Isolation of galectin-1 from human platelets: its interaction with actin.

Galectins are a family of animal lectins defined by their ß-galactoside-binding specificity and a consensus sequence in their carbohydrate-recognition domain. Galectin-1 (Gal-1) is expressed as a non-covalently linked homodimer present in a variety of tissues. Here we describe its isolation from human platelets by a procedure involving ionic exchange chromatography and affinity chromatography on lactose-agarose. Platelet Gal-1 co-purifies with actin, forming an actin-Gal-1 complex which does no dissociate even after treatment with sodium dodecyl sulfate. The presence of both proteins was confirmed by Western blot and by trypsin digestion followed by mass spectrometry identification. By hemagglutination assays we studied the response of recombinant Gal-1/actin, mixed and pre-incubated in different proportions, and then tested against neuraminidase treated rabbit red blood cells. The complex formation was confirmed by confocal microscopy, showing that both proteins co-localised in resting platelets as well as in thrombin-activated ones. These results suggest that endogenous Gal-1 forms an intracellular complex with monomeric actin and that, after platelet activation, Gal-1 could play a role in the polymerization-depolymerization process of actin, which concludes in platelet aggregation.

Galectins: matricellular glycan-binding proteins linking cell adhesion, migration, and survival.

Galectins are a taxonomically widespread family of glycan-binding proteins, defined by at least one conserved carbohydrate-recognition domain with a canonical amino acid sequence and affinity for beta-galactosides. Because of their anti-adhesive as well as pro-adhesive extracellular functions, galectins appear to be a novel class of adhesion-modulating proteins collectively known as matricellular proteins (which include thrombospondin, SPARC, tenascin, hevin, and disintegrins). Accordingly, galectins can display de-adhesive effects when presented as soluble proteins to cells in a strong adhesive state. In this context, the de-adhesive properties of galectins should be considered as physiologically relevant as the proadhesive effects of these glycan-binding proteins. This article focuses on the roles of mammalian galectins in cell adhesion, spreading, and migration, and the crossregulation of these functions. Although careful attention should be paid when examining individual galectin functions due to overlapping distributions, these intriguing glycan-binding proteins offer promising possibilities for the treatment and intervention of a wide variety of pathological processes, including cancer, inflammation, and autoimmunity.

Identification of gp17 glycoprotein and characterization of prostatic acid phosphatase (PAP) and carboxypeptidase E (CPE) fragments in a human seminal plasma fraction interacting with concanavalin A.

The decapacitating fraction of human seminal plasma, which strongly interacts with concanavalin A, is constituted by high mannose-type N-linked glycoproteins, most of them of less than 44 kDa. Each component with apparent molecular mass of 30, 18, and 17 kDa respectively, as judged by SDS-PAGE, was submitted to "in gel" digestion with trypsin followed by HPLC separation of the peptides and sequencing. They were characterized at microscale as gp17, an aspartyl protease that possibly contributes to liquefaction of the seminal plasma coagulum, two fragments of human acid phosphatase (17 and 30 kDa, respectively), and a 17-kDa fragment of carboxypeptidase E. Neither the fragments of prostatic acid phosphatase nor that of carboxypeptidase E had been described before in the human seminal fluid. Very weak bands, of apparent molecular masses 44 and 52 kDa, are consistent with presence of small amounts of parent compounds, prostatic acid phosphatase and carboxypeptidase E.

Inhibition of acrosine-like protease activity by a lectin affinity chromatographic bovine seminal plasma fraction containing the PDC-109 and aSFP proteins.

These studies showed that the fractionation of bovine seminal plasma based on lectin agarose affinity chromatography, employing lectins specific to asparagine linked oligosaccharides, and a lectin specific for fucosylated glycans, lead to products with an inhibitory effect on the acrosine-like protease activity. This effect decreases when glycocompounds containing fucosylated Lewis(x) structures are removed, suggesting that these compounds might have some role in the modulation of this activity in the bull. In the fraction devoid of high mannose, hybrid and non-bisecting lactosaminic oligosaccharide-containing glycocompounds, PDC-109 and aSFP proteins were detected and characterized at microscale.

Ovine placental lactogen and ovine prolactin: partial proteolysis and conformational stability.

The high-resolution structure of ovine placental lactogen (oPL) and ovine prolactin (oPRL), not yet established in detail, was probed by limited proteolysis with the Glu-specific protease from Staphylococcus aureus V8. While in hGH there were no cleavage sites inside of any of the four alpha-helices, the analysis of the fragments obtained after partial proteolysis of oPL showed a site of cleavage at the putative third helix, suggesting that this helix is partially unwound at this point. The partial proteolysis of the rest of the molecule was compatible with a similar folding pattern for oPL, hGH and pGH, on the basis of the crystal structure of these last hormones. In the case of oPRL, proteolytic cleavage occurred at Glu residues which would be located at the end of the first helix and the beginning of the second in the hGH folding model, suggesting that these helices are shorter in oPRL than in hGH. In order to gain further insight on the folding of these molecules, circular dichroism and intrinsic fluorescence measurements were used to examine the effect of denaturing conditions on oPL and oPRL. After exposure to 6 M guanidine the unfolding of both proteins was completely reversed upon elimination of the denaturing agent. In contrast, exposure to pH 3.0 caused an irreversible decrease in the alpha-helical content in both hormones, most striking for oPL, indicating that this hormone is less stable than oPRL or hGH.

Ovine placental lactogen and human growth hormone bind to different regions of the same receptors.

Anti-human growth hormone (hGH) polyclonal and monoclonal antibodies (MAb) failed to recognize ovine placental lactogen (oPL), indicating that the antigenic topographies of both hormones are different. Binding assays showed that oPL completely inhibited hGH binding to lactogenic receptors from Nb2-cells and to somatogenic receptors from rabbit or sheep liver; in contrast, oPL only bound to a subpopulation of rat liver receptors. Zinc ion increased hGH and oPL binding to Nb2-cell receptors and slightly inhibited both hormones' recognition by somatogenic receptors. However, ZnCl(2) increased hGH binding to rat liver microsomes but prevented that of oPL. Furthermore, MAb R7B4, recognizing lactogenic as well as somatogenic receptors, entirely blocked hGH binding to the various receptor systems but not affected oPL binding. Therefore, results presented in this paper suggest that oPL and hGH bind to different regions of the same receptors.

Purification of galectin-3 from ovine placenta: developmentally regulated expression and immunological relevance.

Galectins, beta-galactoside-binding lectins, are extensively distributed in the animal kingdom and share some basic molecular properties. Galectin-3, a member of this family, is generally associated with differentiation, morphogenesis, and metastasis. In this study, galectin-3 was isolated from ovine placental cotyledons round the middle of the gestation period by lactose extraction followed by affinity chromatography on lactosyl-agarose, and separated from galectin-1 by size exclusion chromatography on a Superose 12 column. Under native conditions this lectin behaved as a monomer with an apparent molecular weight of approximately 29,000 and an isoelectric point of 9.0. The partial amino acid sequence of the peptides obtained by tryptic digestion of this protein followed by HPLC separation showed striking homology with other members of the galectin-3 subfamily. Furthermore, ovine placental galectin-3 exhibited specific mitogenic activity toward rat spleen mononuclear cells. Besides, this protein strongly reacted with a rabbit antiserum raised against a chicken galectin. Results obtained by Western blot analysis showed that its expression was greatly decreased in term placenta with respect to the middle of the gestation period, suggesting a regulated expression throughout development.

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.

Galectin-1 from ovine placenta--amino-acid sequence, physicochemical properties and implications in T-cell death.

In the present study we report the amino-acid sequence, carbohydrate specificity and overall biochemical and physicochemical properties of galectin-1, a beta-galactoside-binding lectin from ovine placenta. The complete amino-acid sequence, obtained by tryptic and chymotryptic digestion, revealed that this carbohydrate-binding protein shares all the absolutely preserved and critical residues found in other members of the mammalian galectin-1 subfamily. Moreover, conformational changes induced by protein interaction with its specific disaccharide were investigated by fourth-derivative spectral analysis, intrinsic tryptophan fluorescence measurements and circular dichroism. The first two methods indicated changes in the environment of aromatic residues, in agreement with the role of Trp in carbohydrate binding. The quenching of the fluorescence emission upon addition of lactose, allowed us to calculate the Kd for its interaction with the galectin, which was 0.157 +/- 0.02 mM. The far-ultraviolet CD spectra is consistent with the large extent of beta-sheet structure described for other galectins. Addition of lactose produced no significant changes, suggesting that it causes no modifications in the secondary structure of the lectin. In addition, we explored its potential cell-growth inhibitory activity and implications in T-cell death. Finally, we also provide evidence showing that antagonic properties of galectins-1 and -3 are reciprocally neutralized in a natural mixture of both proteins, suggesting that they could play an important role in the regulation of cell proliferation and death, according to physiological requirements at particular developmental stages of the placenta, thus allowing successful pregnancy to occur.

The heparin-binding lectin from ovine placenta: purification and identification as histone H4.

The heparin-binding lectin complex from ovine placental cotyledons was purified by affinity chromatography on heparin-agarose column. It showed three protein bands, which had molecular weights of 13000, 15000 and 17000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the presence of DNA by agarose gel electrophoresis. The protein components of the complex were separated by reverse-phase HPLC. The minimum inhibitory concentrations of glycosaminoglycans were significantly different for the lectin complex and the separated proteins, suggesting affinity changes upon DNA binding. The haemagglutinating activity specificity allowed the characterization of the fraction with a molecular weight of 13000 as the heparin-binding lectin. This protein was identified as histone H4 by internal sequencing, thus showing that this is the histone responsible for the heparin-binding property of the complex. The accompanying proteins were tentatively identified as histones H2A and H2B.

A sialic acid-binding lectin from ovine placenta: purification, specificity and interaction with actin.

A sialic-acid-specific lectin from ovine placental cotyledons was purified by affinity chromatography on bovine submaxillary mucin-agarose followed by gel filtration, and it showed a molecular weight of 65000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis. This lectin has the capacity to interact with actin, since it binds to actin-F in a cosedimentation assay and it acts as a mediator in the binding of actin to the affinity column. The lectin agglutinated rabbit and rat erythrocytes, but not human A, B or O erythrocytes. Haemagglutination inhibition assays of different saccharides, glycoproteins and glycolipids indicate that this lectin has affinity for sialic acid, which is enhanced by its O-acetylation. The N-terminal sequence of the protein shows 92% identity with rabbit and porcine uterine calreticulin.

Detection and characterization of an ovine placental lactogen stable intermediate in the urea-induced unfolding process.

The urea-induced equilibrium unfolding of ovine placental lactogen, purified from ovine placenta, was followed by size-exclusion chromatography, far-UV CD, and intrinsic tryptophan fluorescence. The data obtained by each of these methods showed a poor fit to a two-state model involving only a native and an unfolded form. A satisfactory fit required, instead, a model that involved a stable, partially folded form in addition to the native and unfolded ones. The results obtained from the best-fitting theoretical curves for the three-state model indicated that this intermediate state, which is the predominant species in solution at 3.6 M of urea activity, is compact, largely alpha-helical, and changes considerably the native-like tertiary packing around its tryptophan residues. These findings suggest that this stable intermediate exhibits properties similar to those that characterize the molten globule state.

Identification of a tyrosine residue in ovine placental lactogen as essential for its binding to receptors.

Nitration of ovine placental lactogen (oPL) with a 10-fold molar excess of tetranitromethane over protein content resulted in the modification of 0.8 tyrosine residue. No conformational changes were observed by either fourth-derivative spectral analysis or circular dichroism. Nitration significantly decreased the binding capacity of the hormone to lactogenic and somatogenic rat liver receptors. This binding capacity was not restored by reduction of the nitro groups, thus indicating that the decrease was not due to the difference in pK between tyrosine and nitrotyrosine. The nitrotyrosine-containing peptide was isolated from a tryptic digest by HPLC and its modification extent was of 67%, which is consistent with the decrease in binding capacities (65% and 70%). Its amino acid sequence was determined and the modified tyrosine residue was identified as Tyr-46. These results provide the first evidence of the involvement of a tyrosine residue in the binding of oPL to both lactogenic and somatogenic receptors. This tyrosine appears to be a shared binding epitope between oPL and the prolactins.

Modification of arginine residues in ovine prolactin by 1,2-cyclohexanedione. Effect on binding capacity to lactogenic receptors.

The reactivity of arginine residues in ovine prolactin was studied by reaction with 1,2-cyclohexanedione. Kinetic analysis of the data showed a good fit with two simultaneous pseudo-first-order equations with apparent velocity constants of 0.28 and 1.2 x 10(-2) min-1, corresponding to 1.8 'fast' and 8.7 'slow' residues, respectively. Modification led to a decrease in binding capacity to lactogenic rat liver receptors, and apparently the modification of the two 'fast' reacting arginine residues is responsible for the rapid loss of this capacity. The presence of a non-reacting arginine has been described in human and bovine growth hormones, and it is located near the carboxy-terminus. This lack of reactivity is probably due to the formation of a salt bridge, since the arginine residue becomes susceptible to modification once the peptide is separated from the rest of the molecule. This salt bridge is absent in ovine prolactin, since the homologous arginine residue is reactive with cyclohexanedione. This result suggests that there could be a difference between the three-dimensional structure of ovine prolactin and of the growth hormones, at least near the carboxy-terminal region of the molecule.

Selective modification of tryptophan-150 in ovine placental lactogen.

1. Ovine placental lactogen was modified by reaction with o-nitrophenylsulfenyl chloride. Fluorescence measurements indicated that one of the two tryptophan residues of the molecule had reacted. Besides, there was some reagent not covalently bound. 2. The reagent was covalently bound to Trp-150. No evidence of modification of Trp-90 was found. 3. Binding capacity to lactogenic as well as somatogenic receptors was diminished but not abolished upon modification, indicating that absolute molecular integrity of Trp-150 is not required for binding. 4. This behavior is similar to that of the tryptophan residues of ovine prolactin.

Chemical modification of ovine prolactin with N-acetylimidazole.

Reaction of ovine prolactin (oPRL) with a 150-fold molar excess of N-acetylimidazole over protein content resulted in the modification of 2.5 tyrosine residues and 1.2 lysine residues. Acetylation greatly decreased the in vitro binding capacity to lactogenic sites. This binding capacity was partially restored by ammonium bicarbonate treatment, which removes O-acetyl groups from tyrosine residues but not N-acetyl groups from lysine residues. The modification extent of the tyrosine residues was determined. The results suggest that acetylation of tyrosine 44 or of tyrosine 96 is likely to be responsible for the decrease in binding activity of acetylated oPRL, and that one of these residues may play a role in the interaction of oPRL with lactogenic receptors.

Chemical modification of human growth hormone with N-acetylimidazole. Effect on binding capacity to lactogenic and somatogenic receptors.

The effect of acetylation of tyrosine residues on the binding capacity of human growth hormone (hGH) to rat liver lactogenic and somatogenic receptors was studied. When 3.7 tyrosine and 4.8 lysine residues were acetylated with N-acetylimidazole, both the in vivo and the in vitro capacities of hGH to compete with 125I-labeled bovine growth hormone for somatogenic binding sites greatly decreased. Acetylation also affected the in vitro binding capacity to lactogenic sites. Most of the somatogenic binding activity was recovered by hydroxylamine treatment, which removes O-acetyl groups from tyrosine residues but not N-acetyl groups from lysine residues. The same treatment partially restored lactogenic binding capacity. The reactivity of hGH tyrosine residues to N-acetylimidazole, together with previous evidence, suggests that: (a) Tyrosine residues 160 and 164, when acetylated, are likely to be responsible for the low binding activity of acetylated hGH. (b) Tyrosine 160 may play a significant role in hGH interaction with lactogenic receptors.

Modification of arginine residues in human growth hormone by 1,2-cyclohexanedione: effects on the binding capacity to lactogenic and somatogenic receptors.

Reactivity of arginine residues in human growth hormone was studied by reaction with 1,2-cyclohexanedione. Kinetic analysis of the data showed a good fit to a pseudo first order curve, with an apparent velocity constant k = 1.26 x 10(-2) min-1 and a maximum modification of 9.6 out of the 11 arginines of the molecule. Modification led to a decrease in binding capacity to both lactogenic and somatogenic rat liver receptors. In either case Tsou plots suggest that the modification of two arginine residues is responsible for this behavior, although it cannot be ascertained whether the two relevant residues are the same for both receptor types. Circular dichroism studies indicated no apparent changes in protein conformation in the modified hormone. Binding capacity was restored upon regeneration of arginines by incubation with Tris-HCl buffer. Only the carboxy-terminal peptide was isolated by HPLC from a tryptic digest of succinylated Arg-modified hGH, indicating that 183 is the nonreacting arginine residue.

Ovine prolactin and human growth hormone derivatives. Specific modification of their alpha-amino groups.

The alpha-amino group of ovine prolactin (oPRL) and human growth hormone (hGH) was selectively modified by transamination with glyoxylic acid. No difference was found in the binding capacity of transaminated oPRL to rat liver lactogenic receptors with respect to its control, although both samples showed a decrease in its binding capacity with reference to the native hormone. This decrease was due to conformational changes caused by the reaction conditions and not by the transamination itself, as shown by the circular dichroism spectra. Transaminated hGH retained the full binding capacity of the hormone. These results suggest that the alpha-amino group is not relevant for the binding to lactogenic liver receptors in both lactogenic hormones.

An approach to the three-dimensional structure of bovine growth hormone based on chemical modification and secondary structure prediction.

Three different methods have been applied to the prediction of secondary structure. The prediction that better fitted the chemical data was chosen. Two regions of the bovine growth hormone molecule (111-117 and 166-174) appear to be exposed to the solvent, according to hydropathic analysis but have several charged residues not reactive towards their specific reagents. Two molecular domains are postulated, each one bearing a region with charged residues on its surface and interacting with the other in the molecule by means of saline bridges. The hydrophobic core of the molecule is formed by the ensemble of the hydrophobic region predicted between residues 81 and 108, and the hydrophobic faces of the amphiphilic helices 109-127 and 9-33.