Journal of Protein Chemistry, Vol. 15, No. 1, 1996

Interaction of Apolipoprotein[a] with ApolipoproteinB-100 Cys3734 Region in Lipoprotein[a] Is Confirmed Immunochemically

Juan Guevara, Jr., 1~ Natal ia V. Valent inova, 1 Oscar Garcia, 1 Anton io M. Gotto, 1 Chao-yuh Yang, 1 Susana Legal, 1 John Gaubatz, ~ and James T. Sparrow ~

Received August 30, 1995

Monospecific polyclonal antibodies (MPAbs) to apoB-100 regions Cys3734 and Cys4190 were isolated by affinity chromatography using the synthetic polypeptides, Q3730VPSSKLDFREIQIYKK3746 and G41s2IYTREELSTMFIREVG419s, respectively, coupled to a hydrophilic resin. Molecular modeling and fluroescence labeling studies have suggested that Cys67 located in kringle type 9 (LPaK9, located between residues 3991 and 4068 of the apo[a] sequence inferred by cDNA) of the apo[a] molecule is disulfide linked to Cys3734 of apoB-100 in human lipoprotein[a] (Lp[a]). This possibility has been further explored with MPAbs. Four species of MPAbs directed to a Cys3734 region of apoB-100 (3730-3746) were isolated from goat anti-human LDL serum by a combination of synthetic peptide (Q3730VPSSKLDFREIQIYKK3746) affinity chromatography and preparative el- ectrophoresis (electrochromatography). MPAbs to the Cys4190 region of apoB-100, a second or alternative disulfide link-site between apo[a] and apoB-100, were also isolated using a synthetic peptide (G41jYTREELSTMFIREVG41qs) affinity resin. Results of immunoassays showed that binding of these four MPAbs to Lp[a] was significantly lower than to LDL. In contrast, MPAbs to the apoB-100 region 4182-4198 which contains Cys4190, a second or alternative disulfide link-site between apo[a] and apoB-100, displayed a less significant difference in binding to Lp[a] and LDL. These results provide additional evidence that the residues 3730-3746 of apoB-100 interact significantly with apo[a] in Lp[a], and that Cys3734 is a likely site for the disulfide bond connecting apo[a] and apoB-100.

KEY WORDS: Apo[a]; apoB-100; funnel; electrochromatography; Cys3734; monospecific; disulfide.

1. I N T R O D U C T I O N

Human lipoprotein[a] (Lp[a]) 3 contains two major and distinctly different high molecular weight apoproteins, apo[a] and apoB-100 (Ehnholm et al., 1972; Gaubatz, et al., 1983, 1987). Apo[a] is a highly glycosylated, hydrophilic protein (Gaubatz et al., 1983, 1987; Armstrong et al., 1985; Fless et al.,

~Department of Medicine, Division of Atherosclerosis and Lipoprotein Research, Baylor College of Medicine, Houston, Texas 77030.

2 To whom correspondence should be addressed.

1986; Seman and Breckenridge, 1986; Eaton et al., 1987; Kratzin et al., 1987) with little apparent affinity for lipid. It is disulfide linked to one molecule of apoB-100 (Gaubatz et al., 1987; Fless et

17

3 Abbreviations: amino acids, single letter, e.g., alanine, A, etc.; BSA, bovine serum albumin; d, density (g/rot); eaca, e- aminocaproic acid; ELISA, enzyme-linked immunosorbant as- say; DTT, dithiothreitol; HRP, horseradish peroxidase; MAb, monoclonal antibody; MPAb, monospecific polyclonal anti- body; PAGE, polyacrylamide gel electrophoresis: PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate: NazEDTA, sodium ethylenediaminetetraacetate: NAN3, so- dium azide; TRIS, (hydroxymethyl)aminomethane.

0277-8033/96/0100-0017509.50/0 �9 1996 Plenum Publishing Corporation

18 Guevara, Valentinova, Garcia, Gotto, Yang, Legal, Gaubatz, and Sparrow

al., 1986; Utermann and Weber, 1983), a highly hydrophobic apolipoprotein which is partly im- bedded in a lipid-rich, pseudomicellar particle. Based on the inferred sequence (McLean et al., 1987) of a 530-kD polymorph, apo[a] consists of a kringle domain and a serine protease-like domain. The amino acid sequence of the kringle domain indicates the presence of 11 different types of kringles (Morrisett et al., 1990), each separated by proline-rich interkringle regions. The kringle motif is a highly conserved, triloop structure typified by three disulfide bonds with a 1-6, 2-4, 3-5 pairing pattern (Park and Tulinsky, 1986; Tulinsky et al., 1988a, b; Mulichak and Tulinsky, 1990; Seshadri et at., 1991; Mulichak et al., 1991); Wu et al., 1991; de Vos et al., 1992). Several proteins involved in hemostasis contain kringle motifs (Furie and Furie, 1988; Hedner and Davie, 1989). One important fibrinolytic zymogen, plasminogen, contains five distinct kringles, two of which (PGK1 and PGK4) are involved in ligand binding (Magnusson et al., 1975, 1976). Apo[a] kringle types 1-10 (LPaK1, etc.) are similar but not identical to plasminogen kringle 4. Only kringles type 10, which is most like PGK4, and type 11 appear to retain the amino acids necessary to form a ligand binding pocket with the capacity to bind lysine. Apo[a] is thought to impart atherogenic and thrombogenic properties to Lp[a] because of its high homology with plasminogen. A positive role for this lipoprotein in mammalian physiology has not been elucidated.

It is now established that apo[a] is disulfide linked to apoB-100 in Lp[a] and that this linkage involves Cys67 of LPaK9 (Brunner and MUller, 1992; Koschinsky et al., 1993), but the location of the corresponding Cys residue on apoB-100 involved in forming this disulfide linkage has yet to be identified. Yang and co-workers (1990) first showed that there are 25Cys residues in the sequence of apoB-100, 16 of which are involved in intramolecular disulfides near the amino end of the molecule. Only two of the remaining sulfhydryl cysteines, Cys3734 and Cys4190, appear to occur on the surface of the LDL particle (Coleman et al., 1990) and hence to be available for forming a disulfide with Cys67 of LPaK9.

Parallel studies involving fluorescence labeling of surface-oriented cysteine residues in Lp[a] and autologous LDL have suggested that Cys3734 of apoB-100 exists as a reactive sulthydryl cysteine in LDL but not in Lp[a] (Guevara et al., 1993). Mol- ecular modeling was used concurrently to explore

possible noncovalent interactions between LPaK9 and seven putative sulfhydryl cysteine-containing regions of apoB-100 (Guevara et al., 1993). The energy-minimized molecular structure of apoB-100 segment 3732-3745 (PSCKLDFREIQIYK) docked with LPaK9 displayed the best fit and the most van der Waals and hydrogen bond contacts. Docked models of the other six apoB-100 peptides containing sulfhydryl cysteines were significantly less compatible.

Results of irnmunochemical studies with monoclonal antibodies (MAbs) to apoB-100 dem- onstrate that the presence of the apo[a] molecule affects the immunoreactivity of several epitopes of apoB-100 in Lp[a] (Zawadzki et al., 1988; Gries et aL, 1988). Theoretically, binding of MAbs specific to apoB-100 epitopes which contain Cys3734 or Cys4190 should be influenced by the presence of the apo[a] molecule in Lp[a]; these reagents would be useful in determining the location of the disulfide linkage between the two apoproteins. However, MAbs with narrow specificity to regions of apoB-100 containing Cys3734 and Cys4190 are not available. MAb Bsol 16, specific to the region of apoB-100 (4154-4189), has been used to study sites near Cys4190 (Zawadzki et al., 1988). However, this MAb has not be utilized to study this epitope as it relates to the disulfide linkage in Lp[a]. An immunochemical approach for the identification of the apoB-100 Cys residue involved in the disulfide bond with apo[a] has not been reported.

In the present report, we describe results which show that apo[a] masks and/or alters the structure of the apoB-100 molecule in the region containing Cys3734, but less significantly the region containing Cys4190. Polyclonal monospecific antibodies aga- inst two regions of apoB-100 that include Cys3734 and Cys4190 were isolated from goat anti-human LDL serum using synthetic peptide affinity chromatography and/or preparative electroch- romatography. These results strongly suggest that the principal disulfide bond joining these proteins is formed between Cys67 of LPaK9 and apoB-100 Cys3734.

2. MATERIALS AND METHODS

2.1. Materials Phenylmethylsulfonyl fluoride (PMSF), Tris,

sodium sulfate, 6-aminohexanoic and (e- aminocaproic acid, eaca), glycerol, and sodium

Apo[a] and ApoB-100 Interactions 19

ethylenediaminetetraacetate (Na2EDTA), sodium azide (NAN3) and dithiothreitol (DTT) were from Sigma Chemical Company (St. Louis, MO). Aprotinin (Trasylol) was from Miles Inc./FBA Pharmaceuticals (West Haven, CN). Aquacide II tm was purchased from CalBioChem, Inc. Lysine- Sepharose was from Pharmacia-LKB (Alameda, CA). MAb 5F8-HRP was purchased from the Cell Engineering Group, Institute of Experimental Cardiology, Moscow, Russia. All other chemicals used were of the highest quality commercially available.

2.2. Preparation of Lp[a], Lp[a-], LDL, and Reduced LDL

Purified Lp[a] and LDL were prepared by methods described previously (Gaubatz et al., 1987). Lp[a] was isolated by a combination of ultracentrifugation at 1.063-1.130g]ml immuno- affinity chromatography, and lysine-Sepharose chromatography. LDL was isolated from plasma of healthy donors by ultracentrifugation at d 1.019- 1.050, then repreated ultracentrifugation at d 1.090. Purity was determined by reduced SDS-PAGE using 3-16% gradient gels. A mixture of protease inhibitors containing aprotinin (0.55 unit/ml), Na2EDTA (1.0raM), PMSF (0.01%w/v), and NaN3 (0.01% w]v) was added to the plasma at the time of collection to give the concentration indicated in parentheses. Protein concentrations were determined by the method of Lowry in the presence of SDS (Markwell et al., 1978). Quant- ification of apoB-100 in Lp[a] and LDL prepara- tions was accomplished by ELISA using a pan-monoclonal antibody, MAb 5F8, as described below. Lp[a-] and reduced LDL were prepared by treating 1-2mg of the lipoprotein in PBS with 100 mM DTT for 90 rain at ambient temperature in a closed vessel followed by ultracentrifugation at d 1.21. An anti-apo[a] ELISA, used routinely in our laboratory, was used to assure the complete removal of the apo[a] molecule from Lp[a-].

2.3. Solid-Phase Synthesis of ApoB-100 Peptides

ApoB-100 peptides 3730-3746 and 4182-4198 were synthesized on a polyamide-based support by methods described previously (Kanda et al., 1991). Cys3734 was replaced with a serine residue to prevent oxidation to the dimeric disulfide. The

affinity gel was prepared by synthesizing the apoB-100 fragment on a large-pore polyamide resin by attaching the first amino acid, lysine, directly without the cleavable linker molecule. The completed synthetic polypeptide polymer was deprotected using anhydrous hydrogen fluoride containing 10% anisole and 1.0% ethanedithiol at -20~ for 3 hr. The synthetic polypeptide polymer was then washed with ether, methanol, water, 1.0% acetic acid, water, 1.0M Tris, water, and 0.1M sodium phosphate buffer, pH7.4. It was then treated with 50 ml of 10% mercaptoethane at 4~ for about 16 hr, washed with 100 ml of nanopure- grade water, and treated with 10mM iodoaceta- mide at 4~ for about 16hr to react with any residual ethanedithiol or mercaptoethane. This was followed by washing with water and with 100 ml of 6 M guanidine-HC1. Finally, the polypeptide poly- mer was equilibrated with 10 mM sodium borate (NazB4O7) , pH 9.0, 1.0 mM Na2EDTA, and 0.01% NaN3 prior to its use for affinity chromatog- raphy of the goat antiserum.

2.4. Isolation of Monospecific Antibodies by Synthetic-Polypeptide Affinity Chromatography

Goat anti-human LDL serum was prepared by immunizing a goat with purified LDL (d 1.030- 1.050) (Albers et aL, 1975). The goat antiserum (100ml) was treated with the protease inhibitor mixture and dialyzed against four changes of 4 L of 10 mM NazB407, pH 9.0, 150 mM NaCI, 1.0 mM NazEDTA, and 0.01% NAN3. The antiserum was loaded on t h e synthetic polypeptide polymer column (1.5 cm i.d. • 4.0 cm) at a flow rate of about 1.0 ml/min. The column was eluted with borate buffer until the eluent optical density was <0.05, then with borate buffer containing 1.5 M NaC1 until <0.01. Bound proteins were eluted with 6.0M guanidine-HCl. Protein-containing fractions were collected, then dialyzed exhaustively in borate buffer without NaCI to remove the guanidine-HCL. Fractions were then concentrated using Aquacide I[ tm.

2.5. Purification of Four Monospecific Polyclonal Antibodies by Preparative Funnel Electrochromatography

Preparative electrophoresis was performed using the funnel electrochromatography apparatus

20 Guevara, Valentinova, Garcia, Gotto, Yang, Legal, Gaubatz, and Sparrow

illustrated in Fig. 1 (Guevara, 1988). The apparatus, designed to process up to 150 mg of protein, was used to purify monospecific polyclonal antibodies initially obtained by synthetic peptide affinity chromatography as described above. Briefly, a 5% polyacrylamide funnel gel was formed which contained 75 mM Tris-C1 buffer, pH 8.6. The gel was preelectrophoresed in Tris-glycine, 50 mM and 250mM, respectively, pH8.3, by applying 200V. The 6.0-ml sample was prepared by dialyzing against 15 mM Tris-C1, pH 8.6, then overlaid on the

surface of the funnel gel and electrophoresed at 500V constant. Fractions (3 ml) were collected from the anodic end of the gel by passing Tris-glycine buffer across the base of the funnel in a direction perpendicular to the current flow. The electrochromatogram illustrated in Fig. 2 shows the protein peaks which were identified by monitoring the optical density at 280 nm. Fractions containing IgG (peaks 1-4) were identified by dot-blot analysis using Protein-G conjugated with horseradish peroxidase (Protein-G-HRP).

S a m p l e Load ing S u r f a c e

~15 cm 2)

1 II T o Fract ion

~ . . = . _ _ - " - ! , " ii~ii'ii ~'~Collector

P r o t e i n �9 : : : : : : : : �9 �9 A g a r o s e or

nno ii . . . . . . . . . . - ...... gel

S t e m ( 1 cm z)

S e m i - p e r m e a b l e / m e m b r a n e

(+)

Fig. 1. The funnel electrochromatography apparatus. Preparative-scale electrochromatography for sample loads greater than 1 mg protein is possible with the funnel apparatus, which is designed to handle as much as 150 mg of protein. Fractions of 3-6 ml containing the proteins such as immunoglobulins are obtained by passing a buffer across the base of the funnel gel in a perpendicular direction to the current flow. The optical density at 280nm for each fraction is monitored as in conventional liquid column chromatography using a detector, and a chromatographic profile of the electrophoretic run is obtained, hence electrochromatography.

Apo[a] and ApoB-100 Interactions 21

E c

co t~

~0

>- ,

c

go

+ J

o _ C I

0.3

0.2

0.1

0 .0

0 I I

50 100 150

F r a c t i o n Number

Fig. 2. Electrochromatogram of monospecific polyclonal anti- bodies to the region of apoB-100 containing Cys3734. Funnel electrochromatography under nondenaturing conditions was performed using a funnel apparatus (Fig. 1) to isolate sub- populations of immunoglobulins which bind to the synthetic epitope polypeptide of apoB-100 which includes residues 3730- 3746. The less negatively charged IgG fractions eluted from the funnel gel in fractions 125-145, peaks 1-4. Fractions containing MPAbs were identified using Protein-G-HRP in dot-blot analysis.

2.6. Purification of Monospecific Polyclonal Antibodies by Protein G Immunoaflinity Chromatography

The Protein-G affinity gel column (5 ml) was equilibrated with phosphate-buffered saline (PBS), p H 7.4; the fraction which bound to the synthetic polypeptide polymer was applied. The gel was eluted with PBS until an absorbance at 280 nm of <0.01 was observed, then the IgG fraction was eluted with 100 mM glycine, p H 2.5. This p H was then immediately neutralized with 1.5 M Tris-C1 p H 8.6.

2.7. Quantification of Lipoproteins Using MAb 5F8-HRP

LDL and Lp[a] concentrations were based on the quantification of apoB-100 using MAb 5F8- HRP as described previously (Yang et al., 1993; Yanushevskaya et al., 1993) which is specific for the T4 fragment of thrombin-digested apoB-100. OMEGA Lipid Fraction Control Serum from Technicon with an apoB-100 concentration of 0.59mg/ml was used as a standard in ELISA. Briefly, microtitration plates (96-well) from Corn- ing were coated overnight at 4~ with LDL (20/xg/ml in 100/xl of PBS). Plates were washed

with PBS/0.5% BSA, then incubated for 1 hr at ambient temperature with 300 txl of PBS/BSA and washed once with PBS/BSA/0.1% TWEEN-20. Then 50 Ixl of sample or standard serum, serially diluted within the range of apoB-100 concentrations (0.9-11.8/xg/ml), was added to the wells and mixed with 50/xl of 5F8-HRP at appropriate dilution, which was determined from 5F8-HRP-dose- dependent binding to immobilized LDL. Plates were inclubated at room temperature for 2hr, washed with PBS/BSA/TWEEN, then with PBS, and finally assayed for peroxidase activity. The apoB-100 concentration in LDL and Lp[a] prepara- tions were estimated from calibration curves established for the standard serum. Coefficients of variance for intra- and interassays were less than 10%.

2.8. Assay of MPAbs Binding to Lipoproteins

LDL, reduced LDL, Lp[a], and Lp[a-] preparations were diluted in PBS to an apoB-100 concentration of 1~0/xg/ml, and 100/xl per well was used for coating ELISA plates by incubating at 4~ overnight. Plates were washed with PBS/BSA, incubated with PBS/BSA (300/M/well) for 1 hr at room temperature, washed once with PBS/BSA/TWEEN, and incubated with serially diluted MPAb for 2 hr. After washing of plates with PBS/BSA/TWEEN, 100/xl of the second antibody, HRP-conjugated donkey anti-goat IgG, at ap- propriate concentration was added to each well. Plates were then incubated at room temperature for 1 hr, washed with PBS/BSA/TWEEN and with PBS, and assayed for peroxidase activity. Relative affinity was estimated as MPAb concentration at 50% of maximal binding (Hamilton and Adkinson, 1988).

3. RESULTS

Monoclonal antibody 5F8-HRP was used in quantification and standardization of LDL and Lp[a] stocks based on their concentration of apoB-100. Although the epitope for 5F8 is known to be between residues 1 and 1297 in the amino terminus of apoB-100, its exact location has not been determined. Only one sulfhydryl-cysteine residue is known to occur in this region (position

22 Guevara, Valentinova, Garcia, Gotto, Yang, Legal, Gaubatz, and Sparrow

r ' -

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@

r-i

-6

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I I I

I

0.5 J

0.0 I I I I I I I

-5 -4 -5 -2 -I 0 1 2 3

Log [MAb, ug/ml]

Fig. 3. Binding of monoclonal antibody 5F8 to the amino- terminus region of apoB-100 is apparently unaffected by apo[a] in Lp[a] (open circles), Lp[a-] (closed circles), and LDL (open triangles and closed triangles).

I I I I I I

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1084), but it is believed not to be on the surface of the LDL particle (Yang et al., 1993). Results obtained with this MAb were within 93+6% (n=10) of LDL and reduced LDL protein concentrations determined by the SDS-Lowry method. The binding curves shown in Fig. 3 for 5F8 were useful controls indicating that, for different samples, the same amount of apoB-100 is bound to the microtiter plate surface. Fifty percent of maximal binding of 5F8-HRP to LDL was reproducible between 30 and 50 ng/ml of conjug- ated antibody. No significant difference was observed in binding of 5F8-HRP to Lp[a] carrying any one of three different molecular weight apo[a] polymorphs, Lp[a-], or LDL.

Monospecific polyclonal antibodies to the region of apoB-100 located between residues 3730 and 3746 were isolated from goat anti-human LDL using an affinity column containing this synthetic peptide. Bound protein was eluted and analyzed for binding to purified preparations of Lp[a] and LDL. Only a slight difference in binding of this population of immunoglobulins to these lipop- roteins was observed (data not shown). When this bound immunoglobulin fraction was further pur- ified using Protein-G affinity chromatography, a significant difference in the binding of the Protein-G-bound MPAbs to apoB-100 in Lp[a], Lp[a-], LDL, and reduced LDL was observed (Fig. 4A). The relative binding affinity of purified

-,3 - 2 - 1 0 1

Log [MPAb, ug/ml]

A

2 5

1 . 5 i i i t i i

1.0

@

2

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I I I I I

4 3 4

Log [MPAb, ug/ml]

Fig. 4. Binding of MPAbs to apoB-100 regions containing Cys3734 (A, apoB-100 region 3730-3746) and Cys4190 (B, apoB-100 region 4182-4198). Gamma-immunoglobulin fractions of goat anti-human LDL serum were isolated using synthetic epitope polypeptide affinity chromatography and Protein-G affinity chromatography. Binding of MPAbs to the region containing Cys3734 was influenced by the presence of apo[a] in Lp[a] (open circles). About 350% more antibody was required to reach 50% maximal binding capacity in the case of Lp[a] as compared to LDL. The binding curves generated for these antibodies to Lp[a-] (closed circles), LDL (open triangles), and reduced LDL (closed triangles) are almost identical. A less significant difference (only 50% more antibody was required to reach 50% maximal binding) in binding of MPAbs to the region containing Cys4190 in apoB-100 of Lp[a], Lp[a-], LDL, and reduced LDL is evident.

Apo[a] and ApoB-100 Interactions 23

MPAbs was three- to four-fold lower for Lp[a] than that seen for Lp[a-], LDL, and reduced LDL. In contrast, experiments performed with MPAbs isolated using a synthetic polypeptide correspond- ing to apoB-200 region 4182-4198 showed less difference in the binding of those immunoglobulins to Lp[a], Lp[a-], LDL, and reduced LDL (Fig. 4B). Approximately 50% more antibody was required to obtain a 50% maximal binding with Lp[a] as compared to LDL. In contrast, about 350% more antibody to the Cys3734 region was required to attain 50% maximal binding.

Monospecific polyclonal immunoglobulins iso- lated by synthetic polypeptide affinity chromatog- raphy were also purified by preparative funnel electrochromatography. The electrochromatogram indicated that a significant number of proteins were eluted from the gel before the less electronegatively charged immunoglobulins. These results suggest that the synthetic peptide containing Cys3734 attracts other plasma proteins with high affinity which were not removed with high-ionic-strength buffer but were eluted from the polymer with 6.0 M guanidine-HC1. Protein which eluted from the funnel gel earlier than the IgG fractions did not bind Protein-G-HRP :in the dot-blot experiments and showed no reaction with donkey anti-goat- IgG-HRP in ELISA. The identity of these plasma proteins which recognize this region of apoB-100 remains undetermined. The four MPAb peaks were identified by dot-blot analysis, then dialyzed against borate buffer prior to their study in binding immunoassays with different Lp[a] and LDL preparations. Results shown in Fig. 5A-D demons- trate that binding of the MPAb peaks 1-4 to Lp[a] preparations was significantly lower than binding to autologous LDL samples. These data indicate that immunoreactivity of this apoB-100 region is different in these two lipoproteins.

4. DISCUSSION

Only two sulfhydryl-cysteine residues of apoB-100 in LDL have been shown to be reactive, Cys3734 and Cys4190 (Coleman et al., 1990), implying that these residues are available for the formation of disulfide linkages with other proteins such as apo[a]. In earlier studies using the fluorescent probe fluorescein-5-maleimide to label free sulfhydryl moieties in Lp[a] and LDL (Guevara et aL, 1993), Cys3734 and Cys4190 of

apoB-100 were labeled in LDL, but only Cys4190 of apoB-100 was labeled in Lp[a]. Those results strongly suggesetd that Cys3734 of apoB-100 might form a disulfide bond with Cys67 of LPaK9. Concurrent studies involving molecular modeling provided additional indirect evidence that the interactions between apoB-100 and apo[a] in Lp[a] included a single covalent bond between Cys3734 on apoB-100 and LPaK9 Cys67 on apo[a], plus other noncovalent bonding between the apoB-100 segment 3732-3745 (PSCKLDFREIQIYK) and the surface of LPaK9 (Guevara et al., 1993).

In the present report, we describe direct immunochemical evidence which strongly suggests that the association between apoB-100 and apo[a] in Lp[a] involves multiple interactions: (1) A point of contact is in the region of Cys3734 on apoB-100 which may form a disulfide bond with Cys67 of LpaK9. This association is based on the significant difference in binding of antibodies specific to this region of apoB-100 which were obtained using funnel electrochromatography. ( 2 ) A less detec- table association may actually occur between apo[a] and the Cys4190 region of apoB-100. Binding assays using polyclonal antibodies purified by, 4190-synthetic polypeptide resin and Protein-G gef affinity chromatography methods showed only a slight different between LDL and Lp[a]. When MPAbs to the region of apoB-100 containing Cys3734 were purified by 3734-synthetic polypep- tide resin and Protein-G gel affinity chromatog- raphy, their binding to apoB-100 on Lp[a] was obstructed by the presence of apo[a]. These immunoassays reveal a small "footprint" for LPaK9 on apoB-100 around the disulfide bond. These differences in binding were enhanced significantly using these antibodies, which were further purified using funnel electrochromatography. In contrast, the region of apoB-100 containing Cys4190, the other available surface-oriented sulfhydryl cysteine, was less altered by the apo[a], and binding of MPAbs to the corresponding region in Lp[a-], LDL, and reduced LDL was not significantly different. Although chemical identification of the disulfide bond between apo[a] and apoB-100 has not yet been reported, the accumulated indirect evidence is now substantially supported by direct immunochemical results.

The relative affinity of the polyclonal anti- bodies specific to apoB-100 region 3730-3746, which were prepared by synthetic peptide affinity chromatography and Protein-G affinity chromatog- raphy, was significantly higher than the affinity of

24 Guevara, Valentinova, Gareia, Gotto, Yang, Legal, Gaubatz, and Sparrow

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Log EM'PAb, ug,/ml]

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Fig. 5. Binding of IgG fractions purified by electrochromatography. Four protein fractions, peaks 1-4 of Fig. 2, containing electrophoretically different immunoglobulins were assayed for binding with LDL (closed circles) and LP[a] (open circles). Normal binding to LDL was exhibited by all four different subpopulations of the monospecific polyclonal antibodies to the Cys3734 of apoB-100. Binding of these antibodies of Lp[a] was significantly lower.

electrophoretically purified subpopulations of poly- clonal antibodies, MPAbs. The IgG fractions isolated by funnel gel electrochromatography were separated according to their intrinsic surface charge. In this case, proteins with a relatively high negative surface charge migrate through the gel more rapidly than a protein possessing a relatively

high positive surface charge. IgGs characteristically possess more positive surface charge than negative surface charge typically migrate to the cathode in native gel electrophoresis (Stanworth and Turner, 1973). Accordingly, we used the same nondenatur- ing buffer which was used in the funnel gel experiment to separate the MPAbs in a 1.0%

Apo[a] and ApoB-100 Interactions 25

agarose submarine gel. The immunoglobulins were identified using Protein-G-HRP as an activity stain and the majority of the antibodies were shown to have migrated cathodically (date not shown). These results demonstrate that the MPAb species purified by funnel electrochromatogrpahy represent at least two, perhaps four, small unique subfractions of the total population of antibodies which recognize the apoB-100 by these MPAbs as compared to the total fraction of MPAbs isolated by Protein-G affinity chromatography does not diminish the significance of the observation that these MPAbs showed lower binding for Lp[a] than for LDL. However, these results do suggest that further development of an appropriate buffer system for funnel electroch- romatography will be necessary to isolate effectively the major species of the MPAbs which possess a more positively charged surface. Electr- ochromatography experiments show that while affinity chromatography methods involving the use of synthetic polypeptide resins and Protein-G gel may enrich the antibody concentration, there are other proteins with higher electronegative mobility still present.

REFERENCES

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