Exon 10 Coding Sequence Is Important for Endoplasmic Reticulum Retention of Endoplasmic Reticulum Aminopeptidase 1

Abstract

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a final trimming enzyme of N-extended antigenic peptides bound to major histocompatibility complex class I molecules in the endoplasmic reticulum (ER). In our previous work, we found that ERAP1 is secreted from macrophages in response to activation by lipopolysaccharide and interferon-γ. In this paper, we searched for the amino acid sequence of ERAP1 protein important for ER retention by constructing chimeric proteins and found that the sequence between 485 and 615 was significant. Moreover, by comparing the genomic organizations of oxytocinase subfamily members, the exon 10 coding sequence, which might be inserted into the common ancestral gene of the oxytocinase subfamily enzymes during evolution, was found to be important for ER retention of ERAP1. Taken together, our data indicate that ERAP1 contains amino acid sequence important for ER retention.Key words aminopeptidase; oxytocinase subfamily; endoplasmic reticulum; chimeric protein Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an aminopeptidase belonging to the M1 family and is initially cloned by searching in a database for adipocyte-derived leucine aminopeptidase (A-LAP).1,2) ERAP1 is also designated puromycin-insensitive leucine specific aminopeptidase (PILSAP), endoplasmic reticulum (ER) aminopeptidase associated with antigen presentation (ERAAP), and aminpeptidase regulator of tumor necrosis factor receptor type 1 shedding (ARTS-1).3-5) From structural and phylogenetic analyses, ERAP1 is most closely related to placental leucine aminopeptidase (P-LAP)/insulin-regulated aminopeptidase (IRAP)/ oxytocinase and ERAP2, also designated as leukocyte-derived arginine aminopeptidase (L-RAP). Therefore, we propose that these three enzymes should be classified into "the oxytocinase subfamily of M1 aminopeptidases," which share the HEXXH(X) 18 E Zn 2+ -binding motif and GAMEN substrate recognition motif. 6)It is well established that ERAP1 is localized in the ER lumen as a soluble protein and functions as a final processing enzyme of antigenic peptides presented to major histocompatibility complex class I molecules.2,4) On the other hand, we found in a recent work that ERAP1 is secreted from macrophages in response to lipopolysaccharide and interferon-γ and enhances their phagocytic activity. 7) Our results suggest that secreted ERAP1 plays a role in the defense mechanisms of infectious diseases via activation of macrophages; thus, it is important to elucidate the ER retention mechanism of the enzyme.In the current study, we searched for the amino acid sequence of ERAP1 required for ER retention. By constructing expression vectors for ERAP1/P-LAP chimeric proteins, we have identified a sequence required for ER retention, the exon 10 coding sequence of ERAP1. Our results suggest that insertion of the sequences corresponding to exon 10 of the ERAP1 gene into the common ancestral gene of the oxytocinase subfamily enzymes occurred during evolution, which provides a scaffold for its unique property of ER retention....

TLR-Mediated Secretion of Endoplasmic Reticulum Aminopeptidase 1 from Macrophages

“…In our previous work, we reported that the exon 10 coding sequence of ERAP1 was critical for its ER retention (38). Certain proteins binding to the exon 10 sequence might be required for the ER retention of ERAP1.…”

Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2

“…As a result, it has been hypothesized that they are retained through the interaction with other ER resident proteins (4). In ERAP1, a 23-amino acid sequence encoded by exon10 of the gene and predicted to be unstructured was initially implicated in that function (45). Recently, Erp44, an ER resident protein disulfide isomerase, has been shown to retain ERAP1 inside the ER through the formation of a mixed disulfide bond with a cysteine residue in the exon 10 loop (46).…”

Endoplasmic Reticulum Aminopeptidase 1 beyond Antigenic Peptide-Processing Enzyme in the Endoplasmic Reticulum

“…GXMEN and HEXXH motifs have been reported to be encoded by exon 6 while 19 amino acids downstream from the HEXXH motif encoded by exon 7 is the glutamic acid residue, which together with the two histidine residues in HEXXH motif binds the zinc ion. 12) Phylogenetic analysis of M1-aminopeptidase family also implies a divergence in ERAP from P-LAP/IRAP 2) ( Fig. 1).…”

“…2). While we identified and initially designated these two isoforms as A-LAPa and A-LAPb, 12) another group termed them as ERAP1b and ERAP1a, respectively. 14) To avoid nomenclature confusion, the isoforms encoded by these mRNAs were re-designated as ERAP1-19E and ERAP1-20E, respectively.…”