Activated rat macrophages produce a galectin-1-like protein that induces apoptosis of T cells: biochemical and functional characterization.
Galectins, a family of closely related beta-galactoside-binding proteins, show specific immunomodulatory properties. We have recently identified the presence of a galectin-like protein in rat peritoneal macrophages by means of a cross-reactivity with a polyclonal Ab raised against a galectin purified from adult chicken liver. Galectin expression was up-regulated in inflammatory and activated macrophages, revealing a significant increase in phorbol ester- and formylmethionine oligopeptide-treated cells. In an attempt to further explore its functional significance, rat macrophage galectin was purified from activated macrophages by a single-step affinity chromatography on a lactosyl-Sepharose matrix. The eluted fraction was resolved as a single protein band of approximately 15,000 Da by SDS-PAGE that immunoreacted strongly with the anti-chicken galectin serum. Gel filtration studies revealed that the protein behaved like a dimer under native conditions, and saccharides bearing a beta-D-galactoside configuration were able to inhibit the hemagglutinating activity displayed by the purified galectin. In agreement with its isoelectric point of approximately 4.8, the amino acid analysis showed a definitive acidic pattern. Internal amino acid sequencing of selected peptides obtained by proteolytic cleavage revealed that this carbohydrate-binding protein shares all the absolutely preserved and critical residues found in other members of the mammalian galectin-1 subfamily. Finally, biochemical and ultrastructural evidence, obtained by genomic DNA fragmentation and transmission electron microscopy, are also provided to show its potential implications in the apoptotic program of T cells. This effect was quantified by using the terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling assay and was found to be associated to the specific carbohydrate-binding properties of galectin.
Rabinovich
G A
GA
Department of Clinical Biochemistry, Faculty of Chemical Sciences, National University of Cordoba, Argentina. grabino@bioclin.Fcq.unc.edu.ar
Iglesias
M M
MM
Modesti
N M
NM
Castagna
L F
LF
Wolfenstein-Todel
C
C
Riera
C M
CM
Sotomayor
C E
CE
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D005260:Female; D037483:Galectin 1; D006388:Hemagglutinins / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology*; D007526:Isoelectric Point; D008262:Macrophage Activation; D008264:Macrophages / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D017208:Rats, Wistar; D013601:T-Lymphocytes / Q000502:physiology*
D037483:Galectin 1; D006388:Hemagglutinins