Aspartyl protease inhibitor pepstatin binds to the presenilins of Alzheimer's disease.

Mutations in the presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease by altering gamma-secretase cleavage of the amyloid precursor protein, the last step in the generation of Abeta peptide. Ablation of presenilin (PS) genes, or mutation of two critical aspartates, abolishes gamma-secretase cleavage, suggesting that PS may be the gamma-secretases. Independently, inhibition experiments indicate that gamma-secretase is an aspartyl protease. To characterize the putative gamma-secretase activity associated with presenilins, lysates from human neuroblastoma SH-SY5Y and human brain homogenates were incubated with biotin derivatives of pepstatin, followed by immunoprecipitation of PS and associated proteins, and biotin detection by Western blotting. Precipitation with PS1 antibodies, directed to either N-terminal or loop regions, yielded the same 43 kDa band, of apparent molecular mass consistent with that of full-length PS1, although it may represent an aspartyl protease complexed with PS1. Incubation of cell lysates with pepstatin-biotin, followed by streptavidin precipitation and PS1 Western blotting, revealed PS1 fragments and full-length protein, indicating that pepstatin-biotin bound to both cleaved and uncleaved PS1. Binding could be competed by gamma-secretase inhibitor L-685,458 and could not be achieved with a PS1 mutant lacking the two transmembrane aspartates. Pepstatin-biotin was also shown to bind to PS2. PS1 was specifically absorbed to pepstatin-agarose, with an optimal pH of 6. Binding of pepstatin-biotin to PS1 from lymphocytes of a heterozygous carrier of pathologic exon 9 deletion was markedly decreased as compared to control lymphocytes, suggesting that this PS1 mutation altered the pepstatin binding site.

Why certain antibodies cross-react with HLA-A and HLA-G: epitope mapping of two common MHC class I reagents.

Antigen presentation at the maternal-fetal interface has been characterized by a reported lack of classical MHC class I products and the presence of a tissue-restricted, non-classical class I product with limited polymorphism, HLA-G. The lack of HLA-A, -B, and -C products at this interface would allow escape from T-cell mediated attack, while the presence of HLA-G may enable evasion of NK cell-mediated destruction. We provide evidence that in addition to HLA-G, the classical class I product HLA-C is also present in trophoblast. Specifically, cDNA from the trophoblast-derived JEG 3 cell line encodes the HLA-C-locus product, HLA-Cw*0401. This protein, obtained by in vitro transcription/translation, has biochemical characteristics identical to MHC class I products immunoprecipitated directly from the same cells. These findings are in agreement with RNA analysis and immunohistochemistry on both cell lines and primary trophoblast tissues. We report here the preferential reactivity in JEG 3 cells of two widely used monoclonal anti-MHC class I heavy chain antibodies, HC10 and HCA2, with HLA-C and HLA-G, respectively. We have mapped the epitopes recognized by these reagents to distinct areas of the alpha1 domain of the MHC class I heavy chain. HCA2 recognizes the motif xLxTLRGx spanning amino acids 77 84 present in both HLA-A and HLA-G. In contrast, HC10 may recognize a discontinuous epitope, with essential elements of the recognized motif surrounding residue 60 in the alpha1 domain of the class I heavy chain, as shown by truncation analysis. These results adequately explain the immunochemical cross-reactivity of HLA-A and HLA-G.

Construction and characterization of a fusion protein of single-chain anti-CD20 antibody and human beta-glucuronidase for antibody-directed enzyme prodrug therapy.

The CD20 antigen is an attractive target for specific treatment of B-cell lymphoma. Antibody-directed enzyme prodrug therapy (ADEPT) aims at the specific activation of a nontoxic prodrug at the tumor site by an enzyme targeted by a tumor-specific antibody such as anti-CD20. We constructed a fusion protein of the single-chain Fv anti-CD20 mouse monoclonal antibody (MoAb) 1H4 and human beta-glucuronidase for the activation of the nontoxic prodrug N-[4-doxorubicin-N-carbonyl(-oxymethyl) phenyl] O-beta-glucuronyl carbamate to doxorubicin at the tumor site. The cDNAs encoding the light- and heavy-chain variable regions of 1H4 were cloned, joined by a synthetic sequence encoding a 15-amino acid linker and fused to human beta-glucuronidase by a synthetic sequence encoding a 6-amino acid linker. An antibody-enzyme fusion protein-producing cell line was established by transfection of the construct into human embryonic kidney 293/EBNA cells. The yield of active fusion protein was 100 ng/mL transfectoma supernatant. Antibody affinity, antibody specificity, and enzyme activity were fully retained by the fusion protein. Immunoprecipitation and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the fusion protein has a relative molecular weight (Mw) of 100 kD under denaturing conditions. Gel filtration analysis indicated that the enzymatically active form of the fusion protein is a tetramer with an Mw of approximately 400 kD. The nontoxic prodrug N-[4-doxorubicin-N-carbonyl(-oxymethyl) phenyl] O-beta-glucuronyl carbamate was hydrolyzed by the fusion protein at a hydrolysis rate similar to that of human beta-glucuronidase. When the fusion protein was specifically bound to Daudi lymphoma cells, the prodrug induced similar antiproliferative effects as doxorubicin. Thus, it is feasible to construct a eukaryotic fusion protein consisting of a single-chain anti-CD20 antibody and human beta-glucuronidase for future use in the activation of anticancer prodrugs in B-cell lymphoma.