Moonjae Cho

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 ∼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 ≈ 7 μM and an equilibration time of t1/2 ≈ 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.

Dimeric galectin-1 induces surface exposure of phosphatidylserine and phagocytic recognition of leukocytes without inducing apoptosis

We report that human galectin-1 (dGal-1), a small dimeric β-galactoside-binding protein, induces phosphatidylserine (PS) exposure, measured by Annexin V staining, on human promyelocytic HL-60 cells, T leukemic MOLT-4 cells, and fMet-Leu-Phe-activated, but not resting, human neutrophils. This effect of dGal-1 on HL-60 and MOLT-4 cells is enhanced by pretreatment of the cells with neuraminidase, but treatment of resting neutrophils with neuraminidase does not enhance their sensitivity to dGal-1. Although the induction of staining with Annexin V is often associated with apoptosis, the dGal-1-treated HL-60 cells, MOLT-4 cells, and activated neutrophils do not undergo apoptosis, and there is no detectable DNA fragmentation. HL-60 and MOLT-4 cells treated with dGal-1 continue to grow normally. By contrast, camptothecin-treated HL-60 cells, etoposide-treated MOLT-4 cells, and anti-Fas-treated neutrophils exhibit extensive DNA fragmentation and/or cell death. Lactose inhibits the dGal-1-induced effects, indicating that dGal-1-induced signaling requires binding to cell surface β-galactosides. The dimeric form of Gal-1 is required for signaling, because a monomeric mutant form of Gal-1, termed mGal-1, binds to cells but does not cause these effects. Importantly, dGal-1, but not mGal-1, treatment of HL-60 cells and activated human neutrophils significantly promotes their phagocytosis by activated mouse macrophages. These dGal-1-induced effects are distinguishable from apoptosis, but like apoptotic agents, prepare cells for phagocytic removal. Such effects of dGal-1 may contribute to leukocyte homeostasis.

Galectin-1, a beta-galactoside-binding lectin in Chinese hamster ovary cells. II. Localization and biosynthesis.

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, ≈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 ≈ 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.

Noble tandem-repeat galectin of Manila clam Ruditapes philippinarum is induced upon infection with the protozoan parasite Perkinsus olseni.

The galectin family of lectins plays crucial roles in the innate immunity systems of vertebrates and invertebrates. Noble galectin (MCGal) was cloned from the marine invertebrate Ruditapes philippinarum and characterized. This protein has an open reading frame of 918 nucleotides, with 309 amino acid residues, and a predicted molecular weight of 33.9kDa. Similar to other galectins, MCGal has neither a signal peptide nor a transmembrane domain, but it contains tandemly repeated carbohydrate recognition domains (CRDs), with typical conserved motifs that are important for carbohydrate recognition. Carbohydrate recognition by the recombinant MCGal (rMCGal), as determined by hapten inhibition of hemagglutination, revealed that rMCGal has features common to the galectin family, i.e., significant affinity for galactose and N-acetylgalactosamine. MCGal mRNA expression was detected mainly in the heart, mantle, foot, adductor, palp, and siphon tissues. Immunohistochemistry (IHC) using an anti-MCGal antibody confirmed MCGal expression in these tissues and in hemocytes. Temporal expression of MCGal mRNA in Manila clams challenged with Perkinsus or Vibrio species was up-regulated as compared with non-challenged healthy clams. rMCGal agglutinated Vibrio tapetis, and agglutination was inhibited by incubation with alpha-lactose. rMCGal also bound to the surface of Perkinsus olseni. MCGal plays a crucial role in Manila clam defense, particularly with respect to pathogen recognition.

Lectin from the Manila Clam Ruditapes philippinarum Is Induced upon Infection with the Protozoan Parasite Perkinsus olseni

Glycan-binding proteins (lectins) are widely expressed in many invertebrates, although the biosynthesis and functions of the lectins are not well understood. Here we report that Manila clam (Ruditapes philippinarum) synthesizes a lectin termed Manila clam lectin (MCL) upon infection with the protozoan parasite Perkinsus olseni. MCL is synthesized in hemocytes as a ∼74-kDa precursor and secreted into hemolymph where it is converted to 30and 34-kDa polypeptides. The synthesis of MCL in hemocytes is stimulated by one or more factors in Perkinsus-infected hemolymph, but not directly by Perkinsus itself. MCL can bind to the surfaces of purified hypnospores and zoospores of the parasite, and this binding is inhibitable by either EDTA or GalNAc. Fluorescent beads coated with purified MCL were actively phagocytosed by hemocytes from the clam. Immunohistochemistry showed that secreted MCL is concentrated within cyst-like structures. To define the glycan binding specificity of MCL we examined its binding to an array of biotinylated glycans. MCL recognizes terminal non-reducing β-linked GalNAc as expressed within the LacdiNAc motif GalNAcβ1–4GlcNAcβ1-R and glycans with terminal, non-reducing β-linked Gal residues. Our results show that the synthesis of MCL is specifically up-regulated upon parasite infection of the clams and may serve as an opsonin through recognition of terminal GalNAc/Gal residues on the parasites.

Ligand reduces galectin-1 sensitivity to oxidative inactivation by enhancing dimer formation.

Galectin-1 (Gal-1) regulates leukocyte turnover by inducing the cell surface exposure of phosphatidylserine (PS), a ligand that targets cells for phagocytic removal, in the absence of apoptosis. Gal-1 monomer-dimer equilibrium appears to modulate Gal-1-induced PS exposure, although the mechanism underlying this regulation remains unclear. Here we show that monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidation. A mutant form of Gal-1, containing C2S and V5D mutations (mGal-1), exhibits impaired dimerization and fails to induce cell surface PS exposure while retaining the ability to recognize carbohydrates and signal Ca2+ flux in leukocytes. mGal-1 also displayed enhanced sensitivity to oxidation, whereas ligand, which partially protected Gal-1 from oxidation, enhanced Gal-1 dimerization. Continual incubation of leukocytes with Gal-1 resulted in gradual oxidative inactivation with concomitant loss of cell surface PS, whereas rapid oxidation prevented mGal-1 from inducing PS exposure. Stabilization of Gal-1 or mGal-1 with iodoacetamide fully protected Gal-1 and mGal-1 from oxidation. Alkylation-induced stabilization allowed Gal-1 to signal sustained PS exposure in leukocytes and mGal-1 to signal both Ca2+ flux and PS exposure. Taken together, these results demonstrate that monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidative inactivation and provides a mechanism whereby ligand partially protects Gal-1 from oxidation.

Transcriptional Regulation of 1,3-Galactosyltransferase in Embryonal Carcinoma Cells by Retinoic Acid MASKING OF LEWIS X ANTIGENS BY α-GALACTOSYLATION

Treatment of mouse teratocarcinoma F9 cells with all-trans-retinoic acid (RA) causes a 9-fold increase in steady-state levels of mRNA for UDP-Gal:β-D-Gal α1,3-galactosyltransferase (α1,3GT) beginning at 36 h. Enzyme activity rises in a similar fashion, which also parallels the induction of laminin and type IV collagen. Nuclear run-on assays indicate that this increase in α1,3GT in RA-treated F9 cells, like that of type IV collagen, is transcriptionally regulated. Differentiation also results in increased secretion of soluble α1,3GT activity into the growth media. The major α-galactosylated glycoprotein present in the media of RA-treated F9 cells, but not of untreated cells, was identified as laminin. Differentiation of F9 cells is accompanied by an increase in α-galactosylation of membrane glycoproteins and a decrease in expression of the stage-specific embryonic antigen, SSEA-1 (also known as the Lewis X antigen or Le), which has the structure Galβ1-4(Fucα1-3)GlcNAcβ1-R. However, flow cytometric analyses with specific antibodies and lectins, following treatment of cells with α-galactosidase, demonstrate that differentiated cells contain Le antigens that are masked by α-galactosylation. Thus, RA induces α1,3GT at the transcriptional level, resulting in major alterations in the surface phenotype of the cells and masking of Le antigens.

Cell Adhesion-dependent Cofilin Serine 3 Phosphorylation by the Integrin-linked Kinase·c-Src Complex

Integrin-linked kinase (ILK) is involved in signal transduction by integrin-mediated cell adhesion that leads to dynamic actin reorganization. Actin (de)polymerization is regulated by cofilin, the Ser3 phosphorylation (pS3cofilin) of which inhibits its actin-severing activity. To determine how ILK regulates pS3cofilin, we examined the effects of ILK on pS3cofilin using normal RIE1 cells. Compared with suspended cells, fibronectin-adherent cells showed enhanced pS3cofilin, depending on ILK expression and c-Src activity. The ILK-mediated pS3cofilin in RIE1 cells did not involve Rho-associated kinase, LIM kinase, or testicular protein kinases, which are known to be upstream of cofilin. The kinase domain of ILK, including proline-rich regions, appeared to interact physically with the Src homology 3 domain of c-Src. In vitro kinase assay revealed that ILK immunoprecipitates phosphorylated the recombinant glutathione S-transferase-cofilin, which was abolished by c-Src inhibition. Interestingly, epidermal growth factor treatment abolished the ILK effects, indicating that the linkage from ILK to cofilin is biologically responsive to extracellular cues. Altogether, this study provides evidence for a new signaling connection from ILK to cofilin for dynamic actin polymerization during cell adhesion, depending on the activity of ILK-associated c-Src.

Blockade of four‐transmembrane L6 family member 5 (TM4SF5)‐mediated tumorigenicity in hepatocytes by a synthetic chalcone derivative

We previously reported that the four‐transmembrane L6 family member 5 (TM4SF5) was highly expressed in hepatocarcinoma, induced morphological elongation and epithelial‐mesenchymal transition, and caused abnormal cell growth in multilayers in vitro and tumor formation in vivo. In this study, we identified a synthetic compound, 4′‐(p‐toluenesulfonylamido)‐4‐hydroxychalcone (TSAHC) that antagonized both the TM4SF5‐mediated multilayer growth and TM4SF5‐enhanced migration/invasion. TSAHC treatment induced multilayer‐growing cells to grow in monolayers, recovering contact inhibition without accompanying apoptosis, and inhibited chemotactic migration and invasion. Tumor formation in nude mice injected with TM4SF5‐expressing cells and the growth of cells expressing endogenous TM4SF5, but not of TM4SF5‐null cells, was suppressed by treatment with TSAHC, but not by treatment with its analogs. The structure‐activity relationship indicated the significance of 4′‐p‐toluenesulfonylamido and 4‐hydroxy groups for the anti‐TM4SF5 effects of TSAHC. Point mutations of the putative N‐glycosylation sites abolished the TM4SF5‐specific TSAHC responsiveness. Conclusion: These observations suggest that TM4SF5‐enhanced tumorigenic proliferation and metastatic potential can be blocked by TSAHC, likely through targeting the extracellular region of TM4SF5, which is important for protein‐protein interactions. (HEPATOLOGY 2009.)

Expression of Human H-type α1,2-Fucosyltransferase Encoding for Blood Group H(O) Antigen in Chinese Hamster Ovary Cells EVIDENCE FOR PREFERENTIAL FUCOSYLATION AND TRUNCATION OF POLYLACTOSAMINE SEQUENCES

The human H(O) blood group is specified by the structure Fucα1-2Galβ1-R, but the factors regulating expression of this determinant on cell surface glycoconjugates are not well understood. To learn more about the regulation of H blood group expression, cDNA encoding the human H-type GDPFuc:β-D-galactoside α1,2-fucosyltransferase (α1,2FT) was stably transfected into Chinese hamster ovary (CHO) cells. The new cell line, designated CHO(α1,2)FT, expressed surface neoglycans containing the H antigen. The structures of the fucosylated neoglycans in CHO(α1,2)FT cells and the distribution of these glycans on glycoproteins were characterized. Seventeen percent of the [3H]Gal-labeled glycopeptides from CHO(α1,2)FT cells bound to the immobilized H blood group-specific lectin Ulex europaeus agglutinin-I (UEA-I), whereas none from parental CHO cells bound to the lectin. The glycopeptides from CHO(α1,2)FT cells binding to UEA-I contained polylactosamine [3Galβ1-4GlcNAcβ1-]n with the terminal sequence Fucα1-2Galβ1- 4GlcNAc-R. Fucosylation of the polylactosamine sequences on complex-type N-glycans in CHO(α1,2)FT cells caused a decrease in both sialylation and length of polylactosamine. Unexpectedly, only small amounts of terminal fucosylation was found in diantennary complex-type N-glycans. The O-glycans and glycolipids were not fucosylated by the H-type α1,2FT. Two major high molecular weight glycoproteins, one of which was shown to be the lysosome-associated membrane glycoprotein LAMP-1, preferentially contained the H-type structure and were bound by immobilized UEA-I. These results demonstrate that in CHO cells the expressed H-type α1,2FT does not indiscriminately fucosylate terminal galactosyl residues in complex-type N-glycans, but it favors glycans containing polylactosamine and dramatically alters their length and sialylation.