Abstract
In this study we report the cloning and characterization of a novel human aminopeptidase, which we designate leukocyte-derived arginine aminopeptidase (L-RAP). The sequence encodes a 960-amino acid protein with significant homology to placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase. The predicted L-RAP contains the HEXXH(X)18E zinc-binding motif, which is characteristic of the M1 family of zinc metallopeptidases. Phylogenetic analysis indicates that L-RAP forms a distinct subfamily with placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in the M1 family. Immunocytochemical analysis indicates that L-RAP is located in the lumenal side of the endoplasmic reticulum. Among various synthetic substrates tested, L-RAP revealed a preference for arginine, establishing that the enzyme is a novel arginine aminopeptidase with restricted substrate specificity. In addition to natural hormones such as angiotensin III and kallidin, L-RAP cleaved various N-terminal extended precursors to major histocompatibility complex class I-presented antigenic peptides. Like other proteins involved in antigen presentation, L-RAP is induced by interferon-gamma. These results indicate that L-RAP is a novel aminopeptidase that can trim the N-terminal extended precursors to antigenic peptides in the endoplasmic reticulum.
Highlights
In this study we report the cloning and characterization of a novel human aminopeptidase, which we designate leukocyte-derived arginine aminopeptidase (LRAP)
Phylogenetic analysis indicates that leukocyte-derived arginine (R) aminopeptidase (L-RAP) forms a distinct subfamily with placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in the M1 family
It should be noted that a sequence in which eight nucleotides and one amino acid are different from L-RAP was submitted to the data bank as mouse- and human-specific aminopeptidase
Summary
P-LAP, placental leucine aminopeptidase; L-RAP, leukocyte-derived arginine aminopeptidase; A-LAP, adipocyte-derived leucine aminopeptidase; ER, endoplasmic reticulum; MHC, major histocompatibility complex; IFN, interferon; ERAP, endoplasmic reticulum-aminopeptidase; PSA, puromycin-sensitive aminopeptidase; aminoacyl-MCA, aminoacyl-4-methylcoumaryl-7-amide; HA, hemagglutinin; EST, expressed sequence tag; PBS, phosphatebuffered saline; HPLC, high pressure liquid chromatography; HEK, human embryonic kidney; HIV-1, human immunodeficiency virus, type 1. The peptides are first cleaved from endogenously synthesized proteins by proteasome or tripeptidyl peptidase II in the cytoplasm, transported into ER lumen, and trimmed by aminopeptidase (28 –30). Our results indicate that like A-LAP/ ERAP1, L-RAP is an aminopeptidase normally retained in the ER and trims certain precursors to MHC class I-presented antigenic peptides
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