Identification and Characterization of Galectin-9, a Novel β-Galactoside-binding Mammalian Lectin

Abstract

A 36-kDa ␤-galactoside mammalian lectin protein, designated as galectin-9, was isolated from mouse embryonic kidney by using a degenerate primer polymerase chain reaction and cloning strategy. Its deduced amino acid sequence had the characteristic conserved sequence motif of galectins. Endogenous galectin-9, extracted from liver and thymus, as well as recombinant galectin-9 exhibited specific binding activity for the lactosyl group. It had two distinct N-and C-terminal carbohydrate-binding domains connected by a link peptide, with no homology to any other protein. Galectin-9 had an alternate splicing isoform, exclusively expressed in the small intestine with a 31-amino acid insertion between the N-terminal domain and link peptide. Sequence homology analysis revealed that the C-terminal carbohydrate-binding domain of mouse galectin-9 had extensive similarity to that of monomeric rat galectin-5. The presence of galectin-5 in the mouse could not be demonstrated by polymerase chain reaction or by Northern or Southern blot genomic DNA analyses. Sequence comparison of rat galectin-5 and rat galectin-9 cDNA did not reveal identical nucleotide sequences in the overlapping C-terminal carbohydrate-binding domain, indicating that galectin-9 is not an alternative splicing isoform of galectin-5. However, galectin-9 had a sequence identical with that of its intestinal isoform in the overlapping regions in both species. Southern blot genomic DNA analyses, using the galectin-9 specific probe derived from the N-terminal carbohydrate-binding domain, indicated the presence of a novel gene encoding galectin-9 in both mice and rats. In contrast to galectin-5, which is mainly expressed in erythrocytes, galectin-9 was found to be widely distributed, i.e. in liver, small intestine, thymus > kidney, spleen, lung, cardiac and skeletal muscle > reticulocyte, brain. Collectively, these data indicate that galectin-9 is a new member of the galectin gene family and has a unique intestinal isoform.There is growing evidence that specific carbohydrate moieties and their putative binding proteins, i.e. lectins, play diverse roles in mammalian physiology and development and in various pathological states (1). The mammalian lectins are classified into four categories, C-type lectins (including selectins), P-type lectins, pentraxins, and galectins; the latter are referred to as S-type or S-Lac lectins (2, 3). Galectins are endowed with two essential biochemical properties: 1) characteristic amino acid homologous sequences; and 2) affinity for ␤-galactoside sugars, i.e. carbohydrate-binding domain. In addition, all the known galectins lack a signal peptide, have a cytoplasmic localization, and are secreted as soluble proteins by a nonclassical secretory pathway (4). Seven mammalian galectins, i.e. galectins-1 (5), -2 (6), -3 (7), -4 (8), -5 (9), -7 (10, 11), and -8 (12), have been cloned and characterized. Structural analyses of various galectins indicate the presence of homodimers of carbohydrate-binding domains in galectin-1 and galectin-2, a monom...

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