Investigation of Protein Paucimannosylation in Liver Cancer
By Nivedita Medum, Novi High School, Novi, Michigan, USA
There were about 40,000 cases of liver cancer in 2022 alone. Of these, 30,000 were fatal. The liver breaks down and stores many of the nutrients that are absorbed from the intestine, makes clotting factors that keep you from bleeding excessively, and breaks down toxic waste from the bloodstream. Therefore, when an individual has liver cancer, some, if not all of these vital functions may be compromised. Glycobiology, a newer branch of science, is the study of the structure, biosynthesis and biology of glycans. Recently it is becoming very popular in the context of cancer research as there is new research that glycans have associations with cancerous cells.
Glycans, or complex carbohydrates, are known to be altered in human cancers. Glycosylation, an important post-translational modification, plays key roles in several intracellular processes. One type of glycosylation, N-linked or asparagine-linked glycosylation, is the attachment of glycans to the asparagine residue of the polypeptide chain of a protein. Among different classes of N-glycans (oligomannose, complex and hybrid types), a lesser known glycan class called paucimannose has recently received recognition for its role in cancer. The purpose of this research paper is to identify and investigate the role of pacumannosylation in cancer. Analysis of data from Chatterjee et al (2019) shows that there is an increase of paucimannose in liver cancer cells when compared to healthy cells. Tumorous cells have a relative abundance of paucimannose of 29.17% as opposed to 4.87% in healthy cells. Therefore, there is a significant positive correlation between an increase in paucimannose and liver tumor micro-environment.
Investigation of Protein Paucimannosylation in Liver Cancer
Cancer is a disease of abnormal cell growth. A major upcoming field in cancer research is glycobiology which is the field of studying glycans. Protein glycosylation is the addition of glycans or complex carbohydrates to protein molecules (Chatterjee et al., 2019). This process is vital for providing the structural framework of DNA & RNA, cell to cell communication, and various other intercellular and intracellular processes. Glycosylation, an important post-translational modification studied for decades has recently shown to resonate with cancer and metastasis—a development of tumors far away from the initial site of cancer—by promoting or inhibiting tumor cell invasion and resulting in oncogenic transformation (Hakomori, 2002).
N-Glycosylation, which is the attachment of glycans to the asparagine residues of protein sequences, is mainly associated with immunity and has been exceptionally useful in the background of cancer research. The three prominent classes of N-glycosylation are paucimannose, oligomannose, and hybrid structures with promising implications such as cancer diagnosis, prevention and cure. Paucimannose, a lesser known N-glycosylation type, has had recent strides in cancer related research (Chaterjee et al. 2021). The purpose of this research is to examine the extent in which paucimannose plays a role in human liver cancer.
Materials and Methods
Data was extracted from paper Chatterjee et al (2019). The data set uses tissues from three healthy donors and three liver cancer patients. Tissue samples were obtained ethically using simple metadate. Denatured proteins from the tissue samples were immobilized on a hydrophobic membrane. Next, hydrophilic glycans were then cleaved using PNGase F enzyme. Released N-glycans were reduced with sodium borohydride and desalted with C18 tips. Lastly, reduced and desalted N-glycans were analyzed on a liquid chromatography setup coupled to a mass spectrometer. The data analyzed is calculated for the area under each curve which represents relative abundance of each glycan.
Results and Discussion
It is not uncommon for cancer environments to have a high abundance of paucimannose-type glycans. For oligomannose, there is almost no difference between the graphs but for the complex structure there is a decrease in the amount in tumor cells compared to healthy cells. However, there is almost no paucimannose in the healthy cells but in the tumorous cell there is an approximately 25% increase. This dramatic difference in the amount of paucimannose in cancer cells compared to healthy cells means that paucimannose glycans have an association with liver cancer cells.
Figure 1: The average relative abundance of each glycan class in healthy cells and liver cancer cells from all three of the donors (Chatterjee et al., 2019).
Figure 2: The average relative abundance of each type of paucimannose in healthy cells and cancer cells of all three donorsNote: Subtypes are labels specifically: “M” is mannose and “F” is fucose. The numbers refer to the linkages (eg: M3F is three mannose and one fucose) (Chatterjee et al., 2019).
Figure 3: The average relative abundance of specific types of paucimannose for each of the three donors (Chatterjee et al., 2019).
Although it is clear that protein paucimannosylation is a characteristic of liver cancer, it is not guaranteed that the existence of an abundance of paucimannose is of cancer origin. Other studies have proven that immune cells have been covered with numerous paucimannose glycans. In the cancer setting, a lot of new cells may come into play because cancer cells are a foreign substances that the body does not recognize. Because of this, the body’s immune cells come to this location to fight off cancer cells, which are also decorated with paucimannosylation. So it is not uncommon for cancer environments to have a high abundance of paucimannose-type glycans. However, whether paucimannose glycans are really immune specific or cancer-specific, is still unknown (Loke et al., 2017).
Paucimannose-type glycans can be of different types based on their linkages and isomeric structures. Figure 2 shows the abundances of the different paucimannose types in tumor vs non tumor cells. Here, we can conclude that paucimannose types M2Fa and M3F (M, mannose; F, fucose) with an average relative abundance of 15.25% and 10.37% respectively. This shows that these specific subtypes are a lot more abundant than the other paucimannose subtypes represented in the graphs. Additionally, the graph implies that paucimannose subtypes with fucose in them also have a tendency to play a greater role in cancerous cells. Other studies have proven that fucose itself, without its other counterparts, has been abundant in the places with heavy concentrations of cancer cells. Although, few studies have investigated the functional contribution to fucose on tumor cells (Keeley et al., 2019). In addition, mannose, a sugar that is prevalent in the subtypes of paucimannose could be another clue as to the body’s cancer response; research has proven that mannose plays a beneficial role in being a therapeutic strategy for cancer, as it is effective in promoting immune tolerance, and more importantly efficient in suppressing tumors (Nan et al., 2022).
Figure 3 states that although each donor has liver cancer, the relative abundance of each of the paucimannose species in each donor is varying. This shows that even though there is a general increase in paucimannose in tumor cells compared to healthy cells, each person's cancer is different and this disease affects their body in a different way. Each donor may also have different conditions setting them apart from each other, such as underlying health conditions, causing their glycan data to look different. These differences can be further explained through neoplastic transformation.
Neoplastic transformation is a process in which certain exposures such as carcinogenic chemicals, oncogenic viruses, or radiations alter a cell function including growth, survival and general cell behavior. Epigenetics influences, which are influences from the environment that change the way genes function, have been linked to neoplastic transformation. Among epigenetic transformations, glycosylation has recently received attention as a vital constituent of neoplastic progression. Altered glycosylation has been proved to alter cell growth and survival but more importantly facilitate metastasis and tumor inducing activities. This research would explain the difference in glycoprotein data from people with liver cancer because everyone has different exposure to environments therefore having different epigenetic alterations and glycosylation alterations. These differences can even be applied to broader implications such as the differences in glycans across different types of cancers. Understanding the differences in glycosylation among cancer patients is vital to provide novel diagnostics and therapeutic targets (Stowell et al., 2015).
Conclusion and Future Directions
This research has uncovered there is a clear difference between the amounts of paucimanose in cancer cells when compared to healthy cells. In addition there is evidence that there are certain subtypes of paucimannose that are prominent in cancerous cells, mostly subtypes with fucose. Other research has supported the notion of the abundance of fucose in cancer as well (Keeley et al., 2019). However there are factors that need to be taken into account when further researching this topic to understand paucimannosylation better and to discover what the role of paucimannose and its subtypes play in cancer.
For example, having more donors, having the protein information and including other cancer types could help scientists get a better broader understanding of paucimannosylation. In addition knowing certain information about the donors such as their metastatic statuses, demographics, or underlying health information could also give much more information. Glycosylation for all cancer patients does not happen the same way, therefore other information on the patients will give a broader and more accurate embodiment of paucimannosylation on cancer patients.
Nonetheless, expanding the knowledge between the association of paucimannosylation and liver cancer could help us better understand, treat, and diagnose cancer. Applications of this research could include new technologies that would help us fight cancer. Overall, paucimannose has important implications for the future of cancer research.
I would like to thank Dr. Sayantani Chatterjee for helping me with the research process as well as the writing process of this research paper. Data used for this research was also taken from a published dataset she and her colleagues wrote.
American Cancer Society. (2022). Liver and intrahepatic bile duct statistics. Cancer Statistics Center. Retrieved February 23, 2023, from https://cancerstatisticscenter.cancer.org/?_ga=2.263484364.191247590.1661797562-2135378298.1661797560#!/cancer-site/Liver%20and%20intrahepatic%20bile%20duct
Chatterjee, S., Lee, L. Y., Kawahara, R., Abrahams, J. L., Adamczyk, B., Anugraham, M., Ashwood, C., Sumer‐bayraktar, Z., Briggs, M. T., Chik, J. H. L., Everest‐dass, A., Förster, S., Hinneburg, H., Leite, K. R. M., Loke, I., Möginger, U., Moh, E. S. X., Nakano, M., Recuero, S., . . . Molloy, M. P. (2019). Protein paucimannosylation is an enriched<i>n</i>‐glycosylation signature of human cancers. PROTEOMICS, 19(21-22), 1900010. https://doi.org/10.1002/pmic.201900010
Chatterjee, S., Ugonotti, J., Lee, L. Y., Everest-dass, A., Kawahara, R., & Thaysen-andersen, M. (2021). Trends in oligomannosylation and α1,2-mannosidase expression in human cancers. Oncotarget, 12(21), 2188-2205. https://doi.org/10.18632/oncotarget.28064
Free microscope public domain CC0 photo [Photograph]. (n.d.). Raw Pixel. https://www.rawpixel.com/image/5924826
Hakomori, S. (2002). Glycosylation defining cancer malignancy: New wine in an old bottle. Proceedings of the National Academy of Sciences of the United States of America, 99(16), 10231-10233.
Keeley, T. S., Yang, S., & Lau, E. (2019). The diverse contributions of fucose linkages in cancer. Cancers, 11(9), 1241. https://doi.org/10.3390/cancers11091241
Loke, I., Østergaard, O., Heegaard, N. H.h., Packer, N. H., & Thaysen-andersen, M. (2017). Paucimannose-Rich n-glycosylation of spatiotemporally regulated human neutrophil elastase modulates its immune functions*. Molecular & Cellular Proteomics, 16(8), 1507-1527. https://doi.org/10.1074/mcp.M116.066746
Nan, F., Sun, Y., Liang, H., Zhou, J., Ma, X., & Zhang, D. (2022). Mannose: A sweet option in the treatment of cancer and inflammation. Frontiers in Pharmacology, 13. https://doi.org/10.3389/fphar.2022.877543
Stowell, S. R., Ju, T., & Cummings, R. D. (2015). Protein glycosylation in cancer. Annual Review of Pathology: Mechanisms of Disease, 10(1), 473-510. https://doi.org/10.1146/annurev-pathol-012414-040438