Dimeric galectin-1 binds with high affinity to alpha2,3-sialylated and non-sialylated terminal N-acetyllactosamine units on surface-bound extended glycans.
Galectin-1 is a member of the galectin family of glycan-binding proteins and occurs as an approximately 29.5-kDa noncovalent homodimer (dGal-1) that is widely expressed in many tissues. Here, we report that human recombinant dGal-1 bound preferentially and with high affinity (apparent K(d) approximately 2-4 microM) to immobilized extended glycans containing terminal N-acetyllactosamine (LN; Galbeta1-4GlcNAc) sequences on poly-N-acetyllactosamine (PL; (-3Galbeta1-4GlcNAcbeta1-)(n)) sequences, complex-type biantennary N-glycans, or novel chitin-derived glycans modified to contain terminal LN. Although terminal Gal residues are important for dGal-1 recognition, dGal-1 bound similarly to alpha3-sialylated and alpha2-fucosylated terminal LN, but not to alpha6-sialylated and alpha3-fucosylated terminal LN. The binding specificity of human recombinant dGal-1 was similar to that observed with purified bovine heart-derived dGal-1. Unexpectedly, dGal-1 bound free ligands in solution with relatively low affinity and displayed no preference for extended glycans, indicating that dGal-1 preferentially recognizes extended glycans only when they are surface-bound, such as found on cell surfaces. Human dGal-1 also bound to both native and desialylated human promyelocytic HL-60 cells with similar affinity as observed for immobilized long chain PL. Binding to these cells was reduced upon treatment with endo-beta-galactosidase, which cleaves PL sequences, indicating that cell-surface PLs are ligands. To test the role of dimerization in dGal-1 binding, we examined the binding of a mutated form of dGal-1 that weakly dimerizes (monomeric Gal-1 (mGal-1)) and a covalently dimerized (chemically cross-linked) form of mGal-1 (cd-mGal-1). dGal-1 and cd-mGal-1 had similar affinities that were both approximately 3.5-fold higher for immobilized PL than observed for mGal-1, suggesting that dGal-1 acts as a dimer to cross-link terminal LN units on immobilized PL. These results indicate that dGal-1 functions as a dimer to recognize LN units on extended PLs on cell surfaces.
Leppänen
Anne
A
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, 975 N. E 10th St., Oklahoma City, OK 73104, USA.
Stowell
Sean
S
Blixt
Ola
O
Cummings
Richard D
RD
D000595:Amino Acid Sequence; D000606:Amino Sugars / Q000378:metabolism*; D000818:Animals; D019921:Biotinylation; D002240:Carbohydrate Sequence; D002417:Cattle; D002850:Chromatography, Gel; D019281:Dimerization; D005453:Fluorescence; D005643:Fucose / Q000378:metabolism; D005690:Galactose / Q000032:analysis; D037483:Galectin 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D013379:Substrate Specificity
D000606:Amino Sugars; D037483:Galectin 1; D008024:Ligands; D011134:Polysaccharides; D005643:Fucose; C000458:N-acetyllactosamine; D019158:N-Acetylneuraminic Acid; D005690:Galactose