RESUMEN
IgA nephropathy is the most common cause of chronic renal failure among primary glomerulonephritides. During the last decade, there was a remarkable progress in understanding its pathogenesis. A number of therapeutic trials has been published that shed light on its treatment. ACEI and AT1R antagonists (sartans) or their combination represent the cornerstone of therapy of IgA nephropathy. However, this treatment is not given to patients having optimal blood pressure, normal glomerular filtration rate, proteinuria less than 0.3 g/24 h, mild abnormalities in renal biopsy, and stationary course of the disease. The medication is administered in a maximal tolerated dose to patients with active, progressing disease. ACEI and AT1R antagonists are also drugs of the first choice in patients with proteinuric IgA nephropathy. However, if proteinuria does not decrease significantly within 3 months from the beginning of this treatment, administration of glucocorticosteroids is recommended. On the basis of prospective, controlled clinical trials and metaanalyses of other therapeutic studies, it has been concluded that glucocorticosteroids decrease proteinuria and slow down the decline of renal function. A complete remission of proteinuria is the aim of the treatment. The effectiveness of cyclophosphamide in active forms of IgA nephropathy, described in some studies, was not confirmed by metaanalyses. Nevertheless, cyclophosphamide may be effective in some patients with rapidly deteriorating renal function and active morphological findings with cellular extracapillary proliferation.
Asunto(s)
Glomerulonefritis por IGA/tratamiento farmacológico , Glomerulonefritis por IGA/diagnóstico , Glomerulonefritis por IGA/fisiopatología , HumanosRESUMEN
Studies of the properties of immune complexes (IC) in the circulation, urine, and mesangium of IgA nephropathy (IgAN) patients have provided data relevant to the pathogenesis of this disease. IC contain predominantly polymeric IgA1 molecules which are deficient in galactose (Gal) residues on O-linked glycan chains in the hinge region (HR) of their heavy (H) chains. As a result of this aberrancy, a novel antigenic determinant(s) involving N-acetylgalactosamine (GalNAc) and perhaps sialic acid (SA) of O-linked glycans is generated and recognized by naturally occurring GalNAc-specific antibodies. Thus, IC in IgAN consist of Gal-deficient IgA1 molecules as an antigen, and GalNAc-specific IgG and/or IgA1 as an antibody. IgG antibodies to Gal-deficient IgA1 are probably induced by cross-reactive microbial antigens; they are present at variable levels not only in humans with or without IgAN but also in many phylogenetically diverse vertebrate species. Incubation of human mesangial cells with IC from sera of IgAN patients indicated that stimulation of cellular proliferative activity was restricted to the large (>800 kDa) complexes. These findings suggest that experimental approaches that prevent the formation of large Gal-deficient IgA1-IgG IC may be applied ultimately in an immunologically mediated therapy.
Asunto(s)
Glomerulonefritis por IGA/inmunología , Glomerulonefritis por IGA/metabolismo , Inmunoglobulina A/metabolismo , Animales , Glomerulonefritis por IGA/patología , Glicosilación , Humanos , Inmunoglobulina A/fisiologíaRESUMEN
Immunoglobulin A (IgA) nephropathy is the most prevalent form of glomerulonephritis worldwide. A renal biopsy is required for an accurate diagnosis, as no convenient biomarker is currently available. We developed a serological test based upon the observation that this nephropathy is characterized by undergalactosylated IgA1 in the circulation and in mesangial immune deposits. In the absence of galactose, the terminal saccharide of O-linked chains in the hinge region of IgA1 is terminal or sialylated N-acetylgalactosamine. A lectin from Helix aspersa, recognizing N-acetylgalactosamine, was used to develop an enzyme-linked immunosorbent assay that measures galactose-deficient IgA1 in serum. The median serum lectin-binding IgA1 level was significantly higher for 153 Caucasian adult patients with IgA nephropathy without progression to end-stage renal disease as compared with that for 150 healthy Caucasian adult controls. As the lectin-binding IgA1 levels for the controls were not normally distributed, the 90th percentile was used for determination of significant elevation. Using a value of 1076 U/ml as the upper limit of normal, 117 of the 153 patients with IgA nephropathy had an elevated serum lectin-binding IgA1 level. The sensitivity as a diagnostic test was 76.5%, with specificity 94%; the positive predictive value was 88.6% and the negative predictive value was 78.9%. We conclude that this lectin-binding assay may have potential as a noninvasive diagnostic test for IgA nephropathy.
Asunto(s)
Galactosa/deficiencia , Glomerulonefritis por IGA/sangre , Glomerulonefritis por IGA/diagnóstico , Inmunoglobulina A/sangre , Acetilgalactosamina/química , Adolescente , Adulto , Animales , Western Blotting , Secuencia de Carbohidratos , Pruebas Diagnósticas de Rutina/métodos , Ensayo de Inmunoadsorción Enzimática , Femenino , Galactosa/química , Mesangio Glomerular/inmunología , Glicosilación , Caracoles Helix/química , Humanos , Inmunoglobulina A/química , Inmunoglobulina A/inmunología , Lectinas/química , Masculino , Persona de Mediana Edad , Antígenos O/química , Antígenos O/metabolismo , Sensibilidad y EspecificidadRESUMEN
Immunoglobulin A (IgA) is a dominant immunoglobulin of the mucosal surfaces, but it is also present in plasma. In men and in hominoid primates it occurs in two subclasses: IgA1 and IgA2. Circulating IgA is mostly IgA1 monomer, secretory IgA is mostly dimer or tetramer with varying content of IgA1 and IgA2 on individual mucosal surfaces. Its main physiological function is a defence of the mucosal surfaces against infection. It binds either specifically to bacterial antigens or through its O-linked glycosidic chains, it binds to the lectins of bacterial cells and thus protects mucosal surfaces against bacterial adhesion and infection. On each of its heavy chain, IgA1 has at least two N-glycosidically bound oligosaccharides and 3 to 5 O-linked side-chains. The occurrence of O-glycosidically bound glycans on other circulating immunoglobulins is rare. An aberrant composition of these glycans may be an antigenic determinant for naturally occurring circulating antibodies. The resulting IgA-containing immune complexes, which are deposited in the glomeruli, may be the cause of IgA nephropathy. IgA glomerular deposits are also frequently present in many other primary and systemic glomerulonephritides.
Asunto(s)
Inmunoglobulina A/análisis , Enfermedades Renales/inmunología , Animales , Glomerulonefritis/inmunología , Glomerulonefritis por IGA/inmunología , Humanos , Inmunoglobulina A/química , Infecciones Urinarias/inmunologíaRESUMEN
Periodontal disease is a chronic inflammatory disease of bacterial etiology. In many other chronic inflammatory diseases, IgG glycans are galactose-deficient and thus capable of complement activation through the lectin pathway. In this study, we examined whether IgG in serum and gingival crevicular fluid, and IgG locally produced by plasma cells in gingiva of periodontal disease patients, display altered glycosylation. We developed a lectin-ELISA to measure levels of galactose-deficient IgG in the fluids and immunofluorescence staining to detect galactose-deficient IgG-producing cells in gingiva. Our results indicated higher levels of galactose-deficient IgG in sera and gingival crevicular fluid from periodontal disease patients, compared with levels in healthy controls. Furthermore, gingivae from periodontal disease patients exhibited infiltration of IgG-producing plasma cells; many of them contained galactose-deficient IgG in the cytoplasm. Analysis of our data suggests that IgG secreted by B-cells was aberrantly glycosylated, which resulted in the production of pro-inflammatory galactose-deficient IgG.
Asunto(s)
Líquido del Surco Gingival/inmunología , Inmunoglobulina G/metabolismo , Pérdida de la Inserción Periodontal/inmunología , Periodoncio/inmunología , Células Plasmáticas/citología , Adulto , Anciano , Femenino , Líquido del Surco Gingival/metabolismo , Glicosilación , Humanos , Masculino , Persona de Mediana Edad , Pérdida de la Inserción Periodontal/metabolismo , Bolsa Periodontal/inmunología , Bolsa Periodontal/metabolismo , Periodoncio/citología , Periodoncio/metabolismo , Células Plasmáticas/metabolismo , Conformación Proteica , Estadística como AsuntoRESUMEN
Human immunoglobulin A is represented by two structurally and functionally distinct subclasses: IgA1 and IgA2. IgA1, which is almost exclusively present in the mesangial deposits in IgA nephropathy patients, contains in its hinge region three to five O-lined carbohydrate chains. A fraction of IgA1 molecules in the circulation of IgA nephropathy patients exhibits aberrant glycosylation. As a result of changes in glycosylation, the neoepitopes represented by glycans are exposed and recognized by naturally occurring antibodies with antiglycan specifciities, and immune complexes are generated. The deposits of these immune complexes in the glomerular mesangia elicit inflammatory response known as IgA nephropathy. Epidemiological studies have shown that dominant hematuria, either isolated or combined with mild proteinuria, is the most frequent urinary syndrome in glomerulonephritis. The morphologic finding of this syndrome is most frequently IgA nephropathy. Originally considered a benign disease, IgA nephropathy is now recognized as a frequent cause of chronic renal failure. The progression is signalized by increasing proteinuria and hypertension. Therefore, a control of blood pressure and lowering of proteinuria remain the corner-stones of the treatment. Angiotensin converting enzyme inhibitors and AT1 blockers may lower both blood pressure and proteinuria and are now increasingly promoted even for treatment of normotensive patients. Steroids are administered to patients with severe proteinuria. High-doses of fish oil seem to slow down the rate of renal failure.
Asunto(s)
Glomerulonefritis por IGA/fisiopatología , Inmunoglobulina A/fisiología , Glomerulonefritis por IGA/diagnóstico , Glomerulonefritis por IGA/terapia , Glicosilación , HumanosRESUMEN
Several new findings emerged recently from biochemical, genetic, and molecular studies of patients with IgA nephropathy. It appears that immunoglobulin A1-secreting cells of IgA nephropathy patients produce increased amounts of aberrantly glycosylated IgA1 in which the O-linked glycans in the hinge region are deficient in the content of galactose. The galactose-deficient IgA1 in the circulation is recognized by naturally occurring antibodies with anti-glycan specificity, and immune complexes are formed. These circulating immune complexes escape hepatic degradation and eventually are deposited in the kidney mesangium. Resident mesangial cells bind the IgA-containing immune complexes with the involvement of a novel IgA receptor and become activated. A familial form of IgA nephropathy has been linked to chromosome 6q22-23. Recent progress in molecular analyses of IgA nephropathy thus defines this disease as an autoimmune process with a novel IgA mesangial receptor and certain genetically determined traits.
Asunto(s)
Glomerulonefritis por IGA/inmunología , Inmunoglobulina A/inmunología , Animales , Canales de Cloruro/inmunología , Galactosa , Mesangio Glomerular/citología , Glomerulonefritis por IGA/genética , Humanos , Inmunoglobulina A/biosíntesis , Polisacáridos/biosíntesisRESUMEN
Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was applied to studies of the molecular heterogeneity of desialylated human IgA1 hinge region glycopeptides released with two IgA1 proteases. Typically, the hinge region of an alpha1 chain contains three to five O-linked glycan chains. Variants of the hinge region peptides released from IgA1(Kni) myeloma protein carrying 0, 1, 2, or 3 GalNAc residues were observed in the mass spectra as well as the nonglycosylated peptide. Variable numbers of Gal residues indicated additional heterogeneity in O-glycosylation of IgA1. In the hinge region preparation from normal human serum IgA1, glycopeptides carrying 2, 3, 4, or 5 GalNAc residues with variable numbers of Gal residues were detected. In conclusion, our new approach using the site-specific cleavage with two IgA1 proteases allowed precise and sensitive MALDI-TOF mass spectrometric analysis of O-glycosylation heterogeneity in IgA1 hinge region.
Asunto(s)
Inmunoglobulina A/química , Secuencia de Aminoácidos , Glicosilación , Humanos , Inmunoglobulina A/inmunología , Espectrometría de Masas , Datos de Secuencia MolecularRESUMEN
Despite 31 years of clinical and laboratory investigation, the pathogenesis of IgAN remains largely enigmatic. However, it seems clear that IgA1 accumulates in the glomerular mesangia due to a systemic process rather than an abnormality intrinsic in the kidney. Based on recent studies, investigators have proposed a wide range of pathogenetic mechanisms: nonspecific attachment because excessive IgA1 is synthesized in a short or prolonged immune response, increased binding due to an IgA-specific receptor (perhaps with altered characteristics) on mesangial cells, and increased glomerular deposition because an abnormal structure of the antibody (particularly of the glycan moieties in the hinge region) allows it to escape its normal degradative pathways or facilitates binding to a receptor on mesangial cells. Whether one or more of these postulates fully explains IgAN remains to be determined. Until then, treatment of the many affected patients worldwide will continue to lack a disease-specific approach.
Asunto(s)
Glomerulonefritis por IGA/etiología , Animales , Formación de Anticuerpos , Antígenos/inmunología , Citocinas/fisiología , Glomerulonefritis por IGA/inmunología , Glomerulonefritis por IGA/fisiopatología , Humanos , Inmunoglobulina A/química , Inmunoglobulina A/inmunología , Inmunoglobulina A/metabolismo , Glomérulos Renales/metabolismo , Polisacáridos/químicaRESUMEN
Circulating immune complexes (CICs) isolated from sera of patients with IgA nephropathy (IgAN) consist of undergalactosylated, mostly polymeric, and J chain-containing IgA1 and IgG antibodies specific for N-acetylgalactosamine (GalNAc) residues in O-linked glycans of the hinge region of IgA1 heavy chains. Antibodies with such specificity occur in sera of IgAN patients, and in smaller quantities in patients with non-IgA proliferative glomerulonephritis and in healthy controls; they are present mainly in the IgG (predominantly IgG2 subclass), and less frequently in the IgA1 isotype. Their specificity for GalNAc was determined by reactivity with IgA1 myeloma proteins with enzymatically removed N-acetylneuraminic acid (NeuNAc) and galactose (Gal); removal of the O-linked glycans of IgA1 resulted in significantly decreased reactivity. Furthermore, IgA2 proteins that lack the hinge region with O-linked glycans but are otherwise structurally similar to IgA1 did not react with IgG or IgA1 antibodies. The re-formation of isolated and acid-dissociated CICs was inhibited more effectively by IgA1 lacking NeuNAc and Gal than by intact IgA1. Immobilized GalNAc and asialo-ovine submaxillary mucin (rich in O-linked glycans) were also effective inhibitors. Our results suggest that the deficiency of Gal in the hinge region of IgA1 molecules results in the generation of antigenic determinants containing GalNAc residues that are recognized by naturally occurring IgG and IgA1 antibodies.
Asunto(s)
Acetilgalactosamina/inmunología , Complejo Antígeno-Anticuerpo/química , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Galactosa/análisis , Glomerulonefritis por IGA/inmunología , Inmunoglobulina A/inmunología , Polisacáridos/inmunología , Adulto , Especificidad de Anticuerpos , Autoanticuerpos/aislamiento & purificación , Autoantígenos/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Epítopos/inmunología , Femenino , Mesangio Glomerular/inmunología , Glicosilación , Humanos , Inmunoglobulina A/aislamiento & purificación , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas de Mieloma/inmunología , Neuraminidasa/farmacología , Polisacáridos/química , Procesamiento Proteico-PostraduccionalRESUMEN
IgA1 proteins from sera of patients with IgA nephropathy (IgAN) are galactosylated to a lesser degree than those from healthy controls. The increased reactivity of intact or de-sialylated serum IgA1 with N-acetylgalactosamine (GalNAc)-specific lectins, Helix aspersa (HAA) and Caragana arborescens (CAA) and de-sialylated IgA1 with Helix pomatia (HPA) and Bauhinia purpurea (BPA) indicated that the Gal deficiency is in glycans located in the hinge region of IgA1 molecules. De-sialylated IgA from sera of 81 IgAN patients bound biotin-labeled lectin HAA more effectively than did de-sialylated IgA from 56 healthy controls (P < 0.0001). Similar results were observed for 67 IgAN patients and 52 controls with second lectin, CAA (P < 0.001). The binding patterns for 9 patients with mesangial proliferative glomerulonephritis of non-IgA origin were similar to those for controls. Incompletely galactosylated IgA1 capable of binding GalNAc-specific lectins was detected in complexes with IgG as demonstrated by ELISA, size-exclusion chromatography and sucrose gradient ultracentrifugation. The formation of IgA1-IgG complexes may affect the serum level of IgA1 by reducing the rate of its elimination and catabolic degradation by the liver.
Asunto(s)
Galactosa/deficiencia , Glomerulonefritis por IGA/sangre , Inmunoglobulina A/sangre , Inmunoglobulina G/metabolismo , Adulto , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Femenino , Galactosa/química , Glomerulonefritis por IGA/inmunología , Humanos , Inmunoglobulina A/química , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/metabolismo , Lectinas , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteoglicanos/química , Proteoglicanos/metabolismoAsunto(s)
Proteínas del Sistema Complemento/fisiología , Inmunoglobulina A/fisiología , Fagocitos/inmunología , Linfocitos B/inmunología , Complemento C3b/fisiología , Humanos , Inmunoglobulina A Secretora/fisiología , Mucosa Intestinal/inmunología , Modelos Inmunológicos , Membrana Mucosa/inmunología , Neutrófilos/inmunología , Fagocitosis , Transducción de SeñalAsunto(s)
Glomerulonefritis por IGA/inmunología , Inmunoglobulina A/química , Oligosacáridos/análisis , Lectinas de Plantas , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Carcinoma Hepatocelular/patología , Glomerulonefritis por IGA/sangre , Glicosilación , Humanos , Inmunoglobulina A/administración & dosificación , Inmunoglobulina A/metabolismo , Cadenas Pesadas de Inmunoglobulina/química , Glomérulos Renales/metabolismo , Lectinas/metabolismo , Neoplasias Hepáticas/patología , Linfoma de Células B Grandes Difuso/patología , Ratones , Datos de Secuencia Molecular , Farmacocinética , Distribución Tisular , Células Tumorales CultivadasRESUMEN
In contrast to antigen-antibody complexes containing native human IgA, solid-phase-deposited IgA activates the alternative complement pathway and binds C3b. To investigate the role of carbohydrate chains in this, various human IgA preparations were treated with neuraminidase alone or together with N-glycanase or O-glycanase, or with mixed glycosidases from the oral bacterium, Streptococcus mitis. Depletion of oligosaccharides was determined by carbohydrate analysis. Removal of sialic acid and N-linked glycan chains greatly increased the C3b-fixing properties of normal serum IgA1 and IgA2. Myeloma IgA1 and IgA2 proteins and secretory IgA had higher C3b-binding activity than normal serum IgA, and this was further increased by removal of sialic acid and N-linked glycans. Fc alpha and Fc alpha-SC fragments of myeloma and secretory IgA1, respectively, but not Fab alpha fragments, obtained by cleavage with bacterial IgA1 proteases and also free secretory component, fixed C3b by the alternative pathway.
Asunto(s)
Complemento C3b/inmunología , Vía Alternativa del Complemento/inmunología , Inmunoglobulina A/inmunología , Oligosacáridos/inmunología , Electroforesis en Gel de Poliacrilamida , Glicósido Hidrolasas , Humanos , Inmunoglobulina A Secretora/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulinas/inmunología , Proteínas de Mieloma/inmunología , Péptido Hidrolasas , Plásticos , Polisacáridos , Ácidos SiálicosRESUMEN
Polyclonal human secretory IgA1 and IgA2 antibodies to a bacterial protein antigen Streptococcus mutans AgI/II, and polyclonal human serum IgA1 and IgA2 antibodies to staphylococcal alpha-toxin, were found to interfere with antigen-mediated C3b fixation. In fluid phase, immune complexes of antigen and IgA failed to fix C3b, whereas antigen-IgG complexes did fix C3b. Partial removal of glycan chains with Streptococcus mitis SK96 glycosidases diminished the capacity of IgA antibodies to interfere with antigen-mediated C3b fixation by the alternative complement pathway. The authors conclude that native serum or secretory IgA antibodies suppress C3b fixation, and that the glycan chains play a significant role in maintaining this property.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Complemento C3b/metabolismo , Inmunoglobulina G/farmacología , Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Antígenos/inmunología , Sitios de Unión de Anticuerpos , Pruebas de Fijación del Complemento , Vía Alternativa del Complemento/inmunología , Humanos , Inmunoglobulina A Secretora/efectos de los fármacos , Inmunoglobulina A Secretora/farmacología , Mieloma Múltiple/inmunología , Oligosacáridos/farmacologíaRESUMEN
The effect of IL-7 on the growth of thymic T lymphocytes was investigated by adding recombinant IL-7 into cell suspension cultures and submersion organ cultures (SOC) of murine fetal thymuses (FT) and newborn thymuses (NBT). FT and NBT were obtained from C57BL/6 mice at day 15 of gestational age and at day 3 after birth, respectively. In both cell suspension cultures and SOC, addition of IL-7 highly improved the cell recovery. In cell suspension cultures, addition of IL-7 resulted in the growth of gamma delta T-cells from FT-cells, whereas the same cytokine promoted the growth of both alpha beta and gamma delta T-cells from NBT-cells. These results may indicate that this cytokine is able to support the proliferation of T-cells of both alpha beta and gamma delta lineages. In marked contrast, in SOC, addition of IL-7 resulted in the growth of gamma delta T-cells not only in FT but also in NBT, despite the fact that the SOC of NBT without exogenous cytokine exclusively promoted the growth of alpha beta T-cells. A similar effect was also seen when IL-2 was added to NBT-SOC, though the skewing to gamma delta lineage was not so strong as in the case of IL-7. In addition, we found that IL-7 mRNA is expressed in the day 15 FT at a much higher level than in the adult thymus. These results strongly suggested that the production of a large amount of IL-7 synthesized in the FT is one of the major factors leading to the generation of gamma delta T-cells in FT.
Asunto(s)
Interleucina-7/fisiología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Subgrupos de Linfocitos T/inmunología , Timo/citología , Animales , Diferenciación Celular , Células Cultivadas , Femenino , Citometría de Flujo , Expresión Génica , Técnicas In Vitro , Interleucina-2/genética , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Timo/embriologíaRESUMEN
Detection of the activity of beta-1,4-galactosyltransferase (beta-1,4-GT) in suspensions of viable mouse hepatocytes, the human hepatoma cell line Hep G2, the human colonic adenocarcinoma cell line HT-29, the monocyte-like cell line U937, and human splenic B and T lymphocytes suggested the presence of beta-1,4-GT, in an enzymatically active form, on plasma membranes. The presence of beta-1,4-GT on cell surfaces was also indicated from the effect of trypsinization of live cells, which significantly reduced cell surface beta-1,4-GT activity, but did not affect the activity associated with cytoplasmic membranes. Furthermore, the presence of beta-1,4-GT on the cell surface was demonstrated by indirect immunofluorescence staining of cells with anti-beta-1,4-GT antibody. The detection of radioactivity in immunoglobulins (Ig) and their component chains after incubation with suspensions of intact cells in the presence of Mn2+ and UDP-[3H]-galactose, indicated that Ig molecules were galactosylated. In the absence of UDP-[3H]-galactose, beta-1,4-GT on cell surfaces, or immobilized on Sepharose-4B, formed stable complexes with galactose acceptors, including Ig. The efficiency of binding decreased in the order: J chain > alpha chain > mu chain > polymeric IgA2 > monomeric/polymeric IgA1 > IgM > IgG. Thus, beta-1,4-GT could act as a cell-surface receptor for Ig through a cation-dependent, lectin-like association of the beta-1,4-GT with the carbohydrate moieties of the Ig. This was confirmed by indirect surface immunofluorescence and radiolabeled ligand binding assays. The binding was inhibitable by EDTA, alpha-lactalbumin (in the presence of glucose), GlcNAc, or uridine 3',5'dialdehyde. At 37 degrees C, the apparent affinity constants and association rate constants of interaction between cell surface beta-1,4-GT on glutaraldehyde-fixed HT-29 and U937 cells and alpha 2 chain or monomeric IgA1 were in the range from 7.1 x 10(7) to 4.6 x 10(8) M-1 and from 1 x 10(5) to 3 x 10(6) M-1 s-1, respectively. The dissociation rate constants and half time of dissociation calculated from these data were in the range from 2.1 x 10(-2) to 5.0 x 10(-4) s-1 and from 33 to 1380 s, respectively. The number of alpha 2 or IgA1 molecules bound per HT-29 and U937 cell were in the range from 1.9 x 10(5) to 1.3 x 10(6). The binding of IgA by the cell surface beta-1,4-GT was not associated with internalization or the catabolic degradation of the ligand.
Asunto(s)
Galactosiltransferasas/metabolismo , Inmunoglobulinas/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Humanos , Inmunoglobulina A/metabolismo , Lectinas/metabolismo , RatonesRESUMEN
Assay of the activity of beta-1,4-galactosyltransferase (beta-1,4-GT) revealed that in addition to serum, milk, colostrum, amniotic and cerebrospinal fluids and malignant effusions, this enzyme is present also in tears and saliva. Molecular-sieve chromatography of human colostral whey and serum and subsequent assay of beta-1,4-GT activity have shown that beta-1,4-GT was present as a free enzyme (55 kDa) and associated with components of larger molar mass. The elution pattern did not change when the chromatography was carried out in a buffer devoid of, or enriched with, Mn2+, a cofactor of beta-1,4-GT activity. However, the activity associated with the large molar mass components was absent when the chromatography was carried out in the presence of a chelating agent (EDTA). Analyses of the eluted material by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and by immunodiffusion indicated that the major colostral component in beta-1,4-GT activity-containing fractions was secretory IgA (S-IgA); in addition, the beta-1,4-GT activity was detected in fractions that contained lactoferrin and alpha-lactalbumin. Interactions of beta-1,4-GT with S-IgA and lactoferrin in colostrum were also demonstrated by the detection of radioactivity in precipitin lines obtained by immunoelectrophoresis and autoradiography of the colostral whey after it had been incubated with UDP-[3H]-galactose. Furthermore, radioactively labeled S-IgA and alpha-chain were detected when colostral whey incubated with UDP-[3H]-galactose was analyzed by SDS-PAGE under non-reducing and reducing conditions, respectively. In serum, the beta-1,4-GT-binding components identified in fractions after molecular-sieve chromatography were IgG, IgA, IgM and transferrin. The binding of beta-1,4-GT to immunoglobulins (Ig) was also demonstrated by assaying the beta-1,4-GT activity associated with Sepharose-4B-immobilized Ig of various isotypes and molecular forms, which were incubated with colostral beta-1,4-GT in the presence of Mn2+. Beta-1,4-GT measured by enzyme activity was bound to these Ig in order: polymeric IgA2 > monomeric IgA1 = polymeric IgA1 = secretory IgA = pentameric IgM > IgG. Immobilized component chains, namely alpha, mu and J chains, bound beta-1,4-GT more effectively than native Ig. Incubation of the IgA1 myeloma protein with crude human colostral galactosyltransferase in the presence of UDP[3H]-galactose and Mn2+ resulted in galactosylation of both N- and O-linked carbohydrate side chains.(ABSTRACT TRUNCATED AT 400 WORDS)