Intracellular Metabolism of Human Apolipoprotein(a) in Stably Transfected Hep G2 Cells

Abstract

Lipoprotein(a) [Lp(a)] consists of LDL and the glycoprotein apolipoprotein(a) [apo(a)], which are covalently linked via a single disulfide bridge. The formation of Lp(a) occurs extracellularly, but an intracellular assembly in human liver cells has also been claimed. The human apo(a) gene locus is highly polymorphic due to a variable number of tandemly arranged kringle IV repeats. The size of apo(a) isoforms correlates inversely with Lp(a) plasma concentrations, which is believed to reflect different synthesis rates. To examine this association at the cellular level, we analyzed the subcellular localization and fate of apo(a) in stably transfected HepG2 cells. Our results demonstrate that apo(a) is synthesized as a precursor with a lower molecular mass which is processed into the mature, secreted form. The retention times of the precursor in the ER positively correlated with the sizes of apo(a) isoforms. The mature form was observed intracellularly at low levels and only in the Golgi apparatus. No apo(a) was found to be associated with the plasma membrane. Under temperature-blocking conditions, we did not detect any apo(a)/apoB-100 complexes within cells. This finding was confirmed in HepG2 cells transiently expressing KDEL-tagged apo(a). The precursor and the mature forms of apo(a) were found in the ER and Golgi fractions, respectively, also in human liver tissue. From our data, we conclude that in HepG2 cells the apo(a) precursor, dependent on the apo(a) isoform, is retained in the ER for a prolonged period of time, possibly due to an extensive maturation process of this large protein. The assembly of Lp(a) takes place exclusively extracellularly following the separate secretion of apo(a) and apoB.

New Insights into the Assembly and Metabolism of ApoB-Containing Lipoproteins from in vivo Kinetic Studies: Results on Healthy Subjects and Patients with Chronic Kidney Disease

“…This conclusion has to be, however, critically evaluated since baboon hepatocytes secrete most of their apoB as VLDL, which does not associate with apo(a) [32]. Similar studies in apo(a)-transfected HepG2 cells could not demonstrate an intracellular apo(a)-apoB assembly for this human hepatocyte model and thus confirmed the results from the baboon studies [24,33,34]. Nevertheless, there is also evidence for intracellular assembly of Lp(a) in cell culture systems.…”

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

“…In our previous studies (24,30) as well as in studies reported by others (20), stably transfected HepG2 cells exhibited low synthesis rates for the larger apo[a] isoforms, as evidenced by the extended labeling periods (several hours) required for apo [a] detection in those studies (20). Others have also commented on the lack of suitable models of human liver-derived cell lines expressing apo[a] (22).…”

“…The 13-K4 apo[a] construct was provided by H-J. Müller (20). These expression constructs are annotated (Figs.…”

“…The most informative insight into the potential mechanisms regulating hepatic production of apo [a] derives from a series of studies demonstrating that variations at the APOA gene locus result in transcription of a variable number of copies of a repeating domain that resembles that of the kringle 4 (K4) found in plasminogen (14-20). Apo [a] contains varying numbers of these K4-like repeats and, in general, plasma levels of Lp[a] are inversely related to the number of these repeat domains (20,21). Studies by White and colleagues have demonstrated that the most plausible mechanism for this relationship is that apo[a] isoforms with larger numbers of K4 repeats undergo a size-dependent maturation process, with retention and folding in the endoplasmic reticulum representing an important and possibly dominant restriction point in the processing, folding, and secretion process (11,22).…”

“…These differences in production rate appear to be reproducible within individuals and are not subject to significant modulation by diet or pharmacologic manipulations directed at lowering plasma cholesterol levels (12,13). The most informative insight into the potential mechanisms regulating hepatic production of apo [a] derives from a series of studies demonstrating that variations at the APOA gene locus result in transcription of a variable number of copies of a repeating domain that resembles that of the kringle 4 (K4) found in plasminogen (14)(15)(16)(17)(18)(19)(20). Apo [a] contains varying numbers of these K4-like repeats and, in general, plasma levels of Lp [a] are inversely related to the number of these repeat domains (20,21).…”