Double-band Phenotypes Research Articles

A single amino acid mutation alters the capsid protein electrophoretic double-band phenotype of the Plum pox virus strain PPV-Rec

Plum pox virus (PPV) isolates differ by their capsid protein (CP) mobility in SDS-PAGE. These electrophoretic phenotypes are likely to result from post-translational modifications of the CP. We demonstrated that the CP mobility was solely determined by the CP N-terminal region. Sequence comparison pinpointed a possible role of mutations at position 66 in determining the CP phenotype of PPV-Rec isolates. Site-directed mutagenesis of a chimeric clone demonstrated that Gly(66) in the CP resulted in the double-band phenotype, while Arg(66) led to a single-band CP pattern, possibly by preventing the phosphorylation of a nearby Ser residue by steric hindrance.

Different apoprotein(a) isoform proportions in serum and carotid plaque

Introduction Cardio- and/or cerebro-vascular risk are associated with high lipoprotein (a) [Lp(a)] levels and low-molecular-weight (LMW) apo(a) isoforms. Aims of this study were to evaluate the deposition of apo(a) isoforms and apoprotein B (apo B) in atherosclerotic plaque from patients (males and females) who had carotid endarterectomy for severe stenosis, and to identify differences between patients classified by gender and divided according to the stability or instability of their plaques. Materials and methods We determined lipids, apo B and Lp(a) in serum and plaque extracts from 55 males and 25 females. Apo(a) was phenotyped and isoforms were classified by number of kringle IV (KIV) repeats. Results Lp(a) levels were higher in female serum and plaque extracts than in male samples, while apo B levels were lower. More Lp(a) than apo B deposition was observed in plaque after normalization for serum levels. Thirty-one different apo(a) isoforms were detected in our patients, with a double band phenotype in 94% of cases. In both sexes, the low/high (L/H) molecular weight apo(a) isoform expression ratio was significantly higher in plaque than in serum. Females with unstable plaques had higher Lp(a) levels in both serum and tissue extracts, and fewer KIV repeats of the principal apo(a) isoform in the serum than the other female group or males. Conclusions In both sexes, the same apo(a) isoforms are found in serum and atherosclerotic plaque, but in different proportions: in plaque, LMW apo(a) is almost always more strongly accumulated than HMW apo(a), irrespective of any combination of apo(a) isoforms in double band phenotypes or Lp(a) serum levels. Moreover, serum and tissue Lp(a) levels were higher in females than in males, and particularly in the group with unstable plaques.

Heterozygous apolipoprotein (a) status and protein expression as a risk factor for premature coronary heart disease

Exactly how apolipoprotein a [APO(a)] isoform size affects the degree of cardiovascular risk associated with high lipoprotein a [LP(a)] levels is not fully understood. Using a sodium dodecyl sulfate-agarose APO(a) & LP(a) phenotyping method, we assessed the role of APO(a) size heterogeneity according to the number of kringle 4 repeats and the differential APO(a) protein expression in 91 male Spanish patients with premature coronary heart disease (CHD) compared with 99 healthy Spanish men. CHD patients had significantly increased median plasma LP(a) levels (0.31 g/L) and a higher percentage of subjects with LP(a) levels of 0.30 g/L or greater (51%) than controls (0.15 g/L and 23%, respectively). Patients with the double-band phenotype had significantly higher plasma LP(a) levels (median 0.37 g/L) compared with those expressing a single-band phenotype (median 0.20 g/L; P =.018) and with their corresponding controls (median 0.15 g/L; P <.001). The double-band phenotype and LP(a) values of 0.30 g/L or greater had a significant association with CHD (odds ratio [OR] 6.47, 95% confidence interval [CI] 2.51-16.7), stronger than that observed for the entire group (OR 4.19, 95% CI 1.97-8.90). The adjusted OR for the APO(a) protein pattern that equally expressed both isoforms indicates an independent association with premature CHD (OR 3.33; 95% CI 1.08-10.3). These results suggest that APO(a) phenotyping might be used in subjects with hyperlipoproteinemia a as a powerful marker to assess the risk of premature CHD because heterozygous status, mainly when both isoforms are equally expressed, is associated with higher cardiovascular risk. (J Lab Clin Med 2002;139:181-7)

Differential expression of double-band apolipoprotein(a) phenotypes in healthy Spanish subjects detected by SDS–agarose immunoblotting

A sodium dodecyl sulphate-agarose apolipoprotein(a) [apo(a)] phenotyping method was set up to attain accurate scanning densitometry of proteins. Serum samples from 99 healthy Spanish men were analysed and twenty-five different apo(a) isoforms (12 to 37 kringle 4 repeats) were detected. Double-band phenotypes accounted for 39.4% ( n=39) and three different patterns of protein expression were identified: pattern A (20.5% of double-band phenotyped samples) predominantly expressed the highest molecular weight isoform; pattern B (53.9%) mainly the lowest molecular weight isoform, and pattern AB (25.6%), expressed both isoforms equally. A significant linear association between expression pattern and lipoprotein(a) [Lp(a)] concentration ≥0.30 g/l was observed. Single-band phenotyped samples ( n=60) were stratified according to apo(a) kringle 4 repeat categories and showed that 90% of isoforms <20 K4 repeats had high Lp(a) concentrations (≥0.30 g/l), whereas isoforms with 20 to 24 or more than 24 kringle 4 repeats had Lp(a) concentrations ≥0.30 g/l in 47% and 14%, respectively. A logistic regression model was fitted to test the association between apo(a) size, expression pattern and Lp(a) concentration. In this model, apo(a) isoform <25 kringle 4 repeats was significantly associated with serum Lp(a) concentration ≥0.30 g/l in both single and double-band phenotyped samples (odds ratio=8.9, p<0.001). In the latter, a differential expression pattern with respect to smaller size isoforms (pattern AB vs A) was significantly associated with Lp(a) concentration ≥0.30 g/l (odds ratio=17.97, P=0.045). Heterogeneity in protein apo(a) size expressed according to kringle 4 repeat number could be categorized in heterozygous phenotypes as three patterns. When small-sized isoform was expressed (pattern B) or both isoforms were equally expressed (pattern AB), the probability of having Lp(a)≥0.30 g/l is higher.

Interactions among Lp(a) phenotypes, Lp(a) concentrations and lipoprotein response to fat-modified diets

Lipoprotein(a) (Lp(a)), an LDL-like particle containing apo(a), a highly glycosylated protein, is a significant genetic risk factor for coronary heart disease (CHD). Lp(a) phenotypes are characterized into single-band and double-band phenotypes according to electrophoretic mobility compared to that of apo B-100. The first goal was to assess whether Lp(a) phenotype influences the concentrations and metabolism of other serum lipoproteins. A second focus was to evaluate the effect on Lp(a) concentrations of substituting medium chain saturated fat for a polyunsaturated, baseline diet. In this two-way cross-over study 18 females are a baseline, polyunsaturated fat diet ( Poly Sat en% ratio = 10.5 11.9 ) for 1 week, and then a high saturated fat diet for 4 weeks ( Poly Sat en% ratio = 3.4 19.8 ) providing either 14 energy % medium chain triglycerides (MCT) 8:0 + 10:0 or 12:0, whereas monounsaturated fat was held constant. Subjects with double-band Lp(a) phenotypes had higher ( P = 0.000) Lp(a) levels on the baseline diet compared to single-band phenotypes. Both diets decreased serum Lp(a) concentration about 30% ( P < 0.05) but raised serum LDL-C about 11%. On the baseline diet, Lp(a) polymorphism did not affect serum LDL-cholesterol levels or receptor-mediated uptake and degradation of LDL. In a two-way ANOVA 8:0 + 10:0 and 12:0 had significantly different effects on change in serum HDL-C concentrations and LDL receptor activity in MNC, but Lp(a) polymorphism had no effect on the variables measured in this study. These results suggest that the response of LDL and Lp(a) levels to the two saturated fat diets were independent of each other. Lp(a) polymorphism did not seem to influence LDL metabolism.

Chimpanzee lipoprotein(A): Relationship between apolipoprotein(A) isoform size and the density profile of lipoprotein(A) in animals with different heterozygous apo(A) phenotypes.

In a previous study [C. Doucet et al., J. Lipid Res 35:263-270, 1994], we have shown that plasma lipoprotein (a) [Lp(a)] levels were significantly elevated in a population of unrelated chimpanzees as compared to those in normolipidemic human subjects. Nonetheless, the inverse correlation between Lp(a) levels and apolipoprotein (a) [apo(a)] isoforms typical of man was maintained in the chimpanzee. In the present study, we describe the density profiles of apo B- and apo A1-containing lipoproteins and of Lp(a) in chimpanzee plasmas heterozygous for apo(a) isoforms after fractionation by single spin ultracentrifugation in an isopycnic gradient. The distribution of apo(a) isoforms in the density gradient was also examined by SDS-agarose gel electrophoresis and immunoblotting using chemiluminescence detection. In all double-band phenotypes examined, the smallest isoform was present along the entire length of the density gradient. The density distribution of the second isoform varied according to the size difference between the respective isoforms. Two isoforms close in size (difference in apparent molecular mass = 60 kDa) were present together in every gradient subfraction. On the contrary, when the two isoforms displayed distinct molecular mass (maximal difference in apparent molecular mass = 340 kDa), then the largest was principally present in the densest fractions of the gradient (d > 1.1 mg/ml). These observations suggest that Lp(a) particles with small apo(a) isoforms are more susceptible to interact with other lipoproteins than are Lp(a) particles with large isoforms.

Identification of Apo(a) Phenotypes in a Korean Population Using a Standardized Nomenclature System Based on the Number of Kringle IV Repeats

We determined serum lipoprotein(a) [Lp(a)] concentrations and apolipoprotein(a) [apo(a)] phenotypes in 193 healthy Koreans. We analysed the apo(a) phenotypes by a simplified sodium dodecyl sulphate-polyacrylamide gel electrophoresis method and classified apo(a) isoforms objectively using an apo(a) phenotype standard with a known number of kringle IV repeats. The frequency distribution of Lp(a) levels showed marked positive skew with a mean of 0.143 g/L and a median of 0.052 g/L. Of the 193 subjects tested, no bands were detected in three, and single- and double-band phenotypes were observed in 103 and 87, respectively. Among the Koreans, the most frequent phenotype was S5(39.4%), followed by S4S5(17.1%), S5S5(14.0%), S4(11.4%), S3S5(5.2%), and the remaining phenotypes (13.0%). The calculated apo(a) allele frequencies were LpF, 0.003; LpS1, 0.013; LpS2, 0.010; LpS3, 0.048; LpS4, 0.198; LpS5, 0.563 and Lp0, 0.165. We found that the serum Lp(a) concentration in Koreans was similar to that of Caucasians, but the apo(a) allele size distribution was shifted toward higher molecular weights.

Liprotein(a) Phenotypes in Japanese Children: A Cohort Study

Elevated serum lipoprotein(a) [Lp(a)] concentrations have been demonstrated to be associated with cardiovascular diseases due to premature atherosclerosis. However, the association of Lp(a) phenotypes with the development of these diseases remains largely unexplored. We analyzed the population-based frequencies of serum Lp(a) phenotypes in 269 Japanese children aged 8-13 years in one community. According to the different apolipoprotein(a) [apo(a)] electrophoretic mobilities, Lp(a) was classified into seven single-band and respective double-band phenotypes. Each individual expressed a single (homozygotic) or a double band (heterozygotic). The serum Lp(a) concentration frequency distribution was skewed toward lower levels with a mean +/- SD of 15.5 +/- 18.0 mg/dl and a median of 11.0 mg/dl. The Lp(a) phenotype frequencies revealed that the frequency of double-band phenotype expression (55%) was higher than that of single bands (44%) and that the frequency of phenotypes representative of low molecular weight apo(a) was very low (2%). The mean serum Lp(a) concentration of the double-band-expressing subjects was higher than that of subjects with the single-band phenotype (20.1 +/- 19.9 vs. 10.5 +/- 15.9 mg/dl, p < 0.01). These findings of Lp(a) phenotypes in children seemed to differ from those in Japanese adults in another study; contrary to expectation, the predominant Lp(a) phenotypes found in children were those frequently associated with cardiovascular diseases in adults. Thus, it is speculated that children whose Lp(a) phenotypes remain unchanged during the transition to adulthood may show an increased susceptibility to cardiovascular disease, although the nutritional effects on the Lp(a) phenotypes cannot be neglected.

Genetic basis for multimodal relationship between apolipoprotein (a) size and lipoprotein (a) concentration in Mexican-Americans

The reported general inverse relationship between apolipoprotein (a) (apo(a)) size and plasma lipoprotein (a) (Lp(a)) concentrations was further characterized using 927 samples taken from members of 42 Mexican-American families. When all samples were displayed in a scatter plot of apo(a) size versus natural log of Lp(a) concentration, the expected inverse relationship was observed ( r 2 = 0.24). However, the scatter plot revealed a multimodal pattern with at least two distinct modes of the inverse relationship between apo(a) size and Lp(a) concentration. Plots of 148 single-banded samples also showed the multimodal pattern, indicating that this pattern did not result from a difference between double- and single-banded samples. Also measured was the Lp(a) concentration associated with each of 508 apo(a) isoforms in samples from 254 double-banded phenotype individuals. These separated isoforms also showed the multimodal pattern. Using pedigree information, 29 different alleles were identified which were found in three or more family members. About 85% of variation in Lp(a) was explained by allele information, suggesting a genetic basis for the multimodal pattern. Thus, the data demonstrate at least two series of apo(a) alleles that have distinct relationships between apo(a) size and Lp(a) concentration.

Coronary artery disease is associated with increased lipoprotein(a) concentrations independent of the size of circulating apolipoprotein(a) isoforms.

Lipoprotein(a) [Lp(a)] concentration and apolipoprotein(a) [apo(a)] isoforms (identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] and Western blotting) were determined in a group of 508 asymptomatic Caucasian members of the community and in 318 Caucasian patients with angiographically defined coronary artery disease (CAD). Conventional risk factors for CAD were also measured. Lp(a) concentration was almost twice as high in subjects with CAD (geometric mean, 152 mg/L [geometric SD, 10 to 1398 mg/L]) as in asymptomatic control subjects (geometric mean, 84 mg/L [geometric SD, 21 to 334 mg/L]). Asymptomatic women had higher concentrations of Lp(a) than asymptomatic men. Patients with CAD were older and were more likely to have smoked and to have a first-degree relative with premature CAD (< 55 years of age), and a higher proportion were male. Patients with CAD had higher concentrations of Lp(a) independently of the number of isoform bands expressed. When apo(a) isoforms were allocated to 1 of 10 classes on the basis of their molecular size (Rf versus apoB in SDS-PAGE), patients with CAD did not express an excess of low-molecular-mass (higher concentration) isoforms but did express a higher proportion of double-band phenotypes with fewer "null" phenotypes. The relationship between the two isoform bands in a double-band phenotype was the same in both populations. Isoform mobility was defined as a continuous variable equal to the mobility of a single isoform band (single-band phenotypes) or the mean of the two isoforms in a double-band phenotype. Two variables, isoform mobility and the number of isoform bands expressed, were used to summarize the large range of isoform patterns (at least 45) that could be identified. Isoform mobility, the number of isoform bands expressed, and the presence of CAD were the three most important independent predictors of Lp(a) concentration (descending order). Only sex and LDL cholesterol were additional independent predictors of Lp(a) concentration in step-wise regression models including a wide range of demographic factors and lipid and glycemic risk factors. We conclude that Lp(a) concentration is associated with CAD independently of the isoform pattern expressed. The apo(a) gene locus exerts a strong control over circulating Lp(a) concentration, and a better understanding of the control of expression of the apo(a) gene will be essential to understand the relationship between Lp(a) and CAD.

Evidence that apolipoprotein(a) phenoptype is a risk factor for coronary artery disease in men < 55 years of age

Whereas the importance of plasma lipoprotein(a) [Lp(a)] levels as a risk factor for premature coronary artery disease (CAD) is certain, it is not clear if the apolipoprotein(a) [apo(a)] phenotype plays an additional and independent role. To investigate the possible effect of apo(a) phenotype on premature CAD (in patients < 55 years of age), plasma Lp(a) concentrations, the apo(a) phenotypes, and their relation with many recognized CAD risk factors were examined in 96 non-diabetic male patients with angiographically defined CAD and in 83 age-matched male control subjects with no angiographic evidence of CAD. Results demonstrate that patients with premature CAD are characterized by higher Lp(a) levels (24 ± 21 vs 17 ± 15 mg/dl, p < 0.01) and a higher frequency of S2 phenotype (32% vs 15%, p < 0.01). Patients with an S2 phenotype exhibited significantly higher plasma Lp(a) concentrations than control subjects with the same isoform (37 ± 22 vs 22 ± 17 mg/dl, p < 0.05). A significant correlation was found between apo B and Lp(a) levels in patients with an S2 phenotype. In addition, patients had a low frequency of S1 and S4, and a high frequency of double-band phenotypes of apo(a). Muttivariate analysis did not demonstrate an independent role for apo(a) phenotype as a risk factor for premature CAD. In conclusion, CAD patients < 55 years of age have a very different pattern of apo(a) phenotypes than subjects with no angiographic evidence of CAD; this study confirms the hypothesis that apo(a) phenotype may play an additional role in the etiology of premature CAD.

Characterization of apo(a) polymorphism by a modified immunoblotting technique in an italian population sample

Apo(a), the specific lipoprotein(a) (Lp(a)) apolipoprotein, is characterized by different isoforms (from 6 to 11 on SDS-PAGE) encoded by a system of autosomal codominant alleles. Electrophoresis on agarose gel displays a better resolving power than SDS-PAGE (a larger number of apo(a) isoforms is detected). The aim of this work was to set up a simple technique that uses a capillary blotting apparatus and a polyvinylidene difluoride membrane for protein transfer. We tested an Italian population sample of 202 healthy subjects (123 men and 79 women) and we detected 22 apo(a) isoforms varying from 280 to 775 kDa. In our sample, 135 subjects (66.5%) had a single-band phenotype, 64 (31.7%) had a double-band phenotype and 3 subjects (1.5%) had no detectable bands (‘null’ phenotype). This simple and reproducible technique could be applied in the genetic screening of apo(a) polymorphisms and for clinical investigations of the risk of developing cardiovascular diseases.

Studies on apolipoprotein(a) phenotypes. Part 2. Phenotype frequencies and Lp(a) concentrations in different phenotypes in patients with angiographically defined coronary artery diseases

In the present paper, we have evaluated serum Lp(a) concentrations, the frequencies of Lp(a) phenotypes and alleles and the association between the Lp(a) phenotypes and serum Lp(a) levels in 470 patients with angiographically defined coronary artery disease (CAD). Serum Lp(a) concentrations were significantly increased in proportion to the number of diseased vessels in the CAD patients. The frequencies of Lp(a) phenotypes in the CAD patients were significantly different from those in healthy subjects. In particular, the frequency of double-band phenotypes was higher in the CAD group. The frequencies of Lp(a) alleles in the CAD patients, however, were not significantly different from those in the healthy subjects. There was a strong inverse relationship between the apparent molecular weights of apo(a) isoforms and serum Lp(a) concentrations. Lp(a) levels in the CAD patients were higher than those in the healthy subjects with the same phenotype. The present results suggest that it is important to consider some posttranslational or environmental modifications and other factors, in addition to the genetic factor, when assessing contributions to plasma Lp(a) levels.

A simple, sensitive technique for classification of apolipoprotein(a) isoforms by sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Lipoprotein(a) (Lp(a)) is a lipoprotein containing a unique glycoprotein, apolipoprotein(a) (apo(a)), which shows considerable heterogeneity of apparent molecular mass on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). A unifying classication of isoforms has been lacking. A simple sensitive procedure for classifying apo(a) isoforms was developed in which the relative mobility of apo(a) on SDS-PAGE was related to that of apolipoprotein (apo) B-100 ( R f vs B ). After Western blotting apo(a) bands were visualised by a sensitive double antibody technique employing commercial polyclonal antibodies (sheep antihuman Lp(a) antibody, alkaline phosphatase-linked donkey antisheep antibody). The technique was sensitive (lower limit of detection 0.02 μg apo(a)) and had good reproducibility (coefficient of variation 0.9–6.4%). Ten isoform mobilites are described (<0.35, 0.40, 0.50, 0.60, 0.70, 0.80, 1.0, 1.10, >1.15). Individuals may have single or double band phenotypes. This classification is compatible with those previously described and the method is suitable for many laboratories, as it employs standard equipment and commercially available materials.

Contribution of the apo[a] phenotype to plasma Lp[a] concentrations shows considerable ethnic variation.

Apolipoprotein[a] polymorphism has been investigated by sodium dodecyl sulfate polyacrylamide (5.37%) gel electrophoresis and immunoblotting using a standardized sample load in four ethnic groups: German, Ghanaian, Chinese, and San (Kalahari Bushmen). A total of 10 different apparent molecular weight (Mr) polymorphs, designated 1 to 10 with increasing Mr, were detected in greater than 99% of all individuals tested (German, 99%; Ghanaian, 99%; Chinese, 100%; San 100%). A null allele is therefore at most an infrequent variant in all populations. Polymorphs 6-10 were common to all four populations, while polymorphs 1-5 appeared to be relatively rare variants not universally detected in each group in the present study. The Chinese had the highest proportion of double-band phenotypes and the observed frequencies were not significantly different from those expected according to simple Mendelian inheritance, whereas the observed apo[a] phenotype distributions of the other three groups did not concur with those expected for Hardy Weinberg equilibrium. The German and Ghanaian groups displayed similar distributions of apo[a] phenotypes while the Chinese and San had significantly higher frequencies of polymorphs 9 and 10. Mean plasma Lp[a] concentrations in Ghanaians (36.2 +/- 31.5 mg/dl) were almost 2-fold greater than in Germans (18.7 +/- 23.1 mg/dl) and ca 1.65-fold greater than in either Chinese (22.9 +/- 18.3 mg/dl) or San (21.1 +/- 19.3 mg/dl). A strong inverse correlation was observed between apo[a] Mr and plasma Lp[a] concentration in Germans but this was much less pronounced in Ghanaians. While the mean plasma Lp[a] levels associated with polymorphs 1-6 were similar in both Germans (43.4 +/- 30.0 mg/dl) and Ghanaians (49.2 +/- 37.6 mg/dl), those Ghanaians with any combination of the polymorphs 9 and 10 had an almost 3-fold greater mean plasma Lp[a] level (20.6 +/- 11.3 mg/dl) than their German counterparts (7.8 +/- 5.7 mg/dl). It is therefore apparent that: 1) differences in apo[a] allele frequencies are not primarily responsible for differences in Lp[a] levels between populations; and 2) the greatest ethnic variation is observed in plasma Lp[a] concentrations associated with the high molecular weight apo[a] polymorphs.

Plasma lipoprotein(a) concentration is controlled by apolipoprotein(a) (apo(a)) protein size and the abundance of hepatic apo(a) mRNA in a cynomolgus monkey model

The cynomolgus macaque was used as a model to study lipoprotein(a) (Lp(a)). Antibodies to Lp(a) were used in Ouchterlony and Western blot analysis to show that cynomolgus monkey and human Lp(a) were similar immunochemically. Monkey Lp(a) levels were measured by a quantitative sandwich enzyme-linked immunosorbent assay in 117 animals, and Lp(a) varied in concentration from 1 to 64 mg/dl. Individual monkeys had apo(a) glycoprotein sizes as either single- or double-band phenotypes that ranged from 400 to 750 kDa. Monkey apo(a) transcript lengths varied from 8.5 to 13.6 kilobases. The Lp(a) concentration, apo(a) glycoprotein size, and apo(a) transcript length distributions were similar to those in humans. In the monkeys, there was a very high correlation between apo(a) transcript size and apo(a) protein size (R = 0.93, p = 0.0001). This variation in apo(a) transcript and protein size was shown to be due to the number of kringle IV repeats in apo(a) mRNA and DNA. Monkey plasma Lp(a) concentrations correlated inversely with apo(a) glycoprotein size (R = 0.43, p = 0.0016) and directly with hepatic apo(a) mRNA abundance (R = 0.54, p = 0.004). Apo(a) transcript lengths did not correlate with hepatic apo(a) mRNA levels. This suggests that apo(a) size and mRNA levels have major independent effects on plasma Lp(a) concentration. In multivariate analysis, they account for up to 58% of the variability in Lp(a) concentration. In summary, these data provide insight into the regulation of Lp(a) levels and suggest that the cynomolgus monkey is a suitable model in which to study the role of Lp(a) in the pathogenesis of atherosclerosis.

Changes in relative mobility of pancreatic amylase variants in isoelectric focusing

Using isoelectric focusing with one ampholytic solution, double- and single-banded amylase phenotypes were found in a sample of rhesus monkeys,Macaca mulatta. When applying different ampholytic solutions, these variants were shown to change their position relative to each other. Single-banded phenotypes showed either a position corresponding to one of the bands of the double-banded phenotype or to an intermediate one. Family studies, however, suggested that the differences between the observed patterns were not caused by genetic differences. This discloses a problem with respect to the interpretation of electrophoretic data, i.e. bands with different positions produced by isoelectric focusing may not necessarily represent genetic differences.

Polymorphic forms of human apolipoprotein[a]: inheritance and relationship of their molecular weights to plasma levels of lipoprotein[a

The plasma concentration of human lipoprotein[a], Lp[a], is highly correlated with coronary artery disease. The protein moiety of Lp[a], apoLp[a], consists of two apoproteins, apo[a] and apoB-100, linked by one or more disulfide bonds(s). Apo[a], the protein unique to Lp[a], exists in polymorphic forms that exhibit different apparent molecular weights (Mr). Polyacrylamide gel electrophoresis in sodium dodecyl sulfate followed by immunoblotting was used to separate and visualize these different forms and to determine the polymorphic pattern of apo[a] in the plasma samples of 692 individuals. A total of 11 different polymorph bands ranging in Mr from 419 kD to 838 kD could be resolved, but only 1 or 2 bands were present per individual. The polymorphic band pattern for an individual was assigned to 1 of the 66 different phenotype designations representing the total number of possible single- and double-band combinations of the 11 detectable bands. All 11 of the possible single-band phenotypes but only 32 of the 55 possible double-band phenotypes were represented. There were 412 plasma samples (59.5%) that contained a single band, 274 (39.6%) contained two bands, and only 6 (0.9%) had no detectable apo[a] band. A highly significant inverse correlation was found between the Mr of the band(s) present and the plasma apoLp[a] concentration (r = -0.461; rho = 0.0001). The correlation was better between apoLp[a] and single-band (r = -0.495; rho = 0.0001) than double-band (r = -0.382; rho = 0.0001) phenotypes. Of the 274 individuals exhibiting double-band phenotypes, the lower Mr band was more intense in 141 (51.4%), the two bands were equally intense in 85 (31.0%), while the higher Mr band was more intense in 48 (17.5%). Based upon the hypothesis that apo[a] polymorphism is controlled by different alleles at a single locus, the frequency of the 11 alleles determined from the observe phenotypes (low Mr----high Mr) was: band 1) 419 kD, 0.00875; band 2) 489 kD, 0.00510; band 3) 536 kD, 0.0555; band 4) 553 kD, 0.0758; band 5) 613 kD, 0.135; band 6) 680 kD, 0.0824; band 7) 705 kD, 0.104; band 8) 742 kD, 0.151; band 9) 760 kD, 0.246; band 10) 796 kD, 0.128; band 11) 838 kD, 0.00802. The observed distribution of phenotypes in the population was compared by chi-square analysis to that predicted on the basis of simple Mendelian inheritance, and the hypothesis was rejected (chi 2 = 921.7; rho less than 0.001). Significantly, the singleband phenotypes are over-represented in the population compared to that predicted.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of five mouse monoclonal antibodies to apolipoprotein[a] from human Lp[a]: evidence for weak plasminogen reactivity.

We describe the development of five murine monoclonal antibodies (14A12, 39A1, 53A9, 73A7, and 128A6) specific to human apolipoprotein[a] (Mr approximately 570,000), and their characterization by a number of procedures including cotitration, competition and inhibition enzyme-linked immunosorbent assays (ELISA), immunoblotting of native lipoproteins and of SDS-solubilized apolipoproteins electrophoresed in polyacrylamide gels, and dot immunobinding assays. The patterns of immunoreactivity of these antibodies were similar. Each reacted in ELISA assays and upon electroimmunoblotting with purified apo[a], with apo[a] liberated by reduction of Lp[a], and with delipidated Lp[a] solubilized in SDS, but by contrast, they reacted with native Lp[a] to a significant degree only upon electroimmunoblotting. No reactivity was seen with LDL-apoB-100 or with other apolipoproteins. The cross-reactivity of these antibodies with the homologous protein, plasminogen, was examined by comparison of the amount of plasminogen or apo[a] required for 50% inhibition of antibody binding to apo[a], and by an ELISA assay. The inhibition assay showed reactivity with plasminogen to be 37- to 50-fold lower than with apo[a], while dot immunobinding showed the lower limit of detection of plasminogen and of apo[a] to be approximately 320 and 31 micrograms, respectively. In an ELISA sandwich assay based on monoclonal antibodies LHLP-1, 14A12, and 53A9, the lower limit of Lp[a] detection (approximately 1 ng/ml protein) was about 100-fold less than that of plasminogen. Chemical modification of apo[a] revealed a significant contribution of arginine residues to the epitopes of 14A12, 39A1, and 53A9. Modification of cysteine residues with iodoacetamide was without effect, thereby distinguishing these antibodies from LHLP-1. Each antibody reacted with the six major size forms of apo[a] (Mr approximately 450,000-750,000) in immunoblots of human sera electrophoresed in SDS-polyacrylamide gels. Marked heterogeneity in apo[a] phenotype was detected and both single and double band phenotypes were observed in a randomized study. Cotitration and competition binding studies showed varying degrees of interaction between all five epitopes, with the exception of 128A6 which appeared to be independent of 39A1 and 53A9 (and vice versa). These data suggest that our five monoclonal antibodies recognize epitopes on apolipoprotein[a] that are exposed and accessible on the native Lp[a] particle. We conclude that our monoclonal antibodies recognize a specific region of apo[a], and that this region undergoes a conformational change upon adsorption of Lp[a] to plastic thereby diminishing epitope recognition.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetics of the quantitative Lp(a) lipoprotein trait. I. Relation of LP(a) glycoprotein phenotypes to Lp(a) lipoprotein concentrations in plasma.

The Lp(a) lipoprotein is a complex particle composed of a low density lipoprotein (LDL)-like lipoprotein and the disulfide bonded Lp(a) glycoprotein. The complex represents a quantitative genetic trait. SDS gel electrophoresis under reducing conditions of sera followed by immunoblotting with affinity-purified polyclonal anti-Lp(a) demonstrated inter- and intra-individual size heterogeneity of the glycoprotein with apparent Mr in the range 400-700kDa. According to their relative mobilities compared to apo B-100 the Lp(a) patterns were categorized into phenotypes F, B, S1, S2, S3 und S4 and into the respective double-band phenotypes. This size heterogeneity seems to be controlled by multiple alleles designated LpF, LpB, LpS1, LpS2, LpS3, LpS4 and a null allele (LpO) at a single locus. Phenotype frequencies observed in 441 unrelated subjects were in good agreement with those expected from the genetic hypothesis. Comparison of Lp(a) lipoprotein concentrations in the different phenotypes revealed a highly significant association of phenotypes B, S1 and S2 with high, and phenotypes S3 und S4 with intermediate Lp(a) concentrations. A third mode is represented by the null phenotype were no Lp(a) band is detected upon immunoblotting and Lp(a) lipoprotein is low or absent. We conclude that the same gene locus is involved in determining Lp(a) glycoprotein phenotype and Lp(a) lipoprotein concentrations in plasma. This major gene seems to be the Lp(a) glycoprotein structural gene locus.