Studies of MUC1 - Gal3 lectin interaction by the sensitive force probe Optical Tweezers (OT)

Aberrant glycosylation patterns of membrane-bound proteins have been observed to be a universal feature of malignant cells. Members of the mucin family of glycoproteins form one of the major carriers of aberrant glycosylation, where there in breast tissue cells and other carcinomas have been found an upregulation of the Mucin1 (MUC1) protein with an increase in several cancer-associated antigens such as the ST (alpha-Neu5Ac(2,3)beta-Gal(1,3)-GalNAc-Ser/Thr), T (beta-Gal(1,3)-GalNAc-Ser/Thr), Tn (GalNAc-Ser/Thr) and STn (alpha-Neu5Ac(2,6)-GalNAc-Ser/Thr) antigens. The presence of the different antigens are not fully understood, but an increase of sialic acid-glycostructures have been found in breast cancer cells. The galactose binding family of lectins called galectins have been found to be present both extracellularly and intracellularly. A member of the beta-galactose binding family, Galectin-3(Gal3), have further been associated with processes such as acute inflammation as well as cancer increase and metastasis. Due to its important role in cancer development, the binding specificity of Gal3 is of interests. Gal3 have further been observed to interact with MUC1(T), but not with the MUC1(Tn). In a study published in 2007, interaction between MUC1(ST) and Gal3 was reported not to occur, but an unpublished study from London revealed informal information about a possibility of an interaction to occur. The experimental data presented in this master thesis were recorded by using a dual beam setup of the sensitive force probe Optical Tweezers. The aim of this study was to provide new information concerning the interaction between the different MUC1-antigens and Gal3. The results obtained provide evidence that MUC1(ST) and Gal3 interact with a rupture strength of 11-31 pN when investigated using loading rates in the range of 39-72 pN/s. The energy landscape of the interactions gave a k(off)-range from 1.3-2.9 1/s and x(beta)-value of 0.54 nm. A rupture frequency analysis was included in order to estimate the occurrence of interaction. MUC1(ST) and Gal3 showed a main rupture frequency estimate of 42.8 % +/- 26. Parallel, interaction between the positive control MUC1(T) and Gal3 gave a rupture frequency estimate of 32.0 % +/- 16. The negative controls MUC1(Tn) and MUC1(Na) showed no specific interaction with Gal3. A parallel study was performed by another masterstudent at NTNU, Ragna Emilie Bugge, where she also reported interaction between MUC1(ST) and Gal3 to occur by use of the sensitive force probe atomic force microscopy(AFM)