Rudolf Grimm

Modulation of IgE reactivity of allergens by site-directed mutagenesis: potential use of hypoallergenic variants for immunotherapy

Specific immunotherapy is an efficient treatment for patients suffering from type I allergy. The mechanisms underlying successful immunotherapy are assumed to operate at the level of T helper cells, leading to a modulation of the immune response to allergens. During immunotherapy, increasing doses of allergens are given on a regular basis, and the beneficial effects for the patient depend on the concentration of allergen used. On the other hand, the risk of IgE‐mediated anaphylactic side effects also increase with the amount of allergen applied per injection. Therefore, we have proposed the use of hypoallergenic (low IgE binding activity) forms of allergens for immunotherapy. We evaluated by site‐directed mutagenesis the contributions of individual amino acid residues/positions for IgE binding to Bet v 1, the major allergen of birch pollen. We found that IgE binding to Bet v 1 depended on at least six amino acid residues/positions. Immunoblot analyses and inhibition experiments showed that the multiple‐point Bet v 1 mutant exhibited extremely low reactivity with serum IgE from birch pollen‐allergic patients. In vivo (skin prick) tests showed that the potency of the multiple‐point mutant to induce typical urticarial type I reactions in pollen‐allergic patients was significantly lower than for wild‐type Bet v 1. Proliferation assays of allergen‐specific T cell clones demonstrated that these six amino acid exchanges in the Bet v 1 sequence did not influence T cell recognition. Thus, the Bet v 1 six‐point mutant displayed significantly reduced IgE binding activity, but conserved T cell activating capacity, which is necessary for immunomodulation. The approach described here may be generally applied to produce allergen variants to be used in a safe therapy form of immediate‐type allergies.—Ferreira, F., Ebner, C., Kramer, B., Casari, G., Briza, P., Kungl, A. J., Grimm, R., Jahn‐Schmid, B., Breiteneder, H., Kraft, D., Breitenbach, M., Rheinberger, H.‐J., Scheiner, O. Modulation of IgE reactivity of allergens by site‐directed mutagenesis: potential use of hypoallergenic variants for immunotherapy. FASEB J. 12, 231–242 (1998)

Contribution of Proteasome-Mediated Proteolysis to the Hierarchy of Epitopes Presented by Major Histocompatibility Complex Class I Molecules

Major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL) recognize peptide epitopes of protein antigens in a hierarchical fashion. We investigated whether proteolytic cleavage, in particular by proteasomes, is important in determining epitope hierarchy. Using highly purified 20S proteasomes, we find preferred cleavage sites directly adjacent to the N- and C-terminal ends of the immunodominant epitope of chicken ovalbumin, Ova257-264, while most of the subdominant epitope, Ova55-62, is destroyed by a major cleavage site located within this epitope. Moreover, we show that variations in amino acid sequences flanking these epitopes influence proteasomal cleavage patterns in parallel with the efficacy of their presentation. The results suggest that proteasomal cleavage within and adjacent to class I-restricted epitopes contributes to their level of presentation.

The chloroplastic protein import machinery contains a Rieske‐type iron–sulfur cluster and a mononuclear iron‐binding protein

Transport of precursor proteins across the chloroplastic envelope membranes requires the interaction of protein translocons localized in both the outer and inner envelope membranes. Analysis by blue native gel electrophoresis revealed that the translocon of the inner envelope membranes consisted of at least six proteins with molecular weights of 36, 45, 52, 60, 100 and 110 kDa, respectively. Tic110 and ClpC, identified as components of the protein import apparatus of the inner envelope membrane, were prominent constituents of this complex. The amino acid sequence of the 52 kDa protein, deduced from the cDNA, contains a predicted Rieske‐type iron–sulfur cluster and a mononuclear iron‐binding site. Diethylpyrocarbonate, a Rieske‐type protein‐modifying reagent, inhibits the translocation of precursor protein across the inner envelope membrane, whereas binding of the precursor to the outer envelope membrane is still possible. In another independent experimental approach, the 52 kDa protein could be co‐purified with a trapped precursor protein in association with the chloroplast protein translocon subunits Toc86, Toc75, Toc34 and Tic110. Together, these results strongly suggest that the 52 kDa protein, named Tic55 due to its calculated molecular weight, is a member of the chloroplastic inner envelope protein translocon.

Purification, cDNA Cloning, and Expression of GDP-l-Fuc:Asn-linked GlcNAc α1,3-Fucosyltransferase from Mung Beans

Substitution of the asparagine-linked GlcNAc by α1,3-linked fucose is a widespread feature of plant as well as of insect glycoproteins, which renders the N-glycan immunogenic. We have purified from mung bean seedlings the GDP-l-Fuc:Asn-linked GlcNAc α1,3-fucosyltransferase (core α1,3-fucosyltransferase) that is responsible for the synthesis of this linkage. The major isoform had an apparent mass of 54 kDa and isoelectric points ranging from 6.8 to 8.2. From that protein, four tryptic peptides were isolated and sequenced. Based on an approach involving reverse transcriptase-polymerase chain reaction with degenerate primers and rapid amplification of cDNA ends, core α1,3-fucosyltransferase cDNA was cloned from mung bean mRNA. The 2200-base pair cDNA contained an open reading frame of 1530 base pairs that encoded a 510-amino acid protein with a predicted molecular mass of 56.8 kDa. Analysis of cDNA derived from genomic DNA revealed the presence of three introns within the open reading frame. Remarkably, from the four exons, only exon II exhibited significant homology to animal and bacterial α1,3/4-fucosyltransferases which, though, are responsible for the biosynthesis of Lewis determinants. The recombinant fucosyltransferase was expressed in Sf21 insect cells using a baculovirus vector. The enzyme acted on glycopeptides having the glycan structures GlcNAcβ1–2Manα1–3(GlcNAcβ1–2Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAcβ1-Asn, GlcNAcβ1–2Manα1–3(GlcNAcβ1–2Manα1–6)Manβ1–4GlcNAcβ1–4(Fucα1–6)GlcNAcβ1-Asn, and GlcNAcβ1–2Manα1–3[Manα1–3(Manα1–6)Manα1–6]Manβ1–4GlcNAcβ1–4GlcNAcβ1-Asn but not on, e.g. N-acetyllactosamine. The structure of the core α1,3-fucosylated product was verified by high performance liquid chromatography of the pyridylaminated glycan and by its insensitivity to N-glycosidase F as revealed by matrix-assisted laser desorption/ionization time of flight mass spectrometry.

Bell Peppers (Capsicum annuum) Express Allergens (Profilin, Pathogenesis-Related Protein P23 and Bet v 1) Depending on the Horticultural Strain

Background: Little is known about the role of bell peppers in food allergy. We collected sera from 11 patients with food allergy to bell peppers to analyze bell pepper extracts for allergen composition. Methods: Proteins of mature fruits of eight horticultural strains of bell peppers were extracted and tested with patients’ sera for IgE binding and with monoclonal and polyclonal antibodies in immunoblot. Results: Profilin was detected in bell pepper extracts by an anti-celery profilin antibody. It showed high IgE binding activity in all extracts, which could be inhibited by recombinant birch pollen profilin. Anti-birch pollen monoclonal antibody BIP3, directed against birch pollen proteins between 30 and 69 kD, bound to bell pepper antigens of comparable molecular weights. A homologue of the major birch pollen allergen Bet v 1 was detected in four of eight horticultural strains of bell peppers, and was shown to bind IgE in 1 of the 11 patients. A 23-kD allergen of bell peppers was shown to correspond to the 23-kD major paprika allergen by IgE absorption experiments. Its N-terminal sequence showed 100% identity to P23 from tomatoes. Conclusion: The appearance of profilin in all and Bet v 1 in 50% of the tested horticultural strains indicates that bell peppers have to be considered potentially dangerous for Bet v 1- and profilin-sensitized patients. Moreover, in 4 of 8 horticultural strains of bell peppers a homologue of the osmotin-like protein P23 from tomatoes is responsible for substantial IgE binding. Contact with Bet v 1 and P23 homologues in bell peppers can therefore be minimized by avoidance of the respective horticultural strains.

Allergens in pepper and paprika. Immunologic investigation of the celery-birch-mugwort-spice syndrome.

Mugwort and birch pollen allergy are frequently associated with IgEmediated hypersensitivity to celery and spices. We analyzed 22 sera from patients with the mugwort‐birch‐celery‐spice syndrome for IgE binding to the spices pepper and paprika by immunoblotting. Immunoblot results revealed two major allergens of 28 and 60 kDa in pepper and a 23‐kDa allergen together with allergens of higher molecular weight in paprika. In immunoblot‐inhibition studies, crude mugwort, birch pollen, and celery extracts significantly reduced the IgE binding to pepper and paprika allergens. However, no inhibition was achieved with rBet v 1 and rBet v 2, suggesting that no homologs of these birch proteins act as allergens in pepper or paprika extracts, N‐terminal sequence analysis of the 14‐ and 28‐kDa pepper and 23‐kDa paprika allergens revealed no homology to known allergens. The 28‐kDa pepper allergen showed homology to a wheat germin protein, and the 23‐kDa paprika allergen was identified as a homolog of a osmotin‐like or pathogenesis‐related protein in tomato. Therefore, we conclude that the IgE cross‐reactivity in the mugwort‐birch‐celery‐spice syndrome to the spices pepper and paprika is not caused by homologs of Bet v 1 and profilin, N‐terminal amino acid sequence analysis of the main allergens in pepper and paprika indicate a relation to frequently occurring plant proteins.

Members of the glutathione S-transferase gene family are antigens in autoimmune hepatitis.

BACKGROUND & AIMS Autoimmmune hepatitis (AIH), a chronic liver disorder, can be classified into two subtypes on the basis of the specificities of circulating autoantibodies. Type I AIH is defined by antibodies to nuclear and/or smooth muscle antigens (SMA), and type II is characterized by antibodies to cytochrome P450IID6. There is an additional type of AIH characterized by antibodies to a cytosolic soluble liver antigen (SLA), which can occur alone or in combination with antinuclear antibodies and SMA. The aim of this study was to identify the reactive antigen in SLA, a heterogenous cytosolic fraction consisting of at least 100 extremely soluble proteins. METHODS Sera from 31 patients with AIH reacting with SLA and from 30 disease controls were tested. The immunoreactive antigens were determined using immunoprecipitation and immunoblotting after one- and two-dimensional polyacrylamide gel electrophoresis. The antigens were identified by microsequencing of the corresponding protein spots. RESULTS Twenty-five of 31 anti-SLA-positive sera (80, 7%) reacted with a set of proteins ranging from 25 to 27 kilodaltons that were identified as three subunits of glutathione S-transferases: Ya, Yb1, and Yc. CONCLUSIONS Glutathione S-transferase subunit proteins represent the major autoantigen in anti-SLA-positive AIH. This new finding permits the establishment of standardized immunoassays for routine diagnosis.

Similarity between nuclear matrix proteins of various cells revealed by an improved isolation method

Comparative analysis of nuclear matrix proteins by two‐dimensional electrophoresis may be greatly impaired by copurifying cytoskeletal proteins. The present data show that the bulk of adhering cytofilaments may mechanically be removed by shearing of nuclei pretreated with vanadyl ribonucleoside complexes. Potential mechanisms of action not based on ribonuclease inhibition are discussed. To individually preserve the integrity of nuclear structures, we developed protocols for the preparation of nuclear matrices from three categories of cells, namely leukocytes, cultured cells, and tissue cells. As exemplified with material from human lymphocytes, cultured amniotic cells, and liver tissue cells, the resulting patterns of nuclear matrix proteins appeared quite similar. Approximately 300 spots were shared among the cell types. Forty‐nine of these were identified, 21 comprising heterogeneous nuclear ribonucleoproteins. Heterogeneous nuclear ribonucleoproteins L and nuclear lamin B2 isoforms were identified by amino acid sequencing and mass spectrometry. However, individually expressed proteins, such as the proliferating cell nuclear antigen, also pertained following application of the protocols. Thus, enhanced resolution and comparability of proteins improve systematic analyses of nuclear matrix proteins from various cellular sources. J. Cell. Biochem. 71:363–374, 1998.

Differential nuclear localization and nuclear matrix association of the splicing factors PSF and PTB

A monoclonal antibody raised against nuclear matrix proteins detected a protein of basic pI in human nuclear matrix protein samples of various cellular origin. The ubiquitously occurring (common) nuclear matrix protein was identified as splicing factor PSF (PTB associated splicing factor). The interaction between the splicing factors PSF and PTB/hnRNP I was confirmed by co‐immunoprecipitation from nuclear salt extracts. However, the nuclear localization of PSF and PTB and their distribution in subnuclear fractions differed markedly. Isolated nuclear matrices contained the bulk of PSF, but only minor amounts of PTB. In confocal microscopy both proteins appeared in speckles, the majority of which did not co‐localize. Removing a large fraction of the soluble PTB structures by salt extraction revealed some colocalization of the more stable PTB fraction with PSF. These PTB/PSF complexes as well as the observed PSF‐PTB interaction may reflect the previously reported presence of PTB and PSF in spliceosomal complexes during RNA processing. The present data, however, point to different cellular distribution and nuclear matrix association of the majority of PSF and PTB. J. Cell. Biochem. 76:559–566, 2000.

Δ5-3β-Hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. – a multifunctional enzyme in steroid metabolism?

Abstract.Δ5-3β-Ηydroxysteroid dehydrogenase (Δ5-3β-HSD; EC 1.1.1.145), an enzyme converting pregn-5-ene-3β-ol-20-one (pregnenolone) to pregn-5-ene-3,20-dione (isoprogesterone), was isolated from the soluble fraction of suspension-cultured cells of Digitalis lanata L. strain VIII. Starting with acetone dry powder the enzyme was purified in three steps using column chromatography on Fractogel-TSK DEAE, hydroxyapatite and Sephacryl G-200. Fractions with highest Δ5-3β-HSD activity were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After in-situ digestion the resulting bands were sequenced N-terminally. The 29-kDa band yielded three fragments with high sequence homology to members of the superfamily of short-chain dehydrogenases/reductases. High similarity was found to microbial hydroxysteroid dehydrogenases. The band may therefore represent the Δ5-3β-HSD. The purified enzyme was characterized with respect to kinetic parameters, substrate specificity and localization. The function of the enzyme in steroid metabolism is discussed.