Intra- and intermolecular interactions of human galectin-3: assessment by full-assignment-based NMR

Abstract

Galectin-3 is an adhesion/growth-regulatory protein with a modular design comprising an N-terminal tail (NT, residues 1-111) and the conserved carbohydrate recognition domain (CRD, residues 112-250). The chimera-type galectin interacts with both glycan and peptide motifs. Complete (13)C/(15)N-assignment of the human protein makes NMR-based analysis of its structure beyond the CRD possible. Using two synthetic NT polypeptides covering residues 1-50 and 51-107, evidence for transient secondary structure was found with helical conformation from residues 5 to 15 as well as proline-mediated, multi-turn structure from residues 18 to 32 and around PGAYP repeats. Intramolecular interactions occur between the CRD F-face (the 5-stranded β-sheet behind the canonical carbohydrate-binding 6-stranded β-sheet of the S-face) and NT in full-length galectin-3, with the sequence P(23)GAW(26)…P(37)GASYPGAY(45) defining the primary binding epitope within the NT. Work with designed peptides indicates that the PGAX motif is crucial for self-interactions between NT/CRD. Phosphorylation at position Ser6 (and Ser12) (a physiological modification) and the influence of ligand binding have minimal effect on this interaction. Finally, galectin-3 molecules can interact weakly with each other via the F-faces of their CRDs, an interaction that appears to be assisted by their NTs. Overall, our results add insight to defining binding sites on galectin-3 beyond the canonical contact area for β-galactosides.

The intrinsically disordered N-terminal domain of galectin-3 dynamically mediates multisite self-association of the protein through fuzzy interactions

“…This is because the corresponding peaks are weaker and/or broader in the spectrum of the higher concentration sample. The chemical shift changes and intensity ratios suggest that galectin-3 self-associates at higher protein concentrations, in agreement with previous observations (18).…”

“…6J). The results from these temperature-dependent experiments are in agreement with those obtained using pulse field gradient NMR to measure the diffusion coefficient of self-associated galectin-3 (18).…”

Galectin-3 Induces a Pro-degradative/inflammatory Gene Signature in Human Chondrocytes, Teaming Up with Galectin-1 in Osteoarthritis Pathogenesis

“…Gal-3 has a trimodular design with a non-triple helical collagen-like stalk, fundamentally different from homodimeric Gal-118. In situ , its interaction with and cross-linking of the glycoprotein lubricin contributes to cartilage lubrication19.…”

Synthetic Mucin‐Like Glycopeptides as Versatile Tools to Measure Effects of Glycan Structure/Density/Position on the Interaction with Adhesion/Growth‐Regulatory Galectins in Arrays

“…Three sugar (headgroup) structures, Tn, TF,a nd core 2, were arranged systematically on thef ivep otential glycosylation sites( compounds 2-16). Combinations of di-a nd tri-Tn antigen modifications (compound [17][18][19][20][21][22][23][24], aTFantigen with twoTnantigens on adjacent Serr esidues( compound 25), as well as aS ialyl-TF antigen( compound 33)a nd itsh eterogeneous modification model( compounds 34 and 35) [6d] were also addedt o this panel. They were synthesizedb yu sing as tandards olidphasep eptide synthesis( SPPS)m ethodology based on the Fmoc/tBu strategy, [13] assistedb ym icrowave irradiation, [14] on ap olyethyleneg lycol-based resin( NovaPEG) with aR inkl inker.…”

“…This protein is the chimera-typeg alectin with at rimodular design composed of an N-terminalp eptide with two sites for Ser phosphorylation and non-triple-helical collagen-like repeats (these two regionsf ormingt he N-terminal tail) as well as the carbohydrate recognition domain (CRD), as shown in Figure 1a.W hile monomeric in solution, galectin-3c an aggregate in the presence of ligands by engaging contacts via the tail and the CRD (for ar ecent review on ligand-induced self-aggregation,see ref. [19]).…”