93
Atherosclerosis, 28 (1977) 93-100 0 EIsevier/North-Holland Scientific
Publishers,
Ltd.
BIOCHEMICAL AND IMMUNOLOGICAL EVIDENCE FOR THE PRESENCE OF AN APOLIPOPROTEIN B-LIKE COMPONENT IN THE SERUM LOW-DENSITY LIPOPROTEINS OF SEVERAL ANIMAL SPECIES
SONIA
GOLDSTEIN,
M. JOHN CHAPMAN
and GERVASE
L. MILLS
Unite’ 35, Laboratoire de Recherche sur le Mhtabolisme des Lipides, I.N.S. E. R.M., H8pital Henri Mondor, 94010 Cre’teil (France) and the Courtauld Institute of Biochemistry, Middlesex Hospital Medical School, London WIP 5PR (U.K.) (Received (Revised, (Accepted
21 January, 1977) received 25 March, 20 June, 1977)
1977)
Summary The major component of the protein moiety of-human LDL, i.e. apolipoprotein B, has been compared biochemically and immunologically with its counterpart in the LDL of several groups of animals (mammals, birds, snakes and fish). A marked resemblance was found in the amino acid composition of the apo-B fractions from all the phylogenetic groups, although immunological crossreactivity with human apolipoprotein B occurred only in the case of nonhuman primate (Old World monkey), non-primate mammalian (pig and guinea pig) and bird (chicken) apo-B components (63%, 24% and about 8% respectively). The cross-reactivity of each animal apo-B component with its human counterpart was 7-14s lower than that observed between the parent LDL’s. The resemblance in amino acid composition between the various apo-B preparations suggests that certain structural characteristics are required in this protein in order for it to bind and stabilise the lipid complement of serum LDL. Key words:
Amino
acid - Apolipoprotein
B -Immunological
cross-reactivity
Introduction Atherosclerosis is at present a major’ factor in morbidity and extensive evidence has accumulated for a principal role of the serum low-density lipoproteins in its pathogenesis [l] . LDL. low-density lipoproteins: Apo, apolipoprotein; lipoprotein nomenclature is according to Alaupovic [51.
Abbreviations:
SDS, sodium dodecyl sulphate. APo-
94
Relatively little knowledge of the structure of the low-density lipoprotein class is available [2], and less still of its counterpart in animals [3,4]. In this regard, recent comparative immunological studies of the serum lowdensity lipoproteins (LDL) from mammals, birds, snakes and fish have indicated that the antigenic structure(s) of LDL has been relatively highly conserved during evolution [ 61. Recent data have revealed that the detergent-solubilised apoprotein of LDL from several of these species contains a major component(s) of high molecular weight which corresponds to apolipoprotein B in man [7-lo]. These studies led to the present work in which the major component of the protein moiety of human LDL, (i.e. apo-B), and its counterpart (i.e. an apo-B-like component) in several animal species have been isolated and compared immunologically and biochemically. Such investigations have permitted an evaluation of the relationship between the various apo-B fractions, which as the major lipid carrier in the LDL class, is implicated in the strong interactions occurring between LDL and the arterial wall [ 111. Materials and methods Blood samples were obtained from the following animals as outlined earlier monkey, baboon, pig, guinea pig, chicken, rat snake (Ptyas rnz~osus), shark (Centrophorus squamosus) and hagfish. Isolation of LDL from the sera was performed by ultracentrifugation [12], in density intervals appropriate to the LDL distribution in the different animal species [6]. The preparation of apolipoprotein B (from LDL) and C (from VLDL) was based on that of Herbert et al. [7,13]. The conditions of amino acid analysis and of electrophoresis in SDS-polyacrylamide gel were described elsewhere [ 71; protein determination was performed according to Lowry et al. [ 141. Antisera were prepared in rabbits against the whole serum and the LDL of each species [6], with the exception of reptilian and fish LDL. Antisera to human apo-LDL, to human apo-B and to human apo-C were prepared in the same manner, using 500-800 I-18protein antigen for the immunisation. Antisera to whole human serum and serum albumin, and to chicken whole serum and egg albumin were purchased from Behringwerke (A.G., Marburg a. Lahn, West Germany). The monospecificity of the antisera to each native LDL, apo-B and apo-C preparation was tested by immunodiffusion and immunoelectrophoresis against the homologous whole serum. APO-C antisera which cross-reacted with apo-B were used after exhaustive absorption with the crossreacting antigen. Double immunodiffusion (ID) and immunoelectrophoresis (IEP) were carried out by the techniques of Ouchterlony [ 151 and Scheidegger [16] respectively, including appropriate controls for the possible formation of artefactual precipitation lines due to SDS [7]. The degree of cross-reactivity between human apo-B and the animal apo-Blike components was established by the microprecipitation [ 171 and inhibition of the precipitation [183 techniques under similar conditions to those employed for LDL [6]. Errors may be introduced into this procedure by the presence of SDS; thus, precipitation may be reduced [17], while the degree of reaction may be artefactually increased by formation of non-specific precipi-
[61: rhesus
95
tates. We therefore included controls consisting of samples of the SDS solutions themselves and of serum albumin and LDL solubilised in detergent, in addition to the conventional antigen and antibody controls. It is however noteworthy that the maximum final concentration of detergent in our assays was as low as 0.002%. Results Electrophoresis in SDS-polyacrylamide gel of the apoprotein of LDL from the rat snake revealed the presence of a polypeptide(s) of high mol. wt. (250,000) as its major component, thereby resembling the LDL of mammals [7,8], birds [lo] and fish [9]. The purity of the human and animal apo-B components was determined by electrophoresis in SDS-polyacrylamide gel and by immunological studies, which showed in each case that only components of mol. wt. 150,000 or more remained. These counterparts to human apo-B cross-reacted with antisera to their homologous LDL (except for the fish and reptilian LDL’s against which antisera were not available), but failed to react with antisera to human or chicken albumin; no additional reactions could be detected between these components and antisera to homologous whole serum. The preparations also failed to give a reaction with antiserum to apo-C. The amino acid composition of the mammalian apo-B’s are compared to those of the apo-B-like components of chicken, rat snake and hagfish LDL’s in Table 1. The amino acid profile typical of human apo-B, i.e. high contents (some 8% or greater of lysine, aspartic and glutamic acids, serine and leucine, and low proportions (
Atherosclerosis, 28 (1977) 93-100 0 EIsevier/North-Holland Scientific
Publishers,
Ltd.
BIOCHEMICAL AND IMMUNOLOGICAL EVIDENCE FOR THE PRESENCE OF AN APOLIPOPROTEIN B-LIKE COMPONENT IN THE SERUM LOW-DENSITY LIPOPROTEINS OF SEVERAL ANIMAL SPECIES
SONIA
GOLDSTEIN,
M. JOHN CHAPMAN
and GERVASE
L. MILLS
Unite’ 35, Laboratoire de Recherche sur le Mhtabolisme des Lipides, I.N.S. E. R.M., H8pital Henri Mondor, 94010 Cre’teil (France) and the Courtauld Institute of Biochemistry, Middlesex Hospital Medical School, London WIP 5PR (U.K.) (Received (Revised, (Accepted
21 January, 1977) received 25 March, 20 June, 1977)
1977)
Summary The major component of the protein moiety of-human LDL, i.e. apolipoprotein B, has been compared biochemically and immunologically with its counterpart in the LDL of several groups of animals (mammals, birds, snakes and fish). A marked resemblance was found in the amino acid composition of the apo-B fractions from all the phylogenetic groups, although immunological crossreactivity with human apolipoprotein B occurred only in the case of nonhuman primate (Old World monkey), non-primate mammalian (pig and guinea pig) and bird (chicken) apo-B components (63%, 24% and about 8% respectively). The cross-reactivity of each animal apo-B component with its human counterpart was 7-14s lower than that observed between the parent LDL’s. The resemblance in amino acid composition between the various apo-B preparations suggests that certain structural characteristics are required in this protein in order for it to bind and stabilise the lipid complement of serum LDL. Key words:
Amino
acid - Apolipoprotein
B -Immunological
cross-reactivity
Introduction Atherosclerosis is at present a major’ factor in morbidity and extensive evidence has accumulated for a principal role of the serum low-density lipoproteins in its pathogenesis [l] . LDL. low-density lipoproteins: Apo, apolipoprotein; lipoprotein nomenclature is according to Alaupovic [51.
Abbreviations:
SDS, sodium dodecyl sulphate. APo-
94
Relatively little knowledge of the structure of the low-density lipoprotein class is available [2], and less still of its counterpart in animals [3,4]. In this regard, recent comparative immunological studies of the serum lowdensity lipoproteins (LDL) from mammals, birds, snakes and fish have indicated that the antigenic structure(s) of LDL has been relatively highly conserved during evolution [ 61. Recent data have revealed that the detergent-solubilised apoprotein of LDL from several of these species contains a major component(s) of high molecular weight which corresponds to apolipoprotein B in man [7-lo]. These studies led to the present work in which the major component of the protein moiety of human LDL, (i.e. apo-B), and its counterpart (i.e. an apo-B-like component) in several animal species have been isolated and compared immunologically and biochemically. Such investigations have permitted an evaluation of the relationship between the various apo-B fractions, which as the major lipid carrier in the LDL class, is implicated in the strong interactions occurring between LDL and the arterial wall [ 111. Materials and methods Blood samples were obtained from the following animals as outlined earlier monkey, baboon, pig, guinea pig, chicken, rat snake (Ptyas rnz~osus), shark (Centrophorus squamosus) and hagfish. Isolation of LDL from the sera was performed by ultracentrifugation [12], in density intervals appropriate to the LDL distribution in the different animal species [6]. The preparation of apolipoprotein B (from LDL) and C (from VLDL) was based on that of Herbert et al. [7,13]. The conditions of amino acid analysis and of electrophoresis in SDS-polyacrylamide gel were described elsewhere [ 71; protein determination was performed according to Lowry et al. [ 141. Antisera were prepared in rabbits against the whole serum and the LDL of each species [6], with the exception of reptilian and fish LDL. Antisera to human apo-LDL, to human apo-B and to human apo-C were prepared in the same manner, using 500-800 I-18protein antigen for the immunisation. Antisera to whole human serum and serum albumin, and to chicken whole serum and egg albumin were purchased from Behringwerke (A.G., Marburg a. Lahn, West Germany). The monospecificity of the antisera to each native LDL, apo-B and apo-C preparation was tested by immunodiffusion and immunoelectrophoresis against the homologous whole serum. APO-C antisera which cross-reacted with apo-B were used after exhaustive absorption with the crossreacting antigen. Double immunodiffusion (ID) and immunoelectrophoresis (IEP) were carried out by the techniques of Ouchterlony [ 151 and Scheidegger [16] respectively, including appropriate controls for the possible formation of artefactual precipitation lines due to SDS [7]. The degree of cross-reactivity between human apo-B and the animal apo-Blike components was established by the microprecipitation [ 171 and inhibition of the precipitation [183 techniques under similar conditions to those employed for LDL [6]. Errors may be introduced into this procedure by the presence of SDS; thus, precipitation may be reduced [17], while the degree of reaction may be artefactually increased by formation of non-specific precipi-
[61: rhesus
95
tates. We therefore included controls consisting of samples of the SDS solutions themselves and of serum albumin and LDL solubilised in detergent, in addition to the conventional antigen and antibody controls. It is however noteworthy that the maximum final concentration of detergent in our assays was as low as 0.002%. Results Electrophoresis in SDS-polyacrylamide gel of the apoprotein of LDL from the rat snake revealed the presence of a polypeptide(s) of high mol. wt. (250,000) as its major component, thereby resembling the LDL of mammals [7,8], birds [lo] and fish [9]. The purity of the human and animal apo-B components was determined by electrophoresis in SDS-polyacrylamide gel and by immunological studies, which showed in each case that only components of mol. wt. 150,000 or more remained. These counterparts to human apo-B cross-reacted with antisera to their homologous LDL (except for the fish and reptilian LDL’s against which antisera were not available), but failed to react with antisera to human or chicken albumin; no additional reactions could be detected between these components and antisera to homologous whole serum. The preparations also failed to give a reaction with antiserum to apo-C. The amino acid composition of the mammalian apo-B’s are compared to those of the apo-B-like components of chicken, rat snake and hagfish LDL’s in Table 1. The amino acid profile typical of human apo-B, i.e. high contents (some 8% or greater of lysine, aspartic and glutamic acids, serine and leucine, and low proportions (
