We report that human galectin-1 (dGal-1), a small dimeric ␤-galactoside-binding protein, induces phosphatidylserine ( It is believed that the turnover of neutrophils and other leukocytes in tissues involves programmed cell death (apoptosis) and then phagocytosis by tissue macrophages (1-4). However, the factors regulating turnover of leukocytes are unclear. Although Fas and Fas ligand (FasL) 1 induce apoptosis of mature, circulating neutrophils in vitro (5-7), mice deficient in FasL (gld) or Fas (lpr) have essentially normal numbers of circulating mature granulocytes (8). FasL/Fas-mediated apoptosis is not essential in regulating the clearance of neutrophils during inflammation (9). Fas and FasL may promote, rather than decrease, inflammatory responses in vivo (10 -12). In transgenic mice expressing bcl-2 in mature neutrophils, apoptosis of circulating cells is inhibited, but neutrophil homeostasis is unaltered, and macrophage-mediated phagocytosis of neutrophils is normal (13). Phagocytosis is required for resolution of the inflammatory process and leukocyte homeostasis in vivo (14 -16). These results suggest that factors not yet defined may regulate leukocyte turnover in tissues. Such observations led us to explore whether the basement membrane and extracellular matrix might harbor other proteins capable of binding to leukocytes and inducing their apoptosis or phagocytic recognition. A candidate protein is the ␤-galactoside-binding protein termed galectin-1 (dGal-1), which binds to most leukocytes. dGal-1 is a widely expressed dimeric protein (subunit ϳ14.6 kDa), which is a member of the galectin family of lectins (17)(18)(19). It is secreted by many cell types, including human endothelial cells (20,21), and is found in the basement membrane and extracellular matrices around capillary walls (22,23). dGal-1 has been reported to have various biological activities, including effects on neurite outgrowth (24, 25), growth inhibition of non-neural cells (26 -28), cell growth stimulation (29,30), and apoptosis of immature thymocytes (31, 32), and to activate human T cells and T cell lines (33,34).To explore the biological activity of dGal-1 toward leukocytes, we prepared a recombinant form of dimeric human dGal-1 and a mutated, monomeric form of galectin-1 (mGal-1). We explored the interactions of these lectins with HL-60 cells, MOLT-4 cells, and both resting and activated human neutrophils. Our results show that dGal-1, but not mGal-1, rapidly enhances surface staining with Annexin V (phosphatidylserine (PS) exposure) in desialylated HL-60 cells, desialylated MOLT-4 cells, and activated, but not resting, human neutrophils. The exposure of PS is often associated with apoptosis * This work was supported by National Institutes of Health Grants AI48075 (to R. D. C.) and HL34363 and RR15577 (to R. P. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate ...

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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.

In the accompanying study (Cho, M., and Cummings, R. D. (1995) J. Biol. Chem. 270, 5198-5206), we reported that Chinese hamster ovary (CHO) cells synthesize galectin-1. We have now used several approaches to define the subcellular location and biosynthesis of galectin-1 in these cells. Galectin-1 was present on the cell surface, as assessed by immunofluorescent staining with monospecific antibody to the protein. Quantitation of the surface-localized galectin-1 was achieved by metabolically radiolabeling cells with [35S]Met/Cys and measuring the amount of lectin (i) sensitive to trypsin, (ii) accessible to biotinylating reagents, and (iii) accessible to the haptenic disaccharide lactose. By all three procedures, approximately 1/2 of the radiolabeled galectin-1 associated with cells was shown to be on the cell surface with the remainder intracellular. The kinetics of externalization of galectin-1 was monitored by pulse-chase radiolabeling, and it was shown that cells secrete the protein with a t1/2 approximately 20 h. The cell surface form of galectin-1 in CHO cells was active and bound to surface glycoconjugates, but lectin accumulating in the culture media was inactive. Lectin synthesized by mutant Lec8 CHO cells, which are unable to galactosylate glycoproteins was not found on the surface and quantitatively accumulated in the media in an inactive form. Taken together, our results demonstrate that galectin-1 is quantitatively externalized by CHO cells and can associate with surface glycoconjugates where the lectin activity is stabilized.

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