The carbohydrate-binding domain on galectin-1 is more extensive for a complex glycan than for simple saccharides: implications for galectin-glycan interactions at the cell surface.
gal-1 (galectin-1) mediates cell-cell and cell-extracellular matrix adhesion, essentially by interacting with beta-galactoside-containing glycans of cell-surface glycoconjugates. Although most structural studies with gal-1 have investigated its binding to simple carbohydrates, in particular lactose and N-acetyl-lactosamine, this view is limited, because gal-1 functions at the cell surface by interacting with more complex glycans that are heterogeneous in size and composition. In the present study we used NMR spectroscopy to investigate the interaction of human gal-1 with a large (120 kDa) complex glycan, GRG (galactorhamnogalacturonate glycan), that contains non-randomly distributed mostly terminal beta(1-->4)-linked galactose side chains. We used 15N-1H-HSQC (heteronuclear single quantum coherence) NMR experiments with 15N-enriched gal-1 to identify the GRG-binding region on gal-1 and found that this region covers a large surface area on gal-1 that includes the quintessential lactose-binding site and runs from that site through a broad valley or cleft towards the dimer interface. HSQC and pulsed-field-gradient NMR diffusion experiments also show that gal-1 binds GRG with a gal-1:GRG stoichiometry of about 5:1 (or 6:1) and with average macroscopic and microscopic equilibrium dissociation constants (Kd) of 8 x 10(-6) M and 40 x 10(-6) M (or 48 x 10(-6) M) respectively, indicating stronger binding than to lactose (Kd=520 x 10(-6) M). Although gal-1 may bind GRG in various ways, the glycan can be competed for by lactose, suggesting that there is one major mode of interaction. Furthermore, even though terminal motifs on GRG are Gal-beta(1-->4)-Gal rather than the traditional Gal-beta(1-->4)-Glc/GlcNAc (where GlcNAc is N-acetylglucosamine), we show that the disaccharide Gal-beta(1-->4)-Gal can bind gal-1 at the lactose-binding domain. In addition, gal-1 binding to GRG disrupts inter-glycan interactions and decreases glycan-mediated solution viscosity, a glycan decongestion effect that may help explain why gal-1 promotes membrane fluidity and lateral diffusion of glycoconjugates within cell membranes. Overall, our results provide an insight into the function of galectin in situ and have potential significant biological consequences.
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Health Sciences Center, 6-155 Jackson Hall, 321 Church Street, Minneapolis, MN 55455, USA.
Michelle C
MC
Miller
Irina V
IV
Nesmelova
David
D
Platt
Anatole
A
Klyosov
Kevin H
KH
Mayo
D001665:Binding Sites; D037483:Galectin 1 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007785:Lactose / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D037483:Galectin 1; D011134:Polysaccharides; D007785:Lactose