Abstract
The tumor-associated carbohydrate Tn antigens include two variants, αGalNAc- O-Thr and αGalNAc- O-Ser. In solution, they exhibit dissimilar shapes and dynamics and bind differently to the same protein receptor. Here, we demonstrate experimentally and theoretically that their conformational preferences in the gas phase are highly similar, revealing the essential role of water. We propose that water molecules prompt the rotation around the glycosidic linkage in the threonine derivative, shielding its hydrophobic methyl group and allowing an optimal solvation of the polar region of the antigen. The unusual arrangement of αGalNAc- O-Thr features a water molecule bound into a "pocket" between the sugar and the threonine. This mechanism is supported by trapping, for the first time, such localized water in the crystal structures of an antibody bound to two glycopeptides that comprise fluorinated Tn antigens in their structure. According to several reported X-ray structures, installing oxygenated amino acids in specific regions of the receptor capable of displacing the bridging water molecule to the bulk-solvent may facilitate the molecular recognition of the Tn antigen with threonine. Overall, our data also explain how water fine-tunes the 3D structure features of similar molecules, which in turn are behind their distinct biological activities.
Highlights
The Tn antigens are among the most specific human tumorassociated carbohydrate antigens (TACAs), present in approximately 90% of tumors.[1,2] In general, the aggressiveness of the carcinoma and the occurrence of these antigens has a clear direct correlation,[3] promoting their use as biomarkers and potential therapeutic targets against cancer.[4]
The factors governing the preferred conformations of the Tn antigens in the gas phase, free of any interference of solvent, were investigated using mass- and conformerselected infrared laser spectroscopy conducted under molecular beam conditions and coupled with quantum chemical computations
These variants displayed a comparable behavior in solution than that observed for the methylated variants, confirmed by 2D-ROESY spectra and experiment-guided Molecular Dynamics (MD) simulations[15]
Summary
The Tn antigens are among the most specific human tumorassociated carbohydrate antigens (TACAs), present in approximately 90% of tumors.[1,2] In general, the aggressiveness of the carcinoma and the occurrence of these antigens has a clear direct correlation,[3] promoting their use as biomarkers and potential therapeutic targets against cancer.[4]. 200 ns MD simulations performed on glycopeptide 3 in the SM3-bound state[6,22] revealed an unusually high water density located between the sugar and the peptide units (Figure 4c), strongly suggesting that the fluorinated N-acetyl group configures a more hydrophilic water pocket between the carbohydrate−peptide interface In light of these theoretical predictions and to provide certain experimental evidence for the proposed solvent-mediated conformational transition in Tn-Thr antigen, we determined the X-ray structures of derivatives 2 and 3 bound to the antibody scFv-SM322 at high resolution (
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