Abstract: According to the Center for Disease Control, in 2020, over 1.6 million new cancer cases were reported and 600 thousand people died of cancer. While cancer cases are decreasing, for every 100,000 people, there are still 403 new cancer cases. G3BP was identified as an interacting partner for extracellular 6-O-endosulfatase Sulf-2. Interaction with G3BP inhibits Sulf2 activity, leading to changes of heparan sulfation that is involved in numerous biological processes, including cancer cell proliferation, migration and/or invasion. Generation of Gal3BP F357W mutant is recommended by C. Barinka for interaction analyses; we will generate the F357W mutant and test the impact of the mutation on Sulf-2-G3BP interactions and the effects on Sulf-2 activity. This research entails the generation of F357W G3BP mutant by site-directed mutagenesis, subcloning wild-type and mutant sequence into lentiviral transfer vector, generation of lentiviral particles in HEK293T cells, transduction of target production cell line, purification by His/Twin-Strep affinity, impact on interaction, and impact on enzymatic activity by HPLC-UV-based assay using heparan sulfate mimetic.
Research Objectives and Questions: Sulf-2 modifies the sulfation patterns of heparan sulfate proteoglycans (HSPGs) through the hydrolysis of sulfate ester bonds. HSPGs are involved in numerous biological processes associated with cancer, including cancer cell proliferation, migration, and invasion. Gal3BP interacts with extracellular 6-O-endosulfatase Sulf-2 and inhibits Sulf-2 activity. Thus, my primary objective is to further define the interaction between Gal3BP and Sulf-2 to explore possibilities for cancer therapies involving Sulf-2 inhibition.
Background: Gal3BP was co-purified with Sulf-2 from conditioned media of HEK293F cell lines overexpressing Sulf-2. Immunoprecipitation with Sulf-2 and Gal3BP specific monoclonal antibodies confirmed the interaction. This interaction was further confirmed by immunoprecipitation of conditioned media of head and neck squamous cell carcinoma (HNSCC) cell lines (Panigrahi et al., 2024). Gal3BP-Sulf-2 interaction was shown to inhibit Sulf-2 enzymatic activity (Benicky, 2024). Molecular modeling by C. Barinka predicted that 357Phe (Phenylalanine, TTC) plays an important role in the binding process of Gal3BP and Sulf-2. This project will explore the possibility of 357Phe as a component of the binding site of Gal3BP-Sulf-2 interaction.
Methodology: Testing F357W mutant impact on Gal3BP inhibition of Sulf-2→ generate plasmids with the F357W mutated sequences of Gal3BP by site-directed mutagenesis in Origene vector (RC204918), F357W (TTC→TTT, phenylalanine→tryptophan) mutated sequence of Gal3BP; Transformation of plasmids (mutated & wildtype sequence) in E.coli; In-fusion cloning (generate final plasmid), seamless insertion of PCR amplified G3BP mutant & wildtype sequences into lentiviral vector; Lentiviral transduction into human embryonic kidney (HEK293F) cells’ with lentiviral particles containing mutated & wildtype plasmids (Panigrahi et al., 2024); Testing F357W mutant impact on Gal3BP inhibition of Sulf-2→ Purify the protein via nickel-affinity column to isolate mutated & wildtype Gal3BP protein via histidine tags; Sulf-2 activity assays→arylsulfatase activity assay (4-MUS) and 6-O-desulfation activity assay with ion exchange chromatography (HPLC).
Potential Impact: Through this research, I hope to characterize the interaction between G3BP and Sulf-2 and generate resources (recombinant proteins) for further studies including migration/invasion assays, utilizing the above resources and data.
Resources & Support Needed: Improving scientific writing, and how to integrate other aspects of advocacy into my research.
I am an incoming Junior double-majoring in Biology and Chinese. My research project for this summer focuses on the galectin-3 binding protein (G3BP). According to the Center for Disease Control, in 2020, over 1.6 million new cancer cases were reported and 600 thousand people died of cancer. While cancer cases are decreasing, for every 100,000 people, there are still 403 new cancer cases.
My research this summer focuses on G3BP, a protein that was identified as an interacting partner for extracellular 6-O-endosulfatase Sulf2. Interaction with G3BP inhibits Sulf2 activity leading to changes of heparan sulfation that is involved in numerous biological processes including cancer cell proliferation, migration and/or invasion. Generation of Gal3BP F357W mutant is recommended by C. Barinka for interaction analyses; we will generate the F357W mutant and test the impact of the mutation on Sulf2-G3BP interactions and the effects on Sulf2 activity.
This research entails the generation of F357W G3BP mutant by site-directed mutagenesis, subcloning wild-type and mutant sequence into lentiviral transfer vector, generation of lentiviral particles in HEK293T cells, transduction of target production cell line, purification by His/Twin-Strep affinity, impact on interaction, impact on enzymatic activity by HPLC-UV-based assay using heparan sulfate mimetic. Through this research, I hope to characterize the interaction between G3BP and Sulf-2 and generate resources (recombinant proteins) for further studies including migration/invasion assays, and utilize the above resources and data.
I am also interested in researching health disparities concerning cancer treatment; unlike other racial groups, cancer is the leading cause of death for Asian Americans. Due to language and cultural barriers, Asian Americans often have lower rates of cancer screening and a lack of proper treatment. Beyond my research in the lab, I am also interested in healthcare access regarding cancer diagnosis and treatment.
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