Anti-CD43 and anti-galectin-1 autoantibodies in patients with systemic lupus erythematosus.

OBJECTIVES: Systemic lupus erythematosus (SLE) is characterized by the production of multiple autoantibodies and also by T-cell dysfunction. CD43 is expressed by most immune cells, is involved in lymphocyte adhesion and activation, and interacts with galectin-1 (Gal-1). The aim of this work was to evaluate the plasma levels of autoantibodies against CD43 and Gal-1 as well as the levels of soluble Gal-1 in SLE Mexican mestizo patients, with the aim of establishing a correlation between these parameters and the clinical profile. METHODS: Serum levels of immunoglobulin (Ig)G autoantibodies against CD43 and Gal-1 and levels of soluble Gal-1 were measured by enzyme-linked immunosorbent assay (ELISA) in 55 patients with SLE and 71 healthy controls. RESULTS: We found significantly enhanced titres of anti-CD43 and anti-Gal-1 antibodies in sera from SLE patients compared to controls. In addition, the serum levels of Gal-1 were significantly higher in SLE patients than in healthy individuals. However, we could detect no correlation of these parameters with disease activity [using the Mexican Systemic Lupus Erythematosus Disease Activity Index (MEX-SLEDAI)], age, or a variety of different clinical or laboratory features. Similarly, no significant correlation with immunosuppressive or glucocorticoid therapy was observed. By contrast, a significant association was found between anti-CD43 titres and time of disease evolution, complement levels, and the presence of anti-Gal-1 antibodies. CONCLUSIONS: As CD43 and Gal-1 participate in modulating the immune system, we suggest that the presence of autoantibodies against these molecules may contribute to the immune deregulation observed in SLE.

Ligand binding and functional properties of betaglycan, a co-receptor of the transforming growth factor-beta superfamily. Specialized binding regions for transforming growth factor-beta and inhibin A.

Betaglycan, also known as the transforming growth factor-beta (TGF-beta) type III receptor, is a membrane-anchored proteoglycan that binds TGF-beta via its core protein. Deletion mutagenesis analysis has revealed two regions of betaglycan ectodomain capable of binding TGF-beta: one at the amino-terminal half, the endoglin-related region (López-Casillas, F., Payne, H., Andres, J. L., and Massagué, J. (1994) J. Cell Biol. 124, 557-568), and the other at the carboxyl-terminal half, the uromodulin-related region (Pepin, M.-C., Beauchemin, M., Plamondon, J., and O'Connor-McCourt, M. D. (1994) Proc. Natl. Acad. Sci. U. S. A 91, 6997-7001). In the present work we have functionally characterized these ligand binding regions. Similar to the wild type receptor, both regions bind TGF-beta2 with higher affinity than TGF-beta1. However, only the endoglin-related region increases the TGF-beta2 labeling of the TGF-beta type II receptor, the so-called "TGF-beta -presentation" function of the wild type receptor. Despite this preference, both regions as well as the wild type receptor mediate the TGF-beta2-dependent Smad2 phosphorylation, indicating that they can function indistinguishably as TGF-beta-enhancing co-receptors. On the other hand, we found that the recently described ability of the wild type betaglycan to bind inhibin A is a property of the core protein that resides in the uromodulin-related region. Binding competition experiments indicate that this region binds inhibin and TGF-beta with the following relative affinities: TGF-beta2 > inhibin A > TGF-beta1. All together, the present results suggest that betaglycan ectodomain is endowed with two bona fide independent ligand binding domains that can perform specialized functions as co-receptors of distinct members of the TGF-beta superfamily.

Recombinant soluble betaglycan is a potent and isoform-selective transforming growth factor-beta neutralizing agent.

Betaglycan is an accessory receptor of members of the transforming growth factor-beta (TGF-beta) superfamily, which regulates their actions through ligand-dependent interactions with type II receptors. A natural soluble form of betaglycan is found in serum and extracellular matrices. Soluble betaglycan, prepared as a recombinant protein using the baculoviral expression system, inhibits the actions of TGF-beta. Because of its potential use as an anti-TGF-beta therapeutic agent, we have purified and characterized baculoviral recombinant soluble betaglycan. Baculoviral soluble betaglycan is a homodimer formed by two 110 kDa monomers associated by non-covalent interactions. This protein is devoid of glycosaminoglycan chains, although it contains the serine residues, which, in vertebrate cells, are modified by these carbohydrates. On the other hand, mannose-rich carbohydrates account for approximately 20 kDa of the mass of the monomer. End-terminal sequence analysis of the soluble betaglycan showed that Gly(24) is the first residue of the mature protein. Similarly to the natural soluble betaglycan, baculoviral soluble betaglycan has an equilibrium dissociation constant (K(d)) of 3.5 nM for TGF-beta1. Ligand competition assays indicate that the relative affinities of recombinant soluble betaglycan for the TGF-beta isoforms are TGF-beta2>TGF-beta3>TGF-beta1. The anti-TGF-beta potency of recombinant soluble betaglycan in vitro is 10-fold higher for TGF-beta2 than for TGF-beta1. Compared with a commercial pan-specific anti-TGF-beta neutralizing antibody, recombinant soluble betaglycan is more potent against TGF-beta2 and similar against TGF-beta1. These results indicate that baculoviral soluble betaglycan has the biochemical and functional properties that would make it a suitable agent for the treatment of the diseases in which excess TGF-beta plays a central physiopathological role.

[Usefulness of prothrombin fragment 1 + 2 and fibrin dimer in the detection of hypercoagulation in obstetrical patients with thrombosis risk factors]. / Utilidad del fragmento 1 + 2 de protrombina y del dímero de fibrina en la detección de hipercoagulabilidad en pacientes obstétricas con factores de riesgo de trombosis.

We measured prothrombin 1 + 2 fragment (f1 + 2p) and dimer D (dD) in plasma from 100 pregnant women at high risk for thromboembolic disease (TED) and in 23 non-pregnant control. Measures of f1 + 2p were made by immunoassay analysis in 75 patients and dD by semiquantitative analysis of plate agglutination in 97 cases. F1 + 2p was significantly elevated in 85% of cases, but its levels was not predictive value for TED. Dimer D was not found in 40 cases, in 33 patients its values were between 500 and 1000 ng/ml. and in the other 24 cases were higher than 2000 ng/ml. Values higher than 1000 ng/ml. were founded in 78% of patients with history of TED, in 60% of cesarean section patients, in 37% of hypertensive patients and in the 23% of diabetic patients. Dimer D, that was higher than 500 ng/ml. in 59% of pregnant and puerperal patients, have predictive value for TED, because 25% of 24 patients that had dD higher than 2000 ng/ml. developed TED and/or coagulation anomalies suggestive of thrombotic activity. These findings were not found in the rest of patients (n 73) which had negative dD or less than 1000 ng/ml.

Differential interactions of the growth factor receptor-bound protein 2 N-SH3 domain with son of sevenless and dynamin. Potential role in the Ras-dependent signaling pathway.

In this paper, we show that the 36-45 surface-exposed sequence WYKAELNGKD of growth factor receptor-bound protein 2 (Grb2) N-SH3 domain inhibits the interaction between Grb2 and a 97-kDa protein identified as dynamin. Moreover, the peptide GPPPQVPSRPNR from dynamin also blocks the binding of dynamin to the proline-rich recognition platform of Grb2. Mutations in the 36-45 motif show that Glu-40 is critical for dynamin recognition. These observations were confirmed by immunoprecipitation experiments, carried out using ER 22 cells. It was also observed that the proline-rich peptide from dynamin was unable to dissociate the Grb2.Sos complex, whereas the proline-rich peptide from Son of sevenless (Sos) inhibited Grb2. dynamin interaction. A time-dependent stimulation of epidermal growth factor receptor overexpressing clone 22 (ER 22) cells by epidermal growth factor resulted in an immediate increase of the Grb2.Sos complex and a concomitant decrease in Grb2.dynamin. This suggests that the recruitment of Grb2.Sos to the membrane, triggered by epidermal growth factor stimulation, activates the Ras-dependent signaling and simultaneously enhances free dynamin levels, leading to both receptor internalization and endocytotic processes.

Nociceptin/orphanin FQ metabolism: role of aminopeptidase and endopeptidase 24.15.

The endogenous opioid receptor-like1 (ORL1) ligand, nociceptin/orphanin FQ (FGGFTGARKSARKLANQ), a heptadecapeptide structurally resembling dynorphin A, has recently been identified. The wide distribution of ORL1 mRNA and nociceptin/orphanin FQ precursor in the CNS, particularly in the limbic system regions and in several areas known to be involved in pain perception, suggests that nociceptin/orphanin FQ is potentially endowed with various central functions. In general, activation and/or inactivation of regulatory peptides occur through the action of cell surface peptidases. The physiological mechanisms under which nociceptin/orphanin FQ is metabolized should lead to a better understanding of its physiological functions. Mouse brain cortical slices were incubated in medium containing the heptadecapeptide in the presence or in the absence of peptidase inhibitors. The critical sites of enzymatic cleavage are Phe1-Gly2, Ala7-Arg8, Ala11-Arg12, and Arg12-Lys13 bonds. The major role played by metallopeptidases was confirmed by the complete protection of metabolism in the presence of EDTA. Aminopeptidase N and endopeptidase 24.15 are the two main enzymes involved in nociceptin/orphanin FQ metabolism, whereas endopeptidase 24.11 (involved in enkephalin [YGGFM(L)] catabolism) does not appear critically involved in nociceptin/orphanin FQ metabolism. The physiological relevance of aminopeptidase N and endopeptidase 24.15 in the heptadecapeptide metabolism remains to be determined.

Identification of growth hormone molecular variants in chicken serum.

It has been described that pituitary growth hormone shows molecular and functional heterogeneity. In birds, size and charge variants of chicken growth hormone (cGH) have been shown in the chicken pituitary gland and in purified preparations of the hormone. Here we demonstrate the existence of cGH molecular isoforms in chicken serum, thus suggesting that they are secreted from the gland. The isolation of total cGH present in sera was performed by immunoaffinity chromatography employing a specific monoclonal antibody against cGH. Different analytical electrophoretic methods (SDS-polyacrylamide gel electrophoresis, isoelectric focusing, bidimensional polyacrylamide gel electrophoresis) followed by Western blot and immunostaining were employed to characterize the serum cGH isoforms, and compared to those present in a fresh pituitary extract. Several identical immunoreactive bands comigrated in both serum and the gland extract in the different systems (SDS-PAGE, MW 16, 22, 26, 29, 52, 62, 66 kDa; IEF, pIs 8.1, 7.5, 7.1, 6.8, 6.2), thus revealing a high correspondence of molecular isoforms of the hormone in the two tissues. Additionally, a glycosylated variant of chicken growth hormone (G-cGH) was also revealed in the serum after concanavalin A-Sepharose chromatography.

Phosphorylation of prolactin and growth hormone.

To determine whether GH and prolactin could be phosphorylated, turkey GH, chicken GH, chicken prolactin and turkey prolactin were incubated in vitro with the catalytic subunit of protein kinase A and [gamma-32P]ATP. Phosphorylation was assessed after sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting and autoradiography. Polyacrylamide electrophoresis showed that both purified native chicken GH and turkey GH were phosphorylated under the conditions employed. However, the glycosylated variant of chicken GH did not appear to be labelled. Chicken prolactin, turkey prolactin and the glycosylated variant of turkey prolactin were all intensely phosphorylated by protein kinase A. Ovine and rat prolactins could also be phosphorylated by protein kinase A. The phosphate content of different native prolactin (turkey, ovine and rat) and GH (ovine and chicken) preparations was also determined and found to be significant. Chicken pituitary cells in primary culture incorporated 32P in GH- and prolactin-like bands isolated by non-denaturing polyacrylamide gel electrophoresis, and this was stimulated by phorbol myristate acetate. Phosphorylation of GH and prolactin may thus explain some of the charge heterogeneity of these hormones.

Purification and electrophoretic analysis of glycosylated chicken growth hormone (G-cGH): evidence of G-cGH isoforms.

It has been shown that chicken growth hormone (cGH) exhibits functional and molecular heterogeneity. Mass and charge variants have been described in fresh pituitary extracts and in pure preparations of the hormone. In an attempt to further study the molecular heterogeneity of cGH we have purified the glycosylated variant of this hormone by affinity chromatography and analyzed it by different electrophoretic methods. Purification was achieved by homogeneizing chicken pituitaries in a protease inhibitor solution (0.5 mM PMSF and aprotinin, 50 KIU/ml); the supernatant of the alkaline extract (pH 9.5) was precipitated with 0.15 M ammonium sulfate and metaphosphoric acid, pH 4.0. The supernatant from this step was further precipitated with 80% ammonium sulfate, pH 6.5. After dialysis and lyophilization, the extract was chromatographed in a Con A-Sepharose column. The fraction eluted with 10 mM alpha-methylmannoside (which contained the glycoproteins) was passed through an immunoaffinity column (anticGH). Glycosylated cGH (G-cGH) was obtained pure after this step. Pure G-cGH was analyzed by nondenaturing electrophoresis (ND-PAGE), SDS-PAGE, isoelectrofocusing (IEF), and bidimensional electrophoresis (2D-PAGE) followed by Western blot and staining either with a specific antibody or with peroxidated Con A. Results showed that monomeric G-cGH has a MW of 29 kDa (under reducing conditions) and is heterogeneous, showing at least three important charge variants with pIs 6.5, 6.7, and 7.2. Mass variants of G-cGH were also detected under nonreducing conditions. Bidimensional analysis revealed that the charge variants had a similar MW (29 kDa).

Molecular isoforms of chicken growth hormone (cGH): different bioactivities of cGH charge variants.

It has been suggested that the functional diversity of growth hormone (GH) is related to its molecular complexity. Here we report a characterization of charge and mass variants of chicken growth hormone (cGH) through a variety of electrophoretic systems [nondenaturing (ND-PAGE), denaturing (SDS-PAGE), under reducing and nonreducing conditions, isoelectrofocusing (IEF), and bidimensional electrophoresis] followed by Western blot and immunostaining with a specific antibody directed against pure cGH. We also report the biological properties of two charge variants on two homologous assays. The studies were carried out with purified cGH and with fresh chicken pituitary extracts. Three charge variants were obtained by ND-PAGE (Rf = 0.23, 0.30, and 0.35), which showed the same molecular weight (26 kDa), while in IEF eight isoforms were observed, the most conspicuous being those with pI = 6.86, 7.5, 7.9, 8.05, and 8.18. In SDS-PAGE under reducing conditions four immunoreactive bands were observed: the monomer (26 kDa), a dimer (52 kDa), a fragment (16 kDa), and a minor band at 22 kDa. Higher MW variants were found under nonreducing conditions. Bidimensional analysis also showed several charge variants for the monomer and the dimer. Bioactivity of two charge variants (0.23 and 0.3) was evaluated with a lipolytic and an antilipolytic assay on chicken adipose tissue explants. It was shown that variant 0.23 was mainly lipolytic, in a dose-dependent response, but lacked antilipolytic effect. On the other hand, variant 0.30 did not show lipolytic effect but presented a clear antilipolytic activity.

Phosphorylation of chicken growth hormone.

The possibility that chicken growth hormone (cGH) can be phosphorylated has been examined. Both native and biosynthetic cGH were phosphorylated by cAMP-dependent protein kinase (and gamma -32P-ATP). The extent of phosphorylation was however less than that observed with ovine prolactin. Under the conditions employed, glycosylated cGH was not phosphorylated. Chicken anterior pituitary cells in primary culture were incubated in the presence of 32P-phosphate. Radioactive phosphate was incorporated in vitro into the fraction immunoprecipitable with antisera against cGH. Incorporation was increased with cell number and time of incubation. The presence of GH releasing factor (GRF) increased the release of 32P-phosphate labelled immunoprecipitable GH into the incubation media but not content of immunoprecipitable GH in the cells. The molecular weight of the phosphorylated immunoreactive cGH in the cells corresponded to cGH dimer.