Requirement of divalent galactoside-binding activity of ecalectin/galectin-9 for eosinophil chemoattraction.
We have previously isolated and cloned a novel eosinophil chemoattractant (ECA) from a human T-cell-derived expression library. This ECA, termed ecalectin, is a variant of human galectin-9, a member of a beta-galactoside binding animal lectin family, which contains two conserved carbohydrate recognition domains (CRDs). In the present study, we addressed whether carbohydrate binding activity is required for the ECA activity of ecalectin and whether both CRDs are essential for this activity. Recombinant full-length wild-type ecalectin (ecalectin-WT) and N-terminal and C-terminal CRD (ecalectin-NT and -CT, respectively) were generated. All of these recombinant proteins exhibited affinity for lactose, a property shared by galectins, but ecalectin-WT exhibited substantially higher hemagglutination activities than ecalectin-NT and -CT. Furthermore, ecalectin-WT showed over 100-fold higher ECA activity than ecalectin-NT and -CT; combination of recombinant domain fragments did not reconstitute the ECA and hemagglutination activities of the full-length protein. ECA activity of ecalectin-WT was inhibited by lactose in a dose-dependent manner. Site-directed mutation of positions Arg(65) of ecalectin-NT and Arg(239) of ecalectin-CT to an aspartic acid residue resulted in the loss of both lactose-binding and ECA activities. We conclude that divalent galactoside-binding activity is required for eosinophil chemoattraction by ecalectin.
Matsushita
N
N
Department of Immunology and Immunopathology, Kagawa Medical School, Kagawa 761-0793, Japan.
Nishi
N
N
Seki
M
M
Matsumoto
R
R
Kuwabara
I
I
Liu
F T
FT
Hata
Y
Y
Nakamura
T
T
Hirashima
M
M
D000943:Antigens, Differentiation / Q000494:pharmacology; D001681:Biological Assay; D002631:Chemotactic Factors, Eosinophil / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004804:Eosinophils / Q000187:drug effects; D005697:Galactosides / Q000378:metabolism*; D037483:Galectin 1; D037502:Galectin 3; D037161:Galectins; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D006388:Hemagglutinins / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007785:Lactose / Q000378:metabolism; D037102:Lectins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D000943:Antigens, Differentiation; D002631:Chemotactic Factors, Eosinophil; D005697:Galactosides; D037483:Galectin 1; D037502:Galectin 3; D037161:Galectins; D006388:Hemagglutinins; C091991:LGALS8 protein, human; C105032:LGALS9 protein, human; D037102:Lectins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D007785:Lactose