HiBiT-Based Detection of PCSK9 Secretion: A Powerful Tool for Protein Trafficking Studies

HiBiT is an 11-amino acid peptide tag developed for highly sensitive bioluminescent detection and quantification of proteins. Its small size is ideal for tracking protein expression and localization due to minimal disruption of the tagged protein’s function and structure. HiBiT exhibits high-affinity binding to its complementary partner, LgBiT, forming an active luminescent enzyme. When a bioluminescent substrate is introduced, this interaction generates a strong, quantifiable luminescent signal directly proportional to the concentration of HiBiT-tagged proteins, facilitating precise and real-time protein analysis. Compared to traditional immunoassays such as ELISA, which captures protein levels at a fixed endpoint, or Western blotting, which requires protein separation and transfer, HiBiT allows real-time tracking of protein dynamics with higher sensitivity and reduced background noise.

HiBiT is a powerful tool for studying extracellular protein activity, including the secretion of proteins, due to the impermeability of LgBiT. In the Nano-Glo^® HiBiT Extracellular Detection System, LgBiT is added to live-cell media, where it binds exclusively to HiBiT-tagged proteins outside of the cell, allowing selective quantification of the extracellular protein population. This system enables the study of various extracellular protein activities, including receptor internalization and recycling, protein trafficking to the cell surface, and protein secretion from the cell. 

Figure 1: Workflow demonstrating the Nano-Glo^® HiBiT Extracellular Detection System

Promega scientists sought to demonstrate the power of HiBiT technology for studying the secretion of extracellular proteins. As a model system, they investigated the processing and secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9). PCSK9 is closely associated with lipid regulation: its overexpression is linked to hyperlipidemia, while reduced secretion or gene knockout leads to hypolipidemia (Abifadel et al., 2003; Cohen et al., 2006). Given this strong connection, PCSK9 has emerged as a compelling therapeutic target, inspiring the development of two FDA-approved antibody therapies—Praluent (Sanofi/Regeneron) and Repatha (Amgen).

Prior research has established the secretion behavior of PCSK9. Before secretion, PCSK9 undergoes an autoproteolytic process within the endoplasmic reticulum relying on a protease domain. Modifications in the genome of PCSK9, including those that impact this autoproteolytic process, result in changes in secretion. One of these is the H226A mutation, which blocks auto-proteolytic processing, causing retention in the endoplasmic reticulum (Benjannet et al., 2004). A second is the C679X mutation, which causes PCSK9 misfolding and leads to retention in the endoplasmic reticulum without inhibiting proteolysis (Zhao et al., 2006).

To utilize HiBiT to evaluate the effect of these mutations on the secretion of PCSK9, HEK293 cells were transfected with an expression construct for C-terminal HiBiT-tagged PCSK9 with either the wild type (WT) protein sequence or containing the H226A or C679X mutation. The amount of secreted PCSK9 was quantified using the Nano-Glo® HiBiT Extracellular Detection System (Figure 2). After the extracellular readings were taken, cells were lysed by the addition of digitonin to measure total PCSK9 in the well, including the intracellular protein.  Because wild-type PCSK9 is effectively secreted, virtually 100% of the total PCSK9 in the well was extracellular.  For both PCSK9 mutations, however, only a small percentage of the total PCSK9 in the well was found to be extracellular, while the signal increased greatly upon cell lysis. The ability of the mutations to block effective secretion is apparent, because the percentage of secreted protein was 0.25% for the H226A mutant and 1% for the C679X mutant. These results demonstrate the capability of HiBiT to sensitively detect changes in protein trafficking.

Figure 2. HiBiT luminescent assays quantify how mutations to PCSK9 impair its secretion from the cell. HiBiT-tagged wild-type (WT) PCSK9 is efficiently secreted, as illustrated by the fact that the high signal from extracellular HiBiT represents nearly 100% of the total HiBiT in the well. Not only do the H226A and C679X mutations dramatically reduce the signal from extracellular PCSK9-HiBiT, but lysis of cells with digitonin increases the signal about 100-fold, indicating that only about 1% of the total PCSK9 in the well had been secreted.

To look more closely at how these mutations impact secretion of PCSK9, the protein from cell extracts and the cell culture media were separated by SDS-PAGE, and HiBiT-tagged PCSK9 was detected using the Nano-Glo^® HiBiT Blotting System (Figure 3). Unlike traditional Western Blotting, HiBiT Blotting is an antibody-free system involving HiBiT-LgBiT complementation on the membrane to produce the luminescent NanoBiT enzyme. This method provides a faster, more streamlined workflow than Western Blotting, while dramatically improving specificity and signal-to-background ratios in many cases through the selectivity of enzyme complementation. Visualization of separated cell extracts demonstrated that PCSK9 in the cell is present in two forms, the unprocessed proPCSK9 form and the processed PCSK9 form (Figure 3, lane 1). ProPCSK9 is self-cleaving through its subtilisin/kexin auto-proteolytic activity, and it is the smaller, processed form of the protein that is secreted and accumulates in the cell medium (Figure 3, lane 4). Because the H226A mutation blocks the proteolytic activity,  it is only found in the larger, non-processed form (Figure 3, lane 2), without detectable secretion (Figure 3, lane 5). The PCSK9 C679X mutation, on the other hand, still has auto-proteolytic activity and can be found in cell extracts in both the proPCSK9 and processed forms (Figure 3, lane 3). However, no secreted protein is detectable (Figure 3, lane 6). The PCSK9 C679X mutation truncates the last 14 residues, causing misfolding that blocks secretion without impairing proteolytic processing (Horton et al., 2007).

 

Figure 3. HiBiT Blotting reveals distinct effects of PCSK9 mutations on intracellular processing and secretion.  In cell extracts (lanes 1-3), WT PCSK9 appears in both unprocessed (proPCSK9) and processed forms. The H226A mutation prevents proteolytic processing (lane 2), while the C679X mutation allows processing (lane 3). In the cell culture media (lanes 4-6), only the processed form of WT PCSK9 is detected, confirming that both mutations block secretion.

To assess the dynamics of PCSK9 secretion, HiBiT luminescence was measured over 24 hours using the Nano-Glo^® HiBiT Extracellular Detection System. In untreated cells, extracellular PCSK9 levels steadily increased over time, consistent with active secretion. In contrast, cells treated with Brefeldin A (BFA), a secretion inhibitor, showed no increase in extracellular PCSK9, while total protein levels increased at nearly the same rate as untreated cells. These results confirm that the observed extracellular signal reflects secretion rather than differences in protein synthesis or cell viability, demonstrating how HiBiT enables real-time, quantitative tracking of protein secretion kinetics.

Figure 4. Time-course measurement of PCSK9-HiBiT secretion. Extracellular PCSK9-HiBiT signal (red squares) and total PCSK9-HiBiT signal under lytic conditions (blue triangles) were measured in transfected HEK293 cells over 24 hours. In cells treated with Brefeldin A (BFA), a secretion inhibitor (closed symbols), the total PCSK9-HiBiT levels continued to rise due to new protein synthesis (closed blue triangles), but extracellular PCSK9-HiBiT remained unchanged (closed red squares). In untreated cells (open symbols), both extracellular and total PCSK9-HiBiT signals increased over time, indicating efficient secretion of newly synthesized protein.

This study demonstrates how HiBiT can be used to track protein expression, processing, and secretion with high sensitivity and precision. Using HiBiT-based assays, we confirmed that wild-type PCSK9 is efficiently secreted, while the H226A and C679X mutations cause intracellular retention by distinct mechanisms. The H226A mutation prevents autoproteolytic processing, leading to ER retention, whereas the C679X mutation disrupts proper protein folding, also resulting in intracellular accumulation. These results validate HiBiT as a powerful tool for detecting changes in protein trafficking using both plate-based luminescent detection and HiBiT Blotting, providing a low-background, highly quantitative alternative to traditional methods.

Beyond PCSK9, HiBiT assays are broadly applicable to the study of extracellular protein dynamics, including the monitoring of GPCR trafficking, and for facilitating the measurement in protein trafficking to and from the plasma membrane. The ability to introduce HiBiT via CRISPR-mediated genome editing further expands its utility, enabling the study of proteins at endogenous expression levels with appropriate regulation. Promega offers a wide variety of CRISPR Ready-to-Use reporter cell lines premade with HiBiT tags. Additionally, real-time kinetic secretion measurements can be performed by adding LgBiT Protein and a stable substrate directly to the culture media, allowing continuous luminescent tracking of secretion events over time.

With these expanded applications, HiBiT serves as a powerful and versatile tool for investigating protein trafficking, secretion mechanisms, and therapeutic protein production across both basic and applied research.