Gabriele Petersen

Monoclonal Antibodies against the Adeno-Associated Virus Type 2 (AAV-2) Capsid: Epitope Mapping and Identification of Capsid Domains Involved in AAV-2–Cell Interaction and Neutralization of AAV-2 Infection

ABSTRACT The previously characterized monoclonal antibodies (MAbs) A1, A69, B1, and A20 are directed against assembled or nonassembled adeno-associated virus type 2 (AAV-2) capsid proteins (A. Wistuba, A. Kern, S. Weger, D. Grimm, and J. A. Kleinschmidt, J. Virol. 71:1341–1352, 1997). Here we describe the linear epitopes of A1, A69, and B1 which reside in VP1, VP2, and VP3, respectively, using gene fragment phage display library, peptide scan, and peptide competition experiments. In addition, MAbs A20, C24-B, C37-B, and D3 directed against conformational epitopes on AAV-2 capsids were characterized. Epitope sequences on the capsid surface were identified by enzyme-linked immunoabsorbent assay using AAV-2 mutants and AAV serotypes, peptide scan, and peptide competition experiments. A20 neutralizes infection following receptor attachment by binding an epitope formed during AAV-2 capsid assembly. The newly isolated antibodies C24-B and C37-B inhibit AAV-2 binding to cells, probably by recognizing a loop region involved in binding of AAV-2 to the cellular receptor. In contrast, binding of D3 to a loop near the predicted threefold spike does not neutralize AAV-2 infection. The identified antigenic regions on the AAV-2 capsid surface are discussed with respect to their possible roles in different steps of the viral life cycle.

MAPPING OF LINEAR EPITOPES RECOGNIZED BY MONOCLONAL ANTIBODIES WITH GENE-FRAGMENT PHAGE DISPLAY LIBRARIES

Epitope mapping with mono- or polyclonal antibodies has so far been done either by dissecting the antigens into overlapping polypeptides in the form of recombinantly expressed fusion proteins, or by synthesizing overlapping short peptides, or by a combination of both methods. Here, we report an alternative method which involves the generation of random gene fragments of approximately 50–200 by in length and cloning these into the 5′ terminus of the protein III gene of fd phages. Selection for phages that bind a given monoclonal antibody and sequencing the DNA inserts of immunopositive phages yields derived amino acid sequences containing the desired epitope. A monoclonal antibody (mAb 215) directed against the largest subunit of Drosophila RNA polymerase II (RPB215) was used to map the corresponding epitope in a fUSE5 phage display library made of random DNA fragments from plasmid DNA containing the entire gene. After a single round of panning with this phage library, bacterial colonies were obtained which produced fd phages displaying the mAb 215 epitope. Sequencing of single-stranded phage DNA from a number of positive colonies (recognized by the antibody on colony immunoblots) resulted in overlapping sequences all containing the 15mer epitope determined by mapping with synthetic peptides. Similarly, we have localized the epitopes recognized by a mouse monoclonal antibody directed against the human p53 protein, and by a mouse monoclonal antibody directed against the human cytokeratin 19 protein. Identification of positive colonies after the panning procedure depends on the detection system used (colony immunoblot or ELISA) and there appear to be some restrictions to the use of linker-encoded amino acids for optimal presentation of epitopes. A comparison with epitope mapping by synthetic peptides shows that the phage display method allows one to map linear epitopes down to a size only slightly larger than the true epitope. In general, our phage display method is faster, easier, and cheaper than the construction of overlapping fusion proteins or the use of synthetic peptides, especially in cases where the antigen is a large polypeptide such as the 215 kDa subunit of eukaryotic RNA polymerase II.

Structure and Function of the Hetero-oligomeric Cysteine Synthase Complex in Plants

Cysteine synthesis in bacteria and plants is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol)-lyase (OAS-TL), which form the hetero-oligomeric cysteine synthase complex (CSC). In plants, but not in bacteria, the CSC is assumed to control cellular sulfur homeostasis by reversible association of the subunits. Application of size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry revealed a hexameric structure of mitochondrial SAT from Arabidopsis thaliana (AtSATm) and a 2:1 ratio of the OAS-TL dimer to the SAT hexamer in the CSC. Comparable results were obtained for the composition of the cytosolic SAT from A. thaliana (AtSATc) and the cytosolic SAT from Glycine max (Glyma16g03080, GmSATc) and their corresponding CSCs. The hexameric SAT structure is also supported by the calculated binding energies between SAT trimers. The interaction sites of dimers of AtSATm trimers are identified using peptide arrays. A negative Gibbs free energy (ΔG = −33 kcal mol−1) explains the spontaneous formation of the AtCSCs, whereas the measured SAT:OAS-TL affinity (KD = 30 nm) is 10 times weaker than that of bacterial CSCs. Free SAT from bacteria is >100-fold more sensitive to feedback inhibition by cysteine than AtSATm/c. The sensitivity of plant SATs to cysteine is further decreased by CSC formation, whereas the feedback inhibition of bacterial SAT by cysteine is not affected by CSC formation. The data demonstrate highly similar quaternary structures of the CSCs from bacteria and plants but emphasize differences with respect to the affinity of CSC formation (KD) and the regulation of cysteine sensitivity of SAT within the CSC.

The gene upstream of DmRP128 codes for a novel GTP-binding protein of Drosophila melanogaster

Upstream of the gene coding for the second-largest subunit of RNA polymerase III (DmRP128) we have found another gene (128up), which is transcribed in the same direction as the RNA polymerase gene. The intergenic distance between the 3′ end of 128up mRNA and the 5′ end of DmRP128 mRNA is only about 100 bp. Transcripts of 128up are present at a much higher level than DmRP128 RNA in Drosophila Schneider 2 cells, embryos, and adult flies. Two transcription start points, seven nucleotides apart, are found for 128up compared to multiple scattered starts for DmRP128. Sequence analysis of 128up cDNA reveals that the gene codes for a 41 kDa protein with homology to GTP-binding proteins and matching four of the structural sequence motifs characteristic of the superfamily of GTPases. Bacterially expressed 128up protein fused to maltose-binding protein specifically binds GTP. Sequences closely related to the 128up protein are found in species as distant as Halobacterium, yeast or mouse; the murine protein is 80% identical to 128up. This evolutionary conservation is indicative of an important, but as yet unknown, physiological role. In accordance with the sequence conservation, antibodies against 128up specifically cross-react with mouse 3T3 cells and human Hep2 cells where the subcellular localization of the protein is predominantly perinuclear. We propose that 128up is a member of a novel class of GTP-binding proteins.

Identification of the genes coding for the second-largest subunits of RNA polymerases I and III of Drosophila melanogaster

SummaryWe have isolated cDNA and genomic clones of Drosophila melanogaster by cross-hybridization with a 658 by fragment of the yeast gene coding for the second-largest subunit of RNA polymerase III (RET1). Determination of the sequence by comparison of genomic and cDNA regions reveals an ORF of 3405 nucleotides which is interrupted in the genomic sequence by an intron of 48 bp. The deduced polypeptide consists of 1135 amino acids with a calculated molecular weight of 128 kDa. The protein sequence shows the same conserved regions of homology as those observed for all the second-largest subunits of RNA polymerases cloned so far. The gene (DmRP128) obviously codes for a second-largest subunit of an RNA polymerase which is different from DmRP140 and DmRP135. We have purified three distinct RNA polymerase activites from D. melanogaster. By using specific RNA polymerase inhibitors in enzyme assays and by comparing their subunit composition we were able to distinguish between RNA polymerase I, II, and III. RNA polymerase preparations of D. melanogaster were blotted and the second-largest subunits were identified with antibodies raised against polypeptides expressed from DmRP128 and DmRP135. Anti-DmRP135 antibodies react strongly with the second-largest subunit of RNA polymerase I but do not react with the respective subunits of RNA polymerase II and III. The second-largest subunit of RNA polymerase III is only recognized by anti-DmRP128. Previously, we have claimed that DmRP135 codes for the second-largest subunit of RNA polymerase III. Based on the new biochemical data reported here we show that DmRP135 codes instead for the second-largest subunit of RNA polymerase I and that DmRP128 corresponds to the equivalent subunit of RNA polymerase III.

Primary structure and functional aspects of the gene coding for the second-largest subunit of RNA polymerase III of Drosophila

SummaryWe have cloned and sequenced the gene coding for the second-largest subunit of RNA polymerase III of Drosophila melanogaster (DmRP135). The gene, interrupted by two introns of 62 and 59 bp, respectively, codes for an mRNA of 3.6 kb. As for other housekeeping genes transcription initiates at several sites (between positions −98 and −76) none of which is preceded by a clear TATA sequence. The deduced polypeptide consists of 1129 amino acids with an aggregate molecular weight of 128 kDa. The protein sequence features the same regions of similarity as observed for the corresponding subunits of RNA polymerase II of Drosophila and yeast and the Escherichia coli β subunit. As in the second-largest subunit of RNA polymerase II there is a zinc-binding motif which is absent in the β subunit of E. coli. Antibodies directed against a fusion protein expressing 164 amino acids of the (DmRP135) polypeptide cross-react with the second-largest subunit of RNA polymerase III of yeast and generate a distinct banding pattern on Drosophila polytene chromosomes distinguishable from that obtained with anti-RNA polymerase II antibodies.

Analysis of the promoter region of the housekeeping gene DmRP140 by sequence comparison of Drosophila melanogaster and Drosophila virilis.

To analyze the transcriptional control regions of Drosophila melanogaster household genes, we have characterized the promoter of the gene coding for the second-largest subunit of RNA polymerase II (DmRP140). Analysis of cDNA revealed that the coding region of the protein extends beyond the originally assumed transcription start point (tsp) and deduced translation start codon [Falkenburg et al., J. Mol. Biol. 195 (1987) 929-937] and that the tsp determined previously corresponds to an intron/exon boundary of an additional intron. Upstream of the polII gene we found a transcription unit that is transcribed in the opposite direction. The initiating ATGs of the two genes are only 467 nucleotides (nt) apart. The untranslated region is extremely A + T-rich (88%) but none of the transcription units is preceded by a canonical TATA element. It does not feature any other known nt sequence motifs thought to be necessary for the basic transcriptional machinery; yet, this region functions as a bidirectional promoter: a central 309-bp fragment directs transcription of a reporter gene in transiently transfected Drosophila culture cells in both orientations. The gene coding for the second-largest subunit of RNA polymerase II of Drosophila virilis (DvRP140) was isolated and partially analyzed. The gene is located on the second chromosome at 22F/23A which corresponds to the position determined for D. melanogaster.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviruses using the cancer marker EphA2 as a receptor in vitro and in vivo by genetic ligand insertion into different capsid scaffolds

Adenoviral gene therapy and oncolysis would critically benefit from targeted cell entry by genetically modified capsids. This requires both the ablation of native adenovirus tropism and the identification of ligands that remain functional in virus context. Here, we establish cell type-specific entry of HAdV-5-based vectors by genetic ligand insertion into a chimeric fiber with shaft and knob domains of the short HAdV-41 fiber (Ad5T/41sSK). This fiber format was reported to ablate transduction in vitro and biodistribution to the liver in vivo. We show that the YSA peptide, binding to the pan-cancer marker EphA2, can be inserted into three positions of the chimeric fiber, resulting in strong transduction of EphA2-positive but not EphA2-negative cells of human melanoma biopsies and of tumor xenografts after intratumoral injection. Transduction was blocked by soluble YSA peptide and restored for EphA2-negative cells after recombinant EphA2 expression. The YSA peptide could also be inserted into three positions of a CAR binding-ablated HAdV-5 fiber enabling specific transduction; however, the Ad5T/41sSK format was superior in vivo. In conclusion, we establish an adenovirus capsid facilitating functional insertion of targeting peptides and a novel adenovirus using the tumor marker EphA2 as receptor with high potential for cancer gene therapy and viral oncolysis.

Characterization of Hap/BAG‐1 variants as RP1 binding proteins with antiapoptotic activity

The MAPRE protein family (EB1, RP1, EB2) represents a highly conserved group of proteins that localize preferentially to the plus end of microtubules, both in the nucleus and cytoplasm. In addition, MAPRE family members are characterized by their capability to bind to the C‐terminus of the adenomatous polyposis coli (APC) protein and tubulin in order to stabilize microtubules. Apart from the interaction with APC and tubulin, no other direct binding partners are known today. Because the RP1 gene product was identified in activated T cells, we set out to search for new interacting molecules in a yeast 2‐hybrid system. We isolated a cDNA variant encoding for the antiapoptotic Hap/BAG‐1 protein truncated by 34 amino acids at the C‐terminus. In the original Hap/BAG‐1 protein, the C‐terminal domain is responsible for binding to Bcl‐2 and Hsp/Hsc70, which is believed to be the reason for its antiapoptotic activity. Although this putative Hap/BAG‐1 variant protein showed no interaction with Bcl‐2 or Hsp/Hsc70, it was perfectly able to confer resistance to apoptosis. Subcellular distribution analysis revealed that the Hap/Bag‐1 variant protein localized homogenously to the cytoplasm and shuttles into the nucleus in response to stress, a process that could be blocked by RP1 protein overexpression.

Immobilized Peptides to Study Protein-Protein Interactions — Potential and Pitfalls

In recent years, immobilized peptides synthesized on activated cellulose membranes (SPOT synthesis, Frank 1992) have become an important tool in the study of protein-protein interactions and numerous other aspects of molecular recognition (reviewed in Reineke et al. 2001). A broad range of applications has already been described and the rising interest in proteomics is bound to rely on the enormous potential of the (automated) method. When adapted to high-throughput screening, SPOTs, i.e. peptide arrays, will become an invaluable tool in pharmacogenomics and drug discovery.