸⁰ also mediate specificity of this peptide for KIV₈. When binding of KIV₇ and KIV₈ to apoB685–699 was investigated, the opposite result was obtained; KIV₇ bound apoB685–699 with a 200-fold higher affinity than -ACA (Fig. 7B; K_D = 1.2 µM), whereas KIV₈ did not interact with this peptide. Once again, this suggests that sequences flanking Lys⁶⁹⁰ mediate the affinity and specificity that characterize the interaction between KIV₇ and apoB685–699. These results are interesting in the context of our previous study (18), in which we demonstrated that mutation of the Lys⁶⁸⁰ residue reduced the apo(a)-apoB interaction to a much greater extent than was observed upon mutation of the Lys⁶⁹⁰ residue. This is in good agreement with the data in the present study in which the interaction of apo(a) KIV₇ with the peptide containing apoB Lys⁶⁹⁰ is weaker than the interaction of apo(a) KIV₈ with the peptide containing Lys⁶⁸⁰. Furthermore, we have previously demonstrated that an apoB-derived peptide containing Lys⁶⁸⁰ and Lys⁶⁹⁰ (apoB680–704) bound 17K r-apo(a) with a K_D of 83.4 nM (18), which corresponds to a higher affinity than either peptide alone. This suggests that both lysine residues are required for maximally efficient binding of apo(a) to apoB. Support for this notion is provided by the observation that tandem mutation of the WLBS in apo(a) KIV₇ and KIV₈ is required to maximally inhibit the non-covalent association between 17K r-apo(a) and LDL (Table III).

Given the extraordinarily high degree of primary sequence identity between KIV₇ and KIV₈ (88%; Ref. 3), the observed specificity of apoB675–689 for KIV₈ and apoB685–699 for KIV₇ is somewhat surprising. With the exception of the sequences flanking Arg³⁵ (Thr³⁷ and Glu³⁸ in KIV₇ and Pro³⁷ and Leu³⁸ in KIV₈), the amino acid differences between KIV₇ and KIV₈ are conservative substitutions. Understanding of the nature of the specificity of KIV₇ and KIV₈ for sequences in apoB must be validated experimentally using high resolution methodologies such as x-ray crystallography and/or NMR. In this regard, preliminary ¹H-¹⁵N NMR analyses of KIV₇ and KIV₈ suggest that the backbone conformation of KIV₈ is significantly different from that of KIV₇ for a subset of residues,⁴ indicating that these two kringle domains may adopt unique structures in some regions.

We have demonstrated that the initial non-covalent interaction between apo(a) and apoB is mediated by a highly specific interaction between two lysine residues (Lys⁶⁸⁰ and Lys⁶⁹⁰) in apoB and two WLBS (KIV₇ and KIV₈) in apo(a). Taken together, our data are consistent with the hypothesis that this highly specific interaction does not merely anchor apo(a) to apoB, but also plays an important role in orientating the two proteins in a manner that facilitates a secondary non-covalent interaction and/or disulfide bond formation. On a molecular level, our data suggest that a productive orientation between apo(a) and LDL would be provided for by the observed specificity of KIV₇ for Lys⁶⁹⁰ and KIV₈ for Lys⁶⁸⁰; this specificity would provide the appropriate directionality to the initial non-covalent interaction, thereby ensuring the efficiency and fidelity of the process of covalent Lp(a) assembly.

	FOOTNOTES

 
^* This work was supported in part by Canadian Institutes of Health Research Grant 11271 (to M. L. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Career Investigator of the Heart and Stroke Foundation of Ontario. To whom correspondence should be addressed. Tel.: 613-533-6586; Fax: 613-533-2987; E-mail: mk11{at}post.queensu.ca.

¹ The abbreviations used are: Lp(a), lipoprotein(a); LDL, low density lipoprotein; apo(a), apolipoprotein(a); apoB, apolipoprotein B; -ACA, -aminocaproic acid; WLBS, weak lysine-binding sites; 17K, 17-kringle; KIV, kringle IV; flu-LDL, fluorescein-labeled low density lipoprotein.

² L. Becker and M. L. Koschinsky, unpublished results.

³ M. L. Koschinsky, unpublished data.

⁴ S. P. Smith, Queen's University, personal communication.

	ACKNOWLEDGMENTS

 
We acknowledge the Queen's University Protein Function and Discovery Group for subsidizing the use of the analytical ultracentrifuge.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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