Journal of General Virology (2004), 85, 1427–1431
Short Communication
DOI 10.1099/vir.0.79844-0
Identification of membrane proteins differentially expressed in human papillomavirus type 16 E5-transfected human keratinocytes by nanoelectrospray ionization mass spectrometry Kerstin Leykauf, Mojiborahman Salek, Holger Schlu¨ter, Wolf-Dieter Lehmann and Angel Alonso Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany
Correspondence Angel Alonso
[email protected]
Received 25 November 2003 Accepted 13 February 2004
Membrane proteins differentially expressed in human papillomavirus type 16 (HPV-16) E5-transfected HaCaT cells have been identified. Membrane proteins were isolated and separated by two-dimensional gel electrophoresis. Spots showing quantitative differences between E5-transfected and control cells were extracted and the proteins were identified by nanoelectrospray ionization mass spectrometry. A total of 24 spots was analysed. Among the proteins showing differential expression, a decreased amount of calnexin and increased expression of hsp70, proteins both involved in maturation and transport of MHC class I complexes to the plasma membrane, were noticed. These findings correlate with the decreased surface expression of MHC class I molecules described in E5-expressing cells, HPV-positive cervical lesions and cervical carcinomas. These results stress the value of the proteomic approach, as used here in the experimental design, which allows the correlation of changes in host gene expression with biological functions of viral genes.
More than 90 % of human cervical tumours have been found to contain papillomavirus sequences (zur Hausen, 2002; Clifford et al., 2003). In human papillomaviruses (HPVs) of the high-risk type, two oncogenes, E6 and E7, are mainly responsible for their transforming properties (Munger & Howley, 2002). Another gene, E5, has been described to possess weak oncogenic activity and to increase greatly the oncogenic properties of E7 (for reviews, see Bouvard et al., 1994; Faulkner-Valle & Banks, 1995; Auvinen et al., 1997; DiMaio & Mattoon, 2001). The E5 reading frame is strongly transcribed in cervical intraepithelial neoplasias (CIN lesions; Stoler et al., 1992; Durst et al., 1992). Nevertheless, due to the absence of appropriate antibodies, no reliable information exists about the amount and location of the putative protein. Furthermore, E5 seems to be dispensable once malignancy has been established, since the viral DNA frequently loses the E5 coding region upon integration into the host genome (Schwarz et al., 1985; Bauer-Hofmann et al., 1996). HPV-16 E5 is a membrane protein, found mainly in the Golgi apparatus, endosomes and in small amounts at the plasma membrane (Burkhardt et al., 1989; Conrad et al., 1993; Oetke et al., 2000).
may confer resistance to ligand- or UV-mediated apoptosis (Kabsch & Alonso, 2002; Zhang et al., 2002). Furthermore, it has been demonstrated that expression of E5 results in down-regulation of MHC class I at the plasma membrane, probably due to defective transport from the Golgi apparatus (Ashrafi et al., 2001). The underlying mechanism is unknown, yet it is unlikely that it is related to an E5mediated effect on glycosylation, since E5 expression does not grossly affect cellular glycosylation (Oetke et al., 2000). Thus, it seems that most of the effects produced by E5 are mediated by membrane or membrane-associated proteins.
It has been reported that E5 is able to modulate epidermal growth factor (EGF) receptor signalling in a liganddependent manner and to abrogate gap junction-mediated cell–cell communication (Straight et al., 1993; Oelze et al., 1995; Crusius et al., 1998). Recent results suggest that E5
The human keratinocyte cell line HaCaT stably transfected with HPV16 E5 under the control of a dexamethasoneinducible promoter was used in our experiments (Oelze et al., 1995). HaCaT cells transfected with the empty vector were used as controls. Cells were grown in the presence of
0007-9844 G 2004 SGM
Printed in Great Britain
No information exists concerning modulation of the expression pattern of membrane proteins in E5-transfected or in HPV-infected keratinocytes. We therefore decided to analyse changes in the membrane protein composition in E5-expressing cells with the aim of correlating putative changes in the expression pattern with known effects of E5 on cellular physiology. This experimental approach provides an alternative to co-immunoprecipitation assays, which are difficult to perform because of the hydrophobic nature of the protein and the impossibility of producing antibodies.
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1 mM dexamethasone until confluence and serum starved for 48 h. To prepare membrane proteins, cells were lysed in a hypotonic buffer. After separation of the nuclei, membranes were pelleted by centrifugation. Proteins from pelleted membranes were extracted with Protoprep (Sigma) as described previously (Molloy et al., 1998). Soluble and insoluble proteins were separated by another round of centrifugation. This extraction method isolates not only membrane-resident proteins but also membrane-associated proteins. The soluble fraction was used for two-dimensional (2D) gel electrophoresis using pH gradients between 4 and 6. In this narrow pH range, diffusion problems are avoided, thus helping the identification and quantification of protein spots. Moreover, most of the membrane proteins of human keratinocytes identified so far have an isoelectric point (iP) in this pH interval (Gromov et al., 2002). Proteins with a molecular mass between 25 and 120 kDa were analysed. After electrophoresis, spots were visualized by silver staining and quantified by densitometry (Shevchenko et al., 1996; Gharahdagi et al., 1999).
For comparison of the protein patterns in E5-positive versus E5-negative cells, unchanged spots were taken as standards (see Table 1). Differentially expressed spots together with some spots showing similar staining were selected for further identification. Reproducibility was assessed by repeating the protein extraction and the 2D gel electrophoresis three times. Eluted proteins were identified by nanoelectrospray ionization mass spectrometry (nanoESIMS). Mass spectra were recorded using a hybrid Q-TOF mass spectrometer type Q-TOF 2 (Micromass). Spray capillaries were manufactured in house using a micropipette puller type P-87 (Sutter Instruments) and coated with a semi-transparent film of gold in a sputter coater type SCD 005 (BAL-TEC AG; Balzers). Using the automated MS to MS/MS switching option, tandem mass spectra of all peptides present in the survey spectrum were recorded. For each m/z value, tandem MS spectra were obtained by using five collision offset values to ensure formation of both sequence-specific and composition-specific fragment ions. Uninterpreted MS/MS data were used for identification via
Table 1. Identification of proteins in HPV-16 E5-expressing keratinocytes Proteins with an SSP number have been identified in the human keratinocyte IEF-database of the Danish centre for human genome research (http://biobase.dk/cgi-bin/celis). Proteins without an SSP number are given with the theoretical molecular mass. Unidentified proteins (labelled with an asterisk) are shown with iP and molecular mass values as obtained in the 2D gel electrophoresis experiments. The spots are labelled from 1 to 25. Numbers in parentheses after the spot number show the group number, as described in the text. Spot no. 1 (2) 2 (1) 3 (1) 4 (3) 5 (1) 6 (1) 7 (3) 8 (2) 9 (1) 10 (3) 11 (3) 12 13 15 16 17 18 (1) 19 20 21 (3) 22 (1) 23 (3) 24 (3) 25
SSP no.
Protein
iP
Molecular mass (kDa)
ExpressionD
9509 7316
Calreticulin precursor b-Actin b-Actin–profilin-complex Proteasomal subunit Keratin 19 B23 nucleophosmin Hsp60 Calnexin Keratin 1 Hsp70 Hsp gp96 precursor * * * * * Keratin 1, epidermolytic hyper-keratosis * * Hsp 70 kDa, mortalin-2 Keratin 9 Elongation factor 1 (potential) Hsp 60 kDa, mitochondrial precursor *
4?29 5?40 5?50 4?65 4?90 4?56 5?21 4?38 4?90 5?37 4?73 4?90 5?00 4?90 5?50 4?80 4?90 5?05 5?10 5?60 5?14 5?20 5?50 4?60
48?1 43?0 43?5 25?6 39?4 37?2 57?6 107?3 64?8 64?3 90?1 75?0 49?0 42?0 44?0 100?0 31?0 100?0 15?0 73?7 45?5 45?5 57?8 35?0
= + + + 2 2 = 2 = + = = = + = + + 2 2 2 2 2 2 +
8107 8216 8225 6403 9702 0515 5523
7420
DLevels of protein expression in E5-transfected relative to control-transfected cells.
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Differentially expressed HPV-16 membrane proteins
the internet by using the search engine Mascot (Matrix Science, London, UK). The results of the experiments are shown in Table 1. A total of 24 spots was analysed from which 16 spots could be identified by nanoESI-MS (Fig. 1a). Eight proteins could not be identified by comparison with available databases and are shown in Table 1 with the experimental iP and molecular mass values obtained. The identified proteins could be arranged into three groups according to their biological activity (labelled 1–3 in Table 1). The first group represented proteins related to the cell structure-like membrane-bound actin and cytokeratins. In this group, the expression of some proteins was increased,
whereas others were decreased or did not change their steady-state concentration. To confirm the differences scored in the electrophoresis, blots of the 2D gels were incubated with antibodies to b-actin. As shown in Fig. 1(b), E5-expressing cells contain higher amounts of membranebound b-actin compared with vector-transfected cells. These results therefore confirmed the results obtained by direct quantification of the spots in the 2D electrophoresis and indicated that our approach was suitable for further analyses (see Table 1). The second group represented proteins related to MHC class I processing proteins (calnexin, calreticulin, hsp70). In this context, the most interesting observation was the reduced content of calnexin in E5-expressing cells. Calnexin is a
HaCaT-E5 iP ...
HaCaT-pMSG
6.0
4.5 6.0
4.5
Mol. mass 105
50
30
_
+
_
+
EGF
Fig. 1. Differential expression of proteins in HPV-16 E5-expressing keratinocytes. (a) Separation of membrane-soluble proteins by 2D gel electrophoresis. Analysed proteins were labelled from 1 to 25 (see Table 1). (b) Protein extracts of E5- and vector-transfected human keratinocytes were separated on 2D gels and immunoblotted with antibodies to b-actin. (c) Transfected cells were treated or not with EGF and immunoblots were incubated with antibodies to calnexin. http://vir.sgmjournals.org
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protein involved in the formation of the MHC class I complex at the endoplasmic reticulum. To confirm the findings of the 2D electrophoresis, we prepared total protein extracts from E5- and control-transfected HaCaT cells and performed immunoblotting with an antibody against calnexin. As shown in Fig. 1(c), E5-expressing keratinocytes contained less calnexin than the control cells. Quantification of the results from four experiments showed a decrease of between 30 and 45 % in E5-expressing cells compared with controls. Interestingly, we also could demonstrate a similar behaviour in A31 embryonic cells constitutively expressing the HPV-16 E5 gene (Leechanachai et al., 1992; Pim et al., 1992), suggesting that this effect was not associated with the cell type used (data not shown). Furthermore, treatment of the cells with EGF did not have any effect on the amount of calnexin, indicating that overactivation of the EGF receptor through E5 (Straight et al., 1993; Crusius et al., 1998) is not involved in the regulation of calnexin expression at a posttranscriptional level. Incubation of the blots with an antibody against histone H1 did not reveal any differences between E5 and control cells, indicating similar loadings of the gels (results not shown). It has been reported that expression of E5 results in downregulation of membrane-associated MHC class I proteins (Ashrafi et al., 2001; Marchetti et al., 2002; Campo, 2002). Thus, it can be hypothesized that the low amount of calnexin observed in the E5 transfectants resulted in a lower level of formation of MHC class I complexes and concomitant protein degradation. A decrease in MHC class I molecules has been described in HPV-positive cervical carcinomas and pre-malignant lesions (Cromme et al., 1993; Ritz et al., 2001), where a strong transcription of the E5 reading frame has been reported (Durst et al., 1992; Stoler et al., 1992). Thus, these results suggest an active role for E5 in MHC class I down-regulation in HPV-infected cells.
the regulation/degradation of MHC proteins. These results, therefore, give a possible molecular explanation to clinical data showing down-regulation of the MHC molecules in pre-malignant or malignant tissues and strongly suggest the involvement of E5 in this process. This leads to the conclusion that the presented approach is a valuable tool for the analysis of the effects of viral proteins on host gene expression. This approach does not substitute for, but complements, the use of specific antibodies for the analysis of virus–host protein interactions.
Acknowledgements This work has been supported by a grant from the Deutsche Forschungsgemeinschaft (KF052/2-1). We thank the excellent technical assistance of Pola Linzmayer.
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