Differences in the post-translational modifications of human papillomavirus type 6b major capsid protein expressed from a baculovirus system compared with a vaccinia virus system

Virus-like particles (VLPs) are being currently investigated in vaccines against viral infections in humans. There are different recombinant-protein-expression systems available for obtaining the necessary VLP preparation for vaccination. However, the differences in post-translational modifications of the recombinant proteins obtained and their differences in efficacy in eliciting an anti-viral response in vaccines are not well established. In this study we have compared the posttranslational modifications of human papillomavirus type-6b major capsid protein L1 (HPV 6bL1) expressed using recombinant baculovirus (rBV) in Sf9 (Spodoptera frugiperda) insect cells, with the protein expressed using recombinant vaccinia virus (rVV) in CV-1 kidney epithelial cells, Two-dimensional gel electrophoresis of biosynthetically labelled rBV-expressed HPV 6bL1 showed several post-translationally modified variants of the protein, whereas rVV-expressed HPV 6bL1 showed only a few variants. Phosphorylations were detected at threonine and serine residues for the L1 expressed from rBV compared with phosphorylation at serine residues only for the L1 expressed from rVV. HPV 6bL1 expressed using rBV incorporated [H-3]mannose and [H-3]galactose, whereas HPV 6bL1 expressed using rVV incorporated only [H-3]galactose. We conclude that post-translational modification of recombinant HPV 6bL1 can differ according to the system used for its expression. Since recombinant L1 protein is a potential human-vaccine candidate, the implication of the observed differences in post-translational modifications on immunogenicity of L1 VLPs warrants investigation.

Multiple conformational epitopes are recognized by natural and induced immunity to the E7 protein of human papilloma virus type 16 in man

The reactivity of sera from patients with cervical cancer with the E7 protein of human papilloma virus type 16 (HPV16) was estimated using a novel non-radioactive immunoprecipitation assay and four established protein-and peptide-based immunoassays. Six of 14 sera from patients with cervical cancer and 1 of 10 sera from healthy laboratory staff showed repeated reactivity with E7 in at least one assay. Four of the 7 reactive sera were consistently reactive in more than one assay, but only one was reactive in all four assays. Following immunization with E7, 2 of 5 patients with cervical cancer had increased E7-specific reactivity, measurable in one or more assays. No single assay was particularly sensitive for E7 reactivity, or predictive of cervical cancer. Mapping of E7 reactivity to specific E7 peptides was unsuccessful, suggesting that natural or induced E7 reactivity in human serum is commonly directed to conformational epitopes of E7, These results suggest that each assay employed with is study measures a different aspect of E7 reactivity, and that various reactivities to E7 may manifest following HPV infection or immunization. This finding is of significance for monitoring of E7 immunotherapy and for serological screening for cervical cancer. Copyright (C) 2000 S.Karger, AG. Basel.

The human papillomavirus E7 protein is able to inhibit the antiviral and anti-growth functions of interferon-alpha

It is now well recognized that cervical cancer is caused by infection with certain human papillomavirus (HPV) subtypes and while interferon-alpha (IFN-alpha) is used to treat HPV-infected lesions, HPV appears to have developed a means to avoid the effects of IFN-alpha. Clinically, resistance appears to be associated with the expression of the E7 oncoprotein. Here we investigated the effects of expression in cells of the E7 protein from high- and low-risk papillomavirus subtypes on a range of responses to IFN-alpha. 2fTGH, a cell line dependent on IFN-alpha for growth in selection medium, grew significantly less well in the presence of E7, and the antiproliferative effects of IFN-alpha upon epithelial cells was lost upon E7 expression. The antiviral effects of IFN-alpha were abrogated in E7-expressing cells. Loss of response to IFN-alpha was found to occur in both high- and low-risk papillomaviruses. Finally, deletion of amino acids 21-24 of HPV type 16 E7 protein partially reversed repression. We conclude that E7 inhibits the functional effects of IFN-alpha and that this property is shared by all HPV subtypes tested. (C) 2000 Academic Press.

Human cyclophilin 40 is a heat shock protein that exhibits altered intracellular localization following heat shock

The unactivated steroid receptors are chaperoned into a conformation that is optimal for binding hormone by a number of heat shock proteins, including Hsp90, Hsp70, Hsp40, and the immunophilin, FKBP52 (Hsp56). Together with its partner cochaperones, cyclophilin 40 (CyP40) and FKBP51, FKBP52 belongs to a distinct group of structurally related immunophilins that modulate steroid receptor function through their association with Hsp90. Due to the structural similarity between the component immunophilins, FKBP52 and cyclophilin 40, we decided to investigate whether CyP40 is also a heat shock protein. Exposure of MCF-7 breast cancer cells to elevated temperatures (42 degreesC for 3 hours) resulted in a 75-fold increase in CyP40 mRNA levels, but no corresponding increase in CyP40 protein expression, even after 7 hours of heat stress. The use of cycloheximide to inhibit protein synthesis revealed that in comparison to MCF-7 cells cultured at 37 degreesC, those exposed to heat stress (42 degreesC for 3 hours) displayed an elevated rate of degradation of both CyP40 and FKBP52 proteins. Concomitantly, the half-life of the CyP40 protein was reduced from more than 24 hours to just over 8 hours following heat shock. As no alteration in CyP40 protein levels occurred in cells exposed to heat shock, an elevated rate of degradation would imply that CyP40 protein was synthesized at an increased rate. hence the designation of human CyP40 as a heat shock protein. Application of heat stress elicited a marked redistribution of CyP40 protein in MCF-7 cells from a predominantly nucleolar localization, with some nuclear and cytoplasmic staining, to a pattern characterized by a pronounced nuclear accumulation of CyP40, with no distinguishable nucleolar staining. This increase in nuclear CyP40 possibly resulted from a redistribution of cytoplasmic and nucleolar CyP40, as no net increase in CyP40 expression levels occurred in response to stress. Exposure of MCF-7 cells to actinomycin D for 4 hours resulted in the translocation of the nucleolar marker protein, B23, from the nucleolus, with only a small reduction in nucleolar CyP40 levels. Under normal growth conditions, MCF-7 cells exhibited an apparent colocalization of CyP40 and FKBP52 within the nucleolus.

Identification of a minimal promoter sequence for the human N-acetyltransferase Type I gene that binds AP-1 (activator protein 1) and YY-1 (Yin and Yang 1)

Human N-acetyltransferase Type I (NAT1) catalyses the acetylation of many aromatic amine and hydrazine compounds and it has been implicated in the catabolism of folic acid. The enzyme is widely expressed in the body, although there are considerable differences in the level of activity between tissues. A search of the mRNA databases revealed the presence of several NAT1 transcripts in human tissue that appear to be derived from different promoters. Because little is known about NAT1 gene regulation, the present study was undertaken to characterize one of the putative promoter sequences of the NAT1 gene located just upstream of the coding region. We show with reverse-transcriptase PCR that mRNA transcribed from this promoter (Promoter 1) is present in a variety of human cell-lines, but not in quiescent peripheral blood mononuclear cells. Using deletion mutant constructs, we identified a 20 bp sequence located 245 bases upstream of the translation start site which was sufficient for basal NAT1 expression. It comprised an AP-1 (activator protein 1)-binding site, flanked on either side by a TCATT motif. Mutational analysis showed that the AP-1 site and the 3' TCATT sequence were necessary for gene expression, whereas the 5' TCATT appeared to attenuate promoter activity. Electromobility shift assays revealed two specific bands made up by complexes of c-Fos/Fra, c-Jun, YY-1 (Yin and Yang 1) and possibly Oct-1. PMA treatment enhanced expression from the NAT1 promoter via the AP-1-binding site. Furthermore, in peripheral blood mononuclear cells, PMA increased endogenous NAT1 activity and induced mRNA expression from Promoter I, suggesting that it is functional in vivo.

The human sulfotransferase SULT1A1 gene is regulated in a synergistic manner by Sp1 and GA binding protein

Human sulfotransferase SULT1A1 is an important phase II xenobiotic metabolizing enzyme that is highly expressed in the liver and mediates the sulfonation of drugs, carcinogens, and steroids. Until this study, the transcriptional regulation of the SULT1A subfamily had been largely unexplored. Preliminary experiments in primary human hepatocytes showed that SULT1A mRNA levels were not changed in response to nuclear receptor activators, such as dexamethasone and 3-methylcolanthrene, unlike other metabolizing enzymes. Using HepG2 cells, the high activity of the TATA-less SULT1A1 promoter was shown to be dependent on the presence of Sp1 and Ets transcription factor binding sites (EBS), located within - 112 nucleotides from the transcriptional start site. The homologous promoter of the closely related SULT1A3 catecholamine sulfotransferase, which is expressed at negligible levels in the adult liver, displayed 70% less activity than SULT1A1. This was shown to be caused by a two-base pair difference in the EBS. The Ets transcription factor GA binding protein (GABP) was shown to bind the SULT1A1 EBS and could transactivate the SULT1A1 promoter in Drosophila melanogaster S2 cells. Cotransfection of Sp1 could synergistically enhance GABP-mediated activation by 10-fold. Although Sp1 and GABP alone could induce SULT1A3 promoter activity, the lack of the EBS on this promoter prevented a synergistic interaction between the two factors. This study reports the first insight into the transcriptional regulation of the SULT1A1 gene and identifies a crucial difference in regulation of the closely related SULT1A3 gene, which accounts for the two enzymes' differential expression patterns observed in the adult liver.

E7 oncoprotein of human papillomavirus type 16 expressed constitutively in the epidermis has no effect on E7-specific B- or Th-repertoires or on the immune response induced or sustained after immunization with E7 protein

A line of FVB (H-2(q)) mice transgenic for the E6/E7 open reading frames of Human Papillomavirus type 16 driven from the alpha-A crystallin promoter expresses E7 mRNA in lens and skin epithelium. E7 protein is detectable in adult skin, coinciding with the development or inflammatory skin disease, which progresses to papillomata and squamous carcinomata in some mice. By examining the outcome of parenteral immunization with E7 protein, we sought to determine whether endogenous expression of E7 in skin had induced a preexisting immune outcome, i.e., specific immunity or tolerance, or whether the mice remain naive (''ignorant'') to E7. Our data show that the antibody response to defined E7 B-epitopes, the proliferative response to Th epitopes, and the delayed-type hypersensitivity (DTH) response to whole E7 did not differ between groups or young and old E6/E7 transgenic mice (likely having different degrees of lifetime exposure to E7 protein) or between E6/E7-transgenic and nontransgenic parental strain control mice. Although an E7-specific CTL response could not be induced in the H-2(q) background of these mice, incorporation of a D-b allele into the genome allowed comparison of D-b-restricted CTL responses in E6/E7 transgenic and nontransgenic mice. Experiments indicated that the E7-immunization-induced CTL response did not differ significantly between E6/E7 transgenic and nontransgenic mice. We interpret these results to indicate that in spite of expression of E7 protein in adult skin, E6/E7 transgenic mice remain immunologically naive (ignorant) of E7 epitopes presented by immunization. (C) 1997 Academic Press.

Accumulation of human heat shock protein 60-reactive T cells in the gingival tissues of periodontitis patients

Heat shock protein 60s (hsp60) are remarkably immunogenic, and both T-cell and antibody responses to hsp60 have been reported in various inflammatory conditions. To clarify the role of hsp60 in T-cell responses in periodontitis, we examined the proliferative response of peripheral blood mononuclear cells (PBMC), as well as the cytokine profile and T-cell clonality, for periodontitis patients and controls following stimulation with recombinant human hsp60 and Porphyromonas gingivalis GroEL. To confirm the infiltration of hsp60-reactive T-cell clones into periodontitis lesions, nucleotide sequences within complementarity-determining region 3 of the T-cell receptor (TCR) beta-chain were compared between hsp60-reactive peripheral blood T cells and periodontitis lesion-infiltrating T cells. Periodontitis patients demonstrated significantly higher proliferative responses of PBMC to human hsp60, but not to P. gingivalis GroEL, than control subjects. The response was inhibited by anti-major histocompatibility complex class 11 antibodies. Analysis of the nucleotide sequences of the TCR demonstrated that human hsp60-reactive T-cell clones and periodontitis lesion-infiltrating T cells have the same receptors, suggesting that hsp60-reactive T cells accumulate in periodontitis lesions. Analysis of the cytokine profile demonstrated that hsp60-reactive PBMC produced significant levels of gamma interferon (IFN-gamma) in periodontitis patients, whereas P. gingivalis GroEL did not induce any, skewing toward a type1 or type2 cytokine profile. In control subjects no significant expression of IFN-gamma or interleukin 4 was induced. These results suggest that periodontitis patients have human hsp60-reactive T cells with a type I cytokine profile in their peripheral blood T-cell pools.

Influence of ageing, heat shock treatment and in vivo total antioxidant status on gene-expression profile and protein synthesis in human peripheral lymphocytes

Ageing results in a progressive, intrinsic and generalised imbalance of the control of regulatory systems. A key manifestation of this complex biological process includes the attenuation of the universal stress response. Here we provide the first global assessment of the ageing process as it affects the heat shock response, utilising human peripheral lymphocytes and cDNA microarray analysis. The genomic approach employed in our preliminary study was supplemented with a proteomic approach. In addition, the current study correlates the in vivo total antioxidant status with the age-related differential gene expression as well as the translational kinetics of heat shock proteins (hsps). Most of the genes encoding stress response proteins on the 4224 element microarray used in this study were significantly elevated after heat shock treatment of lymphocytes obtained from both young and old individuals albeit to a greater extent in the young. Cell signaling and signal transduction genes as well as some oxidoreductases showed varied response. Results from translational kinetics of induction of major hsps, from 0 to 24 It recovery period were broadly consistent with the differential expression of HSC 70 and HSP 40 genes. Total antioxidant levels in plasma from old individuals were found to be significantly lower by comparison with young, in agreement with the widely acknowledged role of oxidant homeostasis in the ageing process. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Characterization of the human sulfate anion transporter (hsat-1) protein and gene (SAT1; SLC26A1)

Sulfate plays an essential role during growth, development, bone/cartilage formation, and cellular metabolism. In this study, we have isolated the human sulfate anion transporter cDNA (hsat-1; SCL26A1) and gene (SAT1), determined its protein function in Xenopus oocytes and characterized SAT1 promoter activity in mammalian renal cell lines. hsat-1 encodes a protein of 75 kDa, with 12 putative transmembrane domains, that induces sulfate, chloride, and oxalate transport in Xenopus oocytes. hsat-1 mRNA is expressed most abundantly in the kidney and liver, with lower levels in the pancreas, testis, brain, small intestine, colon, and lung. The SAT1 gene is comprised of four exons stretching 6 kb in length, with an alternative splice site formed from an optional exon. SAT1 5' flanking region led to promoter activity in renal OK and LLC-PK1 cells. Using SAT1 5' flanking region truncations, the first 135 bp was shown to be sufficient for basal promoter activity. Mutation of the activator protein-1 (AP-1) site at position 252 in the SAT1 promoter led to loss of transcriptional activity, suggesting its requirement for SAT1 basal expression. This study represents the first functional characterization of the human SAT1 gene and protein encoded by the anion transporter hsat-1.

T-cell clonality to Porphyromonas gingivalis and human heat shock protein 60s in patients with atherosclerosis and periodontitis

Individuals with periodontitis have been reported to have a significantly increased risk of developing coronary heart disease. Several studies have demonstrated that the immune response to heat shock protein 60 (HSP60) may be involved in the pathogenesis of both atherosclerosis and chronic periodontitis. To investigate this possible link between these diseases, cellular and humoral immune responses to HSP60 in atherosclerosis patients were compared with those in periodontitis patients and healthy subjects using human and Porphyromonas gingivalis HSP60 (GroEL) as antigens. Antibody levels to both human and P. gingivalis HSP60s were the highest in atherosclerosis patients, followed by periodontitis patients and healthy subjects. Clonal analysis of the T cells clearly demonstrated the presence of not only human HSP60- but also P. gingivalis GroEL-reactive T-cell populations in the peripheral circulation of atherosclerosis patients. Furthermore, these HSP60-reactive T cells seemed to be present in atherosclerotic lesions in some patients. These results suggest that T-cell clones with the same specificity may be involved in the pathogenesis of the different diseases.

The LFA-1-associated molecule PTA-1 (CD226) on T cells forms a dynamic molecular complex with protein 4.1G and human discs large

Clustering of the T cell integrin, LFA-1, at specialized regions of intercellular contact initiates integrin-mediated adhesion and downstream signaling, events that are necessary for a successful immunological response. But how clustering is achieved and sustained is not known. Here we establish that an LFA-1-associated molecule, PTA-1, is localized to membrane rafts and binds the carboxyl-terminal domain of isoforms of the actin-binding protein 4.1G. Protein 4.1 is known to associate with the membrane-associated guanylate kinase homologue, human discs large. We show that the carboxyl-terminal peptide of PTA-1 also can bind human discs large and that the presence or absence of this peptide greatly influences binding between PTA-1 and different isoforms of 4.1G. T cell stimulation with phorbol ester or PTA-1 cross-linking induces PTA-1 and 4.1G to associate tightly with the cytoskeleton, and the PTA-1 from such activated cells now can bind to the amino-terminal region of 4.1G. We propose that these dynamic associations provide the structural basis for a regulated molecular adhesive complex that serves to cluster and transport LFA-1 and associated molecules.