title: Dimeric Galectin-8 induces phosphatidylserine exposure in leukocytes through polylactosamine recognition by the C-terminal domain.
abstract: Human galectins have distinct and overlapping biological roles in immunological homeostasis. However, the underlying differences among galectins in glycan binding specificity regulating these functions are unclear. Galectin-8 (Gal-8), a tandem repeat galectin, has two distinct carbohydrate recognition domains (CRDs) that may cross-link cell surface counter receptors. Here we report that each Gal-8 CRD has differential glycan binding specificity and that cell signaling activity resides in the C-terminal CRD. Full-length Gal-8 and recombinant individual domains (Gal-8N and Gal-8C) bound to human HL60 cells, but only full-length Gal-8 signaled phosphatidylserine (PS) exposure in cells, which occurred independently of apoptosis. Although desialylation of cells did not alter Gal-8 binding, it enhanced cellular sensitivity to Gal-8-induced PS exposure. By contrast, HL60 cell desialylation increased binding by Gal-8C but reduced Gal-8N binding. Enzymatic reduction in surface poly-N-acetyllactosamine (polyLacNAc) glycans in HL60 cells reduced cell surface binding by Gal-8C but did not alter Gal-8N binding. Cross-linking and light scattering studies showed that Gal-8 is dimeric, and studies on individual subunits indicate that dimerization occurs through the Gal-8N domain. Mutations of individual domains within full-length Gal-8 showed that signaling activity toward HL60 cells resides in the C-terminal domain. In glycan microarray analyses, each CRD of Gal-8 showed different binding, with Gal-8N recognizing sulfated and sialylated glycans and Gal-8C recognizing blood group antigens and polyLacNAc glycans. These results demonstrate that Gal-8 dimerization promotes functional bivalency of each CRD, which allows Gal-8 to signal PS exposure in leukocytes entirely through C-terminal domain recognition of polyLacNAc glycans.
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Sean R
authors.initials: SR
authors.lastname: Stowell
authors.forename: Connie M
authors.initials: CM
authors.lastname: Arthur
authors.forename: Kristin A
authors.initials: KA
authors.lastname: Slanina
authors.forename: John R
authors.initials: JR
authors.lastname: Horton
authors.forename: David F
authors.initials: DF
authors.lastname: Smith
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cummings
mesh_terms: D000606:Amino Sugars / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D037161:Galectins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D007962:Leukocytes / Q000187:drug effects* / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D000606:Amino Sugars; D037161:Galectins; C091991:LGALS8 protein, human; D010718:Phosphatidylserines; D011134:Polysaccharides; C066929:polylactosamine