Rose Kulhavy

Receptor-mediated binding and uptake of immunoglobulin a by human liver

We have studied the molecular mechanisms of the binding and uptake of secretory and serum immunoglobulin A (IgA) of both subclasses (1 and 2) and molecular forms (monomer and polymer) by the particulate fraction of human liver homogenate and by a human hepatoma cell line (HepG2). Inhibition by asialoorosomucoid and the requirement for the presence of calcium indicated that the binding of secretory IgA and polymeric IgA1 was mediated by the asialoglycoprotein receptor. Secretory component, which functions as a receptor for polymeric IgA in several animal species, was detected in the epithelial cells of bile ducts, but not in hepatocytes. Secretory IgA and all molecular forms and subclasses of serum IgA were bound by HepG2 cells, which do not express secretory component. The requirement for the presence of calcium, the presence of a terminal galactose residue in IgA, and the molecular weight of the major plasma membrane protein responsible for binding (41,700 daltons) indicated the involvement of asialoglycoprotein receptor. Immunoglobulin A proteins bound by HepG2 cells were endocytosed and catabolized.

Determination of Aberrant O-Glycosylation in the IgA1 Hinge Region by Electron Capture Dissociation Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry

In a number of human diseases of chronic inflammatory or autoimmune character, immunoglobulin molecules display aberrant glycosylation patterns of N- or O-linked glycans. In IgA nephropathy, IgA1 molecules with incompletely galactosylated O-linked glycans in the hinge region (HR) are present in mesangial immunodeposits and in circulating immune complexes. It is not known whether the Gal deficiency in IgA1 proteins occurs randomly or preferentially at specific sites. To develop experimental approaches to address this question, the synthetic IgA1 hinge region and hinge region from a naturally Gal-deficient IgA1 myeloma protein have been analyzed by 9.4 tesla Fourier transform-ion cyclotron resonance mass spectrometry. Fourier transform-ion cyclotron resonance mass spectrometry offers two complementary fragmentation techniques for analysis of protein glycosylation by tandem mass spectrometry. Infrared multiphoton dissociation of isolated myeloma IgA1 hinge region peptides confirms the amino acid sequence of the de-glycosylated peptide and positively identifies a series of fragments differing in O-glycosylation. To localize sites of O-glycan attachment, synthetic IgA1 HR glycopeptides and HR from a naturally Gal-deficient polymeric IgA1 myeloma protein were analyzed by electron capture dissociation and activated ion-electron capture dissociation. Multiple sites of O-glycan attachment (including sites of Gal deficiency) in myeloma IgA1 HR glycoforms were identified (in all but one case uniquely). These results represent the first direct identification of multiple sites of O-glycan attachment in IgA1 hinge region by mass spectrometry, thereby enabling future characterization at the molecular level of aberrant glycosylation of IgA1 in diseases such as IgA nephropathy.

Increased levels of galactose-deficient IgG in sera of HIV-1-infected individuals.

Background:The IgG from sera of patients with chronic inflammatory diseases of autoimmune character or some chronic microbial infections is frequently deficient in galactose on N-linked glycans. However, this phenomenon has not been investigated at length in human viral infections. Objectives:To evaluate the glycosylation of serum IgG in HIV-1-positive patients. Methods:Psathyrella velutina lectin was used in enzyme-linked immunosorbent and Western blot assays to determine glycosylation. In addition, gas–liquid chromatography and mass spectrometry were utilized to confirm the galactose deficiency observed in the lectin-binding assays. Results:HIV-1-infected individuals had significantly higher levels of galactose-deficient IgG than healthy controls. In fact, the galactose deficiency of the N-linked glycans observed in other diseases was even more profound in HIV-1 infection. This deficiency was primarily restricted to IgG when total serum glycoproteins were evaluated and IgG1 was the subclass most affected in all patients. Also, a significant increase in lectin binding was observed on IgG2 and IgG4 from HIV-1-positive females compared with HIV-1-negative females. Conclusions:Identification of deficient galactosylation of serum IgG from HIV-1-infected patients extended the spectrum of diseases in which this phenomenon has been observed. In addition, the results suggest yet another aspect of immune dysfunction as a result of HIV-1 infection.

Human Male Genital Tract Secretions: Both Mucosal and Systemic Immune Compartments Contribute to the Humoral Immunity

In contrast to numerous studies of female genital tract secretions, the molecular properties of Abs and the magnitude of humoral responses in human male genital tract secretions to naturally occurring Ags and to mucosal and systemic immunizations have not been extensively investigated. Therefore, seminal plasma (SP) collected from healthy individuals was analyzed with respect to Ig levels, their isotypes, molecular forms of IgA, and for the presence of Abs to naturally occurring Ags, or induced by systemic or mucosal immunizations with viral and bacterial vaccines. The results indicated that in SP, IgG and not IgA, is the dominant Ig isotype, and that IgM is present at low levels. IgA is represented by secretory IgA, polymeric IgA, and monomeric IgA. In contrast to the female genital tract secretions in which IgA2 occurs in slight excess, the distribution of IgA subclasses in SP resembles that in plasma with a pronounced preponderance of IgA1. The IgG subclass profiles in SP are also similar to those in serum. Thus, SP is an external secretion that shares common features with both typical external secretions and plasma. Specifically, SP contains naturally occurring secretory IgA Abs to environmental Ags of microbial origin and to an orally administered bacterial vaccine, and plasma-derived IgG Abs to systemically injected vaccines. Therefore, both mucosal and systemic immunization with various types of Ags can induce humoral responses in SP. These findings should be considered in immunization strategies to induce humoral responses against sexually transmitted infections, including HIV-1.

Heterogeneity of O-glycosylation in the hinge region of human IgA1

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.

Studies on human secretory immunoglobulin A—III. J chain☆

Abstract J chain has been detected both immunochemically and by disc electrophoresis in human colostral and salivary IgA, in five polymeric myeloma IgA, and in five macroglobulinemic IgM preparations. Serum 7S IgA, IgG, and myeloma IgD are devoid of J chain. Urinary protein from two patients with IgA myeloma of polymeric type had high levels of Bence-Jones protein but no evidence of J chain. For J chain preparation by ion-exchange chromatography, sulfitolysis was preferable to reduction and alkylation because of a better separation of J chain from other subunits. The proportion of one J chain per 11S secretory IgA and per pentameric IgM molecules was determined. It is argued that J chain is not derived from α chain.

Urinary Immunoglobulins in Healthy Individuals and Children with Acute Pyelonephritis

Urine samples obtained from children with acute pyelonephritis and from healthy children and adults were analysed with regard to the molecular form and specific antibody activity of urinary immunoglobulins. The urinary IgA and IgG levels were quantified in unconcentrated urine by radioimmunoassay. The children with urinary tract infection had significantly higher levels of IgG and IgA than age‐matched controls but not higher than healthy adults. After tenfold concentration, the urine was fractionated on an Ultrogel AcA 22 column, and the IgA, secretory IgA, and IgG in the fractions were determined by radioimmunoassay. IgA in urine from healthy adults was predominantly represented by polymeric IgA linked to secretory component: small quantities of monomeric IgA were also present. IgG eluted in the position of the serum standard. Increased proportions of IgG and monomeric IgA were found in the infected patients. Specific antibody activity of the IgG and IgA classes to antigens of the infecting Escherichia coli strain was detected in whole and in fractionated urine from children with acute pyelonephritis. The specific antibody activity in healthy adults and children was low.

Carbohydrate-mediated clearance of secretory IgA from the circulation.

Radioiodinated human secretory IgA (sIgA) injected intravenously into mice was rapidly cleared from the circulation by the liver. A portion of the sIgA was transported as an intact molecule into the bile. However, this transport was less efficient than that of human serum polymeric IgA (pIgA). The clearance of sIgA from the circulation was inhibited by prior injection of asialofetuin, suggesting that its uptake is mediated by the hepatic binding protein (HBP) specific for asialoglycoproteins. Mouse pIgA did not inhibit the hepatic clearance of sIgA. Results of in vivo studies were confirmed by in vitro experiments. The binding of 125I-asialoorosomucoid to either the particulate fraction (2000 g pellet of the homogenate) or the plasma membrane fraction of mouse liver was inhibited by sIgA. When polypeptide components of sIgA were used as inhibitors, significant inhibition was obtained with secretory component (SC), while inhibition with light and J-chains was not statistically significant. Examination of the inhibitory activity of IgA1 and IgA2 myeloma proteins and heavy chains isolated from these proteins revealed that binding of polymeric IgA1 and alpha 1 heavy chains can also be mediated by HBP. However, these interactions appear to be of lower avidity than those with SC. The inhibitory activity of human IgA2 and alpha 2 heavy chains was not significant. The involvement of HBP in binding of sIgA was also confirmed by measuring the inhibition of binding of 125I-sIgA. The binding of this protein by the particulate fraction of the mouse liver homogenate was inhibited by asialoglycoproteins and SC while inhibition with IgA1 and alpha 1 heavy chains was not significant. These results suggest that the carbohydrate moieties recognized by HBP reside primarily in the SC portion of sIgA.

Carbohydrate heterogeneity of human myeloma proteins of the IgA1 and IgA2 subclasses

Comparative studies of the N-linked carbohydrate chains of human myeloma proteins of the IgA1 and IgA2 subclasses were performed. The N-linked carbohydrate chains were released by hydrazinolysis from the polypeptide backbone, converted to radioactive oligosaccharides by sodium borotritide reduction after N-acetylation and separated into one neutral and two acidic fractions by paper electrophoresis. The acidic oligosaccharides were completely converted to neutral oligosaccharides by sialidase treatment, indicating that they were sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were further fractionated by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed that human myeloma IgA proteins contained significant amounts of biantennary complex-type carbohydrate chains in addition to a small amount of the high mannose-type. The results indicated that the oligosaccharide structures of human IgA1 and IgA2 display a high degree of heterogeneity not only in the number of carbohydrate chains, but also in their composition.

Scarcity or Absence of Humoral Immune Responses in the Plasma and Cervicovaginal Lavage Fluids of Heavily HIV-1-Exposed But Persistently Seronegative Women

To address an existing controversy concerning the presence of HIV-1-specific antibodies of the IgA isotype in the female genital tract secretions of highly-exposed but persistently seronegative (HEPSN) women, 41 samples of plasma and cervicovaginal lavage (CVL) fluid were distributed to six laboratories for their blinded evaluation using ELISA with 10 different HIV-1 antigens, chemiluminescence-enhanced Western blots (ECL-WB), and virus neutralization. HIV-specific IgG or IgA antibodies in plasma samples from HEPSN women were absent or detectable only at low levels. In CVL, 11/41 samples displayed low levels of reactivity in ELISA against certain antigens. However, only one sample was positive in two of five laboratories. All but one CVL sample yielded negative results when analyzed by ECL-WB. Viral neutralizing activity was either absent or inconsistently detected in plasma and CVL. Plasma and CVL samples from 26 HIV-1-infected women were used as positive controls. Irrespective of the assays and antigens used, the results generated in all laboratories displayed remarkable concordance in the detection of HIV-1-specific antibodies of the IgG isotype. In contrast, IgA antibodies to HIV-1 antigens were not detected with consistency, and where present, IgA antibodies were at markedly lower levels than IgG. Although HIV-neutralizing activity was detected in plasma of all HIV-1-infected women, only a few of their CVL samples displayed such activity. In conclusion, frequent HIV-1 sexual exposure does not stimulate uniformly detectable mucosal or systemic HIV-1-specific responses, as convincingly documented in the present blindly performed study using a broad variety of immunological assays. Although HIV-1-infection leads to vigorous IgG responses in plasma and CVL, it does not stimulate sustained IgA responses in either fluid.