Delineating binding modes of Gal/GalNAc and structural elements of the molecular recognition of tumor-associated mucin glycopeptides by the human macrophage galactose-type lectin

Citation:
Delineating binding modes of Gal/GalNAc and structural elements of the molecular recognition of tumor-associated mucin glycopeptides by the human macrophage galactose-type lectin, Marcelo, Filipa, Garcia-Martin Fayna, Matsushita Takahiko, Sardinha João, Coelho Helena, Oude-Vrielink Anneloes, Koller Christiane, André Sabine, Cabrita Eurico J., Gabius Hans-Joachim, Nishimura Shin-Ichiro, Jiménez-Barbero Jesús, and Cañada Javier F. , Chem. Eur. J., Volume in press, (2014)

Abstract:

The human macrophage galactose-type lectin (hMGL) is a key physiological receptor for the carcinoma-associated Tn antigen (GalNAc-α-1-O-Ser/Thr) in mucins. We herein report NMR- and modeling-based data on the molecular recognition features of synthetic Tn-bearing glycopeptides by hMGL. Cognate epitopes on the sugar and matching key amino acids involved in the interaction have been identified by saturation transfer difference (STD) NMR spectroscopy. Only the amino acids close to the glycosylation site in the peptides are involved in lectin contact. Moreover, control experiments with non-glycosylated MUC1 peptides unequivocally showed that the sugar residue is essential for hMGL binding, as is Ca2+. The dissociation constants (Kd) have been estimated by STD titrations and/or STD competition experiments and show that Gal was a poor binder for hMGL, with a Kd in the mM range, while GalNAc and MUC1 Tn-glycopetides reached Kd values in the lower μM range. STD-based results suggested a distinct interacting epitope for the two monosaccharides. NMR data have been complemented with molecular dynamics simulations and Corcema- ST to establish a 3D view on the molecular recognition process between Gal, GalNAc and the Tn-presenting glycopeptides and hMGL. Gal and GalNAc have a dual binding mode with opposite trend of the main interaction pattern and the differences in affinity can be explained by additional hydrogen bonds and CH-π contacts involving exclusively the NHAc moiety.