Abstract
A 36-kDa beta-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.
Highlights
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]
By homology search (National Center for Biotechnology Information), 73 clones were identified as mouse galectin-1, while 28 clones had other unrelated cDNA sequences
U72741) showed that it is an isoform of rat galectin-9 since it had a 32-amino acid insertion between the N-terminal carbohydrate domain and the link peptide, as in the mouse galectin-9 intestinal isoform
Summary
Degenerate Oligonucleotide-based Polymerase Chain Reaction (PCR) and Cloning and Nucleotide Sequencing of PCR Products Isolated from. Bacterial pellets were prepared, and suspended in 100 ml of either MEPBS buffer [9, 12] or Tris-dithiothreitol (DTT) buffer [34] containing 1.25% Triton X-100, 10 mM benzamidine, 10 mM ⑀-amino-n-caproic acid, and 2 mM phenylmethanesulfonyl fluoride. They were lysed by sonication, and the lysate was centrifuged at 20,000 ϫ g at 4 °C for 30 min. Overlapped regions of nucleotide similarity are variable and are adjusted by the Gap program (GCG Wisconsin package) to obtain highest homology scores
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