Stabilization of proteins and peptides in diagnostic immunological assays by the molecular chaperone Hsp25.

Diagnostic assays for proteins devoid of enzymatic activity are becoming increasingly important. Antibodies generated against the respective proteins are used for their detection in enzyme-linked immunosorbent assay or patient sera are used to monitor disease-related antibodies against recombinantly produced antigens. A problem frequently encountered with these assays is that the proteins or fragments thereof used as standards have a limited shelf life. A similar problem arises when activities of labile enzymes are used for diagnostic detection. Here, we present a novel approach to 'stabilizing' enzymatic activity and antigenicity of proteins used for immunogenic detection by molecular chaperones. We have exploited the ability of molecular chaperones to keep proteins in their active conformation to overcome the biotechnological problems encountered in protein-based diagnostics of heart attack, stroke, and viral infections such as hepatitis C. We show that Hsp25, a member of the family of small heat shock proteins, known to act as a molecular chaperone in protein folding reactions, can stably bind labile standard proteins. Complex formation does not interfere with immunogenic detection and, importantly, antigenic as well as enzymatic activity remains constant for weeks. This strategy seems to be applicable to a wide range of assays involving unstable proteins, including the generation of vaccines.

Characterization of a linear epitope in the nonstructural region 4 of hepatitis C virus with reactivity to seroconversion antibodies.

Mapping and possible diagnostic meaning of a highly conserved, linear NS4 epitope (NS4/3), located outside the C100-3 antigen within the carboxyl terminal proportion of the NS4 region, with major immunoreactivity with specimens of patients with HCV infection from various geographic origins is described. Transient, acute-phase IgM anti-HCV NS4/3 was detected coincidentally or earlier than active IgG anti-HCV NS4/3 response with four well-characterized seroconversion panels. GenBank alignment studies identified patch homologies between the NS4/3 sequence and a number of non-HCV proteins, which may explain part of the cross-reactivity of the NS4/3 epitope. Some of the "false positive reactivities" of the NS4/3 epitope with asymptomatic blood donors, not being confirmed with FDA-approved anti-HCV assays without the NS4/3 epitope, may be explained by recognition of very early seroconversion antibodies.

Mapping of linear B-cell epitopes on viral polypeptides by multiple Peptide synthesis and fine tuning sensitivity and specificity of the identified Peptide antigens for application in virus diagnosis.

This chapter focuses on methods for epitope mapping on novel viral polypeptrdes and on fine tuning sensitivity and spectficity of the identified peptide antigens for application in virus diagnosis. Because of the development of efficient methods of multiple peptrde synthesis (1-3), antigenic sates of many proteins could be detected.

Anti-interferon activity of adenovirus-2-encoded VAI and VAII RNAs in translation in cultured human cells.

The mode of anti-interferon action of VAI and VAII RNAs of adenovirus type 2 (Ad2) was studied by transfecting interferon-alpha (IFN-alpha)-treated KB cells in culture with a plasmid construct containing the VAI or VAII RNA gene and an SV40 promoter-chloramphenicol acetyltransferase (CAT) gene construct as reporter (pSV2-CAT). The longer the treatment of KB cells with IFN-alpha (2,000 IU/ml) lasted, the higher was the inhibition of CAT expression. A maximum of 76% inhibition was attained without pronounced cytotoxicity during 48 h of treatment. The earlier the VAI RNA gene was transfected, the higher was the enhancement of CAT expression. CAT activity increased from 113 to 157% in normal cells and 200-400% in IFN-alpha treated cells, as compared with the corresponding controls without VAI RNA transfection. The level of CAT mRNA was neither appreciably decreased by IFN-alpha treatment, nor detectably increased by VAI or VAII RNA. The effect of VA RNA thus appeared to be on translation rather than on transcription. The relative constancy of the level of CAT mRNA indicated that IFN-alpha inhibition of CAT expression was not due to the activation of RNase L, but due mainly to translational repression. The level of VAII RNA expressed was only 9-13% of that of VAI RNA. Nevertheless, VAII RNA gene transfection stimulated CAT activity to 112% of the control in non-IFN-alpha-treated cells, and 126-182% in IFN-alpha-inhibited cells. When IFN-alpha treatment was started late after VAI RNA cotransfection, CAT expression increased to 169% which was higher than the expression in cotransfected control cells without IFN-alpha treatment. The enhanced level of CAT activity was in remarkable contrast to the IFN-alpha inhibited level of 25% without VA RNA co-transfection when IFN-alpha was added upon seeding. The enhanced CAT activity in cells treated late with IFN-alpha could be ascribed to higher levels of VA RNAs.

Boehringer Mannheim modular test concepts in HIV and hepatitis immunoassays.

Anti-HIV-antibody and hepatitis C virus (HCV)-antibody screening tests have to be able to detect a variety of virus antibodies. On the other hand, HIV-antigen specific antibody tests that detect only one kind of antibody are needed for prognosis of disease or for distinguishing infection by different virus subtypes. Usually in an enzyme-linked immunosorbent assay for each individual test an individual solid phase has to be created. For our Boehringer Mannheim Enzymun-Test Diagnostics Assay we used a single universal biotin-binding solid phase in all tests and biotin-labeled specific antigens for the individual tests. The modular system for the antibody tests is a convenient tool for the development of a broad test menu for different viruses. We show that the modular system is suited for screening tests for HIV1, HIV2, or HCV antibodies, as as well as for virus typing or for detection of HIV/p24-specific antibodies in a quantitative assay with high precision.

Protein translocation across mitochondrial membranes.

Protein translocation across biological membranes is of fundamental importance for the biogenesis of organelles and in protein secretion. We will give an overview of the recent achievements in the understanding of protein translocation across mitochondrial membranes. In particular we will focus on recently identified components of the mitochondrial import apparatus.

Protein folding causes an arrest of preprotein translocation into mitochondria in vivo.

With vital yeast cells, a hybrid protein consisting of the amino-terminal third of the precursor to cytochrome b2 and of the entire dihydrofolate reductase was arrested on the import pathway into mitochondria. Accumulation of the protein in the mitochondrial membranes was achieved by inducing a stable tertiary structure of the dihydrofolate reductase domain. Thereby, three salient features of mitochondrial protein uptake in vivo were demonstrated: its posttranslational character; the requirement for unfolding of precursors; and import through translocation contact sites. The permanent occupation of translocation sites by the fusion protein inhibited the import of other precursors; it did, however, not lead to leakage of mitochondrial ions, implying the existence of a channel that is sealed around the membrane spanning polypeptide segment.

Processing of mitochondrial precursor proteins.

The mitochondrial processing enzyme consists of two components, the mitochondrial processing peptidase (MPP) and processing enhancing protein (PEP). MPP and PEP act cooperatively in proteolytic processing of mitochondrial precursor proteins. Most of the mitochondrial precursors possess aminoterminal presequences (also called "targeting sequences" or "signal sequences"), that do not display a common motif and that show only limited similarities of the cleavage sites. The mitochondrial processing peptidase is a metal-dependent endoprotease, sensitive to sulfhydryl-modifying reagents and appears to belong to a new class of proteases. MPP and PEP, together with the core 1 and core 2 proteins of the respiratory complex III, form a new protein family.

Import of apocytochrome c into the mitochondrial intermembrane space along a cytochrome c1 sorting pathway.

The question of whether cytochrome c could be functionally sorted to the mitochondrial intermembrane space along a "conservative sorting" pathway was investigated using a fusion protein termed pLc1-c. pLc1-c contains 3-fold targeting information, namely, the complete bipartite presequence of the cytochrome c1 precursor joined to the amino terminus of apocytochrome c. pLc1-c could be selectively imported into the intermembrane space either directly across the outer membrane along a cytochrome c import route or along a cytochrome c1 route via the matrix. Thus, apocytochrome c could be sorted along a conservative sorting pathway; however, following reexport from the matrix, apo-Lc1-c could not be converted to its holo counterpart. Despite the apparent similarity of structure and functional location of the heme lyases and similarity of the heme binding regions in their respective apoproteins, cytochrome c heme lyase and cytochrome c1 heme lyase apparently have different and nonoverlapping substrate specificities.

Contact sites between inner and outer membranes: structure and role in protein translocation into the mitochondria.

Contact sites between both mitochondrial membranes play a predominant role in the transport of nuclear-coded precursor proteins into mitochondria. The characterization of contact sites was greatly advanced by the reversible accumulation of precursor proteins in transit (translocation intermediates). It was found that the sites are saturable, apparently contain proteinaceous components and mediate extensive unfolding of the polypeptide chain in translocation. Some components of mitochondrial contact sites are currently being identified.

Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites.

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.

A novel method for transfection and expression of reconstituted DNA-protein complexes in eukaryotic cells.

Transfection of foreign DNA into eukaryotic cells has become an important tool in molecular biology. Based on the results of previous studies of the core structure of human adenoviruses, we have developed a novel transfection method. The procedure involves the in vitro reconstitution of foreign DNA-of viral or other origins-with the major core protein VII of adenovirus type 2 (Ad2) or protamine from salmon sperm. Both proteins are rich in basic amino acids and appear to share structural features. The DNA-protein complexes are added directly to the medium of eukaryotic cells. The in vitro formation of specific DNA-protein complexes can be assessed by gel electrophoretic analyses. Bovine serum albumin does not enter into specific complexes with DNA. Transfection of DNA-protein VII or DNA-protamine complexes results in their rapid transport into the cell nuclei. About 2-4 hr after transfection, up to 40% of the DNA added to cell cultures in complexes can be found in the nucleus, as compared with less than 10% of the DNA when other transfection methods are applied or when naked DNA is added to cell cultures. DNAs transfected by the new method into mammalian or insect cells retain their characteristic restriction patterns at least 48 hr after transfection and are expressed efficiently. Supercoiled circular plasmid DNAs are converted to open circular or linear DNA. Expression has been measured both for transiently expressed genes (chloramphenicol acetyltransferase gene, Ad2 DNA in human HeLa cells) and for genes that have been integrated into the host genome and are expressed permanently, such as the gene for neomycin phosphotransferase in hamster BHK21 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of evidence for methylation of parental and newly synthesized adenovirus type 2 DNA in productive infections.

Methylations of highly specific sites in the promoter and 5' regions of eucaryotic genes have been shown to shut off gene activity and thus play a role in the long-term regulation of gene expression. It was therefore of interest to investigate whether site-specific DNA methylations could also play a role in adenovirus DNA in productive infections. It has been reported earlier that adenovirion DNA is not detectably methylated (U. Günthert, M. Schweiger, M. Stupp, and W. Doerfler, Proc. Natl. Acad. Sci. USA 73:3923-3927, 1976). In the present study, evidence for the methylation of cytidine residues in 5'-CCGG-3' and 5'-GCGC-3' sequences or the methylation of adenine residues in 5'-GATC-3' and 5'-TCGA-3' sequences in intranuclear adenovirus type 2(Ad2) DNA isolated and analyzed early (5 h) or late (24 h) after infection could not be obtained. In Ad2 DNA, 22.5% of all 5'-CG-3' dinucleotides reside in 5'-CCGG-3' and 5'-GCGC-3' sequences. Intranuclear viral DNA was examined by restriction endonuclease cleavage by using HpaII, MspI, HhaI, DpnI, or TaqI and Southern blot hybridizations. The HindIII fragments of Ad2 DNA served as hybridization probes. The data rendered it very unlikely that free intracellular adenovirus DNA in productively infected cells was extensively methylated. Thus, DNA methylation was not a likely element in the regulation of free adenovirus DNA expression in productively infected cells.