Ludwig Mauch

Association between Antibodies to the MR 67,000 Isoform of Glutamate Decarboxylase (GAD) and Type 1 (Insulin-Dependent) Diabetes Mellitus with Coexisting Autoimmune Polyendocrine Syndrome Type II

By using an immunoprecipitation assay, we analysed reactivity of autoantibodies to human recombinant GAD65 and GAD67 in sera from patients with autoimmune polyendocrine syndrome Type II (APS II) with and without Type 1 (insulin-dependent) diabetes mellitus (IDDM) compared to patients with organ-specific autoimmunity. Overall antibodies to GAD65 were correlated with IDDM in all study groups, whereas GAD67 antibodies were associated with IDDM when APS II coexists. Antibodies to GAD65 and GAD67 were detected in 13 (44.8%) and 7 (24.1%) out of 29 APS II patients with IDDM, but in only 4 (13.8%) and 2 (6.9%) out of 29 APS II patients without IDDM, respectively (p < 0.05). In short-standing IDDM (< 1 year), antibodies to GAD67 were significantly more frequent in patients with APS II (5 of 9 [55.6%] subjects) compared to matched diabetic patients without coexisting polyendocrinopathy (1 of 18 [5.6%] subjects) (p < 0.02). The levels of GAD65 (142 +/- 90 AU) and GAD67 antibodies (178 +/- 95 AU) were significantly higher in patients with polyglandular disease than in patients with isolated IDDM (91 +/- 85 AU and 93 +/- 57 AU) (p < 0.02). Interestingly, all 11 GAD67 antibody positive subjects also had GAD65 antibodies (p < 0.0001), and in 10 of 11 anti-GAD67 positive sera the GAD67 antibodies could be blocked by either GAD67 or GAD65, suggesting the presence of cross-reactive autoantibodies. No correlation was observed between GAD antibodies and age, sex or any particular associated autoimmune disease, besides IDDM.(ABSTRACT TRUNCATED AT 250 WORDS)

Gain of structure and IgE epitopes by eukaryotic expression of the major Timothy grass pollen allergen, Phl p 1

Approximately 400 million allergic patients are sensitized against group 1 grass pollen allergens, a family of highly cross‐reactive allergens present in all grass species. We report the eukaryotic expression of the group 1 allergen from Timothy grass, Phl p 1, in baculovirus‐infected insect cells. Domain elucidation by limited proteolysis and mass spectrometry of the purified recombinant glycoprotein indicates that the C‐terminal 40% of Phl p 1, a major IgE‐reactive segment, represents a stable domain. This domain also exhibits a significant sequence identity of 43% with the family of immunoglobulin domain‐like group 2/3 grass pollen allergens. Circular dichroism analysis demonstrates that insect cell‐expressed rPhl p 1 is a folded species with significant secondary structure. This material is well behaved and is adequate for the growth of crystals that diffract to 2.9 Å resolution. The importance of conformational epitopes for IgE recognition of Phl p 1 is demonstrated by the superior IgE recognition of insect‐cell expressed Phl p 1 compared to Escherichia coli‐expressed Phl p 1. Moreover, insect cell‐expressed Phl p 1 induces potent histamine release and leads to strong up‐regulation of CD203c in basophils from grass pollen allergic patients. Deglycosylated Phl p 1 frequently exhibits higher IgE binding capacity than the recombinant glycoprotein suggesting that rather the intact protein structure than carbohydrate moieties themselves are important for IgE recognition of Phl p 1. This study emphasizes the important contribution of conformational epitopes for the IgE recognition of respiratory allergens and provides a paradigmatic tool for the structural analysis of the IgE allergen interaction.

Site-directed mutagenesis of the FAD-binding histidine of 6-hydroxy-D-nicotine oxidase: Consequences on flavinylation and enzyme activity

6‐Hydroxy‐D‐nicotine oxidase; Site‐directed mutagenesis; Covalant flavinylation; Flavoenzyme; Covalent modification; Protein modification

Recombinant human preproinsulin expression, purification and reaction with insulin autoantibodies in sera from patients with insulin-dependent diabetes mellitus

A novel prokaryotic expression vector pGEX-6T was designed for high-level expression of recombinant fusion protein with a histidine-hexapeptide and glutathione-S-transferase at its N-terminus and the recombinant human preproinsulin at its C-terminus. Efficiency of expression was investigated in the Escherichia coli strain CAG456. The synthesized protein was sequestered in an insoluble form in inclusion bodies and was purified to homogeneity by one-step affinity chromatography based on the specific complex formation of the histidine-hexapeptide and a chelating matrix which was charged with Ni2+ ions. The antigenic nature of the purified recombinant preproinsulin fusion protein was evaluated by ELISA screening for insulin autoantibodies in selected sera from patients with recent-onset type 1 (insulin-dependent) diabetes mellitus classified by the existence of additional autoantibodies reactive against glutamic acid decarboxylase. 14% of the tested sera (n = 43) contained insulin autoantibodies which strongly recognized the recombinant human preproinsulin. Comparable measurements with both recombinant human preproinsulin and mature insulin suggested that the observed autoantigenicity of preproinsulin was mediated by the C-peptide or/and signal peptide.

Functional analysis of the 5′ regulatory region and the UUG translation initiation codon of the Arthrobacter oxidans 6-hydroxy-d-nicotine oxidase gene

SummaryA functional analysis of the Arthrobacter oxidans 6-hydroxy-d-nicotine oxidase (6-HDNO) gene promoter (−35 region TTGACA and −10 region TATCAAT) and the UUG translation start codon was performed using site-directed mutagenesis. Deletion of the C residue from the −10 promoter region or mutations introduced upstream of the −10 region resulted in an increased 6-HDNO expression in Escherichia coli cells in vivo and in both E. coli and A. oxidans coupled transcription-translation systems in vitro. From the identical behaviour of 6-HDNO promoter mutants in the heterologous and homologous systems, it is concluded that A. oxidans harbours an RNA polymerase functionally homologous to the E. coli σ70 and Bacillus subtilis σ43 polymerases. Replacement of the TTG codon (UUG translation initiation codon) with ATG led to a 3.7-fold increase in 6-HDNO expression in E. coli. This effect was less pronounced at higher promoter strengths, from 3.7 in the case of the 6-HDNO wild-type promoter, to 2.5 in the case of the consensus −10 region and to 1.7 in the case of the tac promoter. A double point mutation introduced close to the ribosome binding site resulted in almost the same increase in 6-HDNO expression (3.1-fold) as the TTG-to-ATG exchange. The failure of cAMP to stimulate 6-HDNO expression in the A. oxidans system indicated that expression of this gene in stationary phase cells is not regulated by cAMP-catabolite repressore protein-mediated mechanism of catabolite repression. ppGpp, a nucleotide involved in the initiation of morphological and physiological differentiation in stationary cells, did not significantly affect 6-HDNO expression in the in vitro transcription-translation system.

Interaction of the regulatory protein NicR1 with the promoter region of the pAO1‐encoded 6‐hydroxy‐D‐nicotine oxidase gene of Arthrobacter oxidans

The d,l‐nicotine catabolism of the Gram‐positive soil bacterium Arthrobacter oxidans is linked to the presence within the cells of the 160 kb catabolic plasmid pAO1. pAO1‐cured cells lost the catabolic enzymes and reintroduction of pAO1 by electroporation into cured cells reestablished the nic+ phenotype. DNA band shift assays with extracts from cured and pAO1+ cells suggested that pAO1 encodes the regulatory protein NicR1. Footprint analysis revealed that two homologous palindromes (IR1 and IR2), present in the 5′‐regulatory region of the 6‐HDNO gene, were protected from DNase I digestion. Binding of NicR1 to the palindromes is symmetrical, co‐operative, and stronger to IR1 containing the 6‐HDNO gene promoter than to IR2. Site‐directed mutagenesis revealed that steric constraints and sequence requirements for NicR1 ‐binding are located exclusively in the palindromic sequences. Deletions and insertions in the interpalindromic region and in the 6‐HDNO promoter ‐10 sequence had no effect on the binding characteristics of NicR1 to the 6‐HDNO regulatory region. Acting as a repressor, NicR1 prevents binding of the E. coli RNA‐polymerase to the consensus σ70 promoter in vitro. However, the Interaction of NicR1 with the 6‐HDNO promoter region in extracts of nicotine‐induced cells from various growth stages did not differ from that observed with extracts of nicotine‐uninduced cells.

Growth stage-dependent expression of 6-hydroxy-d-nicotine oxidase of the nicotine regulon of Arthrobacter oxidans

In the gram-positive soil bacterium Arthrobacter oxidansd,l-nicotine induces specific flavoenzymes involved in the catabolism of the alkaloid. The delayed appearance of 6-HdNO activity as compared to that of the other enzymes of the nicotine regulon was investigated. Immunological methods and specific labeling of the 6-HdNO polypeptide with [14C]riboflavin showed that enzyme activity, occurrence of 6-HdNO antigen and covalent flavinylation of the 6-HdNO polypeptide all coincide with the stationary phase of cell growth. Northern blots, hybridized with an intragenic 6-HdNO probe, revealed a 6-HdNO-specific mRNA species of unique size only in stationary phase cells. Our results suggest that 6-HdNO is expressed from an independent transcription unit in a growth stage-dependent manner.