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 D 2 O 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 D 2 O.

Exploring Glycan Binding Specificity of Odorranalectin by Alanine Scanning Library

“…This effect can be due to the substitution of rigid Pro 4 with more flexible Ala residue in case of 12 , and the loss of hydrogen bonding with the solvent by replacement of Ser 3 with Ala in 13 [37] . Moreover, solvation effects, such as solvent re‐organization, or the release of tightly bound water upon ligand binding can contribute significantly to the entropic term [38,39] …”

“…The concentration of ASF was confirmed by measurements using Epoch (Biotek) microplate spectrophotometer and that of peptides was determined using analytical RP‐HPLC. The raw integrated heat plots were analyzed using the MicroCal PEAQ‐ITC software (Malvern) under the one‐set‐of‐sites model and control parameter as fitted offset was applied to each titration as per the company's guidelines and previous applications [39,54–56] . In all cases, thermodynamic parameters were derived from at least two independent runs that were averaged.…”

“…[37] Moreover, solvation effects, such as solvent reorganization, or the release of tightly bound water upon ligand binding can contribute significantly to the entropic term. [38,39] The obtained binding stoichiometries (n-value) were reflective of titrations reaching full saturation, revealing the functional valency of ASF defined as the inverse of the obtained n value (1/n). [40] A weak binding interaction was observed for OL 1 and analogue 13 (Ser 3 Ala) with tetravalent MUC1 glycopeptide 19 (see the Supporting Information Figure S5a and Figure S5c and Table S3, Page S42].…”

“…The raw integrated heat plots were analyzed using the MicroCal PEAQ-ITC software (Malvern) under the one-set-of-sites model and control parameter as fitted offset was applied to each titration as per the company's guidelines and previous applications. [39,[54][55][56] In all cases, thermodynamic parameters were derived from at least two independent runs that were averaged. Thermograms with the exact concentration of ligand and glycoprotein for each run, integrated heat values, and signature plots of binding interactions are provided in the Supporting Information, Pages S41-43.…”

Tyrosine O-GalNAc Alters the Conformation and Proteolytic Susceptibility of APP Model Glycopeptides

“…We used Koenigs−Knorr activation conditions for the O-glycoside formation. 24,25 The procedure involved coupling of the azido chloride derivative 2 with the pentafluorophenyl ester of Fmoc-protected Tyr to form Fmoc-Tyr(2-azido-1-α-D-Gal)-OPfp 3 (see the Supporting Information, Scheme S1, pages S2−S6). The purity of the Tyr building (Glyco)peptides were prepared using an automated solid phase peptide synthesis (SPPS) approach on Tentagel S RAM resin.…”