GalNAc-T14 may Contribute to Production of Galactose-Deficient Immunoglobulin A1, the Main Autoantigen in IgA Nephropathy.

Introduction: Immunoglobulin A1 (IgA1) with galactose-deficient O-glycans (Gd-IgA1) play a key role in the pathogenesis of IgA nephropathy (IgAN). Mucosal-tissue infections increase IL-6 production and, in patients with IgAN, are often associated with macroscopic hematuria. IgA1-secreting cell lines derived from the circulation of patients with IgAN, compared to those of healthy controls (HCs), produce more IgA1 that has O-glycans with terminal or sialylated N-acetylgalactosamine (GalNAc). GalNAc residues are added to IgA1 hinge region by some of the 20 GalNAc transferases, the O-glycosylation-initiating enzymes. Expression of GALNT2, encoding GalNAc-T2, the main enzyme initiating IgA1 O-glycosylation, is similar in cells derived from patients with IgAN and HCs. In this report, we extend our observations of GALNT14 overexpression in IgA1-producing cell lines from patients with IgAN. Methods: GALNT14 expression was analyzed in peripheral blood mononuclear cells (PBMCs) from patients with IgAN and from HCs. Moreover, the effect of GALNT14 overexpression or knock-down on Gd-IgA1 production in Dakiki cells was assessed. Results: GALNT14 was overexpressed in PBMCs from patients with IgAN. IL-6 increased GALNT14 expression in PBMCs from patients with IgAN and HCs. We used IgA1-producing cell line Dakiki, a previously reported model of Gd-IgA1-producing cells, and showed that overexpression of GalNAc-T14 enhanced galactose deficiency of IgA1, whereas siRNA-mediated GalNAc-T14 knock-down reduced it. GalNAc-T14 was localized in trans-Golgi network, as expected. Conclusions: Overexpression of GALNT14 due to inflammatory signals during mucosal infections may contribute to overproduction of Gd-IgA1 in patients with IgAN.

[IgA nephropathy - research-generated questions]. / IgA nefropatie - otázky, které nastolil výzkum.

IgA nephropathy (IgAN) is the most common type of glomerulonephritis. Its etiology involves an increased production of polymeric immunoglobulin A1 with an abnormal composition of some carbohydrate chains. The reaction of these abnormal forms of IgA1 with specific autoantibodies while circulating immune complexes arise and settle in the renal mesangium with subsequent inflammatory activation of mesangial cells which in up to 50% of cases results in end-stage kidney failure. Pathogenesis involves an interplay of genetic predisposition and environmental effects, mainly of microbial nature. Current therapy is not sufficiently effective and lacks the focus on the cause of the disease, therefore more efficient and specific ways of therapy are being sought to target the individual stages of the pathogenetic process of IgAN development. With the accumulation of knowledge, new questions arise, concerning detailed mechanisms of the pathological processes, as discussed in the text.Key words: autoimmunity - glycosylation of IgA hinge region - IgA nephropathy - immunoglobulin IgA - IgA1 hinge region.

[IgA Nephropathy. Facts, uncertainties, and potential causal therapy approaches]. / IgA nefropatie--jistoty, pochybnosti a výhledy kauzální lécby.

IgA nephropathy is currently the most frequently investigated glomerulonephritis. The disease is defined by the presence of dominant or co-dominant deposits of IgA1 in the glomerular mesangium. Circulating immune complexes are most likely the source of the deposited IgA1. However, it is also possible that the aggregates of structurally altered IgA1 or enhanced binding to IgA receptors expressed on mesangial cells lead to deposition. The cause of the formation of immune complexes responsible for IgA nephropathy lies in the incomplete O-linked oligosaccharide side chains, which, due to the deficiency of corresponding glycosyltransferases, lack terminal galactose residues leading to the exposure of N-acetylgalactosamine. Naturally occurring antibodies of the IgG or IgA1 isotype bind to this sugar antigen. In the clinical course, we differentiate between the early stage usually characterized by hematuria, and a variable late stage characterized either by a clinical remission, by persistence of hematuria, or by increasing proteinuria and blood pressure and decreasing renal function in one third of the patients. In the early stage, it is difficult to predict the prognosis of IgA nephropathy, either on the basis of clinical presentation and morphological findings, or according to the level of galactose-deficient IgA1 in the circulation. The reliable criteria of serious prognosis emerge only in the later stages of the disease and include proteinuria, hypertension, and histologically apparent tubular atrophy and interstitial sclerosis. The dominant trend in the treatment of IgA nephropathy is the emphasis on administration of ACE inhibitors/sartans, which are introduced into the treatment at the time of microalbuminuria. If proteinuria does not decrease below 1 g/24 h, treatment with prednisone is justifiable. New findings concerning the molecular/cellular mechanism involved in the pathogenesis of IgA nephropathy suggest the possible therapeutical interference with the generation of nephritogenic immune complexes by a selective blocking of the IgA1 molecules with altered glycan structures using monovalent reagents.

Molecular adjuvants based on nonpyrogenic lipophilic derivatives of norAbuMDP/GMDP formulated in nanoliposomes: stimulation of innate and adaptive immunity.

PURPOSE: The aim of this work was to demonstrate an immunostimulatory and adjuvant effect of new apyrogenic lipophilic derivatives of norAbuMDP and norAbuGMDP formulated in nanoliposomes. METHODS: Nanoliposomes and metallochelating nanoliposomes were prepared by lipid film hydration and extrusion methods. The structure of the liposomal formulation was studied by electron microscopy, AF microscopy, and dynamic light scattering. Sublethal and lethal γ-irradiation mice models were used to demonstrate stimulation of innate immune system. Recombinant Hsp90 antigen (Candida albicans) bound onto metallochelating nanoliposomes was used for immunisation of mice to demonstrate adjuvant activities of tested compounds. RESULTS: Safety and stimulation of innate and adaptive immunity were demonstrated on rabbits and mice. The liposomal formulation of norAbuMDP/GMDP was apyrogenic in rabbit test and lacking any side effect in vivo. Recovery of bone marrow after sublethal γ-irradiation as well as increased survival of mice after lethal irradiation was demonstrated. Enhancement of specific immune response was demonstrated for some derivatives incorporated in metallochelating nanoliposomes with recombinant Hsp90 protein antigen. CONCLUSIONS: Liposomal formulations of new lipophilic derivatives of norAbuMDP/GMDP proved themselves as promising adjuvants for recombinant vaccines as well as immunomodulators for stimulation of innate immunity and bone-marrow recovery after chemo/radio therapy of cancer.

N-acetylgalactosaminide α2,6-sialyltransferase II is a candidate enzyme for sialylation of galactose-deficient IgA1, the key autoantigen in IgA nephropathy.

BACKGROUND: Galactose-deficient O-glycans in the hinge region (HR) of immunoglobulin A1 (IgA1) play a key role in the pathogenesis of IgA nephropathy (IgAN). O-Glycans of circulatory IgA1 consist of N-acetylgalactosamine (GalNAc) with a ß1,3-linked galactose; both sugars may be sialylated. In patients with IgAN, α2,6-sialylated GalNAc is a frequent form of the galactose-deficient O-glycans. Prior analyses of IgA1-producing cells had indicated that α2,6-sialyltransferase II (ST6GalNAc-II) is likely responsible for sialylation of GalNAc of galactose-deficient IgA1, but direct evidence is missing. METHODS: We produced a secreted variant of recombinant human ST6GalNAc-II and an IgA1 fragment comprised of Cα1-HR-Cα2. This IgA1 fragment and a synthetic HR peptide with enzymatically attached GalNAc residues served as acceptors. ST6GalNAc-II activity was assessed in vitro and the attachment of sialic acid to these acceptors was detected by lectin blot and mass spectrometry. RESULTS: ST6GalNAc-II was active with both acceptors. High-resolution mass spectrometry analysis revealed that up to three sialic acid residues were added to the GalNAc residues of the HR glycopeptide. CONCLUSIONS: Our data provide direct evidence that ST6GalNAc-II can sialylate GalNAc of galactose-deficient IgA1. As serum levels of galactose-deficient IgA1 with sialylated glycoforms are increased in IgAN patients, our data explain the corresponding part of the biosynthetic pathway.

Enzymatic sialylation of IgA1 O-glycans: implications for studies of IgA nephropathy.

Patients with IgA nephropathy (IgAN) have elevated circulating levels of IgA1 with some O-glycans consisting of galactose (Gal)-deficient N-acetylgalactosamine (GalNAc) with or without N-acetylneuraminic acid (NeuAc). We have analyzed O-glycosylation heterogeneity of naturally asialo-IgA1 (Ale) myeloma protein that mimics Gal-deficient IgA1 (Gd-IgA1) of patients with IgAN, except that IgA1 O-glycans of IgAN patients are frequently sialylated. Specifically, serum IgA1 of healthy controls has more α2,3-sialylated O-glycans (NeuAc attached to Gal) than α2,6-sialylated O-glycans (NeuAc attached to GalNAc). As IgA1-producing cells from IgAN patients have an increased activity of α2,6-sialyltransferase (ST6GalNAc), we hypothesize that such activity may promote premature sialylation of GalNAc and, thus, production of Gd-IgA1, as sialylation of GalNAc prevents subsequent Gal attachment. Distribution of NeuAc in IgA1 O-glycans may play an important role in the pathogenesis of IgAN. To better understand biological functions of NeuAc in IgA1, we established protocols for enzymatic sialylation leading to α2,3- or α2,6-sialylation of IgA1 O-glycans. Sialylation of Gal-deficient asialo-IgA1 (Ale) myeloma protein by an ST6GalNAc enzyme generated sialylated IgA1 that mimics the Gal-deficient IgA1 glycoforms in patients with IgAN, characterized by α2,6-sialylated Gal-deficient GalNAc. In contrast, sialylation of the same myeloma protein by an α2,3-sialyltransferase yielded IgA1 typical for healthy controls, characterized by α2,3-sialylated Gal. The GalNAc-specific lectin from Helix aspersa (HAA) is used to measure levels of Gd-IgA1. We assessed HAA binding to IgA1 sialylated at Gal or GalNAc. As expected, α2,6-sialylation of IgA1 markedly decreased reactivity with HAA. Notably, α2,3-sialylation also decreased reactivity with HAA. Neuraminidase treatment recovered the original HAA reactivity in both instances. These results suggest that binding of a GalNAc-specific lectin is modulated by sialylation of GalNAc as well as Gal in the clustered IgA1 O-glycans. Thus, enzymatic sialylation offers a useful model to test the role of NeuAc in reactivities of the clustered O-glycans with lectins.

Aberrant O-glycosylation and anti-glycan antibodies in an autoimmune disease IgA nephropathy and breast adenocarcinoma.

Glycosylation abnormalities have been observed in autoimmune diseases and cancer. Here, we compare mechanisms of aberrant O-glycosylation, i.e., formation of Tn and sialyl-Tn structures, on MUC1 in breast cancer, and on IgA1 in an autoimmune disease, IgA nephropathy. The pathways of aberrant O-glycosylation, although different for MUC1 and IgA1, include dysregulation in glycosyltransferase expression, stability, and/or intracellular localization. Moreover, these aberrant glycoproteins are recognized by antibodies, although with different consequences. In breast cancer, elevated levels of antibodies recognizing aberrant MUC1 are associated with better outcome, whereas in IgA nephropathy, the antibodies recognizing aberrant IgA1 are part of the pathogenetic process.

Enhancement of immune response towards non-lipidized Borrelia burgdorferi recombinant OspC antigen by binding onto the surface of metallochelating nanoliposomes with entrapped lipophilic derivatives of norAbuMDP.

Lyme disease caused by spirochete Borrelia burgdorferi sensu lato, is a tick-born illness. If the infection is not eliminated by the host immune system and/or antibiotics, it may further disseminate and cause severe chronic complications. The immune response to Borrelia is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies associated with Th1 cell activation. A new experimental vaccine was constructed using non-lipidized form of recombinant B. burgdorferi s.s. OspC protein was anchored by metallochelating bond onto the surface of nanoliposomes containing novel nonpyrogenic lipophilized norAbuMDP analogues denoted MT05 and MT06. After i.d. immunization, the experimental vaccines surpassed Alum with respect to OspC-specific titers of IgG2a, IgG2b isotypes when MT06 was used and IgG3, IgM isotypes when MT05 was used. Both adjuvants exerted a high adjuvant effect comparable or better than MDP and proved themselves as nonpyrogenic.

Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells.

The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system.

Metallochelating liposomes with associated lipophilised norAbuMDP as biocompatible platform for construction of vaccines with recombinant His-tagged antigens: preparation, structural study and immune response towards rHsp90.

Hsp90-CA is present in cell wall of Candida pseudohyphae or hyphae-typical pathogenic morphotype for both systemic and mucosal Candida infections. Heat shock protein from Candida albicans (hsp90-CA) is an important target for protective antibodies during disseminated candidiasis of experimental mice and human. His-tagged protein rHsp90 was prepared and used as the antigen for preparation of experimental recombinant liposomal vaccine. Nickel-chelating liposomes (the size around 100nm, PDI≤0.1) were prepared from the mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS-NTA-Ni (molar ratio 95:5%) by hydration of lipid film and extrusion methods. New non-pyrogenic hydrophobised derivative of MDP (C18-O-6-norAbuMDP) was incorporated into liposomes as adjuvans. rHsp90 was attached onto the surface of metallochelating liposomes by metallochelating bond and the structure of these proteoliposomes was studied by dynamic light scattering, AF microscopy, TEM and GPC. The liposomes with surface-exposed C18-O-6-norAbuMDP were well recognised and phagocyted by human dendritic cells in vitro. In vivo the immune response towards this experimental vaccine applied in mice (i.d.) demonstrated both TH1 and TH2 response comparable to FCA, but without any side effects. Metallochelating liposomes with lipophilic derivatives of muramyl dipeptide represent a new biocompatible platform for construction of experimental recombinant vaccines and drug-targeting systems.

Isolation of recombinant cysteine dioxygenase protein from Trichophyton mentagrophytes.

Cysteine dioxygenase (CDO, EC 1.13.11.20) catalyses the oxygenation of cysteine to cysteine sulphinic acid leading to the production of sulphite, sulphate and taurine as the final metabolites of cysteine catabolism. Keratinolytic fungi secrete sulphite and sulphate to reduce disulphide bridges in host tissue keratin proteins as the first step of keratinolysis. In the present study, we describe the identification of cDNA, as well as expression and characterisation of recombinant CDO protein from Trichophyton mentagrophytes. The cDNA was amplified using primers designed on the basis of high conservancy CDO regions identified in other fungi. PCR product was cloned and sequenced. Recombinant CDO was expressed in Escherichia coli, and affinity purified and identified by matrix-assisted laser desorption/ionization - time-of-flight mass spectrometry (MALDI-TOF MS). Enzyme activity was assayed by monitoring the production of cysteine sulphinate using mass spectrometry. The Cdo cDNA encodes for a protein consisting of 219 amino acids. Recombinant CDO protein C-terminally fused with a His tag was purified by affinity chromatography. The CDO purified under native condition was proved to be enzymatically active. Protein identity was confirmed by MALDI-TOF MS. Comparison of cDNA sequence with those identified in other fungi revealed significant homology. Identification of T. mentagrophytes CDO provides indispensable tools for future studies of dermatophyte pathogenicity and development of new approaches for prevention and therapy.

Novel modification of growth medium enables efficient E. coli expression and simple purification of an endotoxin-free recombinant murine hsp70 protein.

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-D-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite, N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other His.

Systemic and mucosal immunization with Candida albicans hsp90 elicits hsp90-specific humoral response in vaginal mucosa which is further enhanced during experimental vaginal candidiasis.

The Candida albicans heat shock protein 90 kDa (hsp90-CA) is an important target for protective antibodies in disseminated candidiasis of experimental mice and humans. Hsp90-CA is present in the cell wall of Candida pseudohyphae or hyphae--typical pathogenic morphotypes in both mucosal and systemic Candida infections. However, the potential protective effects of hsp90-CA-specific antibodies in vaginal candidiasis has not yet been reported. In the present study we used various vaccine formulations (recombinant hsp90-CA protein and hsp90-CA-encoding DNA vaccine) and routes of administration (intradermal, intranasal, and intravenous) to induce both hsp90-CA-specific systemic and vaginal mucosa immune responses in experimental BALB/c mice. The results showed that intradermal recombinant hsp90-CA protein priming, followed by intranasal or intradermal recombinant hsp90-CA protein boosting induced significant increases in both serum and vaginal hsp90-CA-specific IgG and IgA antibodies compared to the control group, as well as enhanced hsp90-CA-specific splenocyte responses in vitro. In the intradermally boosted group, subsequent experimental vaginal Candida infection induced additional increases in the hsp90-CA specific IgG isotype, suggesting that Candida has the ability to induce a local hsp90-specific antibody (IgG) response during vulvovaginal candidiasis. Further work is required to elucidate the importance of immunity to highly conserved antigens during infection of the human female reproductive tract where a balance between immunity to and tolerance for commonly antigens such as hsp90 is necessary for the maintenance of fertility.

DNA vaccines: are they still just a powerful tool for the future?

Vaccination is historically one of the most successful strategies for the prevention of infectious diseases. For safety reasons, modern vaccinology tends toward the usage of inactivated or attenuated microorganisms and uses predominantly subunit vaccines. The antigens need to be clearly defined, pure, stable, appropriately composed, and properly presented to the immune system of the host. Differing ratios of various proportions between specific CD4+ and CD8+ T cell responses are essential for conferring the required protection in the case of individual vaccines. To stimulate both CD4+ and CD8+ T cells, the antigens must be processed and presented to both antigen-presentation pathways, MHC I and MHC II. Protein antigens delivered by vaccination are processed as extracellular antigens. However, extracellularly delivered antigen can be directed towards intracellular presentation pathways in conjugation with molecules involved in antigen cross-presentation, e.g. heat shock proteins, or by genomic-DNA vaccination. In this overview, current knowledge of the host immune response to DNA vaccines is summarized in the introduction. The subsequent sections discuss techniques for enhancing DNA vaccine efficacy, such as DNA delivery to specific tissues, delivery of DNA to the cell cytoplasm or nucleus, and enhancement of the immune response using molecular adjuvants. Finally, the prospects of DNA vaccination and ongoing clinical trials with various DNA vaccines are discussed.

Biological aspects of Lyme disease spirochetes: unique bacteria of the Borrelia burgdorferi species group.

BACKGROUND: Borrelia burgdorferi sensu lato is a group of at least twelve closely related species some of which are responsible for Lyme disease, the most frequent zoonosis in Europe and the USA. Many of the biological features of Borrelia are unique in prokaryotes and very interesting not only from the medical viewpoint but also from the view of molecular biology. METHODS: Relevant recent articles were searched using PubMed and Google search tools. RESULTS AND CONCLUSION: This is a review of the biological, genetic and physiological features of the spirochete species group, Borrelia burgdorferi sensu lato. In spite of a lot of recent articles focused on B. burgdorferi sensu lato, many features of Borrelia biology remain obscure. It is one of the main reasons for persisting problems with prevention, diagnosis and therapy of Lyme disease. The aim of the review is to summarize ongoing current knowledge into a lucid and comprehensible form.