Glycobiology Advance Access originally published online on November 29, 2005
Glycobiology 2006 16(4):305-317; doi:10.1093/glycob/cwj063
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Complex N-glycans are the major ligands for galectin-1, -3, and -8 on Chinese hamster ovary cells
2 Department of Cell Biology, Albert Einstein College of Medicine, New York, NY 10461; and 3 Section MIG (Microbiology, Immunology, Glycobiology), Institute of Laboratory Medicine, Lund University, PO Box 124, SE-221 00, Lund, Sweden
1 To whom correspondence should be addressed; e-mail: stanley{at}aecom.yu.edu
Received on August 18, 2005; revised on November 10, 2005; accepted on November 15, 2005
Galectins are implicated in a large variety of biological functions, many of which depend on their carbohydrate-binding ability. Fifteen members of the family have been identified in vertebrates based on binding to galactose (Gal) that is mediated by one or two, evolutionarily conserved, carbohydrate-recognition domains (CRDs). Variations in glycan structures expressed on glycoconjugates at the cell surface may, therefore, affect galectin binding and functions. To identify roles for different glycans in the binding of the three types of mammalian galectins to cells, we performed fluorescence cytometry at 4deg;C with recombinant rat galectin-1, human galectin-3, and three forms of human galectin-8, to Chinese hamster ovary (CHO) cells and 12 different CHO glycosylation mutants. All galectin species bound to parent CHO cells and binding was inhibited >90% by 0.2 M lactose. Galectin-8 isoforms with either a long or a short inter-CRD linker bound similarly to CHO cells. However, a truncated form of galectin-8 containing only the N-terminal CRD bound only weakly to CHO cells and the C-terminal galectin-8 CRD exhibited extremely low binding. Binding of the galectins to the different CHO glycosylation mutants revealed that complex N-glycans are the major ligands for each galectin except the N-terminal CRD of galectins-8, and also identified some fine differences in glycan recognition. Interestingly, increased binding of galectin-1 at 4deg;C correlated with increased propidium iodide (PI) uptake, whereas galectin-3 or -8 binding did not induce permeability to PI. The CHO glycosylation mutants with various repertoires of cell surface glycans are a useful tool for investigating galectin–cell interactions as they present complex and simple glycans in a natural mixture of multivalent protein and lipid glycoconjugates anchored in a cell membrane.
Key words: CHO / galectin / glycan recognition / glycosylation mutants / lectin
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