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Mob DNA. 2019 Dec 31;11:1. doi: 10.1186/s13100-019-0191-2. eCollection 2020.

LINE-1 ORF2p expression is nearly imperceptible in human cancers.

Author information

1
1McKusick Nathans Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.
2
2Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.
3
3Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, NYU School of Medicine, New York, NY 10016 USA.
4
4Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY 10065 USA.
5
5Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114 USA.
6
6Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY 10065 USA.
7
7Moscow Institute of Physics and Technology, Dolgoprudny, 141701 Russia.
8
8European Research Institute for the Biology of Ageing, University Medical Center Groningen, Groningen, 9713 AV The Netherlands.
#
Contributed equally

Abstract

Background:

Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF1p protein expression, and somatic L1 retrotransposition. ORF2p encodes the endonuclease and reverse transcriptase activities required for L1 retrotransposition. Its expression is poorly characterized in human tissues and cell lines.

Results:

We report mass spectrometry-based tumor proteome profiling studies wherein ORF2p eludes detection. To test whether ORF2p could be detected with specific reagents, we developed and validated five rabbit monoclonal antibodies with immunoreactivity for specific epitopes on the protein. These reagents readily detect ectopic ORF2p expressed from bicistronic L1 constructs. However, endogenous ORF2p is not detected in human tumor samples or cell lines by western blot, immunoprecipitation, or immunohistochemistry despite high levels of ORF1p expression. Moreover, we report endogenous ORF1p-associated interactomes, affinity isolated from colorectal cancers, wherein we similarly fail to detect ORF2p. These samples include primary tumors harboring hundreds of somatically acquired L1 insertions. The new data are available via ProteomeXchange with identifier PXD013743.

Conclusions:

Although somatic retrotransposition provides unequivocal genetic evidence for the expression of ORF2p in human cancers, we are unable to directly measure its presence using several standard methods. Experimental systems have previously indicated an unequal stoichiometry between ORF1p and ORF2p, but in vivo, the expression of these two proteins may be more strikingly uncoupled. These findings are consistent with observations that ORF2p is not tolerable for cell growth.

Conflict of interest statement

Competing interestsJohns Hopkins University has licensed these LINE-1 monoclonal ORF2p antibodies for commercialization to Abcam (Cambridge, UK). D.A., M.S.T., and K.H.B. will receive royalties from sales. K.H.B. and M.S.T. receive royalties from sales of a LINE-1 ORF1p monoclonal antibody licensed to EMD Millipore by Johns Hopkins University.

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