Monoclonal antibodies and the characterization of apolipoprotein structure and function

Prog. Lipid Res. Vol. 23, pp. 169-195, 1985 Printed in Great Britain. All rights reserved

0163-7827/85/$0.00 + 0.50 f~?) 1986 Pergamon Press Ltd

MONOCLONAL ANTIBODIES A N D THE CHARACTERIZATION OF APOLIPOPROTEIN STRUCTURE A N D FUNCTION YVES L. MARCEL, PHILIP K . WELCH, PETER MILTHORP, FRANCOIS TERCE, CAMILLA VEZINA a n d R o s s W . MILNE

Laboratory of Lipoprotein Metabolism, Clinical Research Institute of Montreal, Montreal, Quebec, Canada CONTENTS I. OVERVIEW II. APOLIPOPROTEINSB A. Introduction B. Structural and functional domains of apo B identified by MAB 1. Positions of the antigenic determinants on LDL apo B 2. Positions of the antigenic determinants of LDL apo B relative to the LDL receptor recognition site on apo B 3. Position of antigenic determinants on apo B species and proteolytic fragments 4. Distribution of antigenic determinants on tryptic fragments of apo B C. Relationships between the immunizing antigens and the location of resulting antigenic determinants which are recognized on apo B by different MABs D. Role of lipids in the expression of antigenic determinants on apo B-containing lipoproteins E. Heterogeneity in the expression of apo B antigenic determinants in lipoproteins F. Segregation and characterization of lipoproteins containing apo B100 and apo B48 III. AeOLII'OPROTEINS E A. Introduction B. Characterization of apo E binding site to the cellular receptors IV. APOLIPOPROTEINSAI A. Introduction B. Characterization of apo AI antigenic determinants recognized by series I MAB C. Characterization of apo AI antigenic determinants recognized by series 2 MAB and comparison to those recognized by series I MAB 1. Specificity in RIA 2. Absence of cryptic sites in serum 3. Mapping of antibody binding sites on apo AI 4. Conclusions D. Effect of sample storage on apo AI immunoreactivity I. The immunoreactivity of fresh and stored sera with series 1 MAB 2. The immunoreactivity of fresh and stored sera with series 2 MAB 3. Retrospective E. Effect of pH on apo AI antigenic sites 1. Apo AI isomorphs 2. Alkaline treatment modifies antigenic sites 3. Optimum conditions for modification of antigenic sites 4. Reaction of series 2 MAB with apo AI samples treated with NaOH F. Effect of alkaline pH on apo AI physicochemical properties and immunoreactivity G. Conclusions V. LECITHIN: CHOLESTEROLACYLTRANSFERASEAND APOLIPOPROTEINSD A. Introduction B. Production of MAB against LCAT and apo D C. Characterization of antibodies against LCAT D. Characterization of antibodies against apo D E. Redefinition of apo D as an antigen ACKNOWLEDGEMENTS REFERENCES

[ 70 170 170 171 171

172 172 173

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ABBREVIATIONS VLDL, very low density lipoproteins; IDL, intermediate density lipoproteins; LDL, high density lipoproteins; HDL, high density lipoproteins; apolipoprotein, apo; MAB, monoclonal antibody(ies); RIA, radioimmunoassay; IEF, isoelectricfocusing. J.P.L. 2~4 A

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Although the technology for the production of monoclonal antibodies is recent, it has seen a most rapid expansion and monoclonal antibodies have found applications in most fields of biology. Because of their highly defined specificity, they have been used in many immunoassays and have allowed the development of a new format of assays such as the immunometric or sandwich assays. More importantly, they have found extensive applications in immunocytochemistry, in immunoaffinity systems and as probes for the study of the structure and function of proteins, especially enzymes, hormones and receptors. The monoclonal antibodies provide powerful tools to study cellular membrane proteins allowing the quantitation and the characterization of functional domains of these proteins and providing markers to evaluate their distribution within cells. Applications of monoclonal antibodies to the field of serum lipoproteins only date back to four years ago but yet new information has been obtained which may not have been possible otherwise. It is the purpose of this review to illustrate how monoclonal antibodies can be applied to the characterization of apolipoproteins and more specifically to the delineation of their functional domains. This approach has been particularly successful for apo B and E, which interact with specific cellular receptors, and their respective binding domains have been identified. Applications to other apolipoproteins and related enzymes are more recent and the progress is limited. Nevertheless, the immunologic heterogeneity of apo AI in its native or delipidated forms has been shown and the drastic alterations which occur for certain of its antigenic determinants under in vitro conditions have been documented. Finally, we will also examine how, in the case of apo D, monoclonal antibodies demonstrate the previously unrecognized heterogeneity of their antigen.

ii. APOLIPOPROTEINS B A . Introduction

Apolipoproteins B are the second most abundant proteins of human plasma lipoproteins and they play an essential role in the intravascular transport of lipids, mostly in the form of neutral lipids: triglycerides and cholesteryl esters. Accordingly, one should expect proteins which bind to such lipids to be highly hydrophobic, and indeed they are. It is probably because of this unique property that apo B remains to date one of the most poorly characterized apolipoproteins. Delipidated forms of apo B are highly insoluble in non-denaturing aqueous buffers and tend to become aggregated even in the presence of detergents. To further compound the difficulty of the study, apo B has been shown to be susceptible to proteolysis as well as oxidative cleavage. These serious technical obstacles have so far prevented the identification of the monomeric units of apo B and reports to date range from about 8 x l03 to 550 x 103 daltons. 44 Apo B is a glycoprotein which contains 8 to 10% of carbohydrates, mainly galactose, mannose, glucosamine and neuraminic acid. Two complex oligo saccharide chains have been identified which are characterized by high mannose content. 95 The high carbohydrate content may have contributed to the variations in molecular weight estimate for apo B cited above. Recently progress has been made in the characterization of apo B with the recognition of its structural and metabolic heterogeneity. 46 In the human, the major species of apo B is synthesized by the liver and released into the circulation mostly in the form of VLDL which serve for the intravascular transport of endogenous triglyceride. The V L D L are eventually transformed into IDL and LDL by action of lipoprotein lipase and other factors. It appears that this hepatic apo B remains associated with the same lipoprotein particle during its intravascular cycle. It has an apparent molecular weight in SDS gel electrophoresis of 549,000 and has been termed apo B100 using a centile system of nomenclature based on molecular weight. 46 The other species of apo B which is present in the chylomicrons has an apparent molecular weight of 264,000 and is called apo B48 in the same nomenclature. Apo B48 and apo BI00 differ in amino acid composition 46 and appear

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to be under separate genetic control, as a newly discovered disorder has been described where chylomicrons containing apo B48 are secreted but where none of the apo B100-containing lipoproteins are present28 Two other forms of apo B, apo B74 and apo B26, exist in the LDL of certain individuals and evidence suggests that they represent complementary fragments of apo B10046 and that they originate by proteolytic cleavage of the parent apo B100, possibly by action of Kallikrein}5 Apo B100-containing lipoproteins are catabolized via the LDL receptor which recognizes both apo B100 and apo E (also called the apo B/E receptor) and which is present on the membrane of most mammalian cell types. ~2 Lipoproteins which enter the cell by the LDL receptor pathway are internalized by endocytosis and hydrolyzed in the lysosomes. The cholesterol which is liberated into the cytoplasm by this mechanism controls a series of metabolic events: (1) the rate-limiting enzyme for cholesterol synthesis, 3-hydroxy-3-methyl glutaryl coenzyme A reductase, is inhibited, (2) the activity of acyl-CoA cholesterol acyltransferase is enhanced which allows the intracellular esterification of excess cholesterol, and (3) finally the LDL receptor is down-regulated by inhibition of its synthesis. The interrelated sequence of events is called the LDL pathway and supplies the cell with exogenous cholesterol while also preventing its excessive cellular accumulation and synthesis. In contast, apo B48-containing lipoproteins are catabolized by a different receptor system which is located on hepatocyte membranes and which recognizes apo E as a ligand. 3s This is called the chylomicron remnant receptor or the apo E receptor.

B. Structural and Functional Domaines of apo B Identified by MAB We shall review here the application of MAB as probes for the identification and eventual characterization of apo B structure and heterogeneity, first concentrating on the contribution from our own laboratory and second comparing them with the results of others. By immunization of mice with normal human LDL isolated between the densities of 1.030 and 1.050 g/ml, we have produced a series of murine MAB which react with LDL apo B.71 Seven of these MAB have been studied in detail. All were of the IgG class and had affinity constants in the range 7~ of 10 9.

1. Positions of the Antigenic Determinants on LDL apo B From co-titration experiments, where LDL are immobilized on plastic and where serial dilutions of one or two antibodies are reacted with the ligand, one can determine whether the two MAB react with the same or different antigenic determinants on the antigen. Using this technique, we were able to conclude that five of the seven MAB (3F5, 4G3, 3A10, 3A8, 5El 1) were specific for determinants clustered together in the same region of LDL. For example, 3F5 interferes with the binding of 4G3 and 4G3 blocks the binding of 3A8 and 3A10, but the latter do not prevent the binding of 3F5. In contrast, the determinants recognized by 1DI and 2D8 are distant from one another and from the cluster of determinants recognized by the five other antibodies. From these experiments, we have proposed a theoretical linear map of the spatial relationship of these antigenic determinants on LDL, as illustrated below (Scheme 1). In this map, the placement of

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determinants 1D1 and 2D8 is arbitrary but that of five others is predicted by the co-titration experiments. 7t

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2. Positions of the Antigenic Determinants of LDL apo B Relative to the LDL Receptor Recognition Site on apo B The spatial relationship between the antigenic determinants recognized by the MAB and the site on apo B recognized by the LDL receptor has been evaluated by the ability of Fab fragments from the different antibodies to prevent the binding of LDL to fibroblasts. Even in large molar excess relative to apo B, antibodies 1D1 and 2D8 do not block the binding of LDL, whereas the Fab fragments of 3F5, 4G3, 3A8, 3A10 and 5Eli are capable of blocking both the binding of LDL to its receptor and the LDL-mediated suppression of cellular cholesterol synthesis. 7~ Therefore, the determinants which have been grouped together in the co-titration experiments are also those that are capable of interference with the L D L pathway. They are thus located within or near the domain which on apo B interacts with the LDL receptor (Scheme 2). The fact that 3F5 is intermediate in its ability LDL RECEPTOR RECOGNITION SITE I

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to block both L D L binding and LDL-mediated suppression of cholesterol synthesis compared to the other antibodies may be a reflection of its relative distance from the actual binding site. Likewise, 3F5 is also located on the edge of the cluster formed by these determinants and the two observations are consistent with one another. 3. Position of Antigenic Determinants on apo B Species and Proteolytic Fragments To this point we had identified functional domains on the intact L D L molecule but to position these sites in relation to one another on both intestinal and hepatic apo B, we needed to study apo B species in the absence of their respective lipids which modify the apoprotein configuration and bring about an additional complexity to the immunologic cross-reactivities. For these experiments, delipidated apo B or its proteolytic fragments have been solubilized in the presence of SDS, submitted to SDS gel electrophoresis and the migrated proteins have been transferred electrophoretically to nitrocellulose paper. 62 The apo B species and fragments which uniformly bound to SDS molecules and to nitrocellulose have been tested for reactivity with the MAB. The results of these studies are summarized in the following composite scheme (Scheme 3). Two of the antibodies, I DI and 2D8, cross-react with the intestinal form of apo B, apo B4862 which indicates sequence homologies between apo B48 and apo B100. These results are also compatible with the possibility that the B48 sequence may be contained within that of BI00. With these observations, we were able to improve the map of the theoretical spatial relationship of the determinants. For example, because the presence of determinants ID1 and 2D8 on both apo B48 and apo B I0 indicate that at least part of apo B48 sequence is contained within that of apo Bl00, then determinants 1D1 and 2D8 should be placed on the same side of the map in relation to the cluster of determinants corresponding to the LDL receptor recognition site. Such a displacement of the determinant recognized by 1DI is, in fact, compatible with the co-titration experiments since these had not assigned any specific position to either 1D1 or 2D8. In addition, antibody 1D1 is the only one which cross-reacts with apo B26 and apo BI00 whereas all others from our series react with both apo B74 and apo BI00, a finding which is consistent with the hypothesis that the two species represent complementary fragments of apo B100. 4~ Therefore, in keeping with these results, 1D 1 was placed first on the map of the antigenic determinants and next to 2D8. In the course of these experiments, we also noted the existence in some LDL samples of an apo B fragment with an apparent molecular

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LDR RECEPTOR RECOGNITION SITE r 1

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LDL 8100 B74 826 B48 B50

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equivalent to apo B50 in the centile nomenclature system of Kane et al., 46 which was present in very low concentration but which reacted strongly with antibodies 2D8, 3F5 and 4G3. This result also corroborates the hypothesis that these three determinants are adjacent to one another, provided that the band identified as apo B50 is homogeneous and results from a double cleavage on the parent apo B100. 4. Distribution of Antigenic Determinants on Tryptic Fragments of apo B In order to gain further insights into the spatial distribution of antigenic determinants on apo B100, we submitted LDL to tryptic digestions and the resulting fragments have been separated by SDS gel electrophoresis or by two-dimensional electrophoresis. After transfer to nitrocellulose, the resulting replicas of the separated fragments have been reacted with the different MAB. 99 Under these conditions, the smallest single fragment which bears all the antigenic determinants is a peptide of 125,000 daltons. As this represents about 25~o of LDL apo B, this may be interpreted as evidence that the majority of the apo B molecule is hidden in LDL perhaps by lipids, and is thus nonimmunogenic. The determinant present on apo B26 is dissociated from all other determinants on fragments of less than 125,000 daltons and thus no further information on its spatial location relative to the others could be gained from these experiments. In contrast, the determinants recognized by 3F5 and 4G3, which had been previously shown to be close together on LDL, also appear close together in the primary structure of apo B since they are present together on a peptide of 43,000 daltons. Similarly, the sites recognized by antibodies 3A8 and 5El 1, which have been found close to one another on co-titrations using LDL, are also present together on fragments of 72,000 and 68,000 daltons. Surprisingly, the determinant 2D8 which is present on both apo B48 and apo B100, and the determinant 3A8 which is near the recognition site of the LDL receptor on apo B100, are also present on the same tryptic fragment which has an apparent molecular weight of 43,000. The pattern of reactivity of the MAB with the tryptic fragments is different from that seen with two lectins specific for the two main oligosaccharide chains of apo B, 95 which suggests that the sites recognized by the antibodies are not constituted by the principal carbohydrate moieties of apo B, 99 although the site recognized by 2D8 appears to be close to a carbohydrate chain having a terminal mannose residue. The various reaction of the MAB with the tryptic fragments of LDL and apo B have been summarized on Scheme 4a. In Scheme 4a, the dotted lines illustrate the possibility that the determinants recognized by 3F5 and 4G3 are present in the fragments of 72,000 and 43,000 daltons but that they may not be expressed. However, this is very unlikely because antibodies 3F5 and 4G3 have always reacted very strongly with apo B fragments solubilized in SDS that bear these determinants and which are immobilized on nitrocellulose replica. In order to reconcile the different observations made about the reactions of the apo B fragments with the antibodies, the series of determinants which overlap with the LDL receptor recognition site can also be inverted to give its mirror image without contradiction with the co-titration experiments. The new map thus obtained (Scheme 4B) again contains dotted lines in the fragments B50 and 43,000 daltons which indicate their negative reaction with antibodies

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5El 1, 3A8 and 3A10. Because these antibodies have generally displayed relatively weak reactions with apo B fragments solubilized in SDS, the map represented on Scheme 4b may be more consistent with the experimental results than that of Scheme 4a. However, the real spatial relationship of antigenic determinants on LDL apo B as well as on apo B fragments is a three-dimensional structure, which we have schematized as a linear map. Therefore, this map must be considered and used only as long as it can logically accommodate the experimental results. In addition, because of its voluntary simplication, the linear map of apo B determinants must not be assimilated to a representation of the primary sequence of apo B. Also, the likelihood that long hydrophobic sequences of apo B are imbedded into the lipid phase and thus are nonimmunogenic would prevent such a map to be used as a representation of primary structure. Given these limits and restrictions, it is most rewarding that the spatial relationships of the determinants as delineated by the co-titration experiments of LDL have held through in most of the experiments with partial fragments of apo B. The same organization is also confirmed using a new series of MAB this time raised against delipidated and solubilized apo B (R. W. Milne and Y. L. Marcel, unpublished experiments). These new antibodies, in fact, give us the clue to the reasons behind the similar arrangements of determinants in LDL and in apo B fragments as it appears that LDL apo B and apo B in the presence of SDS assume a similar conformation although the antigenicity of specific areas of the molecule is modulated by iipids.

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C. Relationship Between the lmmuniz&g Antigens and the Location of Result&g Antigenic Determinants which are Recognized on apo B by Different MABs As reviewed in the Introduction, apo B is the major apolipoprotein in a variety of lipoproteins which vary in size from large to small VLDL, to IDL and to LDL. Each of these density classes comprise a spectrum of particles which carry different ratios of lipid relative to protein, different proportions of core neutral lipids, mostly triglycerides and cholesteryl esters and different complements of other apolipoproteins such as apo CII, apo CIII and apo E. As work progresses on the immunochemical characterization of apo B, it has become obvious that the antigenic determinants expressed on this apolipoprotein vary greatly as a function of the hydrated density of the lipoprotein class in which it is present. Conversely, when different classes of apo B-containing lipoproteins are used for immunization, different batteries of monoclonal antibodies are obtained which recognize determinants characteristic for that lipoprotein class. Curtiss, Edgington and colleague23'1°3 have characterized a series of MAB against apo B which were obtained from mice immunized with either VLDL or IDL prepared from plasma pools made up of three or more normal, healthy donors and from which chylomicrons had been removed. The majority of the MAB thus obtained reacted with both BI00 and B48; in addition, all of their antibodies reacted with the B26 fragment of apo B. In contrast, Tikkanen, Schonfeld and colleagues,26'~°°'l°2whoimmunized the mice with LDL (d 1.025-1.050 g/ml) isolated from the plasma of a donor with homozygous familial hypercholesterolemia, obtained MAB which reacted with determinants present on both B100 and B74 but not on B26. Three of these antibodies also crossreacted with B48. The latter series of antibodies is therefore similar in determinant positions to our series of MAB, most of which react with B100 and B74 with the exception of one which reacts with B100 and B26 and with two that cross-react with B100 and B48 and which were obtained after immunization of mice with similar LDL but from a normolipemic donor. Whereas, the antibodies raised by immunization with VLDL or IDL could not block the LDL pathway in cultured human fibroblasts (Curtiss, personal communication), two of the seven antibodies raised by immunization with LDL from familial hypercholesterolemia1°2and five of the seven antibodies resulting from immunization with normal LDL 7~ could block the binding of LDL to its receptor. These results may be, in part, related to the fact that LDL from familial hypercholesterolemia is a larger and lipid-enriched lipoprotein compared to LDL from normal subjects. 97 It indeed appears that the recognition site for the LDL receptor is not expressed on VLDL apo B but that, as lipolysis progresses and the particles become smaller, the conformation of apo B changes or the masking effect exerted by lipids disappears which results in the expression of the LDL receptor binding site on the apo B.5~ It is for this reason that immunization with LDL but not with VLDL can generate antibodies which recognize determinants either close to the receptor binding site or capable of interaction with the binding site. Conversely, the antigenic determinants found on the B26 fragment are best exposed on triglyceride-rich lipoproteins and, in order to generate antibodies reacting with these determinants, the most efficient immunization is done with VLDL rather than LDL.

D. Role of Lipids in the Expression of Antigenic Determinants on apo B-Containing Lipoproteins In order to study the function, if any, of lipids in the antigenicity of apo B, the most direct approach was to prepare a delipidated and solubilized form of apo B by a method described by Cardin et al. ~4 which yields a protein soluble in the absence of denaturing agents or detergents. Of the six antigenic determinants previously characterized by us on LDL apo B 62'71 (Scheme 4B), only one, 1DI, was found to be significantly expressed in the soluble form of apo B 63 In the hope of regenerating immunoreactivity of the other

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determinants on the soluble apo B, the protein was incorporated in various amphipathic lipids or structures such as SDS micelles, cholesterol-lecithin liposomes or microemulsions consisting of cholesteryl oleate core stabilized by a phosphatidylcholine layer. ~4 When apo B was equilibrated with either SDS micelles or with cholesterol-lecithin liposomes, the immunoreactivity of the determinant recognized by antibody 2D8 was partially generated but not that of the others. 6~ In contrast, incubation of apo B with the microemulsion preparation also restored the antigenicity of the determinants reacting with antibodies 3F5, 4G3 and 5El 1. However, the regeneration of these antigenic determinants could only be achieved when solubilized apo B was treated with SDS prior to equilibration with microemulsions. 6~ These results are important in several respects. First, the immunoreactivity of 1DI with soluble apo B was found to be always equal or less than that with L D L thus indicating that delipidation did not uncover any additional 1D1 sites on LDL particles. Also, because the 1D1 determinant is expressed on both soluble and LDL apo B, it is likely to be constituted by the primary or secondary sequence of apo B and may serve as a useful marker for studies of apo B sequence and structure. In other studies, we had shown that the determinant 1DI is present in chylomicrons but requires partial delipidation for its expression. 62 Similarly, in VLDL, the determinant ID1 is expressed but its immunoreactivity increases with partial delipidation. 6~ In contrast, in LDL, the immunoreactivity of 1D1 is unaffected by partial or total delipidation. 6~63 Therefore, although the determinant 1D1 does not require lipid for its expression in LDL apo B, its immunoreactivity is decreased or absent in V L D L or chylomicrons. Such a variation in triglyceride-rich particles may be attributed either to a masking effect by lipids such as triglycerides or to conformational constraints imposed on apo B by the lipid load or by the size of the particle itself. Located next to ID1 on the map, the antigenic site recognized by 2D8 requires for its expression that apo B be equilibrated with minimally amphipathic structures such as those provided by SDS micelles or cholesterol-lecithin liposomes. We also know that the determinant 2D8 is about equally expressed in chylomicrons, VLDL and LDL. 6~62 Therefore, this determinant is located in a domain of apo B which is thermodynamically stable in the presence of lipids but is relatively unaffected by the different lipids or by the size of the particle in which apo B is present. The second and most important finding in these studies was the stringent requirements of apo B for defined lipid structures with a hydrophobic core at the sites recognized by antibodies 3F5, 4G3 and 5El1. These are the sites that are close to the LDL receptor recognition domains, and it follows, therefore, that this domain of apo B must adopt a very specific conformation to express its normal antigenic determinants. Conversely, we propose that this specific conformation is also that which is required for the recognition of LDL apo B by the L D L receptor. Indeed, a number of recent studies suggest that for apo B as well as apo E, the conformation of these apolipoproteins is the crucial element in their abilities to bind to the LDL receptor, j°'42As lipolysis of V L D L by lipoprotein lipase progresses, V L D L particles become denser, We can hypothesize that, as the lipoprotein diameter decreases, the packing of apo B increases which results in the concentration of positive charges in the receptor binding domaine of the protein. At the stage of V L D L (Sf 20-60), these smaller particles start to present the appropriate concentration of positive charges of apo B and begin to express LDL receptor binding activity. ~° While it is likely that a similar conformation of this apo B domain is essential and necessary for the expression of both receptor being activity and antigenic determinants 3F5, 4G3 and 5El 1, these antigenic determinants can be expressed at least in part on lipoproteins which do not bind to the receptor such as VLDL. 6~These observations can be rationalized if one assumes that the domain of apo B which binds to the receptor represents a large portion of the apo B molecule which contains several of the antigenic determinants. In addition, these determinants may be only close to, but not within, the receptor binding domain of apo B. In conclusion, the experiments discussed above have defined three classes of determinants on apo B, based on their lipid requirements: (I) determinants such as that

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recognized by ID1 which have no absolute requirement for lipid although they may be differentially expressed as a function of the lipid load; (2) those which require the minimal presence of amphipathic lipids, such as 2D8 and (3) the determinants which require a lipid structure that mimics LDL and which is characterized by a neutral core of cholesteryl esters surrounded by amphipathic lipids, such as 3F5, 4G3 and 5Ell, the latter determinants are also those which are close to the LDL receptor binding site. These lipid requirements of apo B antigenic determinants are summarized and illustrated in Scheme 5. LIPID REOUIREHENTS FOR EXPRESSIONOF APO B ANTIGENIC DETERI~INANTS

B-48/B-IO0

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SCHEUE5, E. Heterogeneity in the Expression of apo B Antigenic Determinants in Lipoproteins

As we have seen above, most of the determinants recognized by our antibodies require lipids for their expression; this feature should result in a variation of their immunoreactivity in the various plasma lipoproteins. Apo B-containing lipoproteins constitute a heterogeneous populations of particles which vary in size as a function of their lipid load and which contain different types of neutral lipid from chylomicrons to VLDL to IDL to LDL. In addition, the lighter and larger of these particles also contain many other apolipoproteins whereas LDL contains mostly apo B. It is not presently known whether the presence of several apolipoproteins on the same particles cause steric hindrance and tighter packing of the molecules on the surface of the lipoproteins, however, competitive binding and displacement between apo CIII and apo E for VLDL surface indicate that the apoproteins interact with one another) 7 In keeping with these considerations, several groups working with different batteries of monoclonal antibodies have observed variations in the expression of many antigenic determinants in apo B-containing lipoproteins. Schonfeld et al. 88 had first shown that certain polyclonal antisera to LDL could detect a progressive increase in immunoreactivity in radioimmunoassay of the particles as they are transformed by lipase action from the large triglyceride-rich VLDL to the smaller cholesteryl ester-rich LDL. The same phenomenon is also observed with certain monoclonal antibodies against apo B. 6i'i°°'i°3 Tikkanen e t al., 1°° studying different subclasses of VLDL, concluded that as the VLDL become smaller, there is an increase in immunoreactivity due to changes either in relative apparent affinities or number of antigenic determinants or both depending on the individual determinants and VLDL donors. Tsao et al., t°3 who have used monoclonal antibodies raised against VLDL and IDL, also observed the immunological heterogeneity ofVLDL, IDL and LDL. They concluded that there exists at least three patterns of epitope expression. The first, which is characterized by MAB which fail to distinguish amongst apo B determinants on VDL, IDL and LDL; the second pattern which is characterized by MAB which react better with the determinants on VLDL and IDL than with those on LDL, and finally a third pattern characterized by MAB which do not distinguish between determinants expressed on IDL and LDL but have lower affinities for those on VLDL. These results are consistent with those observed with several of our antibodies which expressed differential reactivities with VLDL and LDL. Whereas antibody 2D8 reacts equally with VLDL, IDL and LDL, antibody ID1 which is specific for a determinant

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present on both apo B100 and apo B48, recognizes only 10~ of the apo B present in VLDL and will react with chylomicrons only after partial delipidation. 6~ This differential immunoreactivity of the determinants is further complicated by the observations of intersubject variation. Tikkanen et al. ~"~ have observed with LDL prepared from patients randomly selected at a lipid clinic, immunoreactivities in radioimmunoassays which for certain determinants ranged from 30 to 400')~, of an LDL standard. The immunoreactivity of these LDL with four of the antibodies was positively correlated with the relative phospholipid concentration of the LDL while another antibody detected a negative correlation with triglyceride and a positive correlation with cholesterol content of the LDL. We also observed that the immunoreactivities of certain of our antibodies are highly and positively correlated with the cholesterol to protein ratio of LDL subfractions isolated from normal subjects and from patients with hyperapobetalipoproteinemia? 6 This ratio is an index of particle size as well as density and therefore indicated that, as the L D k size decreased, the conformation of the LDL apo B changed progressively resulting in decreased immunoreactivity with antibodies 2D8, 3F5 and 4G3. Patton et al. 7~ have reported that one anti-LDL monoclonal antibody was more sensitive than a conventional antiserum to apo B in discriminating between patients with and without angiographically documented coronary heart disease. It appears therefore that monoclonal antibodies against apo B could be selected which would be able to identify subjects at risk tbr atherosclerosis. However, it remains to be seen whether these inter-subject differences detected by antibodies represent changes in apo B conformation related to the lipoprotein physicochemical composition or to true apo B polymorphism. A genetic polymorphism of human L D L has been described by Shumaker et al. 9~ based on binding of mouse MAB. 23~°' Population studies indicated the existence of both homozygous and heterozygous populations and, based on family studies, a model was proposed consisting of two alleles at a single locus which segregate in a Mendelian fashion. The polymorphism appeared to be independent of the carbohydrate moiety of apo B and L D L particle size and may therefore be a function of the apo B gene. F. Segregation and Characterization o [ Lipoproteins containing apo B IO0 and apo B48

While in normal subjects chylomicrons and their remnants are catabolized rapidly and cleared by the apo E hepatic receptor, 55 in type III dyslipoproteinemia there is an accumulation of these lipoproteins because the patients are homozygous for an apo E allele coding for an apo E isoform which is poorly recognized by the apo E receptor. 68'82 The chylomicron remnants accumulate in a V L D L fraction which is characterized by a typical broad B-electrophoretic migration. However, the lipoproteins in this fraction contain both apo B100 and apo B48. z745 Using monoclonal antibodies specific for apo B100 (5El 1 and 4G3) coupled to Sepharose, we could separate by immunoaffinity the V L D L from type III patients into retained and non-retained fractions which contained, respectively, apo B100 and apo B48 as their sole apo B species. 72 This result clearly establishes that, in human, the apo B100 and apo B48 lipoproteins which are, respectively, of hepatic and intestinal origin 44 remain segregated and constitute different lipoprotein families. Apo B48 V L D L have an electrophoretic migration characteristic of chylomicrons and are enriched in apo E and cholesteryl esters and depleted in triglycerides. In contrast, apo BI00 VLDL have a B-electrophoretic migration and are triglyceride-rich and cholesteryl ester-poor relative to the apo B48 V L D L fraction. 72 Type IV hyperlipoproteinemia, which is characterized by elevated plasma triglycerides and normal L D L cholesterol, is caused by the hypersecretion of large triglyceride-rich V L D L 13'16't7"35 in association with a saturated or defective catabolism of VLDL.16'35'78 When V L D L from type IV were similarly separated by immunoaffinity on 5El1- and 4G3-Sepharose into apo B48 and apo B100 fractions, the apo B48 VLDL was found to have an increased triglyceride to cholesteryl ester ratio compared to the apo B100 V L D L ~8 in contrast to the results obtained with type III VLDL. 72 Nevertheless, apo B48 VLDL from type IV are similar to those of type III in their electrophoretic migration and relative

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enrichment in apo E. These studies show that, in type III and type IV hyperlipoproteinemia, the lipid composition of the VLDL subclasses reflect the metabolic defects associated with the respective disorders, i.e. the cholesteryl ester enrichment of the apo B48 VLDL is due to its defective recognition by the apo E which results in a longer vascular half-life of the particle and a greater exposure to the cholesteryl ester transfer process. 64 One of the most intriguing findings of these studies is the existence of immunochemical heterogeneity of apo B100 VLDL in both type III and type IV as in normal subjects. 72'98 Indeed, apo Bl00 VLDL could not be totally removed by repeated passage on either 5El l or 4G3 Sepharose whereas it was completely retained by sequential passage on the two immunoadsorbers. This demonstrates that subpopulations of apo B100 VLDL exist which express one but not both antigenic determinants 4G3 and 5El 1. At this stage, we can only speculate on whether this phenomenon represents masking of one of these determinants in some VLDL or the existence of subpopulations with different compositions and different metabolic functions. The ability to isolate apo B48 VLDL also allowed us to test if apo B48 can serve as a ligand for the apo B/E and apo E receptors/2'55 Both apo B48 and apo BI00-VLDL isolated from type III were shown to be capable of binding to receptors on fibroblasts and hepatic cell membranes. However, in the case of apo B48 VLDL, the binding could be totally abolished with the anti-apo E antibody 1D7 which blocks specifically the binding of apo E to the receptors, 1°9whereas under the same conditions, apo B100 VLDL showed residual binding.4° This residual binding of apo B100 VLDL could be blocked completely in the presence of both antibodies 1D7 and 4G3, and it could be increased by treatment of the particles with lipase while the treatment had no effect on the binding of apo B48 VLDL. 4° These studies thus demonstrate that apo B48 does not include the domaine which is recognized by the LDL (apo B/E) receptor and that apo B48 VLDL binds to the apo B/E and apo E receptors only because of its apo E content. III. APOLIPOPROTEINS E A. Introduction Apo E is a 299 amino acid protein which is produced primarily in the liver but also by numerous other peripheral tissues?4'57 It is a component of chylomicrons and their remnants, of VLDL and of certain subclasses of HDL. Like apo B, apo E is recognized by the LDL (apo B/E) receptor and can thus mediate particle uptake by the LDL pathway. In addition, a second receptor specific for apo E has been identified on liver cells which is thought to be responsible for the hepatic capture of chylomicron remnants. Apo E exhibits genetic polymorphism. Variant forms of apo E have been shown to differ from the parent form, apo E3, by amino acid substitutions at various locations in the molecule. Nomenclature of the variant forms is based on their electrophoretic charge and specific amino acid substitution relative to apo E3. Thus, apo E4 (cysll2-arg) differs from apo E3 by the substitution of an arginine for a cysteine at residue 112 whereas the most common form of apo E2, apo E2 (arglss-cys), differs from the parent form by substitution of a cysteine for an arginine at residue 158) 3,H° Amongst other variants which have been identified are apo E2 (arg~as-cys)82and apo E2 (lys~46-gln).sl Variants which result from the replacement of an arginine or lysine by a neutral amino acid in the region of apo E between residues 140 and 160 have an impaired ability to react with both the LDL receptor and hepatic apo E receptor? 6 Homozygosity for alleles which code for such apo E variants appears necessary for the expression of type III hyperlipoproteinemia, a condition characterized by the accumulation in the plasma of remnant particles. B. Characterization of apo E Binding Site to the Cellular Receptors Apo E purified from a pool of plasma VLDL from normal fasted subjects was used as immunogen and screening antigen in the production of a series of anti-apo E MAB. 7° Five

IN()

Y.L. Marcel et

al.

stable clones were obtained, all of which secreted MAB of the igG class with apparent dissociation constants between 2 x 109 and 7 × 101°.m9 When these MAB were used as reagents in radioimmunoassays to measure plasma apo E, an excellent correlation was tbund between values obtained by individual MAB and the levels measured by a mouse polyclonal anti-apo E serum. 7° All MAB and the polyclonal serum indicated a similar distribution of apo E in lipoprotein subtractions. Disruption of lipoprotein particles by chemical or physical means did not alter their immunoreactivity with the MAB which would indicate that the antibodies are directed against apo E determinants which are fully expressed in lipoproteins. When tested for cross-reactivity with chemically modified VLDL, one MAB, 1D7, was distinguished by its reduced reactivity with V L D L in which lysine residues were carbamylated. Weisgraber e t al. ~°~ have used these MAB in an attempt to identify the domain on apo E which is responsible for the interaction with the L D L receptor. The strategy employed was to first identify MAB capable of blocking binding of apo E to the receptor and then to localize their corresponding determinants on the apo E molecule. Of five anti-apo E MAB tested, only one, I D7, was capable of blocking specific apo E binding to cultured human fibroblasts. The determinant for I D7 was shown to be present on a thrombin fragment of apo E which included residues 1 to 191 and on a fragment composed of residues 126 to 218 produced by cyanogen bromide cleavage. MAB 1D7 bound to two synthetic apo E fragments which included, respectively, residues 129 to 169 and 139 to 169, but not to fragments 144 to 169 nor 148 to 169. The four MAB which failed to block binding of apo E to the fibroblast receptor and which appear to react with a determinant on the extreme N-terminal region of apo E (Karl Weisgraber et al., unpublished results) did not recognize any of the synthetic fragments. Apo E2 (argls~-cys) showed the same immunoreactivity with ID7 as did apo E3 whereas apo E2 (argL45-cys) and apo E2 (lys~46-gln) showed 49 and 32~0, respectively, of the immunoreactivity of apo E3. These observations show that the 1D7 determinant is located between residues 139 and 169 and that residues 145 and 146 but not 158 are probably implicated in the determinant. Thus, studies with MAB 1D7 together with the observation that those amino acid substitutions which decrease reactivity of apo E with cell surface are localized between residues 142 and 158 as well as results of cell binding studies using apo E fragments 4~ indicate strongly that the receptor-binding domaine is in the region of residues 140 to 160. It has been recently reported 42 that the arginine residue at position 158 may not be directly implicated in recognition o f a p o E by the receptor; however, a positive charge at this site may be essential to confer the proper conformation of the molecule to permit binding. It is interesting to note that 1D7 which blocks apo E recognition to fibroblasts shows no cross-reactivity with apo B. 7° These anti-apo E MAB have been used in experiments to identify the heparinbinding domaines of apo E employing a strategy similar to that which has been used for the receptor binding domaine (Karl Weisgraber et al., unpublished results). Apo E possesses two heparin-binding domaines, one in the region of the molecule which also includes the receptor binding domaine and the 1D7 determinant and a second in C-terminal region of the molecule. MAB against apo E have been used to assess the relative contributions of apo E and apo B to the binding of lipoproteins to receptors 39'4°'5~ Hui et al. 4" have shown that, while apo B-48 V L D L isolated from type III subjects show some binding to L D L and hepatic receptors, it can be totally eliminated by anti-apo E MAB 1D7 which indicates that apo E and not apo B-48 is responsible for the observed binding. In contrast, the binding of the apo B-100 V L D L fraction appeared to have both a major apo E and a minor apo B component. 394° Lipase treatment of apo B-100 V L D L increased the relative contribution of apo B-100 to the binding whereas lipase treatment of apo B-48 V L D L failed to induce apo B-48 recognition by the receptor. In a recent study, Krul et al. 5~ have used anti-apo E and anti-apo B MAB to show that, in large VLDL, apo E is the primary ligand recognized by the L D L receptor on cultured fibroblasts and that, as the particles become smaller, there is a progressive shift from apo E to apo B as the ligand recognized by the receptor. Bradley et al. > have shown a similar switch from apo E to apo B recognition

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by the LDL receptor concomitant with the conversion of hypertriglyceridemic VLDL to LDL. For this study, the authors have profited from the relative sensitivity of apo E and resistance of apo B to protease inactivation. IV. APOLIPOPROTEINS AI

A. Introduction Apolipoprotein AI is the major apolipoprotein of normal human plasma (about 140 mg/dl) and is found in the HDL, accounting for at least 55~o of the H D L protein. Apo AI structure and its function as a cofactor in the reaction of lecithin:cholesterol acyltransferase are well established; 26,6° however, its exact role in cholesterol transport remains in part speculative. 26 Serum concentrations of apo AI and H D L cholesterol are both negative predictors for cardiovascular disease but apo AI appears to add to the predictive value of H D L cholesterol, 7'52:H a finding which has further increased the interest given to this protein. Various immunoassays for apo AI have been reported which shared to different degrees the difficulties inherent in the determinations of serum apolipoproteins. 8°'93 Self-association of apo AI 3°:°6 has made difficult the development of appropriate standards while apo AI in lipoproteins does not express all its antigenic sites 59'84 indicating that some sites are masked by lipids in intact lipoproteins or that their ability to bind an antibody depends on the conformations of AI as affected by lipids or other proteins. To date, most of the work on the immunoassays of apo AI have been done with polyclonal antisera raised in rabbits or goats in response to immunization with purified apo AI. 93 Most standards used consisted of purified apo AI and the samples assayed have been pretreated by a variety of denaturing and chaotropic agents to simultaneously expose antigenic sites on apo AI and native lipoproteins. However, there is no consensus on the method to be used and even on the need for denaturing treatments. 93 One interesting approach to obviate the problem of differential antigenic determinant expression in isolated apo AI and plasma lipoproteins has been the selection of antibodies to apo AI antigenic sites exposed on the surface of the H D L particle. To this end, a polyclonal antiserum to apo AI is passed over serum lipoproteins coupled to Sepharose. Surface antigen-specific antibodies are retained on the column, subsequently eluted and used in immunoassays.59 Another valid approach to the problems of standardizing AI immunoassays is to use well-characterized monoclonal antibodies to study the expression of antigenic sites in AI, without the confounding effects of uncharacterized antibodies from polyclonal antisera. This will enable us to develop assays that quantitate AI antigenic sites common to all isomorphs and expressed in the presence or absence of lipids. 32'69'76'9°'I°4:13 Furthermore, monoclonal antibodies will provide precise markers to study structure-function relationship in apo AI. In the following pages, we will review the properties of two distinct series of monoclonal antibodies against apo AI which were produced by our laboratory and which have revealed some immunological properties that are unique to apo AI. These two series of antibodies are referred to as series 1 and series 2 MAB.

B. Characterization of apo AI Antigenic Determinants Recognized by Series 1 MAB A first series of monoclonal antibodies against apo AI has been produced from mice immunized with apo AI isolated from normal plasma HDL3 by preparative isoelectric focusing. 76 Likewise, the screening of the clones producing the antibodies 7° was also done using the same purified apo AI. From this first fusion and screening, we selected and cloned four hybridomas which each secreted antibodies of the IgG~ subclass and which are identified as 3D4, 5A6, 6B8 and 5G6. ~°8 Their specificity has been demonstrated, they all react similarly and with only the proteins having the molecular weight and pI characteristics of apo AI. The antibodies react with all known charge-polymorphs of apo AI and

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pro-apo AI, which therefore demonstrate the immunological similarity of these isomorphs/°8 In a solid-phase RIA using competing MAB, we found inhibition, enhancement or lack of effect of antibody binding to apo AI according to the pair of antibodies tested. Antibodies 3D4, 6B8 and 5G6 were different from one another and reacted with different antigenic determinants, but 5A6 was similar to 3D4 and reacted at the same site. ~°~ To further define the position of the determinants recognized by these antibodies, we compared their reactions with fragments of apo AI cleaved by CNBr, ]~ that we separated by polyacrylamide gel electrophoresis and analyzed by Western blots. Three different patterns of reaction with the apo AI fragments were found which corroborate and extend the determinant heterogeneity described above. The antigenic site for 6B8 is in fragment 3 and that for 3D4 and 5A6 is in fragment 2. The 5G6 reactions were not as intense on autoradiography as those of the other antibodies but they were strongest on partial cleavage fragments which included fragments ! and 2. Thus, the site for 5G6 is either on fragment 1, or spans the CNBr cleavage site between fragments I and 2, or requires the presence of both fragments 1 and 2 to form a conformational determinant. For further references, these antigenic determinants on apo AI have been called site B recognized by 5G6 on fragment 1-2, site C recognized by 3D4~5A6 on fragment 2 and site D recognized by 6B8 on fragment 3. 4F7"

566

3D4 3GfO 5A6

2F1" 4HI

\\," , / / / A'

A

C'

C

5F6 6E38

D ~_ COOH

CNBr 1

CNBr 2

243

148

112

86

CNBr 3

CNBr 4

SCHEME 6.

C. Characterization of apo A I Antigenic Determinants Recognized by Series 2 M A B and Comparison to those Recognized by Series I M A B The second series of monoclonal antibodies against apo AI were produced from mice immunized with H D L freshly prepared from normal human seraY As described in subsequent sections, a multiple screening of the clones producing the antibodies was carried out with freshly prepared HDL, H D L stored at 4°C and purified apo AI. From this fusion, seven clones were selected which secrete antibodies against apo A! and which are identified as 5F6, 4H1, 3G10, 2F1, 4F7, 5C7 and 3B12. v3 All MAB are of the IgG class except 5C7, which is an IgM. 3BI2 is an unstable clone which has not been included in most of the experiments reviewed in the following pages. As with series 1 antibodies, we have shown that series 2 MAB react with only one band corresponding to the major protein of H D L with a molecular weight of 28,000 on Western blots of SDS gel electrophoresis of apo HDL. 5C7 reacted relatively poorly, this is perhaps related to the low affinity of binding sites on IgM. 2F1 also bound poorly in this system in contrast to its relatively normal behavior in RIA. Again, with the exception of 5C7 and 2F l, which react also weakly with apo H D L separated by isoelectric focusing, the other antibodies reacted clearly with all apo AI isomorphs including those of pro-apo AI. Therefore, series 2 MAB also react with only the proteins having the molecular weight and pI characteristics of apo AI. 73 1. Specificity in Competitive RIA In the competitive RIA, purified apo AI inhibited the binding of all these MAB to apo H D L fixed to Immulon 2 wells. This suggested that all the antibodies were specific for sites

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on apo AI. However, the magnitude of the reaction of these MAB with purified apo AI allowed them to be separated into two groups. In series 2 MAB, only antibody 2F1 behaved like series l MAB (3D4, 5A6, 6B8) and reacted poorly with purified apo AI. About 20 times as much purified apo AI was required to produce one-half maximal binding compared to a preparation of HDL. This weak reactivity was initially attributed to loss of antigenic sites on apo AI during its purification from apo HDL. However, as will be explained later, this difference in reactivity is an H D L storage phenomenon resulting in an increase in the number of antigenic sites in H D L that are specific for this subgroup of MAB. In contrast, the remaining MAB from series 2 except 5C7 reacted equally well with purified apo AI, apo HDL, H D L and serum. 5C7 was not tested due to its high capacity binding of apo A|. These results suggest that, in at least the second series of MAB, purification o f a p o AI does not result in the loss of antigenic sites. Also, all these antibodies react with all the apo AI present in native serum. This make them potentially useful for quantitative assays as it indicates that there are no cryptic sites on apo AI for these antibodies due to lipid masking or different protein conformations. The experiments which confirm this hypothesis are described below.

2. Absence of Cryptic Sites in Serum We had initially observed an equivalent reactivity of apo AI in apo H D L and serum in the RIA; however, most polyclonal antibody preparations against apo AI have been reported not to react completely with apo AI in serum. 93 To ensure that the maximum number of reactive sites in serum for these MAB had been made available, preparations of serum, H D L and apo H D L have been treated with diisopropylether-butanol, high concentrations of Tween-20 or tetramethyl urea, heated at 52°C for several hours, or frozen and thawed. All these treatments have been shown to delipidate lipoproteins and/or denature apo AI, but none of these changed the reactivity of the apo AI-containing preparations in RIA using 3D4 as a representative antibody. These results coupled with the fact that serum apo AI reacted as well on a proportional weight basis as apo H D L suggests to us that these MAB react with all apo AI molecules present in serum. Also, the lack of effect of pre-treating the molecule with denaturing agents suggest that the MAB reacted with sites that were relatively independent of tertiary conformational constraints.

3. Mapping of Antibody Binding Sites on apo AI The binding sites for series 2 MAB were first mapped by competitive RIA between antibodies. The results for these experiments confirmed and extended those obtained for series 1 MAB. Antibody 3G10 maps next to site C, 5F6 maps at site D, while 4HI and 2F1 compete partially with each other and bind at sites different from those of other MAB. 73 The location of the individual antigenic determinants identified by series 2 MAB on apo AI-CNBr fragments was also identified. Antibodies 4HI and 2FI react with determinants present on CNBr fragment 1 on the amino terminal end of apo AI which are identified as sites A and A'. Antibody 3G10 recognizes a determinant on CNBrfragment 2 but at a site C' different from that of 3D4 and 5A6. In contrast, 4F7 shows the same specificity as 3D4 and 5A6 and reacts at site C on fragment 2. Finally, antibody 5F6 is specific determinant on CNBr fragment 3 and is mapped at site D together with antibody 6B8 from series I. 73 These results are summarized on the map of determinants showed on Scheme 6. Coincidence of binding sites between series 1 and series 2 MAB at apo AI sites C and D indicates that these antibodies are reacting with the same apo AI molecules. In addition, MAB from both series interacted with each other whether by competition or by enhancement of binding at the different sites, an observation which also suggests that all the mapped binding sites are present on the same apo AI molecules. If we consider the secondary structure of apo AI as predicted by Chou and Fasman

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rules j9'2° from apo AI sequence, ~ we observe that a large number of a-turns are located toward the N-terminus of the molecules and, consequently, the chain from residues 1 to 148 represents a densely packed region where :~-helices and fl-sheets are positioned next to one another (Scheme 7). This packing of the molecules in the N-terminal region where all the antigenic determinants identified by MAB from series 1 and 2 are located, explains how binding of antibodies to determinants seemingly remote on the primary structure but closer on the secondary structure may result in partial inhibition or in enhanced binding. I

4

16 ~

65 85 ~

9.4 zo,~

226 l

2141

~

185 200

SCHEME 7.

4. Conclusions

The fact that, after two separate fusions and different methods of selections of clones, only six antigenic sites were unambiguously observed suggests that the number of immunodominant sites on apo AI are limited. Since all the MAB react completely with AI resident in serum HDL, these immunodominant sites are not cryptic in serum. In order in part to verify this hypothesis, all mapping experiments were repeated with H D L instead of apo H D L bound in the solid phase. No differences in mapping were found. This indicates that the relationship between these MAB binding sites on apo AI were the same in apo H D L or H D L bound to solid phase, that is no change in location of M A B - a p o AI interactions occurred that could be related to tertiary conformational differences between apo H D L and H D L produced by apo AI-lipid interactions in HDL. Other workers have produced polyclonal antisera 5'47"53'85"86'89which react with apo AI only when lipid has been removed, indicating that the apo AI-lipid interaction masked some antibody binding site(s). Series 2 MAB were selected with purified apo AI and HDL. Consequently, after fusion, cells producing antibody only to apo AI were not cloned. This probably explains why no MAB to cryptic sites were produced. Schonfeld and colleagues 87 have also described a series of MAB directed against apo AI. Their antibodies also identified determinants located on CNBr fragments 1 and 3. The antibodies which react with the determinant present on fragment 3, recognized equally H D L and apo AI suggesting that this site was equally expressed on all H D L particles and solubilized apo AI. By contrast, the antigenic determinant present on fragment 1 was not expressed on all H D L particles, compared to purified and solubilized apo AI. Therefore, both our studies with two different series of monoclonal antibodies 7~'~°~ and those of Schonfeld and colleagues 87 indicate that the first three CNBr fragments on the N-terminal end of the molecule constitute the most antigenic regions of human apo AI. This is in contrast with earlier results where immunization of rabbits with human apo AI also elicited polyclonal antibodies that reacted with CNBr fragment 4 on the carboxy terminal end of the molecule. 84-85It is likely that these opposite results are related to species specificity and reflect the different antigenicity of human apo AI in the mouse and the rabbit. D. Effect of Sample Storage on apo AI Immunoreaeti~,itv 1. The Immunoreactivity of Fresh and Stored Sera with Series 1 M A B

During the development of apo AI assay, it became evident that series 1 MAB reacted

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very poorly with freshly prepared sera or plasma. TM Initially, a set of ten normal sera were measured with a secondary HDL3 standard which had been stored for several months at 4°C. Compared to the range of 1.2-1.4 mg/ml of apo AI reported by others for normal human sera 93 we detected from 30 to 60 times less apo AI than expected. This result was found using MAB 3D4 and 5A6 which react with apo AI on site B. A similar phenomenon occurred using MAB 6B8 which reacts with apo AI on site C. 5G6 which was known to react weakly with the standard also reacted poorly with fresh sera. It became clear that this was a general phenomenon and not the property of one idiomorphic antigenic site. At this point, we suspected that a component of sera was interfering with the assay, but mixing fresh and stored sera gave us additive immunoreactivity and therefore demonstrated that no inhibitor was present in fresh serum. Preparations of H D L and apo H D L isolated from these sera behaved in the same manner suggesting that cryptic antigenic sites were not involved. A comparison of antibody 3D4 immunoreactivity was then made between serum, H D L and apo H D L from one of the same donors that had been stored at 4°C for longer than two months and the corresponding fractions that had been freshly prepared. The stored samples contained 0.05% sodium azide and no sign of bacterial action or obvious deterioration was evident. In the RIA using 3D4, all the stored samples were at least 60-fold more immunoreactive than the corresponding fresh samples. Therefore, the samples stored at 4°C had gained immunoreactivity, while samples stored frozen for several months showed the same lack of reactivity as the fresh samples. This may be part of the explanation of the lack of reactivity of purified apo AI with series 1 MAB as it was stored frozen or lyophilized immediately after isolation. At present, no systematic study of the time course of generation of antigenic sites on apo AI upon storage has been done; however, preliminary experiments indicate that about two weeks at 4°C is required to detect an increase in activity. Pretreatment of fresh sera or H D L with diisopropylether butanol, high concentrations of Tween-20, tetramethyl urea, heating at 52°C and freezing and thawing had no effect on the reactivity of the fresh material. Based on these results, it was hypothesized that the apo AI has been chemically and/or structurally modified upon storage and that series 1 antibodies expressed a higher affinity with such modified structures. Although these MAB are perfectly adequate for detection of apo AI from fresh or stored samples on Western blots, they were not useful for quantification of apo AI. It was evident that this series of MAB had been produced by immunization and/or selection with a stored antigen that had undergone some undefined modification and that a new set of MAB was required for quantitation of apo AI by RIA. ~4

2. The Immunoreactivity of Fresh and Stored Sera with Series 2 MAB To produce these MAB of series 2, mice were immunized with freshly prepared and/or freshly prepared and frozen HDL. The selection was then made with a combination of fresh HDL, stored H D L and purified apo AI. Selection with apo AI was used to eliminate hybridomas making antibody to apolipoproteins other than apo AI found in HDL. The rationale for using stored H D L was to try and select a clone reacting with a determinant on apo AI that was not modified on storage at 4°C in the aqueous phase. This approach was partially successful. In order to evaluate the immunoreactivities of series 2 MAB, their reactions with freshly prepared H D L and apo AI were compared with that of stored H D L from the same donor. The series 2 antibodies fell into three categories: in the first category, antibody 2FI reacted in a manner similar to but more extreme than 3D4---a representative antibody from series 1; it reacted better with stored H D L than with fresh H D L or purified apo AI. In the second category, antibodies 3G10, 4F7 and 5F6 reacted equally with fresh H D L and apo AI but their immunoreactivities observed with these antigens were much greater than those observed with stored HDL. In the third category, 4HI reacted equally with fresh or stored H D L and with apo AI and thus appeared to be an ideal antibody for application to RIA of apo AI in various sera or lipoprotein classes. JP.LR

234

B

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Y.L. Marcel et al.

3. Retrospective The fact that mapping shows that sites C and D on apo AI react with both series 1 MAB ~°~ and with those in series 2 MAB, 73 which react well with fresh apo AI, suggests that there are specific sites on apo AI alterable by storage which initially reacted with one group of MAB and, after modification, with a second set of MAB. This increased reactivity with time is accompanied by a concurrent decrease in reactivity with some of the series 2 MAB reactive with these sites. TM The presence of a large number of glutamine residues in apo AI and the c o m m o n occurrence of deamidation of proteins on storage suggests that this chemical modification may be responsible for the above change in antigenic specificity. The fact that 4H1 reacts equally well with both fresh and stored apo AI suggests that site A is not modified on storage. However, 2F1, which reacts at a site A' close to site A, reacts poorly with fresh H D L and very poorly with apo AI. This indicates that the modifications of apo AI in vitro affects selectively defined regions of the molecule. For the purpose of quantitative assay of apo AI, MAB 4HI appears ideal and MAB 3G10, 4F7 and 5F6 can be used if special care, such as freezing, is taken with the storage of the apo Al-containing samples. At this point, it became clear that the profound modification of apo AI immunoreactivity which occurred upon storage at 4~'C warranted a detailed investigation of the structure of apo AI before and after such storage.

E. Effect (~[ p H on apo A I Antigenic Sites 1. Apo A I Isomorphs As has been mentioned above, delipidation and denaturing agents have no effect on the reactivity of series 1 MAB with apo AI sites C and D in fresh sera. It was felt, therefore, that a more fundamental chemical reaction was involved in the effect of storage of apo AI at 4~C. Previous results have shown that upon isoelectric focusing apo AI could be resolved into four to six b a n d s . 32"69"76'9°'1°4"t13 Interestingly, some of us had previously observed that apo AI polymorphism could be altered by specific pH conditions: the exposure of isolated apo AI3 alkaline conditions (pH = 8 . 9 ) followed by IEF resulted in regeneration of isomorphs at the pls of apo AI 3, AI4, AIs, AI6. 7~ In order to investigate whether storage also had an effect on this polymorphism, two preparations of apo H D L , one fresh and one that had been stored for several months, both from the same donor, were subjected to two-dimensional IEF and SDS-gel electrophoresis. These twodimensional gels showed that in the stored sample the apo AI isomorph pattern had been modified. In fresh apo AI, most of the stained protein appeared as apo AI 3, the next most abundant isomorph was apo AI 4. The two pro-apo AI isomorphs (apo Al~ and AI~ ) were only apparent in the fresh serum sample. In the stored sample, the protein appeared to be evenly distributed between isomorphs apo AI~ and apo AI 4 and a small amount of protein shifted into the more acidic isomorphs (apo AI5 and AI(,). Thus, it appears that storage of apo AI-containing material at 4 ' C for more than two weeks causes redistribution of apo AI into isomorphs with more acidic p1.74 With these results and based on the earlier observations, 7~ it was decided to try and modify fresh apo Al using alkaline conditions to determine whether enhanced reaction with apo AI sites C and D would also occur in parallel to the generation of apo AI acidic isomorphs.

2. Alkaline Treatment Mod(Oes Antigenic Sites In initial experiments, apo H D L or H D L samples were kept overnight at different pHs ranging from 0.7 to 12.7 and then neutralized prior to testing in the RIA with series I MAB 3D4. Incubation at acidic pH has no effect on the reactivity of antigenic sites but at alkaline p H ( > l l) the apo AI samples acquire a greatly increased immunoreactivity with 3D4. approximately 30-fold. TM

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This suggests to us that generation of additional apo AI isomorphs or redistribution of already existing isomorphs, which occurs with storage at pH 8.9, is insufficient or unrelated to the instability of apo AI sites C and D, and to the modification of the immunoreactivity of these sites with series 1 MAB. The mutability of these sites at very alkaline pH (drastic conditions) which results in the partial digestion of apo AI (see below) indicate that to mimic the effect of storage a severe chemical reaction must occur, and that the resultant antigenic sites are generated directly in this way.

3. Optimum Conditions for Modification of Antigenic Sites Dose-response curves of N a O H treatment showed that a ratio of 1:2 (v/v) of I N N a O H to serum gave the optimum increase in immunoreactivity of apo AI antigenic sites that reacted with series 1 MAB. Higher N a O H concentration that this results in only a partial generation of antigenic sites presumably because a further reaction resulted in the destruction of the newly-formed antigens. The treatment of stored H D L which contained the maximum antigenic activity supports this hypothesis, since treatment with excess N a O H resulted in the continuous loss of antigenic activity. 74 With treatment of fresh H D L under optimum alkaline conditions, there was a rapid increase in immunoreactivity up to 6 hr, followed by a plateau. In contrast to this, treatment of stored H D L for 2 hr or more resulted in a slight but sharp loss in activity. These results suggest that even the optimum conditions used resulted in some loss of sites reactive with series 1 MAB while, at the same time, generating many more reactive sites. Fresh H D L when treated under optimum conditions, increased in activity about eight times, while the difference in activity between untreated fresh and stored material was 16 times. The stored and treated material on the other hand lost about one-half its activity. Although equivalence between stored and fresh material can be reached by treatment with NaOH, it is hard to control so that this approach cannot be totally satisfactory for use in immunoassays to quantify apo AI in plasma samples. 74

4. Reaction of Series 2 MAB with apo AI Samples Treated with NaOH Based on the previously noted antigenic similarity between stored sera and fresh sera treated with NaOH, we expected that the series 2 MAB would react in a similar manner. Only two antibodies behaved as expected. 4H 1 reacted only slightly better with apo H D L when it was treated with NaOH, and 5F6 reacted very poorly with N a O H treated apo HDL. 2F1 which behaved as a series 1 MAB in reference to stored sera reacted poorly with N a O H treated fresh material compared to untreated fresh apo HDL. 3G10 which reacted well with fresh material reacted much better with N a O H treated fresh samples. 4F7 which in the RIA reacted much better with fresh material reacted even better with N a O H treated material. 74 These results suggest that there is a relationship between the alteration of antigenic sites on storage and treatment with N a O H in reference to antibodies 4H1, 5F6 and series 1 MAB. However, the other MAB do not conform according to this model. It must therefore be concluded that N a O H treatment is only a partial model for the effect of storage at 4 ° C . 73'74

F. Effect of Alkaline pH on apo AI Physicochemical Properties and Immunoreactivity Since previous work had shown that alkaline pH generated acidic isomorphs, we were interested in seeing whether the treatment with N a O H that we used to increase the binding of series 1 MAB generated additional isomorphs and/or redistributed the protein content of the naturally occurring isomorphs. 76 It was also of interest to determine whether the antibodies reacted selectively with certain artificially induced isomorphs. When the control and the N a O H treated apo H D L sample were dialyzed using a membrane with a molecular cutoff of 12,000 daltons, losses of immunoreactivity and total

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protein were only observed in the treated samples. In contrast, when a dialysis membrane with a molecular cutoff of 3500 daltons was used, no significant loss of immunoreactive material or protein was detected. This initial differential loss of alkaline treated material through the dialysis membrane indicated that, although apo AI antigenic sites had been activated, some apo A! had undergone cleavage. TM When the same samples were also separated on isoelectricfocusing slab gels and reacted with 5F6, 4HI and 6B8 after transfer to nitrocellulose paper, the control showed the normal isomorph distribution, that is the pro-apo AI bands and several apo AI isomorphs closer to the anode, and the apo AI3 isomorph produced the darkest band on autoradiography. On the other hand, the NaOH treated sample appeared as a large immunoreactive spot without any distinct bands, and shifted towards the anode. The most alkaline immunoreactive protein corresponded in position to the most acid isomorph of control] 4 Previous results showed that stored samples contained the same pattern of isomorphs, with some redistribution, towards the anode but nothing like the pI shift created by NaOH treatment. G. Conclusions

Although the characterization of apo A1 through the use of monoclonal antibodies is still in its infancy, significant progress has already been made. The difficulties inherent to the study of apo AI result, as we have seen, from its self-association and to the fact that apo AI in lipoproteins may not always express all its potential antigenic sites. These properties are governed by the amphipathic nature of this apolipoprotein and by its belonging to a highly heterogeneous class of lipoproteins commonly identified as HDL. This heterogeneity of apo AI-containing lipoproteins, which is well-documented, ~'65 certainly contributes to the variable expression of apo AI antigenic determinants which was first noted with polyclonal antisera. 5"47'53"85'86'89However, the causes for these variations in antigenicity remain highly speculative and protein-lipid as well protein-protein interactions have been invoked. The first generation of MAB to apo AI has verified this heterogeneity of antigenic determinants which have been mapped to CNBr fragments 1, 2 and 3. 73's7 Schonfeld and colleagues have noted that some of these determinants are expressed on all lipoprotein particles whereas others appear to be in part cryptic. 87 We have made the same observations and shown that certain of our antibodies, but not all, recognized equally well apo AI in lipoproteins and in delipidated and/or purified forms. 7374 We have also shown that all determinants recognized by our two series of MAB were common to all isomorphs of apo AI including pro-apo AI. The in vitro induced polymorphism of AI is not to be confused with the genetic polymorphism of AI which has been the object of several reports. It has been proposed earlier that in vitro induced polymorphism of AI is introduced by deamidation reactions. 4377 This theory has been substantiated by the observation that it was enhanced at alkaline pH, 7~a condition which favors deamidation of glutamine residues by nucleophilic reactions with OH . Although glutamine residues within a random peptide chain are relatively resistant to deamidation, 33 this will not be the case in proteins such as apo AI where large segments of the peptide chain are highly hydrophobic: OH ions present within the vicinity of such hydrophobic segments will have a low solvation with water molecules and will thus be highly nucleophilic and capable of active deamidation of adjacent glutamine residues, most of which are indeed present in apo AI within highly hydrophobic sequences or could be positioned in the proximity of these hydrophobic sequences as a function of the protein tertiary structure. Such a deamidation process could involve most of the 19 glutamine residues of apo AI and possibly also some of the asparagine residues: with such a mechanism, each isomorph of apo AI will be composed of a heterogeneous population of molecules, characterized only but its number of deamidated glutamine residues. Such a random heterogeneity would explain the reactivity of the various antibodies with each and every isomorph. A new insight into the immunology of apo AI has been acquired with the demonstration

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that storage of serum, HDL or apo HDL at 4°C results in a profound modification of apo AI antigenicity: antigenic determinants which are expressed on apo AI of freshly-drawn blood are lost upon storage whereas others increase upon storage. 74 While these changes can be stopped by freezing of the samples, they can be accelerated and/or simulated by alkaline treatment of apo AI-containing samples. Having made these observations, we were also able to show that MAB can be selected which react optimally with determinants that are present on both fresh and stored samples or mostly expressed in either fresh or stored samples. This selection together with the choice of sites that are not cryptic makes available to us antibodies that are perfectly suitable for application to quantitative immunoassays of plasma apo AI. Alkaline treatment of serum results in a significant increase in immunoreactivity with our series 1 MAB. Concomitantly this causes the appearance of very acidic isomorphs of apo AI as well as fragmentation of the molecule. Milder treatment such as solubilization of apo HDL at pH 8.9 also results in the formation of acidic isomorphs of apo AI but it is not known whether such treatment also increases apo AI immunoreactivity. Until a correlation is established between the two phenomenons, they can only be considered concurrent but not causative. v. LECITHIN: CHOLESTEROL ACYLTRANSFERASE AND APOLIPOPROTEINS D A . Introduction

Lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) mediates the esterification of cholesterol transported by plasma lipoproteins by hydrolysis of the acyl group esterified at position 2 of lecithin and its transfer to the 3-hydroxyl group of cholesterol. Despite the progress made in the characterization of the enzyme, the exact mechanisms by which LCAT interacts with its two molecules of substrates remains a matter of controversy.6° For the reaction to proceed, the presence of a specific apoprotein which serves as an activator of the reaction is required. Apo AI is the major apoprotein to serve for that purpose 28 although apo CI can also function as an activator.92 Using immunoassays for LCAT, Albers et al. 3 have found that 50~o of plasma LCAT was associated with HDL while Chung et al. 21 have observed that about 6~ of the enzyme was associated with LDL and 20~o with HDL. It is apparent that a significant proportion of plasma LCAT is bound to HDL particles and therefore probably to apo AI-containing particles despite the shearing forces exerted by the centrifugation process and despite the relatively weak interaction between LCAT and apo A131 which might tend to minimize the observed association between LCAT and HDL. While it is well-established that most of the plasma LCAT is found within HDL and VHDL, there is still no final evidence as to the nature of the physiological substrate for the enzyme, although discoidal HDL and, in general, cholesteryl ester-poor HDL are heavily favored.25'6° LCAT associated with large spherical HDL can be displaced by apo All and apo CIII, a mechanism which would favor the reassociation of the enzyme with newly-formed HDL such as the so-called discoidal HDL. "2 Some evidence has also been obtained that LCAT acts not only on HDL but also on LDL. 9 In parallel and not necessarily in contradiction with its association with HDL cited above, LCAT has also been proposed to be part of a complex which contains LCAT, apo AI and apo D, and which may serve not only as a cholesterol esterifying complex but also as a cholesteryl ester transfer complex in plasma; 29 however, it now appears doubtful that the latter function is mediated by apo D . 4"75 We do not know whether the putative LCAT-apo AI-apo D complex remains intact during its intravascular transit and binds to cholesterol and lecithin-rich particles to esterify cholesterol or whether cholesterol and lecithin are transferred to the complex prior to esterification. Alternatively, the LCAT molecule may transfer from one particle to another as a function of its cholesterol and lecithin concentration or by specific displacement by apolipoproteins such as apo All and apo CIII. "2

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Although the role of apo D in cholesteryl ester metabolism remains unsettled~ apo D and LCAT remain physically linked to one another. Indeed, in most complete purifications of L C A T , apo D was found to be the last contaminant of the preparations and its removal required passage on immunoadsorber, hydroxylapatite chromatography or preparative gel electrophoresis.6° McConathy and Alaupovic 66-67 were the first investigators to purify and partially characterize human plasma apo D. Other investigators 4'6 have successfully repeated this purification, and Albers et al. 4 found similar properties to those first described. Apo D has been described as a glycoprotein of molecular weight about 18,130 plus 18"i, carbohydrate 67 giving apparent molecular weights of 19,000-26,00067 or 32,0004 on SDS polyacrylamide gel electrophoresis. On alkaline-urea gel electrophoresis, 4,~',66.67this protein migrates between apolipoproteins AII and CII. Three major isomorphs have been observed on isoelectric focusing with pI 5.20, 5.08 and 5.00. 4 Apo D had previously been referred to as "thin-line polypeptide" owing to the narrow immunoprecipitation line that is formed in immunodiffusion experiments on HDL in agarose. 2,8"48"5° Kostner purified and partially characterized a protein that he named apolipoprotein AII149 which was responsible for the "thin-line" antigen in his experiments. Apo AIII had similar migration to apo D on alkaline-urea gel electrophoresis and similar molecular weight, but the two proteins differed in amino acid composition. Although H D L 3 was used for the isolation of apolipoproteins D and AIII in all reports of its characterization 4'4~'6667 the antigen has been detected or assayed in all lipoprotein density classes VLDL, LDL, HDL and V H D L 422 B. Production of M A B against L C A T and apo D

As mentioned above, LCAT and apo D remain associated through several steps of purification and, as a matter of convenience, mice were immunized with an LCAT preparation which contained about 20~ by weight of apo D in order to generate simultaneously antibodies directed against both antigens. The initial screening for the positive hybridomas was done with the same LCAT preparation and followed by secondary screenings with pure LCAT preparations 94and with two pure apo D samples, j8.~,7 Sixteen hybridomas were identified which secreted antibodies against the LCAT preparation but of these eight were positive and eight were negative with the apo D samples. Therefore, eight of these hybridomas appeared to secrete antibodies against an antigen present in the LCAT preparation while the eight others secreted antibodies against an antigen in the apo D samples. Two MAB identified as 2H11 and 5D4, which are tentatively directed against LCAT and five MAB identified as 4El 1, 5GI0, 2B9, 5H6 and 2G12 and potentially against apo D, have been selected 1°5 for further characterization. C. Characterization of Antibodies against LCA T

Because no LCAT preparation could be obtained that did not react with the antibodies against apo D, the demonstration of specificity for the antibodies against LCAT was difficult. This is due to the very high sensitivity of the immunoassays and probably to the highly antigenic nature of apo D. Nevertheless, in serial dilutions of the purest LCAT preparations, the reactivity with 2 H l l and 5D4 was maintained at dilutions 100-fold higher that those necessitated for reaction with antibodies against apo D. ~°5 When purified LCAT was submitted to two-dimensional electrophoresis and transferred to nitrocellulose paper, antibodies 2 H l l and 5D4 reacted with transferred proteins of 65,000 daltons and with pI reported for LCAT, that is multiple isoforms ranging from 4.4 to 5.0. j°5 In addition, treatment with neuraminidase of the LCAT preparations resulted in an increase of the pI of the immunoreactive isoforms together with a decrease in immunoreactivity with both antibodies 2 H l i and 5D4. ~°5 However, in agreement with the findings of others, z4 neuraminidase treatment did not decrease the activity of the enzyme, Therefore, these results indicate that both 2H11 and 5D4 react with a glycoprotein with

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the molecular weight and pI of LCAT and at sites which probably include sialic acid. In contrast, the other antibodies which are directed against apo D do not react with the glycoprotein identified as LCAT. Additional proof for the specificity of antibodies 2H 11 and 5D4 was obtained when they were immobilized on Sepharose; as immunoadsorbers, they could remove both LCAT activity and their antigen from partially purified LCAT preparations. However, these antibodies, when added in solution to LCAT preparation, did not inhibit LCAT activity and thus they appear to react at sites different from the active site on the enzymes. While the specificity of the reaction of antibodies 2H11 and 5D4 with LCAT is well-established, these antibodies have been of limited interest for application to immunoassays due to the cryptic nature of the determinants which are recognized. ~°5 However, preliminary experiments have shown that these cryptic sites can be exposed when serum or lipoproteins are treated with SDS under reducing conditions. These observations are compatible with the notion that LCAT exists in association with other apolipoproteins. D. Characterization of Antibodies against apo D

The literature describing apo D and its antisera has reporte& '66 that the purified protein did not react with antisera specific for apo AI, AII, B, CI, CII, CIII or albumin, showing that apo D did not contain the antigenic sites of any of these previously characterized apolipoproteins, and so was a newly-discovered protein. Furthermore, antisera raised against apo D did not react with apo AI, AII, B, CI, CII, CIII, E, albumin or LCAT, showing that the antibodies in the anti-apo D serum were specific for antigenic determinants found in apo D but absent in the other characterized apolipoproteins. A single immunoprecipitin line was found between plasma and anti-apo D antiserum on immunoelectrophoresis, showing that all antigenic determinants, recognized by antibodies in these anti-apo D sera, were found together on the same protein or lipoprotein. The studies gave good evidence for the presence of the newly-discovered apo D in human plasma, and for its immunochemical structure being different from that of any of the previously characterized apolipoproteins. We have characterized five monoclonal anti-apo D antibodies. ~°7Antibodies 4El 1 and 2B9 were IgG2a subclass, antibodies 5G10, 2G12 and 5H6 were IgGl subclass. On nitrocellulose sheet replicas of SDS-polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing, the antibodies reacted with protein bands which had the reported properties of apo D, i.e. molecular weight about 28,000 (19,000-35,000 reported for apo D), pl 4.8-5.2 (4.7-5.2 reported for apo D) and migration between apo AII and apo CII on alkaline-urea PAGE. But, in addition, the antibodies reacted with other protein bands of higher molecular weight and pI than apo D, present in human HDL, lipoproteindepleted plasma and LCAT-rich fractions. For now, we consider these newly-discovered immunoreactive bands to be cross-reacting proteins. That is, they contain antigenic determinants which cross-react with antibodies raised against, and selected with apo D, but we do not know that the proteins are homologous in any other regard. Our observation of cross-reaction is original, but we obtained the same result with a goat anti-D antiserum. From the published reports that anti-apo D antisera did not react with any previously known apolipoprotein, or LCAT or albumin, we conclude that these cross-reacting proteins are none of the known, well-characterized apolipoproteins, and probably represent a group of previously unidentified apolipoproteins. E. Redefinition of apo D as an Antigen

The nature of the cross-reacting proteins and their relationship with apo D is the current focus of our studies, in particular to determine the extent of homology between the cross-reacting proteins and apo D. It may be that they share only one or two antigenic determinants in common, e.g. an oligosaccharide antigen or a short amino acid sequence homology, the remaining parts of the protein structure bearing little or no resemblance

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among the different proteins. This would suggest that the proteins have no metabolic relationship one with another. Alternatively, the cross-reacting proteins may contain long amino acid sequences that are homologous to apo D, or may even contain the entirety of the primary structure of apo D. This would suggest a genetic and metabolic similarity between the proteins, the differences between the proteins being due to post-translational modifications of the same protein, or to the production of several proteins from homologous but not identical genes. An analogy to apo D and its cross-reacting proteins is possibly seen in the B26 and B74 fragments of apo B100 presumed to be produced by proteolytic cleavage, or the cross-reactions of some monoclonal anti-apo BI00 antibodies with apo B48. The full extent and nature of homology between apo B48 and apo B100 is not known. Preliminary experiments by RIA and with replicas of gel electrophoresis have shown us that the antigenic determinants of three of the monoclonal anti-apo D antibodies are not expressed under all conditions, but seem to be antigenic in only some protein contbrmations. This appears to be consistent among all the cross-reacting proteins and apo D, suggesting similarity in their structure. All of the cross-reacting proteins are found in the HDL, indicating that either they all have lipid-binding properties, or that they share the property of binding to another apolipoprotein. We have reported ~'5 that apo D, detected immunochemically, was present in the lipoproteins of lowest pI in HDL, but was absent or in very low concentration in most of the H D L particles. This segregation of apo D and the cross-reacting proteins onto a small population of the H D L particles suggests that these proteins may share the same metabolic pathway leading to their incorporation in the HDL. The observation that monoclonal antibodies which react with apo D also cross-react with other, as yet uncharacterized, plasma apolipoproteins brings important implications to the immunoassay of plasma apo D. We have seen a similar cross-reaction using a goat anti-apo D serum; therefore, our monoclonal antibodies appear to have their counterparts in conventional antisera. During immunoassay, such antibodies would react with their antigenic determinants present on both apo D and the cross-reacting proteins. If these assays are calibrated with a standard of pure apo D, then the results of the assay can be correctly reported in terms of the concentration of antigenic determinants present in an equivalent concentration of standard apo D. However, this measurement gives no information on the proportion of antigenic determinants that are present in apo D itself, and the proportion present in the cross-reacting proteins. These proportions could vary genetically or according to the physiological or pathological state of the subject. The proportion could be dependent or independent of the total concentration of antigenic determinants present. Without knowing the chemical and metabolic relationships between apo D and the cross-reacting proteins, we cannot be sure that what appears to be a change in plasma concentration of immunoreactive apo D is, in fact, a change in the chemical concentration of D itself and is related to the metabolism of that protein alone. Monoclonal antibodies directed against apo D offer us probes with which we can study homology between these proteins, determinant by determinant, and without the uncertainty that we have a contamination of our antiserum by antibodies that were raised against a minor, unrelated protein contaminant of the apo D that was immunized, Acknowledgements--Personal research cited by the authors was supported by grants from the Medical Research Council of Canada (Program Grant PG-27), Quebec Heart Foundation and Fondation de la Recherche en Sante du Quebec. Peter Milthorp is a recipient of an Industrial Research Followship (Natural Sciences and Engineering Research Council of Canada) with A-B Biological Supplies Inc., Hamilton, Ontario. Ross W. Milne is a Scholar of the Fondation de la Recherche en Sante du Quebec.

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