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

Mark Sokolowski,Dawn L Deharo,Victoria P Belancio,Cecily B Defreece,Kristine J Kines,Geraldine Servant

doi:10.1186/s13100-014-0029-x

Abstract

BackgroundLINE-1 (L1) retrotransposons are common occupants of mammalian genomes representing about a fifth of the genetic content. Ongoing L1 retrotransposition in the germ line and somatic tissues has contributed to structural genomic variations and disease-causing mutations in the human genome. L1 mobilization relies on the function of two, self-encoded proteins, ORF1 and ORF2. The ORF2 protein contains two characterized domains: endonuclease and reverse transcriptase.ResultsUsing a bacterially purified endonuclease domain of the human L1 ORF2 protein, we have generated a monoclonal antibody specific to the human ORF2 protein. We determined that the epitope recognized by this monoclonal antibody includes amino acid 205, which is required for the function of the L1 ORF2 protein endonuclease. Using an in vitro L1 cleavage assay, we demonstrate that the monoclonal anti-ORF2 protein antibody partially inhibits L1 endonuclease activity without having any effect on the in vitro activity of the human AP endonuclease.ConclusionsOverall, our data demonstrate that this anti-ORF2 protein monoclonal antibody is a useful tool for human L1-related studies and that it provides a rationale for the development of antibody-based inhibitors of L1-induced damage.Electronic supplementary materialThe online version of this article (doi:10.1186/s13100-014-0029-x) contains supplementary material, which is available to authorized users.

Highlights

LINE-1 (L1) retrotransposons are common occupants of mammalian genomes representing about a fifth of the genetic content
Western blot analysis using this custom ORF2p monoclonal antibody detected a product of the expected size in the clarified lysate and the final elution of the human EN protein used for inoculation (Figure 1B, ORF2 monoclonal panel)
Plasmids encoding mouse codon-optimized full-length ORF2 or ORF2 endonuclease sequences were transiently transfected into 293 cells and total cellular lysates were analyzed by SDS-PAGE followed by immunoblotting with the anti-ORF2p monoclonal antibody

Summary

Introduction

LINE-1 (L1) retrotransposons are common occupants of mammalian genomes representing about a fifth of the genetic content. Functional full-length L1 transcripts contain two open reading frames (ORFs) encoding ORF1 and ORF2 proteins (ORF1p and ORF2p, respectively) (Figure 1A). L1, Alu, and SVA form ribonucleoprotein (RNP) particles which reach the nucleus to complete their replication cycles by integrating in the host genome via a process of target-primed reverse transcription [9,10]. This copy-and-paste process has produced approximately 500,000 L1 loci, accounting for about 17% of the human genome, and over 1,000,000 copies of Alu, which comprise about 11% of our genome [11]. The majority of the L1 loci are 5′ truncated with about 80 to 100 full-length L1 copies demonstrated to be retrotranspositionally active [12,13,14,15,16]

Methods

Results

Discussion

Conclusion