Development of a monoclonal antibody specific to the endonuclease domain of the human LINE-1 ORF2 protein

Sokolowski, Mark J.; DeFreece, Cecily; Servant, Géraldine; Kines, Kristine J.; deHaro, Dawn; Belancio, Victoria P.

doi:10.1186/preaccept-2133002533141022

Cited by 3 publications

(4 citation statements)

References 53 publications

(84 reference statements)

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“…western blotting, immunoprecipitation, immunohistochemistry, and immunofluorescence demonstrate the utility of these reagents in experimental systems with ectopic LINE-1 expression. However, we cannot detect endogenous ORF2p using these approaches, corroborating previous studies by Belancio [30].…”

Section: Introductionsupporting

confidence: 92%

“…To our knowledge, to date, two groups have independently reported development of monoclonal antibodies recognizing human ORF2p, both using BALB/c mice. One reagent, developed by Belancio and colleagues [30], was reported to provide detection of ectopically-expressed ORF2p only. A second reagent, developed by Sciamanna, Spadafora, and colleagues [31], was reported to detect endogenous ORF2p in several malignant tissues where ORF1p expression has been reported.…”

Section: Introductionmentioning

confidence: 99%

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LINE-1 ORF2p Expression is Nearly Imperceptible in Human Cancers

Ardeljan

Wang

Oghbaie

et al. 2019

Preprint

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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. References

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Section: Introductionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

LINE-1 ORF2p Expression is Nearly Imperceptible in Human Cancers

Ardeljan

Wang

Oghbaie

et al. 2019

Preprint

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“…Although L1ORF1p is readily detectable [25,27,32], L1ORF2p is very challenging to detect in vitro and in vivo. As the recombinant ORF2p doesn’t express well, short synthetic peptides from the entire length of L1ORF2p protein sequence have been mainly used for the generation of L1ORF2p antibody[28,29, 33–35]. In the past, a number of polyclonal and monoclonal antibodies against human and mouse L1-ORF2p have been reported[28,29,32–35].…”

Section: Discussionmentioning

confidence: 99%

Robust expression of LINE-1 retrotransposon encoded proteins in oral squamous cell carcinoma

Mukherjee

Sur

Singh³

et al. 2020

Preprint

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Retrotransposons are sequences which transpose within genomes using RNA as an intermediate. Long INterpersed Element-1 (LINE1 or L1) is the only active retrotransposon occupying around 17% of the human genome with an estimated 500,000 copies. An active L1 encodes two proteins (L1ORF1p and L1ORF2p); both of which are critical in the process of retrotransposition. In-order to propagate to the nextgeneration, L1s remain active in germ tissues and at an early stage of development. Surprisingly, by some unknown mechanism, L1 also shows activity in certain parts of the normal brain and many cancers. L1 activity is generally determined by assaying L1ORF1p because of its high expression and availability of the antibody. However, due to its lowerexpression and the unavailability of a robust antibody, detection of L1ORF2p has been limited. L1ORF2p is the crucial protein in the process of retrotransposition as it provides endonuclease and reverse transcriptase (RT) activity. Here, we report a novel human L1ORF2p antibody generated using an 80-amino-acid stretch from the RT domain, which is highly conserved among different species. The antibody detects significant L1ORF2p expression in murine germ tissues and human oral squamous cell carcinoma (OSCC) samples. This particular cancer is prevalent in India due to excessive use of tobacco. Here, using our in-house antibodies against L1 proteins, we show that more than fifty percent of samples are positive for L1 proteins. Overall, we reported a novel L1ORF2p antibody that detects L1 activity in germ tissues and OSCC

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“…While several specific antibodies against ORF1p have been raised (Taylor et al, 2013, Wylie et al, 2016, Doucet-O'Hare et al, 2015, a highly effective antibody against ORF2 protein that would allow definitive observation of protein localization is still lacking. Antibodies against LINE-1 ORF2p have been recently developed (De Luca et al, 2016, Sokolowski et al, 2014 but the much lower amount of expressed ORF2p compared to ORF1p makes the study of ORF2p expression and localization difficult. To overcome these difficulties, tagged ORF2ps have been employed (Taylor et al, 2013, Doucet et al, 2010.…”

Section: Introductionmentioning

confidence: 99%

LINE-1 and the cell cycle: protein localization and functional dynamics

Mita¹,

Wudzinska²,

Sun³

et al. 2017

Preprint

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LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology to aid its proliferation. However, a clear understanding of L1's lifecycle and the processes involved in restricting its insertion and its intragenomic spreading remains elusive. Here we identify modes of L1 proteins' entrance into the nucleus, a necessary step for L1 proliferation. Using functional, biochemical, and imaging approaches, we also show a clear cell cycle bias for L1 retrotransposition that peaks during the S phase. Our observations provide a basis for novel interpretations about the nature of nuclear and cytoplasmic L1 ribonucleoproteins (RNPs) and the potential role of DNA replication in L1 retrotransposition.

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Development of a monoclonal antibody specific to the endonuclease domain of the human LINE-1 ORF2 protein

Cited by 3 publications

References 53 publications

LINE-1 ORF2p Expression is Nearly Imperceptible in Human Cancers

LINE-1 ORF2p Expression is Nearly Imperceptible in Human Cancers

Robust expression of LINE-1 retrotransposon encoded proteins in oral squamous cell carcinoma

LINE-1 and the cell cycle: protein localization and functional dynamics

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