Full text
| |
| Author(s): |
Patricia Suemy Kumagai
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | São Carlos. |
| Institution: | Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT) |
| Defense date: | 2016-03-23 |
| Examining board members: |
Antônio José da Costa Filho;
Paulo Mascarello Bisch;
Pietro Ciancaglini;
Ana Paula Valente;
Richard John Ward
|
| Advisor: | Antônio José da Costa Filho |
| Abstract | |
Human galectin-4 (HGal-4), a member of the galectin family, contains two carbohydrate recognition domains (CRDs) with high affinity for β-galactosides and is widely distributed in normal and neoplastic cells of different organisms. Its functions include a wide variety of cellular events such as inflammation, cancer, cell adhesion, tumor progression and metastasis. However, many questions about their interactions with different carbohydrates, the specificity of these interactions and the specific role of galectins remain unanswered. In this study, we propose the investigation of galectin-glycan interactions of human galectin-4 and its independent CRDs (CRD-I and-II) through a combination of biophysical methods. From circular dichroism (CD), measured in different spectral ranges, and fluorescence experiments we were able to understand changes in secondary and terciary structure of the protein while interacting with lactose/sucrose. These results along with hemagglutination assays showed that galectin-4 and its CRDs respond differently to sugar binding. From fluorescence, ITC and MST measurements we determined the dissociation constants for the CRDs (Kd ~0.5 mM) and for HGal-4. These values qualitatively indicated the formation of potential oligomers of CRDs and of HGal-4. The investigation of the HGal-4 interaction with membranes was firstly performed using mimetic membranes and monitored by EPR spectroscopy. The composition of the mimetic membranes was gradually increased so that to span simple compositions (such as DMPC), passing by lipid rafts in the presence of different glycolipds (GM1, LPS) up to interactions with tumor cells (U87MG, T98G e HT-29). These experiments showed that galectin-4 recognizes and binds to membrane models constituted by complex glycans on their surface. We also investigated the involvement of endogenous and exogenous HGal-4 in chemotherapies of tumor cells and found an important role of HGal-4 in the case of HT-29 cells. At last, we presented the work done in an one-year internship at the University of Oxford, during which we investigated the C-terminal region of the GPCR family receptor, the neurotensin receptor NTS1. Here, we used once again the EPR technique combined with the production/spin-labelling of mutants of the receptors, and determined that helix H8 was stabilized upon receptor reconstitution in proteolipossomes. (AU) | |
| FAPESP's process: | 11/21767-2 - Molecular interactions in the mechanism of action of human galectin-4 |
| Grantee: | Patricia Suemy Kumagai |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |