Galectin-1, a beta-galactoside-binding lectin in Chinese hamster ovary cells. I. Physical and chemical characterization.
We report our studies on the characterization of an approximately 14-kDa lectin, termed galectin-1 that we have found to be expressed by Chinese hamster ovary (CHO) cells. cDNA for galectin-1 from CHO cells was prepared and sequenced, and a recombinant form (rGal-1) was expressed in Escherichia coli. A mutated form of the protein that fully retained activity was also constructed (termed C2SrGal-1) in which Cys-2 was changed to Ser-2. rGal-1 was stable in the presence of reducing agent, but it quickly lost all activity in the absence of reducing agent. In contrast, glycoprotein ligands, such as basement membrane laminin, stabilized the activity of rGal-1 in the absence of reducing agent (t1/2 = 2 weeks). C2SrGal-1 was stable in the presence or absence of either ligand or reducing agent. Unexpectedly, galectin-1 was found to exist in a reversible and active monomer-dimer equilibrium with a Kd approximately 7 microM and an equilibration time of t1/2 approximately 10 h. Addition of haptenic sugars did not affect this equilibrium. Galectin-1 isolated from the cytosol of CHO cells was found to exist as monomers and dimers. These studies demonstrate that galectin-1 binding to a biological ligand stabilizes its activity and that the monomer/dimer state of the protein is regulated by lectin concentration.
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University of Oklahoma Health Sciences Center, Department of Biochemistry and Molecular Biology, Oklahoma City 73190.
Cummings
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D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003545:Cysteine; D017931:DNA Primers; D004355:Drug Stability; D004926:Escherichia coli; D005690:Galactose / Q000378:metabolism*; D037483:Galectin 1; D006388:Hemagglutinins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D037102:Lectins / Q000096:biosynthesis / Q000737:chemistry; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D012694:Serine; D013997:Time Factors
D017931:DNA Primers; D037483:Galectin 1; D006388:Hemagglutinins; D037102:Lectins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012694:Serine; D003545:Cysteine; D005690:Galactose