Alfred Engel

Human Monoclonal Antibodies Isolated from Type I Diabetes Patients Define Multiple Epitopes in the Protein Tyrosine Phosphatase-Like IA-2 Antigen

Protein tyrosine phosphatase-like IA-2 autoantigen is one of the major targets of humoral autoimmunity in patients with insulin-dependant diabetes mellitus (IDDM). In an effort to define the epitopes recognized by autoantibodies against IA-2, we generated five human mAbs (hAbs) from peripheral B lymphocytes isolated from patients most of whom had been recently diagnosed for IDDM. Determination and fine mapping of the critical regions for autoantibody binding was performed by RIA using mutant and chimeric constructs of IA-2- and IA-2β-regions. Four of the five IgG autoantibodies recognized distinct epitopes within the protein tyrosine phosphatase (PTP)-like domain of IA-2. The minimal region required for binding by three of the PTP-like domain-specific hAbs could be located to aa 777–979. Two of these hAbs cross-reacted with the related IA-2β PTP-like domain (IA-2β aa 741-1033). A further PTP-like domain specific hAb required the entire PTP-like domain (aa 687–979) for binding, but critical amino acids clustered in the N-terminal region 687–777. An additional epitope could be localized within the juxtamembrane domain (aa 603–779). In competition experiments, the epitope recognized by one of the hAbs was shown to be targeted by 10 of 14 anti-IA-2-positive sera. Nucleotide sequence analysis of this hAb revealed that it used a VH germline gene (DP-71) preferably expressed in autoantibodies associated with IDDM. The presence of somatic mutations in both heavy and light chain genes and the high affinity or this Ab suggest that the immune response to IA-2 is Ag driven.

Chaperone-Aided in Vitro Renaturation of an Engineered E1 Envelope Protein for Detection of Anti-Rubella Virus IgG Antibodies

The envelope glycoproteins of Rubella virus, E1 and E2, mediate cell tropism, and E1 in particular plays a pivotal role in the fusion of the virus with the endosomal membrane. Both are the prime targets of the humoral immune response. Recombinant variants of the E1 ectodomain as well as E1 antigen preparations from virus lysates are commonly used to detect anti-Rubella immunoglobulins in human sera. Hitherto, recombinant E1 for diagnostic applications has been produced chiefly in eukaryotic expression systems. Here, we report the high-yield overproduction of an engineered E1 ectodomain in the Escherichia coli cytosol and its simple and convenient renaturation into a highly soluble and immunoreactive conformation. C-Terminal fusion to one or two units of the E. coli chaperone SlyD enhances expression, facilitates in vitro refolding, and improves the overall solubility of Rubella E1. As part of this fusion protein, the E1 ectodomain fragment of residues 201-432 adopts an immunoreactive fold, providing a promising tool for the sensitive and specific detection of anti-E1 IgG in Rubella serology. Two disulfide bonds in the membrane-adjacent part of the E1 ectodomain are sufficient to generate conformations with a high and specific antigenicity. The covalently attached chaperone modules do not impair antibody recognition and binding of Rubella E1 when assessed in a heterogeneous immunoassay. SlyD and related folding helpers are apparently generic tools for the expression and refolding of otherwise unavailable proteins of diagnostic or medical importance.

Combining EL4-B5-based B-cell stimulation and phage display technology for the successful isolation of human anti-Scl-70 autoantibody fragments

Scl-70 is the major antigen recognised by autoantibodies in the sera of patients with systemic sclerosis (SSc). The autoantibodies that specifically react with Scl-70 are highly characteristic of the disease and represent valuable markers for the diagnosis of SSc. We describe a novel strategy for cloning autoantibody fragments starting with a small blood sample from an SSc patient. B cells isolated from the collected peripheral blood mononuclear cells (PBMCs) were cultured in vitro using the EL4-B5 system. Anti-Scl-70 IgG-producing cells were pooled for RNA preparation followed by the generation of phagemid libraries of approximately 10(7) independent single-chain Fvs (scFvs). The screening of these libraries by phage display allowed us to isolate four anti-Scl-70 scFvs following three rounds of biopanning. About 10 times more starting blood material was needed to generate scFv libraries of similar size from PBMCs of an SSc patient and only two anti-Scl-70 scFvs were isolated after three rounds of phage selection. Together, this work shows that functional autoantibody fragments can be advantageously cloned after in vitro expansion of B cells. The isolated anti-Scl-70 autoantibody fragments represent useful tools for calibrating SSc diagnostic assays.

Rec. ST6Gal-I variants to control enzymatic activity in processes of in vitro glycoengineering

Background Glycosylation is an important posttranslational modification of proteins influencing protein folding, stability and regulation of the biological activity. The sialyl mojety (sialic acid, 5-N-acetylneuramic acid) is usually exposed at the terminal position of N-glycosylation and therefore, a major contributor to biological recognition and ligand function, e.g. IgG featuring terminal sialic acids were shown to induce less inflammatory response and increased serum half-life. The biosynthesis of sialyl conjugates is controlled by a set of sugar-active enzymes including sialyltransferases which are classified as ST3, ST6 and ST8 based on the hydroxyl position of the glycosyl acceptor the Neu5Ac is transferred to [1]. The ST6 family consists of 2 subfamilies, ST6Gal and ST6GalNAc. ST6Gal catalyzes the transfer of Neu5Ac residues to the hydroxyl group in C6 of a terminal galactose residue of type 2 disaccharide (Galb1-4GlcNAc). To our knowledge, the access to recombinant ST6GalI for therapeutic applications is still limited due to low expression and/or poor activity in various hosts (Pichia pastoris, Spodoptera frugiperda and E. coli). The present study describes the high-yield expression of two variants of human beta-galactoside alpha-2,6 sialyltransferase 1 (ST6Gal-I, EC 2.4.99.1; data base entry P15907) by transient gene expression in HEK293 cells with yields >100 mg/L featuring distinct mono(G2 +1SA) as well as bi(G2+2SA) sialylation activity. Materials and methods Two N-terminally truncated fragments of human ST6Gal-I (delta89, residues 89-406, and delta108, residues 109-406) were designed for transient gene expression (TGE): Instead of the natural leader sequence and N-terminal residues, both ST6Gal-I coding regions harbor the Erythropoietin (EPO) signal sequence in order to ensure correct processing of the polypeptides by the secretion machinery. Following cloning into pM1MT, expression of the ST6Gal-I coding sequences is under control of a hCMV promoter followed by an intron A. Sialyltransferase assays: 1. Asialofetuin was used as acceptor and CMP-9F-NANA as donor substrate. Enzymatic activity was determined by measuring the transfer of 9F-NANA to asialofetuin. 2. Recombinant humanized IgG1 and IgG4 monoclonal antibodies (mabs), characterized as G2+0SA, as well as desialylated EPO were used as targets in sialylation experiments (30 μg enzyme/300 μg target protein). Both enzyme variants of ST6Gal-I (delta89 and delta108) were used under identical reaction conditions and the sialylation status was analyzed by mass spectrometry.

Two N-terminally truncated variants of human β-galactoside α2,6 sialyltransferase I with distinct properties for in vitro protein glycosylation.

Sialic acid groups of protein N-glycans are important determinants of biological activity. Exposed at the end of the glycan chain, they are potential targets for glycan remodeling. Sialyltransferases (STs; EC 2.4.99) are the enzymes that catalyze the sialic acid transfer from a CMP-activated donor on to a carbohydrate acceptor in vivo. Recombinant expression of the full-length human β-galactoside α2,6 sialyltransferase I (ST6Gal-I) was hampered and therefore variants with truncated N-termini were investigated. We report on the distinct properties of two N-terminally truncated versions of ST6Gal-I, namely Δ89ST6Gal-I and Δ108ST6Gal-I, which were successfully expressed in human embryonic kidney cells. The different properties of these enzymes result most probably from the loss of interactions from helix α1 in the Δ108ST6Gal-I variant, which plays a role in acceptor substrate binding. The Km for N-acetyl-d-lactosamine was 10-fold increased for Δ108ST6Gal-I (84 mM) as compared to Δ89ST6Gal-I (8.3 mM). The two enzyme variants constitute a suitable tool box for the terminal modification of N-glycans. While the enzyme Δ89ST6Gal-I exhibited both ST (di-sialylation) and sialidase activity on a monoclonal antibody, the enzyme Δ108ST6Gal-I showed only ST activity with specificity for mono-sialylation.

RNA Silencing Suppressors Boost the Production of Recombinant Proteins and Viruses

RNA-mediated gene silencing or RNA silencing is a gene regulation mechanism in eukaryotes involved in RNA-mediated sequence-specific RNA degradation in the cytoplasm followed by chromatin remodelling in the nucleus that plays a crucial role in differentiation and developmental processes. Besides, the RNA silencing machinery serves as an innate defense response against viruses and transposons and is responsible for down regulating transgene expression. Therefore, the induction of RNA silencing in producer cells limits the production of recombinant proteins and of (recombinant) virus particles for use as vaccines or in gene therapies.