Quantitative measurement of human papillomavirus type 16 e5 oncoprotein levels in epithelial cell lines by mass spectrometry

Abstract

The high-risk human papillomavirus type 16 (HPV-16) E5 protein (16E5) induces tumors in a transgenic mouse model and may contribute to early stages of cervical carcinogenesis. Although high-risk E5 expression is generally thought to be lost during the progression to cervical carcinoma following integration of HPV DNA into the host genome, episomal viral DNA has been documented in a subset of HPV-16-positive malignant lesions. Numerous studies have shown that transcripts that could potentially encode 16E5 are present in cervical biopsy specimens and cervical cancer cell lines, but the presence of E5 protein has been demonstrated in only two reports that have not been corroborated. In the present study, we show that trypsin cleavage of 16E5 generates a unique four-amino-acid C-terminal peptide (FLIT) that serves as a marker for E5 expression in transfected cells and epithelial cell lines containing integrated and episomal HPV-16 DNA. Following trypsin cleavage, reversed-phase chromatography and mass spectrometry (MS) were used to detect FLIT. Immunoprecipitation assays using a newly generated anti-16E5 antibody confirmed that 16E5 was solely responsible for the FLIT signal, and deuterated FLIT peptide provided an internal standard that enabled us to quantify the number of 16E5 molecules per cell. We show that 16E5 is expressed in the Caski but not in the SiHa cervical cancer cell line, suggesting that 16E5 may contribute to the malignant phenotype of some cervical cancers, even in cells exclusively containing an integrated HPV genome.

Fig 1

Characterization of the FLIT peptide as a marker for 16E5. (A) Amino acid sequence of 16E5. The protein is cleaved by trypsin after the 2 arginine residues indicated in red, which generates 3 peptides, including C-terminal FLIT. (B) MRM shows breakdown of the parental FLIT ion (493.3 Da) to three major secondary ions during MS: IT (233.1 Da), FL (261.1 Da), and FLI (374.2 Da). (C) FLIT chromatogram showing all 3 transitions eluting at 24.9 min. MRM was performed on synthetic FLIT peptide that was eluted from a C₁₈ UPLC nanocolumn (15-cm length) with an increasing acetonitrile gradient.

Fig 2

Quantification of the 16E5 protein and 16E5 marker FLIT in transfected COS cells. (A) Anti-AU1 Western blot (IB) of COS cells 24 h after transfection with an empty expression vector or the same vector encoding AU1 epitope-tagged wt 16E5 or 16E5 which has been codon modified (cm) to optimize expression. (B) Chromatograms showing the D5-FLIT/IT transition for 0.5 pg of deuterated FLIT standard which was added to each of the tryptic digests described below. (C) Chromatograms showing the FLIT/IT transition for endogenous FLIT peptide in tryptic digests of COS cell lysates prepared 24 h after transfection as for panel A. Note the absence of a FLIT peak in cells transfected with the empty expression vector. Levels of 16E5 expression were calculated from the area under the peak of FLIT/IT transitions compared to the area of corresponding D5-FLIT/FLIT transitions. All retention times of the deuterated internal standards and endogenous FLIT were 24.95 ± 0.1 min (15-cm column).

Fig 3

Chromatograms of FLIT/FL and FLIT/FLI transitions for endogenous FLIT peptide in tryptic digests of COS cell lysates prepared 24 h after transfection with DNA encoding 16E5 which has been codon modified (cm) to optimize expression (A), the empty expression vector (B), or wt 16E5 (C). FLIT retention times were 25.08 ± 0.04 min (15-cm column).

Fig 4

16E5 levels in immortalized and transformed cervical cell lines. (A) Real-time PCR showing levels of E5 and E1^E4 transcripts relative to GAPDH in HPV-16-negative (C33A) and HPV-16-positive (SiHa and Caski) cervical cancer cell lines. Error bars represent means ± standard errors of the means (SEM) (n = 3). (B) Chromatograms of D5-FLIT/FLIT transitions for the deuterated internal standard added to tryptic digests of the cervical cancer cell lines in panel A and for HPV-16 E6/E7-immortalized HECs that stably express wt 16E5. (C) Chromatograms of FLIT/IT transitions for endogenous FLIT peptide in the tryptic digests. No endogenous FLIT was detected in C33A and SiHa cells. All retention times of the deuterated internal standards and endogenous FLIT were 25.08 ± 0.4 min (15-cm column).

Fig 5

Chromatograms of FLIT/FL and FLIT/FLI transitions for endogenous FLIT peptide in tryptic digests of C33A (A), SiHa (B), Caski (C), and HPV-16 E6/E7-immortalized HECs that stably express wt 16E5 (D). FLIT retention times were 25.10 ± 0.4 min (15-cm column).

Fig 6

Immunoprecipitation confirms that the MS-detected FLIT peptide in Caski cells originates from 16E5. (A) 16E5 antiserum immunoprecipitates the 16E5 protein. COS cells were transfected with an empty expression vector or with the same vector encoding 16E5 with or without an AU1 epitope tag. Twenty-four hours later, the cells were lysed and immunoprecipitations (IP) were performed (on equal amounts of cell protein) using 16E5 antiserum or preimmune serum from the same rabbit (P.I.). Immunoprecipitated proteins were analyzed on Western blots labeled with an anti-AU1 monoclonal antibody (IB: AU1). (B to F) 16E5 antiserum “clears” Caski cell lysates of FLIT peptide. A Caski cell lysate was digested with trypsin and analyzed by UPLC and MS without immunoprecipitation (B) or following immunoprecipitation with 16E5 antiserum (C) or preimmune serum (E). Proteins immunoprecipitated by the 16E5 antiserum (D) or preimmune serum (F) were eluted, digested with trypsin, and analyzed by UPLC and MS. The black bar indicates the signal intensity of the blank (no sample).

Fig 7

Levels of 16E5 expressed from epithelial cell lines harboring episomal HPV-16 genomes. (A) Chromatograms of D5-FLIT/FLIT transitions for deuterated internal standard added to tryptic digests of parental NIKS cells or NIKS-4H and NIKS-2L clonal cell lines harboring episomal copies of the HPV-16 genome. (B) Chromatograms of FLIT/IT transitions for endogenous FLIT peptide in the tryptic digests. No endogenous FLIT was detected in the parental NIKS cells. All retention times of the deuterated internal standards and endogenous FLIT were 28.88 ± 0.1 min (25-cm column).

Fig 8

Chromatograms of FLIT/FL and FLIT/FLI transitions for endogenous FLIT peptide in tryptic digests of parental NIKS cells (A) or NIKS-4H (B) and NIKS-2L (C) clonal cell lines harboring episomal copies of the HPV-16 genome. FLIT retention times were 28.93 ± 0.1 min (25-cm column).