Influence of Allelic Variation on Apolipoprotein(a) Folding in the Endoplasmic Reticulum

Abstract

Plasma levels of lipoprotein(a) (Lp(a)) vary over 1000-fold between individuals and are determined by the gene for its unique apolipoprotein, apo(a), which has greater than 100 alleles. Using primary baboon hepatocyte cultures, we previously demonstrated that differences in the ability of apo(a) allelic variants to escape the endoplasmic reticulum (ER) are a major determinant of Lp(a) production rate. To examine the reason for these differences, the folding of newly synthesized apo(a) was analyzed in pulse-chase experiments. Samples were harvested in the presence of N-ethylmaleimide to preserve disulfide-bonded folding intermediates, and apo(a) was analyzed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. Apo(a) required a prolonged period (30 -60 min) to reach its fully oxidized form. Multiple folding intermediates were resolved, including a disulfide-linked, apo(a)-containing complex. Unexpectedly, all allelic variants examined showed similar patterns and kinetics of folding. Even "null" apo(a) proteins, which are unable to exit the ER, appeared to fold normally. The ER glucosidase inhibitor, castanospermine, prevented apo(a) secretion, but did not inhibit folding. This suggests that an event which is dependent on trimming of N-linked glucoses, and which occurs after the folding events detectable in our assay, is required for apo(a) secretion. Differences in the ability to undergo this event may explain the variable efficiency with which apo(a) allelic variants exit the ER. Apolipoprotein(a) (apo(a))1 is a highly polymorphic, high molecular weight glycoprotein that circulates in plasma as a component of lipoprotein(a) (Lp(a)). Lp(a) is composed of low density lipoprotein in which apoB is attached to apo(a) by disulfide linkage (1, 2). As many as 34 different isoforms of apo(a) have been identified in human plasma, which vary in size from Ͻ300 to Ͼ800 kDa (3-6). Apo(a) has a highly complex, repetitive structure and is homologous with plasminogen (7). Apo(a) contains an inactive copy of the plasminogen protease domain and a single plasminogen kringle 5 (K5) domain, preceded by multiple domains with homology to plasminogen K4 (7). The size variation of apo(a) is due to differences in the number of K4 domains encoded in the apo(a) gene (8 -11), which varies from approximately 12 to 51 (3). Each K4 repeat contains 3 internal disulfide bonds, 1 N-linked, and 6 potential O-linked glycosylation sites (7). Thus, a large apo(a) isoform may contain in excess of 150 disulfide bonds, 50 N-linked and 300 O-linked carbohydrate side chains.Lp(a) is only found in the plasma of primates (12) and the hedgehog (13,14). In humans, plasma levels of Lp(a) vary from Ͻ1 to Ͼ100 mg/dl and are highly heritable (15). High plasma levels of Lp(a) are associated with an increased incidence of cardiovascular diseases (for review, see Ref. 16). Greater than 90% of the inter-individual variation in Lp(a) concentration is attributable to the apo(a) gene locus (17). There is an inverse correlation between apo(a) size and plasma...

Apolipoprotein[a] secretion from hepatoma cells is regulated in a size-dependent manner by alterations in disulfide bond formation

“…Our results using nonreducing SDS-PAGE demonstrate size dependent kinetics of apo [a] folding. In addition, no higher molecular weight apo[a] complex was detected in our studies, in contrast to the findings of White and colleagues (35). We suspect that the differences between these finding may be explained by differences in the cell systems (i.e., rat hepatoma cells vs. baboon hepatocytes) used in these studies.…”

“…Earlier studies from White and colleagues using baboon hepatocytes demonstrated that the bulk of disulfide bond formations occurred posttranslationally, with folding being completed 30-60 min after synthesis with a hierarchy of early folding intermediates that might represent incompletely processed forms (35). These authors found no differences in the patterns or kinetics of folding with isoforms of different size.…”

Genetic control of lipoprotein(a) concentrations is different in Africans and Caucasians

“…Molecular biology studies have demonstrated that the effect of the K IV VNTR on Lp(a) levels is direct and causal and exists also in non-human primates. 34 Hence this seems to be a basic mechanism underlying the genetic variation in Lp(a) concentrations in all human ethnic groups. Despite this similarity there exist also significant differences between human populations.…”

Lipoprotein(a) in Homozygous Familial Hypercholesterolemia

“…The mechanism of this association was elucidated in in vitro cell studies that showed longer retention times for larger isoforms in the endoplasmic reticulum. 13 However, isoforms with an identical number of K-IV repeats show a considerable divergence of Lp(a) levels, which is explained by sequence variation at or close to the apo(a) gene locus. Variation at the apo(a) gene locus explains from 70% to Ͼ90% of the variation in Lp(a) concentration.…”