Common antigenicity for two glycosidases

Enzyme replacement therapy (ERT) has proven to be an effective therapy for some lysosomal storage disorder (LSD) patients. A potential complication during ERT is the generation of an immune response against the replacement protein. We have investigated the antigenicity of two distantly related glycosidases, alpha-glucosidase (Pompe disease or glycogen storage disease type II, GSD II), and alpha-L-iduronidase (Hurler syndrome, mucopolysaccharidosis type I, MPS I). The linear sequence epitope reactivity of affinity purified polyclonal antibodies to recombinant human alpha-glucosidase and alpha-L-iduronidase was defined, to both glycosidases. The polyclonal antibodies exhibited some cross-reactive epitopes on the two proteins. Moreover, a monoclonal antibody to the active site of alpha-glucosidase showed cross-reactivity with a catalytic structural element of alpha-L-iduronidase. In a previous study, in MPS I patients who developed an immune response to ERT, this same site on alpha-L-iduronidase was highly antigenic and the last to tolerise following repeated enzyme infusions. We conclude that glycosidases can exhibit cross-reactive epitopes, and infer that this may relate to common structural elements associated with their active sites.     

Taenia solium: a two-dimensional Western blotting method combined with the use of an EST-library for the identification of immunogenic proteins recognized by sera from neurocysticercosis patients

Commercial antigens used to diagnose human neurocysticercosis (NCC) are obtained from either a soluble parasite extract or a parasite-derived glycoprotein fraction. The aim of the present study was to identify antigenic proteins as potential diagnostic candidates in this context. Soluble immunogenic proteins from Taenia solium cysticerci were identified by two-dimensional electrophoresis Western blotting using human sera from Nicaragua confirmed to be positive for NCC by computer tomography. Six antigenic proteins were identified and sequenced by liquid chromatography–mass spectrometry. Among these immunogenic proteins, a novel sequence was found and named Tsol-p27. To determine the antigenicity of Tsol-p27, the previously reported antigen TsolHSP36 and the new Tsol-p27 were expressed as recombinant proteins and evaluated serologically. Immunoblotting demonstrated that Tsol-p27 was recognized by sera from 13 NCC-positive humans, whereas TsolHSP36 was identified by only two of those 13 positive sera. None of the antigens were recognized by negative control sera. Despite the limited number of serum samples evaluated in this study, the results indicate that Tsol-p27 might be a suitable candidate for diagnosis of human NCC.     

Generation of specificity-variant antibodies by alteration of carbohydrate in light chain of human monoclonal antibodies.

A hybridoma line, C5TN, produced human monoclonal antibody of which light chain had N-linked carbohydrate chain within the variable region. Some molecular-weight variants of light chain of the antibody were produced by C5TN variants resistant to cytotoxic effect of concanavalin A. The variant antibodies significantly altered the original cross-reactivity with antigens or lost the ability of antigen binding. The variants variously trimmed their carbohydrate chains by glycosidases, showed the changed reactivity or acquired the ability to bind for antigens. The carbohydrate-deficient antibodies from tunicamycin-treated C5TN and the variant clones behaved in a similar manner on antigen-binding reactivity. Furthermore, comparison of antibodies of which light chains have carbohydrate chains sensitive and resistant to some glycosidases showed that carbohydrate chain in variable region of light chain can influence their reactivity with antigen.     

Diagnostic epitope variability within Taenia solium 8 kDa antigen family: implications for cysticercosis immunodetection

To study diagnostic epitopes within the Taenia solium 8 kDa antigen family, six overlapping synthetic peptides from an 8 kDa family member (Ts8B2) were synthesized and evaluated by ELISA and MABA with sera from patients with neurocysticercosis (NCC), from infected pigs and from rabbits immunized with recombinant Ts8B2 protein. The pre-immune rabbit sera and the Ts8B2 recombinant protein served as negative and positive controls, respectively. A similar analysis was done with the already described antigenic peptides from another member of the 8 kDa family, highly similar to Ts8B2, the CyDA antigen. Surprisingly, neither the Ts8B2 peptides nor the CyDA peptides were recognized by infected human and porcine sera. However, the entire Ts8B2 recombinant, as well as amino and carboxy-terminal halves were recognized by the positive serum samples. The observed lack of recognition of linear Ts8B2 peptides suggests that the principal serological response to the Ts8B2 family is focused on conformational epitopes in contrast to the previously observed antigenicity of the CyDA peptides. This differential antigenicity of 8 kDa family peptides could be related with parasite antigenic variability. The fact that rabbits experimentally immunized with Ts8B2 did make anti-peptide antibodies to peptides Ts8B2-6 and CyDA-6, located in the carboxy-terminal region demonstrated that the Ts8B2 peptides are not intrinsically non-immunogenic.     

Biological roles of blood group antigens

Recognition and application of blood group differences on human red cells permitted the development of safe procedures for blood transfusion. Blood group antigens are markers on surface-exposed red cell proteins or the sugar moiety of glycoproteins or glycolipids. Apart from their presumed biological function, some antigens have been identified as receptors for host/parasite interactions. Thus, carbohydrates that determine P antigenicity are the binding receptor for certain strains of pyelonephritic coliforms. Other pathogenic coliforms bind to the membrane structure that carries the Dra antigen. A structure associated with Duffy antigens is the attachment receptor for the parasite of Plasmodium vivax malaria, while Plasmodium falciparum parasites bind to structures associated with membrane glycophorins. Structure/function relationships have been established by the finding that lack of Rh protein in red cells of Rhnull phenotype is associated with stomatocytic cell morphology and a hemolytic state. Absence of glycophorin C, and the Gerbich blood group antigens that it carries, is associated with elliptocytic red cells. Absence of Kx antigen protein in the Kell system is associated with the McLeod blood group phenotype, with acanthocytic cell morphology and reduced in vivo survival. McLeod individuals also have late-onset muscular dystrophy and neurological disorders.     

Humoral immune response of carp Cyprinus carpio to Myxobolus artus (Myxozoa: Myxobolidae) infection

The circulating antibody which reacted with sonicated spores of Myxobolus artus was detected in some naturally infected carp. However, some other fish had no detectable sign of infection, though they had the antibody. When carp were injected either with intact or sonicated spores, the antibody was not produced, while fish injected either with developing stages (presporogonic and sporoblast stages) of the parasite or sonicated spores with bovine serum albumin elicited the antibody production. The results of the injection experiments suggest that (1) developing stages have antigenicity to carp, and (2) spores have lost the antigenicity; sonicated spores are haptens, with which the antibody can react. In an indirect fluorescent antibody technique, sera positive for the antigen reacted with developing stages of the parasite, but not with the spore.
The mechanism of the host immune response against M. artus is discussed in relation to a previous observation that the parasite sometimes underwent abnormal development, in which host encapsulation was imperfect or even lacking, probably leading to degeneration of pseudocysts before the completion of spore formation. It is plausible that the antibody was produced when pseudocysts which showed abnormal growth ruptured during their developing stages, resulting in exposure of the young parasite to the host immune system.     

Specific bovine antibody response against a new recombinant Cryptosporidium parvum antigen containing 4 zinc-finger motifs

A Cryptosporidium parvum sporozoite and oocyst lambda gt11 cDNA library was screened with a hyperimmune rabbit serum that was developed against insoluble fragments of ultrasonicated oocysts. A clone named Cp22.4.1 encoding a protein of 231 amino acids with 4 zinc-finger domains characterized by a Cys-X2-Cys-X4-His-X4-Cys motif was isolated and characterized. There was a complete match between the sequencing data of the coding region of Cp22.4.1 and the corresponding gene at chromosomal level. Cloning in a pBAD-TOPO-TA expression vector permitted to evaluate the antigenicity of the recombinant His-tagged antigen. This antigen was recognized by 2 out of 5 sera from Cryptosporidium immune calves and not by sera from parasite naive animals.     

The plant lectin wheat germ agglutinin inhibits the binding of pemphigus foliaceus autoantibodies to desmoglein 1 in a majority of patients and prevents pathomechanisms of pemphigus foliaceus in vitro and in vivo

Pemphigus foliaceus (PF) is a life-threatening autoimmune blistering skin disease caused by pathogenic IgG autoantibodies against desmoglein 1 (dg1), a desmosomal cadherin-type adhesion glycoprotein. Using lectins and glycosidases, we have shown that dg1 displays an N-glycosylation pattern of the complex triantennary type. We have found that lectins and glycosidases interfere with N-bound sugar residues on the amino-terminal ectodomain of dg1 and completely abolish, in vitro, the antigenicity of dg1 in most of the patients' sera. Moreover, in an ex vivo model using punch biopsies from normal human skin, we demonstrate that preincubation of the epidermis in wheat germ agglutinin (WGA) prevents PF autoantibody binding, acantholysis, and subcorneal blistering. In addition, we show that topical treatment with WGA inhibits PF autoantibody binding to keratinocytes in both newborn BALB/c mice and in organotypic human epidermis grafted onto the back of SCID mice. The epidermis of these pretreated animals displays a regular morphology, whereas control animals develop the immunopathologic phenotype of PF. These findings suggest that WGA may interfere with autoantibody binding to dg1, preventing experimental PF without affecting the adhesive function of dg1. Our observations may provide a new approach to the therapy of PF.     

Electrophoretic and immunoelectrophoretic study of the enzymatic degradation of human orosomucoid (alpha 1 -acid glycoprotein)

Purified, intact orosomucoid (alpha 1-acid glycoprotein) derived from whole human plasma was incubated with a number of proteolytic and saccharolytic enzymes under a variety of conditions. The unfractionated digests were immediately examined by both agar gel- and immunoelectrophoresis for the presence of antigenically active (precipitating) and/or inactive macrofragments. Despite otherwise clear evidence of rapid degradation by several proteases, no antigenic subunits were detected. Among the glycosidases, almond emulsin produced a suggestion of modification of the carbohydrate moiety of a sialic acid-poor orosomucoid preparation obtained from Cohn Fr. VI, but had no effect on antigenic properties. These results are presented as further evidence for a lack of involvement of the extensive carbohydrate component of orosomucoid in its antigenic reactions, and support previous data implicating the polypeptide chain as solely responsible for its antigenicity.     

Fluorescence study on the conformation of a cyclic enkephalin analog in aqueous solution

Certain patients with ovarian germ cell tumors develop a specific antibody reacting with glycoprotein-bound large carbohydrates of murine teratocarcinoma cells. The antigenic determinant was found to involve an alpha-galactosyl residue, since alpha-galactosidase from coffee bean, but not other glycosidases abolished the antigenic activity of the large glycan isolated from F9 and OTT6050 cells. Several evidences excluded the possibility that the antigen is blood group B or P1 antigen. These results indicate tumor-associated expression of an unusual alpha-galactosyl residue in human ovarian germ cell tumors.     

Early recognized antigen (p34) of Toxoplasma gondii after peroral ingestion of tissue cyst forming strain (Me49 strain) in mice.

Serum from mouse orally ingested with tissue cyst forming strain (Me49) of Toxoplasma gondii was assayed by Western blot and immunofluorescene assay (IFA) to establish early responses in antigenicity of the parasite in mouse model of foodborne toxoplasmosis. Sera were collected weekly to blot the RH antigen transferred onto nitrocellulose paper after being separated by 12% SDS-PAGE. With the second week serum, 34 kDa protein (p34) was detected uniquely, and all antigens of T. gondii were detected with the sera from 3 or 4 weeks. p34 was not a member of the major surface membrane proteins and confirmed to be localized in the rhoptry by IFA. It was secreted into parasitophorous vacuolar membrane (PVM) during the entry into host cells. When applied to the human sera of which the ELISA absorbance was in negative range, 10.3% of sera detected p34, while all the ELISA positive sera detected the band. It has diagnostic usefulness of presumed T. gondii infection. We suggest the name of the p34 protein as ROP9.     

Brugia malayi: detection of parasite antigen in sera from infected jirds

Sera from Brugia malayi-infected jirds were demonstrated to contain a heat-stable, 95- to 105-kDa parasite antigen by immunoblot with rabbit antibody to the parasite and with a monoclonal antibody that binds to phosphorylcholine. This antigen is a major component of B. malayi adult worm excretory/secretory antigen, and it is present in lavage fluid obtained from ip-infected animals. The antigen was detected by enzyme immunoassay in all sera collected from jirds 9-54 weeks after sc injection with 100 or 300 infective larvae (L3). Parasite antigen titers were higher in animals infected with the higher L3 dose. Antiphosphorylcholine antibodies were present in jird sera for the first 12 weeks after larval injection, but thereafter, antibody titers decreased to undetectable levels. Parasite antigen was not detected by immunoblot or enzyme immunoassay in sera from 21 human subjects with B. malayi microfilaremia. Antigen may be cleared from human sera by antiphosphorylcholine antibodies, which were present in all sera tested. The practical significance of B. malayi antigen detection in the jird is that it provides a sensitive means of noninvasively monitoring the status of infection in this important experimental filariasis model.     

An approach to postembedding staining of protein (immunoglobulin) antigen embedded in plastic: prerequisites and limitations

A method is described for performing postembedding staining of protein (immunoglobulin) antigen embedded in styrene-methacrylate resin. Fixation of specimens in a combination of 4% paraformaldehyde and 0.2% picric acid and washing in buffer containing 7% sucrose, followed by abrupt dehydration with absolute acetone in the cold preserved the antigenicity, although in a masked form. The masked antigenicity could be reexposed by treatment with nonspecific protease. Staining with fluorescent-, peroxidase-, or ferritin-labeled antibodies on semi- and ultrathin sections resulted in specific localization of the antigen. We applied this technique to the localization of rabbit immunoglobulin in specimens of renal tissue obtained from rats with anti-glomerular basement membrane nephritis; we also localized human IgG in a renal biopsy specimen. The prerequisites for recovery of antigenicity are such that preservation of tissue structure at the light microscopic level is good, but relatively poor at the electron microscopic level.     

Plasmodium falciparum: characterization of a 33-kDa soluble antigen

A 33-kDa soluble antigen identified in the culture supernatant by patient serum and monoclonal antibodies was present in rings, trophozoites, schizonts, and merozoites of Plasmodium falciparum. The antigen which is released into the culture supernatant by growing parasites was also observed in the host cells of trophozoites and schizonts and could be localized on the host cell surface. Its specificity for the surface of trophozoites and schizonts was observed to decrease with increased duration without subculture. The antigen could then be detected on the surface of noninfected erythrocytes. The antigenicity of the 33-kDa antigen was destroyed by heating at 65 degrees C. Monoclonal and polyclonal specific antibodies weakly inhibited parasite growth in vitro. The antigen was present in both knob positive and knob negative parasites in all the P. falciparum isolates tested.     

Cryofixed, freeze-dried and paraffin-embedded skin enables successful immunohistochemical staining of skin basement membrane antigens.

Conventional chemical fixation and paraffin-embedding procedures give good preservation of morphology, although the antigenicity of many proteins in the tissue sample is destroyed. On the other hand, fresh frozen sections can preserve the antigenicity, but provide poor morphological preservation. To overcome this dilemma, cryofixation and freeze drying were used on human skin tissue, applying methodology which has only been used to study lymphoid tissue. First, fresh human skin was cryofixed in liquid isopentane (-160 degrees C) cooled by liquid nitrogen. The skin was then freeze-dried at -40 degrees C and 10(-2) atmospheric pressure for 72 h, followed by embedding in paraffin. Sections 4 microns thick taken from this cryofixed, freeze-dried, and paraffin-embedded skin were stained with hematoxylin-eosin or used for immunolabeling with antibodies against basement membrane antigen, including type IV and type VII collagen, bullous pemphigoid antigen, epidermolysis bullosa acquisita antigen, and GB3 antigen. The morphological preservation of these sections was as good as that of routine formalin-fixed and paraffin-embedded skin sections. The basement membrane was clearly immunostained with all antibodies used, and the intensity of the reaction was as strong as that seen in frozen sections. Evaluation of antigen distribution in conjunction with the detailed skin structure was therefore possible in the same sections.     

Incorporating significant amino acid pairs to identify O-linked glycosylation sites on transmembrane proteins and non-transmembrane proteins

Background  

While occurring enzymatically in biological systems, O-linked glycosylation affects protein folding, localization and trafficking, protein solubility, antigenicity, biological activity, as well as cell-cell interactions on membrane proteins. Catalytic enzymes involve glycotransferases, sugar-transferring enzymes and glycosidases which trim specific monosaccharides from precursors to form intermediate structures. Due to the difficulty of experimental identification, several works have used computational methods to identify glycosylation sites.     

Production of antibodies specific for double stranded antigen DNA cloned from immune complexes in plasma of a SLE patient.

The antigenicity of antigen DNA isolated from immune complexes in plasma of a SLE patient was examined. DDY mice were immunized with the cloned antigen DNA carrying a sequence homologous with a part of bacteriophage f1 (KS8 DNA) by the coupling method, and the antibody response was estimated by radioimmunoassay. Antibodies specific to double stranded DNA were elicited. Moreover, the antibodies showed preferential binding to KS8 DNA than other DNA derived from Escherichia coli. These results suggest that KS8 DNA has a significant antigenicity in mice.     

Immunochemical Studies on Bacterial Blood Group Substances

Two oligosaccharides were isolated from a partial hydrolysis of the lipopolysaccharide from Shigella dysenteriae which had a common antigen with human red blood cells (RBC). A disaccharide sh-2 had a structure of O-β-D-galactosyl-(l→2)-D-galactose, and was active in the hemagglutination inhibition test between human O RBC and anti-S. dysenteriae chicken serum. A tetrasaccharide sh-12 was composed of galactose and rhamnose and was a more effective inhibitor than sh-2. Both oligosaccharides were assumed to be derived from the determinant structure of a common antigen. Serological specificity of the hemagglutinin in chicken serum was examined using these oligosaccharides and human milk oligosaccharides, and the 1→2 linked galactosyl residue was supposed to be important for determination of the heterophile RBC antigenicity.     

Designing of a Novel Fusion Protein Vaccine Candidate Against Human Visceral Leishmaniasis (VL) Using Immunoinformatics and Structural Approaches

Leishmaniasis is caused by an obligate intracellular protozoan parasite. The clinical forms of leishmaniasis differ from cutaneous leishmaniasis, mucocutaneous leishmaniasis and visceral leishmaniasis (VL) which depend on the parasite species and the host’s immune responses. There are significant challenges to the available anti-leishmanial drug therapy, particularly in severe forms of disease, and the rise of drug resistance has made it more difficult. Currently, no licensed vaccines have been introduced to the market for the control and elimination of VL. A potential target for use in candidate vaccines against leishmaniasis has been shown to be leishmania Kinetoplastid membrane protein-11 (KMP-11) antigen. In this study, we chose KMP-11 antigen as target antigen in our vaccine construct. In addition, B-type flagellin (fliC) was used as an adjuvant for enhancing vaccine immunogenicity. The GSGSGSGSGSG linker was applied to link the KMP-11 antigen and fliC (KMP-11-fliC) to construct our fusion protein. Bioinformatics approaches such as; 3D homology modeling, CTL, B-cell, MHC class I and II epitopes prediction, allergenicity, antigenicity evaluations, molecular docking, fast simulations of flexibility of docked complex and in silico cloning were employed to analysis and evaluation of various properties of the designed fusion construct. Computational results showed that our engineered structure has the potential for proper stimulation of cellular and humoral immune responses against VL. Consequently, it could be proposed as a candidate vaccine against VL according to these data and after verifying the efficacy of the candidate vaccine through in vivo and in vitro immunological tests.

     

Purification and characterization of a high molecular weight glycoprotein detectable in human milk and breast carcinomas

The murine monoclonal antibody (MAb) designated DF3 has defined a high m.w. antigen detectable in human breast carcinomas and in human milk. DF3 antigen is detectable on apical borders of secretory mammary epithelial cells and in the cytosol of less differentiated malignant cells. DF3 antigen expression has been shown to correlate with the degree of human breast tumor differentiation, and the detection of a cross-reactive species in human milk has suggested that DF3 antigen might be useful as a biochemical marker of differentiated mammary epithelial cells. To further characterize DF3 antigen, we have developed an approach to purify the cross-reactive species by using gel filtration and antibody affinity chromatography. The affinity column-purified DF3 antigen was absorbed by wheat germ agglutinin and peanut agglutinin, but not by concanavalin A or lentil lectin. In contrast, wheat germ agglutinin inhibited MAb DF3 reactivity with the purified antigen, whereas there was little, if any, inhibition when using peanut agglutinin. These findings are thus consistent with the involvement of terminal N-acetyl-D-neuraminic acid and/or N-acetylglucosamine residues in the antigenic site. DF3 antigenicity was also sensitive to neuraminidase, but not chondroitinase ABC, chondroitinase AC, chondroitin-4-sulfatase, or hyaluronidase. Furthermore, DF3 antigen was sensitive to Pronase, subtilisin BPN', and alpha-chymotrypsin. The presence of O-glycosidic linkages between carbohydrate and protein in the DF3 antigenic site was further supported by the presence of NaBH4-sensitive sites. Together, these results suggest that sialyl oligosaccharides present on a peptide backbone are required for maintaining DF3 antigenicity. Similar findings have been demonstrated for DF3 antigen purified from both human milk and breast cancer effusions. However, the DF3 antigen in human milk consisted of a single high m.w. species, whereas the tumor-associated antigen consisted of two distinct glycoproteins with m.w. of 330,000 and 450,000. These findings may be relevant to the recent demonstration that distinct high m.w. DF3 antigens are elevated in the circulation of patients with breast carcinoma.