The expression of HaloTag fusion PCSK9 (PCSK9-HT) is a key tool in this study to investigate the fate of PCSK9. We based the study on the premise that in any cell the PCSK9 (and PCSK9-HT) can only originate from one of two sources: either the cell synthesizes new protein (translated and expressed de novo), which will be trafficked throughout the cell and (at least partially) secreted, or the cell takes up external PCSK9, which has been previously secreted by any cell. Based on this belief, we utilized the features of the HaloTag ligands to distinguish the source of PCSK9-HT while studying its distribution. We selected a cell line with a PCSK9-HT expression level close to the endogenous level (~ 2 fold) of PCSK9 (Fig. ). This strategy should help avoid any artifacts caused by over-expression of a recombinant protein.
The various HaloTag ligands covalently bind the HaloTag in a one to one stoichiometry, i.e. an individual HaloTag (and thereby the PCSK9 molecule fused with it) will remain intact with the first label bound to it throughout the remainder of the experimental procedure. Since each label can be restricted to PCSK9-HT of one origin, it will only represent molecules of that particular origin throughout the experiment.
In this study, we utilized the cell-impermeant Alexa Fluor 488 HT ligand (green fluorescence emission) and the cell-permeant TMR HT ligand (red fluorescence emission). If cell permeant TMR HT ligand is added to the cells after wash-out of extracellular secreted PCSK9-HT, then all intracellular PCSK9 will be labeled. One caveat is that the origin of the PCSK9-HT labeled this way is not clear, since previously internalized PCSK9 is also subject to TMR staining. Alternatively, if cell impermeant Alexa Fluor 488 HT ligand is added to cells in PCSK9-HT containing media (secreted by the cells or added after purification), only the PCSK9 outside the cells will be labeled. If the ligand concentration is significantly higher than the PCSK9-HT concentration, almost complete saturation of PCSK9-HT with Alexa Fluor 488 ligand can be achieved. We postulate that any Alexa Fluor 488 labeled PCSK9-HT detected inside a cell has to be the result of PCSK9 uptake, presumably via LDLR internalization. Through sequential addition of the HT ligands at key time-points, the relationship between intra- and extra-cellular PCSK9 has thus been evaluated (Fig. ).
Even though the function of circulating PCSK9 in accelerating the degradation of the LDLR has been well established, two critical questions are still under investigation: (1) The exact mechanism by which PCSK9 causes the LDLR to be targeted for degradation is poorly understood, and (2) It is still unknown whether only external PCSK9 taken up into the cell after LDLR binding can serve this function [11
]. The answer to the second question will require a detailed understanding of the contact points between newly synthesized PCSK9 and LDLR in the cell. This study was designed to provide basic data for that purpose.
Supplemental movies 1
displayed the same cell. In movie 1
, the green channel representing the cell impermeant Alexa Fluor 488 ligand is shown as prevalent color, whereas in movie 2, the red channel representing the cell permeant TMR ligand is emphasized. The translocation of the green signal from the membrane, and the occasional formation of vesicles in the focal plane, can be observed. The red signal shows a very different distribution, emphasizing the difference in the cellular fate of the intracellular vs. internalized PCSK9.
As predicted, PCSK9-HT labeled outside the cell with cell-impermeant Alexa Fluor 488 ligand was taken up into the cell and could be detected on the cell surface, followed by the endosome and lysosome, where it co-localized with the LDLR. Low or null level of Alexa Fluor 488 signal was detected in the ER and Golgi (Figs.
). This phenomenon correlates well with the known pathway of PCSK9 co-internalization with the LDLR, followed by trafficking through the endosome to the lysosome for degradation. Addition of cell permeant TMR HT ligand yielded a different staining pattern. The label was predominantly in the ER and Golgi at the early time points post-labeling, i.e. the TMR ligand bound PCSK9-HT which was inside the protein synthesis and maturation pathway (Figs.
). At the later time points, TMR signal was observed in endosomes and lysosomes; however, we have not yet been able to distinguish whether this is due to re-uptake or direct intracellular trafficking. Therefore, further studies are required to determine whether PCSK9 mediated LDLR degradation occurs exclusively via
the internalization pathway (i.e. intracellularly synthesized PCSK9 is secreted and subsequently endocytosed in conjunction with LDLR) or prefers an intracellular pathway. The model system we have developed allows us to next address this in combination with tools to block cellular uptake, possibly utilizing for example dynasore [24
] or knock down of ARH [16