Arginyl aminopeptidase-like 1 (RNPEPL1): from hypothetical reading frame toward functional characterization

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Maynard, Karen Beasley
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Middle Tennessee State University
Arginyl aminopeptidase-like 1 (RNPEPL1) was first reported as a hypothetical reading frame during various genomic studies to complete the human genome. This study aimed to move the gene from hypothetical status to verified, as well as move toward establishing a biological role or function in inflammation based on gene family similarity.
First, this investigation confirmed the gene was transcribed and the transcript encoded a functional enzyme with M1 aminopeptidase characteristics. RNA was expressed in 20 human tissues, with three splice variants being identified in the process. Protein was expressed, purified, and characterized. RNPEPL1 protein did have enzymatic activity with a high specificity for cleaving methionine, citrulline, and glutamine N-terminal residues. The enzyme also demonstrated a broad pH range and similar inhibitor sensitivities to other members of the M1 family.
The M1 aminopeptidase family was then studied in depth to identify conservations within the gene structures that may indicate potential functional role(s) of RNPEPL1. This study followed the twelve human members, eleven mouse members, and eleven rat members of the M1 aminopeptidase family through multiple alignments, phylogenetic trees, synteny studies, and exon structure analyses. All members shared the conserved HEXXHX<sub>18</sub>E and G/AMEN domains crucial for inclusion into the family. Phylogenetic analyses at the amino acid level indicated the presence of three groups within the M1 family. The first group contained three genes (LTA<sub>4</sub>H, RNPEP, and RNPEPL1), while the second group contained five highly related genes (ENPEP, ANPEP, APQ, TRHDE, and NPEPPS), and the final group contained three related genes (LNPEP, ERAP1, and ERAP2). ERAP2, which was only found in humans, resulted in a synteny study among the three species. Results suggested that ERAP2 may have been destroyed during a chromosomal rearrangement in the rat/mouse lineage, among others. Finally, exon structure analyses showed high conservation of most of the second and third groups, with the LTA<sub>4</sub>H group appearing to have diverged earlier.
Due to the clustering of RNPEPL1 with the LTA<sub>4</sub>H family in the above studies, it was possible that RNPEPL1, like RNPEP and LTA<sub>4</sub>H, might have a role in inflammation. To test this, RNPEPL1 expression levels were analyzed to determine what changes, if any, were present in cells, blood plasma, and tissue sections undergoing an inflammatory response. Results indicated that RNPEPL1 expression does decline between 25% - 30% after 24 hours of treatment.
In conclusion, RNPEPL1 is a true gene that transcribes a functional M1 aminopeptidase. This family is one of great divergence, indicating that it is an old gene family. RNPEPL1 is best placed within the LTA<sub>4</sub>H group, in which the other members play various roles in inflammation. RNPEPL1 does decrease significantly during inflammatory response, however the change is slight.