AFM-based quantification of the single-molecule interaction between Siglec-1 protein and sialic acid

Details

Supervisors

Alsteens, David

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

Tumour-associated macrophages (TAM) play a crucial role in the tumour microenvironment (TME), particularly in breast cancer. These immune cells, which are normally involved in defense against infection and immune surveillance, can be hijacked by cancer cells to promote tumour progression. TAMs contribute to tumour growth, angiogenesis, suppression of the anti-tumour immune response and metastasis. One of the receptors potentially involved in the interaction between TAMs and cancer cells is Siglec-1, a surface molecule expressed by macrophages. Siglec-1 is known for its role in recognizing sialic acids, compounds present on the surface of cells. Although the precise mechanism by which Siglec-1 influences the polarization of macrophages into TAMs in breast cancer remains unknown. To characterize the binding from both a kinetic and thermodynamic perspective, the interactions of two sialic acids (α2,3-SA and α2,6-SA) with Siglec-1 will be studied at the single-molecule level on a model surface. In this study, atomic force microscopy (AFM) is highlighted for its ability to measure forces in the picoNewton range necessary for studying the interaction between sialic acid and Siglec-1. The Single-Molecule Force Spectroscopy (SMFS) method is employed to determine kinetic and thermodynamic parameters. By coupling a functionalized nanoscopic tip with sialic acid and a surface coated with Siglec-1 in a PBS buffer solution, the interaction between Siglec-1 and sialic acid could be analyzed. Firstly, we ensured the specificity of the studied binding to correspond only to the interaction between Siglec-1 and α2,3-SA. Subsequently, dynamic force microscopy (DFS) and contact time experiments were conducted, allowing us to obtain the width of the free energy barrier (xϐ), the kinetic dissociation rate (koff), and association rate (kon). The kinetic parameters obtained provided dissociation constants (KD) between α2,3-SA and Siglec-1 as well as between α2,6-SA and Siglec-1. Both KD values are in the micrometer range, indicating a moderate interaction for both sialic acids with Siglec-1. Now that the binding between Siglec-1 and two sialic acids has been characterized, future studies should aim to characterize the interaction between Siglec-1 and sialic acid in a more relevant physiological environment. Studying the interaction, either on model surfaces or cells, of Siglec-1 with other sugars to discuss its specificity would also be pertinent. This study opens various perspectives regarding Siglec-1 in the context of TAMs.