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J. Biol. Chem., Vol. 280, Issue 7, 5549-5562, February 18, 2005

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Dimeric Galectin-1 Binds with High Affinity to 2,3-Sialylated and Non-sialylated Terminal N-Acetyllactosamine Units on Surface-bound Extended Glycans^*

Anne Leppänen, Sean Stowell, Ola Blixt¶, and Richard D. Cummings||

From the Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 and the ^¶Carbohydrate Synthesis and Protein Expression Core Resource, Consortium for Functional Glycomics, Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037

Galectin-1 is a member of the galectin family of glycan-binding proteins and occurs as an 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 2–4 µM) to immobilized extended glycans containing terminal N-acetyllactosamine (LN; Gal1–4GlcNAc) sequences on poly-N-acetyllactosamine (PL; (–3Gal1–4GlcNAc1–)_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 3-sialylated and 2-fucosylated terminal LN, but not to 6-sialylated and 3-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--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 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.

Received for publication, October 22, 2004 , and in revised form, November 18, 2004.

^* This work was supported by National Institute of General Medical Sciences Grant GM62116 (to the Consortium for Functional Glycomics) and National Institutes of Health Grant AI48075 (to R. D. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Div. of Biochemistry, Dept. of Biological and Environmental Sciences, University of Helsinki, 00790 Helsinki, Finland.

^|| To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, BRC417, 975 N. E. 10th St., Oklahoma City, OK 73104. Tel.: 405-271-2481; Fax: 405-271-3910; E-mail: richard-cummings{at}ouhsc.edu.

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