Protein–Glycan Quinary Interactions in Crowding Environment Unveiled by NMR Spectroscopy

Abstract

Protein-glycan interactions as modulators for quinary structures in crowding environments were explored. The interaction between human galectin 3 (Gal-3) and distinct macromolecular crowders, such as bovine and human serum albumin (BSA and HSA), Ficoll 70 and PEG3350, was scrutinized. The molecular recognition event of the specific ligand, lactose, by Gal-3 in crowding conditions was evaluated. Gal-3 interactions were monitored by NMR analysing chemical shift perturbation (CSP) and line broadening of H N-HSQC signals. The intensity of the Gal-3 H N-HSQC signals decreased in the presence of all crowders, due to the increase in the solution viscosity and to the formation of large protein complexes. When glycosylated containing samples of BSA and HSA were used, signal broadening was more severe than that observed in the presence of the more viscous solutions of PEG3350 and Ficoll 70. However, for the samples containing glycoproteins, the signal intensity of H N-HSQC recovered upon addition of lactose. We show that serum proteins interact with Gal-3, through their α2,3-linked sialylgalactose moieties exposed at their surfaces, competing with lactose for the same binding site. The quinary interaction between Gal-3 and serum glycoproteins, could help to co-localize Gal-3 at the cell surface, and may play a role in adhesion and signalling functions of this protein.

Molecular Recognition of a Thomsen–Friedenreich Antigen Mimetic Targeting Human Galectin‐3

“…A 15 N‐labelled Gal‐3 CRD was expressed and purified as previously reported and titrated with increasing concentrations of the TF mimetic 2 (Figure A,B). Chemical shift perturbation of the cross‐peaks corresponding to the amino acids of Gal‐3 were monitored by 1 H– 15 N heteronuclear single‐quantum coherence ( 1 H, 15 N‐HSQC) experiments.…”

“…A 15 N-labelled Gal-3 CRD was expressed and purified as previously reported [23] andt itrated with increasingc oncentrationso f the TF mimetic 2 (Figure 2A,B). Chemical shift perturbation of the cross-peaks corresponding to the amino acids of Gal-3 were monitored by 1 H- 15 [24] A cutoffl ine wase stimated to better determine the affected and unaffected residues of Gal-3 CRD upon the addition of 2 (Figure 2C and Supporting Information).…”

“…Expression and purification of Gal-3 CRD:T he protocol used for the expression and purification of Gal-3 CRD was based on previously reported work. [23] The gene for human Gal-3 was synthesized by NZYTech using ac odon optimization strategy for E. coli expression. The region corresponding to residues 114-250 (named Gal-3 CRD) was sub-cloned into the expression vector pET21 by NZY-Tech.…”

Protein folding and quinary interactions: creating cellular organisation through functional disorder

“…This folding process results in functional protein structures that act individually or collectively in multiunit complexes (quaternary structure) . Proteins also interact with each other and with other biomolecules, like DNA, RNA, lipids and carbohydrates to form macromolecular assemblies beyond quaternary structure . These so‐called quinary interactions constitute the fifth level of protein organisation inside the cell and so are also designated as quinary structure .…”

Exploring the binding pattern between pepsin and deferasirox using detailed experimental and computer simulation methods

“…Therefore, studying the binding interaction between the drug and pepsin can provide a scientic basis for the treatment of gastric diseases induced by drug. 8,9 Based on the abovementioned discussion, it is necessary to study the interaction between DFX and pepsin. Moreover, as the basic unit of most life activities, protein is the most important component of the body's cells.…”