Lipoprotein lipase (LPL) has long been a focal point in the study of lipid metabolism. Its role in releasing free fatty acids from triglycerides, allowing their transport across cell membranes, is vital for energy production, while dysregulation of this enzyme has been linked to cardiovascular disease [1].
Recent structural findings have challenged the traditional understanding of LPL, suggesting that LPL's regulation is more complex than previously thought, with different structural states possibly influencing its activity [2].
To gain further insight into this research, we spoke to the first author of this study, Kathryn Gunn, PhD – currently a postdoctoral fellow in Dr. Saskia Neher’s lab at UNC Chapel Hill and future principal investigator at Stony Brook University.
Keep reading and register for a free webinar where Kathryn will discuss how she used mass photometry to study LPL oligomerization dynamics.
Outline
Introducing the first author
Dr. Gunn is a postdoctoral fellow in Dr. Saskia Neher’s lab at UNC Chapel Hill and she received her PhD from Northwestern University. Her postdoctoral work on lipase structure and mechanism has been recognized with multiple fellowships and awards, including a K99/R00 and most recently the Porter Prize for Research Excellence from the American Society for Cell Biology. In January 2024, Dr. Gunn will be starting her own lab at Stony Brook University where she will study oligomeric regulation of lipases.
Research question: Does the historically accepted active LPL dimer actually exist?
There has been a longstanding question regarding the oligomeric states of lipoprotein lipase and how its structure relates to its activity. The traditional belief was that LPL primarily existed as an active dimer and an inactive monomer, but previous structural studies by Kathryn and colleagues challenged this notion.
Kathryn explains: "My colleagues and I uncovered the structure of an inactive form where LPL formed a filament, a completely unexpected finding. This raised a fundamental question in the field: Does the historically accepted LPL dimer - considered the active form - actually exist, and if so, what does it look like?"
To answer this question, they employed a multifaceted approach. They utilized mass photometry to analyze the LPL oligomeric states and structural changes in solution, while cryoEM was used to visualize LPL structures in thin ice layers. Cross-linking, mass spectrometry, and western blotting were also employed to further characterize LPL oligomers and protein interactions. These methods collectively provided insights into LPL's structural dynamics, suggesting that while there is added nuance to the traditional active dimeric model, LPL can be an active dimer [2].
Mass photometry assesses LPL oligomerization in solution
LPL oligomerization is concentration dependent.
The research team assessed oligomerization behavior with mass photometry, a powerful single-molecule technique for determining the molecular mass of proteins in solution. This method allowed them to explore how LPL's oligomeric state is influenced by various factors, including concentration and additives such as deoxycholate and heparin2.
At low LPL concentrations (16 nM), mass photometry revealed that LPL presents as a monomer (56 kDa). At higher LPL concentrations (50, 78 and 100 nM), there was a notable shift towards a dimeric form (99 kDa) and a shoulder suggesting a higher-order LPL species (131 kDa). The same pattern was observed using heparin and deoxycholate, additives known to bind to LPL and influence activity2 (Fig. 1).
Fig. 1: LPL oligomerization is dependent on LPL concentration. With increasing LPL concentration there is a mass shift from monomer (~55 kDa) to dimer (~100 kDa). In two conditions (PBS and heparin), a higher-order species also started forming (131 kDa). The same higher-order species is not observed with deoxycholate, although a shift toward dimer formation is seen. Figure source: [2]
LPL crosslinking and mass photometry.
To capture and analyze LPL oligomers from higher concentration solutions, Kathryn and her colleagues used gradient fixation (GraFix). The results of the gradient fractionations were analyzed using mass photometry. The mass photometry histogram showed consistent peaks, matching the molecular mass of LPL monomers, dimers, trimers, and tetramers, with and without the addition of deoxycholate (Fig. 2).
Fig. 2: Mass photometry analysis of crosslinked LPL. With and without the addition of deoxycholate, increase in LPL concentration (1 μM) results in the formation of a wider range of oligomers. These include monomers (∼ 60 kDa), dimers (∼ 100 kDa), trimers (∼ 150 kDa) and tetramers (∼ 210 kDa). Figure source: [2]
Mass photometry complements cryoEM data
CryoEM provided information on detailed structural features of LPL, such as the tail-to-tail dimer arrangement and the presence of a hydrophobic pore near the active site. However, cryoEM data alone could not fully capture the dynamic nature of LPL oligomerization, especially at different LPL concentrations and in the presence of additives.
Kathryn explains: “Initially, we thought that deoxycholate, a bile salt, was the key driver of the oligomeric change. However, our mass photometry and cryoEM experiments revealed a different story. It seems that the air-water interface on our cryoEM grids played a more pivotal role in changing the oligomeric state. This unexpected finding led us to consider the air-water interface as a substrate mimic. While it wasn't our initial plan, this discovery added a fascinating layer to our research.”
The mass photometry data was pivotal to get a quantitative perspective on the LPL oligomeric states, in solution. It confirmed the presence of monomers, dimers, trimers, and tetramers and provided a clearer picture of how these states vary with concentration and the influence of specific additives like deoxycholate and heparin.
Kathryn adds: "The biggest advantage of mass photometry is that we could assay so many conditions in a high throughput way. We were able to characterize the monomer-to-dimer transition in a low concentration range, which would have been difficult to detect with other techniques.”
Looking Ahead: The future of LPL research
In her new lab, Kathryn’s research will focus on the interplay between oligomeric state and enzyme function. She comments: “While my postdoc research mainly centered on one specific lipase, I plan to explore other lipases and, eventually, broaden my research to enzymes beyond lipases. The goal is to gain a comprehensive understanding of structure-dependent enzyme regulation. I suspect that the key to this lies in understanding the role of oligomeric states, which can have implications for conditions like pancreatitis and enzyme secretion control.”
When asked about whether she will continue using mass photometry, Kathryn replied: “I certainly hope to continue using mass photometry in my future research endeavors. Given its unique capabilities and relevance to my work, I see mass photometry as a valuable tool in exploring the relationship between enzymatic activity and oligomeric states."
Further resources
Here you can learn how mass photometry can be used to measure protein oligomerization in different experimental conditions – and even detect rare oligomeric species.
In this webinar, Dr. Margaret Stratton (UMass Amherst) talks about her work on the role of CaMKII in different tissues. Mass photometry has been used to quantitatively determine the stoichiometry of different variants of this crucial oligomeric enzyme.
Learn how mass photometry helped study the conformational variability of the shelterin complex, together with negative stain EM and native mass spectrometry.
References
[1] Kumari, A., Kristensen, K. K., Ploug, M. & Winther, A.-M. L. The Importance of Lipoprotein Lipase Regulation in Atherosclerosis. Biomedicines 9, 782 (2021), DOI: 10.3390/biomedicines9070782
[2] Gunn, K. H. & Neher, S. B. Structure of dimeric lipoprotein lipase reveals a pore adjacent to the active site. Nat Commun 14, 2569 (2023)DOI: 10.1038/s41467-023-38243-9
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