Vergleichende Charakterisierung der Oberflächenproteine von verschiedenen Hepatitis-B-Virus-Genotypen

Für das humane Hepatitis-B-Virus sind acht unterschiedliche Genotypen (GtA–GtH) bekannt, welche sich anhand der geographischen Verteilung, der molekularen Virologie und der Virus-assoziierten Pathogenese unterscheiden. Die für diese Arbeit durchgeführten Experimente stellen eine vergleichende Analyse der verschiedenen HBV-Genotypen – ohne Einfluss von spezifischen Wirtsfaktoren wie z. B. die Immunabwehr – dar. Auf diesem Wege bietet sich zusätzlich die Möglichkeit, virale Proteine in großem Maßstab aufzureinigen und so für eine weitere Nutzung verfügbar zu machen. Die durchgeführten vergleichenden Analysen zeigten, dass es zwischen den verschiedenen untersuchten Genotypen eindeutige Unterschiede bei Bildung und Ausschleusung der diagnostisch wichtigen Oberflächenproteine gibt. Hierbei weist der Genotyp G – auch im Hinblick auf die intrazelluläre Verteilung des HBsAg – die größten Abweichungen auf, da dieser durch eine Sekretionsinkompetenz von subviralen Partikeln charakterisiert wurde. Bei den Glykosylierungsmustern der verschiedenen viralen Oberflächenproteine konnten keine eindeutigen Unterschiede beobachtet werden, doch zeigten 2D-Elektrophorese-Analysen eindeutige Hinweise auf eine Genotypen-spezifische abweichende posttranslationale Modifikation des LHBs. Mit Hilfe des gewählten Expressionssystems konnte in einem speziell etablierten Verfahren HBsAg der verschiedenen untersuchten HBV-Genotypen durch Trennung subviraler und viraler Partikel gereinigt werden und stand so für weitere Analysen zu Verfügung. Die Testung dieses Materials in mehreren quantitativen Nachweissystemen konnte bestätigen, dass eine Eignung des rekombinant hergestellten und nachfolgend aufgereinigten HBsAg als Referenzmaterial zur Eichung von kommerziell erhältlichen Diagnose-Systemen besteht. Die in dieser Arbeit durchgeführten Untersuchungen an unterschiedlichen qualitativen Nachweisverfahren konnten nachfolgend Genotypen-spezifische Unterschiede in der Signifikanz der jeweiligen Nachweisbarkeit aufzeigen. Schließlich konnte so der gesteigerte Nutzen dieses gereinigten rekombinant hergestellten Materials mit einem zwischen den unterschiedlichen HBV-Genotypen diskriminierenden Fokus bestätigt werden. Dies spielt eine große Rolle hinsichtlich angepasster Impfverfahren, optimierter Diagnostik bzw. Therapie, Sicherheit von Nachweisverfahren und darüber hinaus in der Impfstoff- und Antikörperproduktion.For the human hepatitis B virus, eight distinct and two candidate genotypes have been described, which differ with respect to geographic distribution, molecular virology and virus-associated pathogenesis. The performed experiments reflect a comparative analysis of the different HBV genotypes unbiased by host-depending factors like immunological responses. Moreover, this enables a large-scale purification of separate viral proteins, thus allowing their further exploitation. The performed comparative analyses showed significant differences between the investigated genotypes with regard to production and secretion of the diagnostically important surface proteins. The most apparent deviations can be observed with respect to HBV genotype G, especially in connection with the intracellular distribution of the viral surface proteins. Genotype G is characterized by the inability in secreting subviral particles, even though these are produced and secreted in large amounts compared to fully assembled viruses by the other HBV genotypes. No unambiguous differences can be detected with respect to the glycosylation pattern of the viral surface proteins. In contrast to this, 2-D gel electrophoresis revealed significant variances in connection with the specific posttranslational modifications of the viral large surface protein LHBs. In connection with the chosen expression system, HBsAg of the different HBV genotypes could be successfully and reproducibly separated in subviral and viral particles. Thus, highly purified viral material was available for further experiments. The suitability of this enriched HBsAg as a unique reference standard for the calibration of commercially available diagnostic tools was proved by a testing within several independent quantitative HBsAg detection systems and mathematical comparison with the current certified standardization material. In light of the relevance of HBsAg as diagnostic marker, the detectability of purified recombinant HBsAg of various genotypes by HBsAg-specific detection systems licensed in Europe was investigated, showing similar sensitivities for genotypes included in this analysis. These data indicate that recombinant HBsAg reproducibly purified following a defined protocol might be used as an alternative to reference materials currently established. With this, the enhanced benefit of purified and enriched recombinant HBsAg of different HBV genotypes could be confirmed. This finally facilitates further improvements in connection with adapted vaccination studies, optimized diagnostics and therapies, safety of the commonly used detection systems and furthermore the development of new antibodies and vaccines

Non-Secreted Clusterin Isoforms Are Translated in Rare Amounts from Distinct Human mRNA Variants and Do Not Affect Bax-Mediated Apoptosis or the NF-κB Signaling Pathway

<div><p>Clusterin, also known as apolipoprotein J, is expressed from a variety of tissues and implicated in pathological disorders such as neurodegenerative diseases, ischemia and cancer. In contrast to secretory clusterin (sCLU), which acts as an extracellular chaperone, the synthesis, subcellular localization and function(s) of intracellular CLU isoforms is currently a matter of intense discussion. By investigating human CLU mRNAs we here unravel mechanisms leading to the synthesis of distinct CLU protein isoforms and analyze their subcellular localization and their impact on apoptosis and on NF-κB-activity. Quantitative PCR-analyses revealed the expression of four different stress-inducible CLU mRNA variants in non-cancer and cancer cell lines. In all cell lines variant 1 represents the most abundant mRNA, whereas all other variants collectively account for no more than 0.34% of total CLU mRNA, even under stressed conditions. Overexpression of CLU cDNAs combined with <i>in vitro</i> mutagenesis revealed distinct translational start sites including a so far uncharacterized non-canonical CUG start codon. We show that all exon 2-containing mRNAs encode sCLU and at least three non-glycosylated intracellular isoforms, CLU_1‑449, CLU_21‑449 and CLU_34‑449, which all reside in the cytosol of unstressed and stressed HEK‑293 cells. The latter is the only form expressed from an alternatively spliced mRNA variant lacking exon 2. Functional analysis revealed that none of these cytosolic CLU forms modulate caspase-mediated intrinsic apoptosis or significantly affects TNF-α-induced NF-κB-activity. Therefore our data challenge some of the current ideas regarding the physiological functions of CLU isoforms in pathologies.</p> </div

Impact of individual CLU isoforms on apoptosis and on NF-κB-activity.

<p>HEK‑293 cells were transfected with pcDNA6 (mock), unmodified variant 1 (wildtype), variant 1 [Δex2] (Δex2) or point-mutated versions of variant 1 cDNA encoding only sCLU/CLU_1‑449, CLU_21‑449 or CLU_34‑449. (A) 24 hours after transfection the activity of caspases 3 and 7 was determined. Data are expressed relative to the values obtained from Bax cDNA transfected cells (mean ± SD, n = 3), which served as a positive control. In contrast to Bax, the expression of all CLU protein forms does not activate caspase‑3/7. (B) 24 hours after transfection 10 µM MG‑132 or DMSO (control) was added for 10 hours followed by measurement of caspase 3/7 activity. Data are expressed relative to the values obtained from corresponding control cells (mean ± SD, n = 3). MG‑132 treatment causes an increase in caspase 3/7 activity in all cells analyzed. Neither CLU protein form significantly affects the extent of MG‑132-induced caspase‑3/7 activation. (C) HEK-293 were cotransfected with Bax cDNA together with pcDNA6 (mock +Bax), Bcl-x_L or point-mutated versions of variant 1 cDNA. 24 hours after transfection the activity of caspases 3 and 7 was determined. Cells transfected with pcDNA6 alone (mock) served as negative control. Data are expressed relative to the values obtained from mock +Bax transfected cells (mean ± SD, n = 3). While cotransfection with Bcl-x_L cDNA, suppresses caspase 3/7 activation demonstrating the validity of the experimental setup, neither of the CLU isoforms significantly modulates Bax-mediated apoptosis. (D) HEK-293 were cotransfected with pNF-κB-Luc and the indicated versions of variant 1. 18 hours after transfection cells were incubated for 24 hours with either 10 ng/ml TNF-α (+TNF) or with BSA solution (-TNF). Cells were lysed and luciferase activity was determined as a measure of NF-κB-activity. Data are expressed as increase in Luciferase activity relative to mock transfected cells incubated with BSA solution (mean ± SD, n=4). A representative Western Blot of cell lysates is embedded showing CLU expression in the samples analyzed. The order is: mock, wildtype, Δex2, sCLU/CLU_1‑449, CLU_21‑449 and CLU_34‑449. Only CLU_34‑449 expressed from variant 1 [Δex2] reduces NF-κB activity (Δex2).</p

Overview of the human CLU gene and mRNA variants.

<p>The human CLU gene encodes at least 3 different pre-mRNAs which contain unique exons 1 but share exons 2-9. Alternative splicing of variant 1 pre-mRNA generates an mRNA (variant 1 [Δex2]) that lacks exon 2 and the SSCR (black box). The position of the sCLU start codon (framed) is defined as nt = 1. Notice the additional in-frame AUG codons on exon 3 of all mRNAs and on exon 1c of variant 3.</p

Properties of distinct CLU isoforms and their synthesis in unstressed and stressed cells.

<p>(A) A CLU-specific Western blot of cell lysate of MG‑132-treated HEK‑293 cells is shown. All detectable protein bands can be assigned to distinct CLU forms with different properties and subcellular localization (¹ determined MWs of secretory CLU forms according to Kapron et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075303#B55" target="_blank">55</a>]). (B) Model depicting the synthesis of CLU isoforms in unstressed and stressed HEK-293 cells. Under unstressed conditions, the dominant isoform sCLU is translated from variant 1, which accounts virtually for the total CLU mRNA amount. Cytosolic CLU_1‑449 (‘mistranlocated’ sCLU pre-pro-protein) and CLU_21‑449 (translated from exon 2 CUG) are not detectable due to presumably low expression and proteasomal degradation. CLU_34‑449 is translated from exon 3 AUG on variant 1 and from variant 1 [Δex2]. Although CLU_34‑449 is not proteasomally degraded, it is not detectable in unstressed cells reflecting its low expression level. Induction of cellular stress (MG-132, heat) induces transcriptional upregulation of variant 1 and its splicing to variant 1 [Δex2] leading to enhanced synthesis of all CLU isoforms. Further, the ‘mistranslocation’ of CLU_1‑449 to the cytosol is increased. In the case of proteotoxic stress induced by MG-132, diminished proteasomal degradation of CLU_1‑449 and CLU_21‑449 further promotes their accumulation. Altogether these events generate amounts of the rare cytosolic CLU isoforms that are detectable in Western Blot analyses. They account for 3.6% ± 1.6% (mean ± SD, <i>n</i>=5) of total cell-associated CLU in stressed cells as determined by densitometric quantification of corresponding bands. Note that different expression levels (from low to high) are indicated by the different line width of arrows (from dashed to bold). Variants 2 and 3 are not illustrated because they are expressed in very low amounts and virtually do not contribute to the synthesis of any CLU isoform.</p

Proteasomal inhibition and heat stress modulate sCLU and intracellular CLU protein expression in cancer and non-cancer cells.

<p>HEK‑293, PC‑3, MCF‑7 and Caco-2 cells were treated with DMSO as control (C), 10 µM MG-132 (MG) or subjected to heat shock (45°C). Whole cell lysates (upper panel) and cell culture media (lower panel) of cells were analyzed for CLU expression by Western blot. 45-50 kDa CLU protein bands were detected primarily in stressed cells (*). Data shown are representative of three independent experiments.</p

Expression of CLU mRNA variants in cancer and non-cancer cells upon proteotoxic stress.

<p>(A) Semi-quantitative RT‑PCR analyses of the expression of different CLU mRNA variants in control and MG‑132-treated HEK‑293, MCF‑7, Caco-2 and PC‑3 cells. The different numbers of amplification cycles performed reflect the varying amounts of individual CLU mRNA variants expressed. Analysis of Hsp27 mRNA indicates the induction of a heat-shock response upon MG‑132 treatment. GAPDH RT‑PCR served as control to ensure the use of equal amounts of reverse transcribed mRNA. Data shown are representative of at least 3 independent experiments. (B, C, D) Quantification of CLU mRNA expression in control and MG‑132-treated HEK‑293 (B), MCF‑7 (C) and PC‑3 cells (D) by qPCR. The amounts of individual transcripts are expressed as copy number per ng of total RNA (mean ± SD, <i>n</i> = 3). Variant 1 is the pre-dominant CLU mRNA in all cell lines conforming to the amounts of total CLU mRNA. The other variants represent low abundant CLU mRNAs.</p