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Cellular Long Chain Fatty Acid Uptake by CD36: Identifying an Amino Acid That Plays a Critical Role and Assessing Physiological Impact in Bone Marrow Derived Macrophages

  • Author / Creator
    Piche, Julia E
  • Long chain fatty acids (LCFA) serve essential functions in human cells, and the presence of too little or too much could have detrimental effects. LCFA can enter cells through passive diffusion, also known as flip-flop, but the majority enter in association with a protein allowing regulation and unidirectional transport. Proteins associated with LCFA transport are fatty acid transport proteins and cluster of differentiation 36 (CD36). CD36 has many roles in the body, including binding thrombospondin-1 and being a macrophage scavenger receptor, but its connection with LCFA transport has only been investigated since 1981. There is controversy surrounding whether CD36 is independently transporting LCFA across the plasma membrane or involved in a signalling pathway increasing uptake. This research project aimed to identify a single amino acid mutation that does not disrupt the expression of CD36 or its ability to signal while decreasing LCFA uptake. This mutation could then be used to determine if the loss of CD36 LCFA uptake has biological effects in macrophage phenotype expression. Through two different methods, we demonstrated that the substitution of lysine 164 with glutamine (K164Q) significantly decreased LCFA uptake in vitro in HEK293T/17 cells. This mutant had a significant 45.7% decrease in LCFA transport compared to Wildtype CD36 and was not statistically different than cells without CD36. Using CD36-TLR2 signalling with a secretory alkaline phosphatase reporter assay dependent on NFkB activation, this mutation did not significantly decrease CD36’s ability to signal (Wildtype CD36, 1.679 ± 0.163 AU vs K164Q CD36, 1.6436 ± 0.102 AU; No CD36, 1.329 ± 0.161 AU). Low transfection efficiency prevented a conclusion on the impact on macrophage phenotype, but the collected data suggest this can be proven in the near future with an optimized protocol. Of the three cell types activated by IL-4 to M2 expression, Wildtype CD36 cells had the largest increase in an M2 marker, while K164Q CD36 had the smallest increase. This project encourages future work on the topic by confirming decreased M2 expression in macrophages without LCFA uptake via CD36 and opens up the possibility of examining the exclusive loss of CD36 LCFA uptake in a mouse model allowing studies relevant to wound healing and the progression of various conditions related to LCFA transport.

  • Subjects / Keywords
  • Graduation date
    Spring 2021
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/r3-wq98-nc94
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.