Functional analysis of the carbohydrate recognition domains and a linker peptide of galectin-9 as to eosinophil chemoattractant activity.
Human galectin-9 is a beta-galactoside-binding protein consisting of two carbohydrate recognition domains (CRDs) and a linker peptide. We have shown that galectin-9 represents a novel class of eosinophil chemoattractants (ECAs) produced by activated T cells. A previous study demonstrated that the carbohydrate binding activity of galectin-9 is indispensable for eosinophil chemoattraction and that the N- and C-terminal CRDs exhibit comparable ECA activity, which is substantially lower than that of full-length galectin-9. In this study, we investigated the roles of the two CRDs in ECA activity in conjunction with the sugar-binding properties of the CRDs. In addition, to address the significance of the linker peptide structure, we compare the three isoforms of galectin-9, which only differ in the linker peptide region, in terms of ECA activity. Recombinant proteins consisting of two N-terminal CRDs (galectin-9NN), two C-terminal CRDs (galectin-9CC), and three isoforms of galectin-9 (galectin-9S, -9M, and -9L) were generated. All the recombinant proteins had hemagglutination activity comparable to that of the predominant wild-type galectin-9 (galectin-9M). Galectin-9NN and galectin-9CC induced eosinophil chemotaxis in a manner indistinguishable from the case of galectin-9M. Although the isoform of galectin-9 with the longest linker peptide, galectin-9L, exhibited limited solubility, the three isoforms showed comparable ECA activity over the concentration range tested. The interactions between N- and C-terminal CRDs and glycoprotein glycans and glycolipid glycans were examined using frontal affinity chromatography. Both CRDs exhibited high affinity for branched complex type sugar chain, especially for tri- and tetraantennary N-linked glycans with N-acetyllactosamine units, and the oligosaccharides inhibited the ECA activity at low concentrations. These results suggest that the N- and C-terminal CRDs of galectin-9 interact with the same or a closely related ligand on the eosinophil membrane when acting as an ECA and that ECA activity does not depend on a specific structure of the linker peptide.
Sato
Miki
M
Department of Endocrinology, Kagawa Medical University, 761-0793, Japan.
Nishi
Nozomu
N
Shoji
Hiroki
H
Seki
Masako
M
Hashidate
Tomomi
T
Hirabayashi
Jun
J
Kasai Ki
Ken-ichi
K
Hata
Yuiro
Y
Suzuki
Shigehiko
S
Hirashima
Mitsuomi
M
Nakamura
Takanori
T
D001483:Base Sequence; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002630:Chemotactic Factors / Q000737:chemistry*; D017931:DNA Primers; D004804:Eosinophils / Q000502:physiology*; D037161:Galectins; D006801:Humans; D007700:Kinetics; D037102:Lectins / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry*
D002630:Chemotactic Factors; D017931:DNA Primers; D037161:Galectins; C105032:LGALS9 protein, human; D037102:Lectins; D010455:Peptides