Highly glycosylated MUC1 mediates high affinity L-selectin binding at the human endometrial surface

Abstract

Background: Sialyl-Lewis X/L-selectin high affinity binding interactions between transmembrane O-glycosylated mucins proteins and the embryo have been implicated in implantation processes within the human reproductive system. However, the adhesive properties of these mucins at the endometrial cell surface are difficult to resolve due to known discrepancies between in vivo models and the human reproductive system and a lack of sensitivity in current in vitro models. To overcome these limitations, an in vitro model of the human endometrial epithelial was interrogated with single molecule force spectroscopy (SMFS) to delineate the molecular configurations of mucin proteins that mediate the high affinity L-selectin binding required for human embryo implantation.

Results: This study reveals that MUC1 contributes to both the intrinsic and extrinsic adhesive properties of the HEC-1 cellular surface. High expression of MUC1 on the cell surface led to a significantly increased intrinsic adhesion force (148 pN vs. 271 pN, p < 0.001), whereas this adhesion force was significantly reduced (271 pN vs. 118 pN, p < 0.001) following siRNA mediated MUC1 ablation. Whilst high expression of MUC1 displaying elevated glycosylation led to strong extrinsic (> 400 pN) L-selectin binding at the cell surface, low expression of MUC1 with reduced glycosylation resulted in significantly less (≤200 pN) binding events.

Conclusions: An optimal level of MUC1 together with highly glycosylated decoration of the protein is critical for high affinity L-selectin binding. This study demonstrates that MUC1 contributes to cellular adhesive properties which may function to facilitate trophoblast binding to the endometrial cell surface through the L-selectin/sialyl-Lewis x adhesion system subsequent to implantation.

Keywords: Adhesion; Biophysics; Human reproduction; Implantation; Mucin; Single molecule force spectroscopy.

Fig. 1

Endometrial epithelial cell mucins, mechanics and adhesion. a Schematic of transmembrane mucin MUC1 and Epidermal Growth Factor Receptor (EGFR) present on the endometrial cell surface, demonstrating MUC1 extension high above other surface receptor molecules. b AFM QNM surface adhesion images of HEC-1A and HEC-1B cell lines assessed with a non functionalized AFM probe. c Quantitative data of Cell Elasticity (kPa), Adhesion (pN) and Deformation (nm) from AFM QNM data from HEC-1A and HEC-1B cell lines with a non-functionalized probe. Data shown is based on a minimum of three biological repeats, with three cellular areas per repeat (n = 9), analysed as parametric data using 2 tailed T test. Significance given as *** p < 0.001

Fig. 2

Cell adhesion of HEC-1A and HEC-1B cells in the presence/absence of MUC1 specific siRNA. a A population wide screen (minimum 1000 cells) assessed for the expression of MUC1 at the protein level using IN Cell microscope, in the presence and absence of MUC1 siRNA for both cell lines. b and c Topography AFM images of both cell lines in the presence and absence of MUC1 specific siRNA, scale bar 20 µm (b – control and MUC1 siRNA image Z range is 4.5 µm; c – Control image Z range is 8.8 µm, MUC1 siRNA image Z range is 4.0 µm). (d  and e) HEC-1A and HEC-1B cell mechanical and adhesive properties, stiffness (kPa), adhesion (pN) and deformation (nm), achieved with AFM QNM imaging were assessed in the presence and absence of MUC1 siRNA. All data shown is based on a minimum of three biological repeats with three cellular areas per repeat (n = 9), analysed as parametric data using 2 tailed T test. Significance given as * p < 0.05, **p < 0.001

Fig. 3

MUC1 VNTR specific antibody functionalised probe used to interrogate HEC-1A and HEC-1B cell surfaces. a Example SMFS force curves from HEC-1A and HEC-1B cell surfaces b the rupture force (pN) achieved from the surface of HEC-1A and HEC-1B cells achieved with a MUC1 antibody functionalized probe and an anti-GAPDH antibody functionalized probe, in the presence and absence of MUC1 siRNA. c The percentage number of positive and negative adhesion events assessed from the SMFS force curves achieved from HEC-1B cells in the presence and absence of MUC1 siRNA

Fig. 4

Mucin glycoprotein manipulation using inflammatory cytokine combinations.  a and b MUC1 and GLcNAc RNA expression data following treatment with TNFα and IFNγ cytokine treatment alone or in combination. (Models B-1 & A-1 – Control; B-2–200IU IFNγ + 25 ng/ml TNFα; B-3–25 ng/ml TNFα; A-2–200IU IFNγ; A-3–25 ng/ml TNFα). All qPCR data shown as average and SD of a minimum 3 repeats. Significance given as * p < 0.05, **p < 0.001, ***p < 0.0001

Fig. 5

L-selectin adhesion to endometrial cell surface models. L-selectin binding to HEC-1A and HEC-1B cell models (B1-3 and A1-3) was characterized using a L-selectin functionalised AFM probe with SMFS, resulting in measurements of adhesion (nN) and adhesion energy (nJ). SMFS data shown as box plots and frequency histograms produced from a minimum of 8000 curves, 5 cells per sample and minimum of 3 biological repeats (n = 15)

Fig. 6

L-selectin binding events to endometrial cell surface models. a AFM SMFS force map images of individual L-selectin binding events across the HEC-1A and HEC-1B endometrial cell surface models (B1-3 and A1-3), scale bar 1.5 µm. b Corresponding L-selectin binding event data related to the snap-off forces (pN) of the functionalized probe from the HEC-1A and HEC-1B cell models. All SMFS data shown in B is total number of events across a minimum of 6000 curves, across 5 cells, from 3 biological repeats (n = 15) [40]