Affinity of galectin-8 and its carbohydrate recognition domains for ligands in solution and at the cell surface.
Galectin-8 has two different carbohydrate recognition domains (CRDs), the N-terminal Gal-8N and the C-terminal Gal-8C linked by a peptide, and has various effects on cell adhesion and signaling. To understand the mechanism for these effects further, we compared the binding activities of galectin-8 in solution with its binding and activation of cells. We used glycan array analysis to broaden the specificity profile of the two galectin-8 CRDs, as well as intact galectin-8s (short and long linker), confirming the unique preference for sulfated and sialylated glycans of Gal-8N. Using a fluorescence anisotropy assay, we examined the solution affinities for a subset of these glycans, the highest being 50 nM for NeuAcalpha2,3Lac by Gal-8N. Thus, carbohydrate-protein interactions can be of high affinity without requiring multivalency. More importantly, using fluorescence polarization, we also gained information on how the affinity is built by multiple weak interactions between different fragments of the glycan and its carrier molecule and the galectin CRD subsites (A-E). In intact galectin-8 proteins, the two domains act independently of each other in solution, whereas at a surface they act together. Ligands with moderate or weak affinity for the isolated CRDs on the array are bound strongly by intact galectin-8s. Also galectin-8 binding and signaling at cell surfaces can be explained by combined binding of the two CRDs to low or medium affinity ligands, and their highest affinity ligands, such as sialylated galactosides, are not required.
Carlsson
Susanne
S
Section of Microbiology Immunology and Glycobiology (MIG), Sölvegatan 23, 223 62, Sweden. susanne.carlsson@med.lu.se
Oberg
Christopher T
CT
Carlsson
Michael C
MC
Sundin
Anders
A
Nilsson
Ulf J
UJ
Smith
David
D
Cummings
Richard D
RD
Almkvist
Jenny
J
Karlsson
Anna
A
Leffler
Hakon
H
D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D019793:Fluorescein; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D005697:Galactosides / Q000737:chemistry / Q000378:metabolism; D037161:Galectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D007785:Lactose / Q000037:antagonists & inhibitors; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012996:Solutions / Q000737:chemistry; D020298:U937 Cells; D014734:Vibrio cholerae / Q000378:metabolism
D005456:Fluorescent Dyes; D005697:Galactosides; D037161:Galectins; C091991:LGALS8 protein, human; D008024:Ligands; D011994:Recombinant Proteins; D012996:Solutions; D007785:Lactose; D019793:Fluorescein