Calorimetric Analysis of the Interplay between Synthetic Tn Antigen-Presenting MUC1 Glycopeptides and Human Macrophage Galactose-Type Lectin

Abstract

Human macrophage galactose-type lectin (hMGL, HML, CD301, CLEC10A), a C-type lectin expressed by dendritic cells and macrophages, is a receptor for N-acetylgalactosamine α-linked to serine/threonine residues (Tn antigen, CD175) and its α2,6-sialylated derivative (sTn, CD175s). Because these two epitopes are among malignant cell glycan displays, particularly when presented by mucin-1 (MUC1), assessing the influence of the site and frequency of glycosylation on lectin recognition will identify determinants governing this interplay. Thus, chemical synthesis of the tandem-repeat O-glycan acceptor region of MUC1 and site-specific threonine glycosylation in all permutations were carried out. Isothermal titration calorimetry (ITC) analysis of the binding of hMGL to this library of MUC1 glycopeptides revealed an enthalpy-driven process and an affinity enhancement of an order of magnitude with an increasing glycan count from 6-8 μM for monoglycosylated peptides to 0.6 μM for triglycosylated peptide. ITC measurements performed in D₂O permitted further exploration of the solvation dynamics during binding. A shift in enthalpy-entropy compensation and contact position-specific effects with the likely involvement of the peptide surroundings were detected. KinITC analysis revealed a prolonged lifetime of the lectin-glycan complex with increasing glycan valency and with a change in the solvent to D₂O.

Scheme 1. Synthetic Procedures for Fmoc-Thr(Tn) Building Block 5 for Use in SPPS

Reagents and conditions: (a) acetic anhydride, pyridine, DCM, rt, 16 h; (b) FeCl₃, NaN₃, H₂O₂, ACN, −45 °C, 6 h; (c) DIC, Pfp-OH, EtOAc, −15 °C, 4 h; (d) AgClO₄, anhydrous DCM/toluene [1:1 (v/v)], −30 °C, 16 h; (e) zinc powder, acetic acid/acetic anhydride/THF [1:6:6 (v/v/v)], rt, 18 h.

Figure 1

Characterization of hMGL by SDS–PAGE, two-dimensional (2D) gel electrophoresis, and gel filtration. (A) SDS–PAGE (15% polyacrylamide) of 0.1, 0.5, and 1.0 μg of hMGL as well as 0.2 μg of galectin-3 (protein with a similar mass and constitution with stalk and CRD). (B) 2D gel electrophoresis of 20 μg of hMGL in the pI range of 3–10. (C) 2D electrophoresis of a mixture of 20 μg of hMGL and 20 μg of galectin-1 in the pI range of 4–7. (D) Gel filtration of hMGL at the indicated concentrations (applied volume of 50 μL in each case).

Figure 2

Selected ITC binding curves, including thermographic profiles for interaction of (A) GalNAc (1.50 mM) with hMGL (29.6 μM), (B) MUC1-Thr16 (0.50 mM) with hMGL (22 μM), (C) MUC1-Thr4,16 (0.50 mM) with hMGL (20 μM), and (D) MUC1-Thr4,9,16 (0.25 mM) with hMGL (20 μM) in buffered H₂O determined by ITC. Isotherms and thermograms were reproduced in GUSSI version 1.4.2 from the data obtained by MicroCal PEAQ-ITC software.

Figure 3

Left and middle plots showing enthalpy–entropy compensation for hMGL–glycopeptide interaction in (A) H₂O and (B) D₂O. The enthalpy and entropy from each of the individual experiments for the glycopeptides binding with hMGL are plotted. Numbered points indicate individual measurements (see Table 2 for point identification). A linear fit to all of the data points yielded lines with slopes of −1.077 and −1.131 for the glycopeptides in H₂O and D₂O, respectively. Panel C shows transfer between the two solvents. Enthalpy–entropy compensation for the transfer from H₂O to D₂O yielded a line with a slope of −0.92.