From the ‡Laboratory of Cellular Biochemistry and �Division of Biomolecular Characterization, RIKEN, Wako-shi,Saitama 351-0198, ¶Laboratory of Applied Protein Chemistry, Department of Agriculture, Tokyo University of Agricultureand Technology, Fuchu, Tokyo 183-0054, and **Department of Obstetrics and Gynecology, Nagoya University School ofMedicine, Showa, Nagoya 466-8550, Japan
In this study we report the cloning and characteriza-tion of a novel human aminopeptidase, which we desig-nate leukocyte-derived arginine aminopeptidase (L-RAP). The sequence encodes a 960-amino acid proteinwith significant homology to placental leucine amin-opeptidase and adipocyte-derived leucine aminopepti-dase. The predicted L-RAP contains the HEXXH(X)18Ezinc-binding motif, which is characteristic of the M1family of zinc metallopeptidases. Phylogenetic analysisindicates that L-RAP forms a distinct subfamily withplacental leucine aminopeptidase and adipocyte-de-rived leucine aminopeptidase in the M1 family. Immu-nocytochemical analysis indicates that L-RAP is locatedin the lumenal side of the endoplasmic reticulum.Among various synthetic substrates tested, L-RAP re-vealed a preference for arginine, establishing that theenzyme is a novel arginine aminopeptidase with re-stricted substrate specificity. In addition to natural hor-mones such as angiotensin III and kallidin, L-RAPcleaved various N-terminal extended precursors to ma-jor histocompatibility complex class I-presented anti-genic peptides. Like other proteins involved in antigenpresentation, L-RAP is induced by interferon-�. Theseresults indicate that L-RAP is a novel aminopeptidasethat can trim the N-terminal extended precursors toantigenic peptides in the endoplasmic reticulum.
Aminopeptidases hydrolyze N-terminal amino acids of pro-teins or peptide substrates. They are distributed widely inanimal and plant tissues, as well as in bacteria and fungi,suggesting that they play important roles in various biologicalprocesses. They are essential for protein maturation, the acti-vation, modulation, and degradation of bioactive peptides, andthe determination of protein stability (1). In addition, severalaminopeptidases are known as differentiation antigens andcontrol cell proliferation and differentiation (2–5).
In our previous work, we cloned a cDNA for the placentalleucine aminopeptidase (P-LAP)1/oxytocinase, a type II mem-brane-spanning protein that belongs to the M1 zinc metal-lopeptidase (gluzincin) family (6). Subsequently we cloned acDNA encoding adipocyte-derived leucine aminopeptidase(A-LAP), which is also designated as puromycin-insensitiveleucine-specific aminopeptidase or endoplasmic reticulum(ER)-aminopeptidase (ERAP)-1, as a highly homologous pro-tein to P-LAP (7–9). Gluzincin aminopeptidases share the con-sensus HEXXH(X)18E zinc-binding motif essential for enzy-matic activity (10). This growing family of mammalian zinc-containing aminopeptidase includes membrane-bound (P-LAP,aminopeptidase A, aminopeptidase N, and thyrotropin-releas-ing hormone degrading enzyme) (3, 4, 6, 11, 12), cytosolic(puromycin-sensitive aminopeptidase (PSA) and leukotriene A4
hydrolase) (13, 14), secretory (aminopeptidase B) (15), and ERresident (A-LAP/ERAP1) (9, 16) proteins. Mutational analysesrevealed that essential amino acid residues are well conservedamong members of the family (17–20).
Recent evidence facilitates new insights into the biologicalsignificance of the M1 family of aminopeptidases. It was re-ported that aminopeptidase A plays a role in the regulation ofblood pressure by regulating the renin-angiotensin system inthe brain (21). P-LAP/oxytocinase, which is also designated asinsulin-regulated aminopeptidase, was shown to be the angio-tensin IV receptor and may play a role in the memory retentionand retrieval (22). Recently, we and others reported thatA-LAP/ERAP1 is a final processing enzyme of the precursors ofmajor histocompatibility complex (MHC) class I-presented an-tigenic peptides (9, 16). This enzyme was also shown to playroles in blood pressure regulation and angiogenesis (23, 24). Itwas also reported that PSA gene-deficient mice show dwarfismand display increased anxiety and analgesia (25). Furthermore,Osada et al. (26, 27) reported the roles of PSA in reproductionprocess. These results indicate that the mammalian aminopep-tidases belonging to the M1 family of metallopeptidases playroles in the regulation of important biological processes.
Precursors to MHC class I-presented peptides with extraN-terminal residues are trimmed to mature epitopes in the ER.* This work was supported in part by grants-in-aid from the Ministry
of Education, Science, Sports and Culture of Japan and a grant for“Chemical Biology Research Program” from RIKEN. The costs of pub-lication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submittedto the DDBJ/GenBankTM/EBI Data Bank with accession number(s)AB109031.
§ To whom correspondence should be addressed: Laboratory of Cel-lular Biochemistry, RIKEN, The Institute of Physical and ChemicalResearch, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. Tel.:81-48-467-9515; Fax: 81-48-462-4670; E-mail: [email protected].
1 The abbreviations used are: P-LAP, placental leucine aminopepti-dase; L-RAP, leukocyte-derived arginine aminopeptidase; A-LAP, adi-pocyte-derived leucine aminopeptidase; ER, endoplasmic reticulum;MHC, major histocompatibility complex; IFN, interferon; ERAP, endo-plasmic reticulum-aminopeptidase; PSA, puromycin-sensitive ami-nopeptidase; aminoacyl-MCA, aminoacyl-4-methylcoumaryl-7-amide;HA, hemagglutinin; EST, expressed sequence tag; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; HEK,human embryonic kidney; HIV-1, human immunodeficiency virus,type 1.
The peptides are first cleaved from endogenously synthesizedproteins by proteasome or tripeptidyl peptidase II in the cyto-plasm, transported into ER lumen, and then trimmed by amin-opeptidase (28–30). Although A-LAP/ERAP1 was shown to bethe trimming aminopeptidase in the ER, another enzyme isexpected, because there remains trimming activity after re-moval of A-LAP/ERAP1 from ER lumenal protein fraction (9).
In an effort to elucidate the biological significance of the M1family of aminopeptidases further, we have cloned in this studya novel member of this family, termed leukocyte-derived argi-nine (R) aminopeptidase (L-RAP) by searching a data base forhomologous protein to P-LAP and A-LAP. Enzymatic analysesusing synthetic substrates indicate that the enzyme has uniquesubstrate specificity and preferentially cleaves N-terminal ba-sic amino acids. Moreover, our results indicate that like A-LAP/ERAP1, L-RAP is an aminopeptidase normally retained in theER and trims certain precursors to MHC class I-presentedantigenic peptides.
EXPERIMENTAL PROCEDURES
Cloning and Sequencing of the Human L-RAP cDNA—The partialnucleotide sequences of cDNA encoding peptides with homology toP-LAP and A-LAP were found in the EST data base of GenBankTM byuse of the TBLASTN program. The 337-base pair fragment from theEST clone (GenBankTM accession number AA361076) was used as aprobe to screen a human leukocyte cDNA library (Invitrogen). Thelibrary were plated at a density of �5 � 103 colony-forming units/100-mm plate. They were transferred to a nylon membrane (Biodyne B;Pall, East Hills, NY) and then hybridized using 32P-labeled probe asdescribed. DNA sequence was determined using a Taq dye terminatorcycle sequencing kit and an Applied Biosystems model 377 DNAsequencer.
Expression of L-RAP in HeLa S3 Cells—The SalI-NotI fragment ofthe L-RAP-HA or L-RAP(s)-HA cDNA was inserted into the pSport-1vector (Invitrogen). For transfection experiments, HeLa S3 cells platedin 24-well plates were grown to subconfluency in Dulbecco’s modifiedEagle’s medium containing 10% calf serum. Then the cells were trans-fected with either pSport-1/L-RAP-HA or pSport-1/L-RAP(s)-HA em-ploying LipofectAMINE and Plus reagent (Invitrogen) according to themanufacturer’s instruction.
Production of Recombinant L-RAP—The cDNA encoding the signalsequence of bombyxin (i.e. MKILLATALMLSTVMWVSTA) followed bythe open reading frame of L-RAP starting at Ala56 was ligated into theEcoRI sites of pFastbac1 vector. Recombinant baculovirus producingL-RAP was prepared using Bac-to-Bac system (Invitrogen) according tothe manufacturer’s instruction. Then High Five cells (Invitrogen) in-fected with the virus were cultured for 72 h in 3 liters of ExpressFiveserum-free medium (Invitrogen) using a Cellmaster 1700 large scaleculture system (Wakenyaku, Kyoto, Japan) supplied with 8 ppm ofoxygen. Culture medium was collected by centrifugation.
Preparation of Antibody against L-RAP—Synthetic peptides corre-sponding to the C-terminal 12 amino acids of L-RAP including cysteinelinker (i.e. LC12, CLPTLRTWLMVNT) and C-terminal 19 amino acidsof L-RAP(s) (i.e. SC19, CHSDPKMTSNMVRIKRVTE) were coupled tokeyhole limpet hemocyanin according to the method described previ-ously and injected into rabbits for antibody production following stand-ard procedures. Namely, the conjugates mixed with Freund’s completeadjuvant were injected intraperitoneally (500 �g of peptide), followedby an injection every 7 days over a 3-week period of the conjugate (250�g of peptide) with Freund’s complete adjuvant.
Western Blot Analysis—Test samples were separated by SDS-PAGEon an 8% separating gel and transferred to polyvinylidene difluoridemembranes (Pall). The membranes were blocked with Tris/HCl-buff-ered saline (NaCl/Tris) (pH 7.4) containing 0.1% Tween 20 (NaCl/Tris/Tween), 5% skimmed milk for 1 h at room temperature and thenincubated in NaCl/Tris/Tween, 5% skimmed milk, and 2.5 �g/ml rabbitanti-L-RAP antibody for 2 h at room temperature. The filter waswashed three times with NaCl/Tris/Tween and incubated for 1 h withhorseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Pro-mega, Madison, WI), diluted to 1/20,000 in NaCl/Tris/Tween containing5% skimmed milk. After washing the filter three times with NaCl/Tris/Tween, the blots were detected by an enhanced chemiluminescencemethod using an ECL plus Western blotting kit obtained from Amer-sham Biosciences. The results were visualized by fluorography usingRX-U Fuji medical x-ray film.
Immunocytochemical Analysis—HeLa S3 cells grown on a cover glasswere washed three times with PBS and fixed with 4% paraformalde-hyde in PBS for 10 min at room temperature. Cells were then perme-abilized in PBS solution containing either 0.2% Triton X-100 or 25�g/ml digitonin for 2 min. Coverslips were blocked for 1 h with PBScontaining 3% bovine serum albumin (blocking buffer) and incubatedfor 1.5 h at room temperature with 5 �g/ml of affinity-purified ratanti-HA antibody and 5 �g/ml of mouse anti-KDEL monoclonal anti-body in blocking buffer. The cells were then washed six times with PBSand incubated with 0.4 �g/ml of Alexa Fluor 488-labeled goat anti-ratIgG antibody and Alexa Fluor 594-labeled goat anti-mouse IgG anti-body in blocking buffer for 1 h. After washing with PBS six times, cellswere mounted in a drop of PermaFluor aqueous mounting medium(Immunon, Pittsburgh, PA) and viewed with a Leica TCS NT laserscanning microscope (Leica, Wetzlar, Germany).
Isolation of Lumenal Contents—To obtain cell-free extracts, Jurkat-Tcells were suspended in SET buffer (250 mM sucrose, 1 mM EDTA, 20mM Tris/HCl (pH 7.4) containing 100 �M phenylmethylsulfonyl fluo-ride), lysed at 4 °C in a Potter-Elvehjem glass Teflon-type tissue grinderand centrifuged for 10 min at 1,000 � g to remove nuclei and cell debris.The resulting lysate was centrifuged for 1 h at 100,000 � g at 4 °C toseparate microsomal fraction from cytosolic fraction. The microsomallumenal contents were separated by the method described previouslywith slight modification (9). In brief, the lumenal contents were re-leased with 1 mg/ml of digitonin in SET buffer (pH 7.4) and thenseparated from the microsomal membranes by ultracentrifugation(400,000 � g, 4 °C for 1 h). Membrane fraction was then resuspended inSDS sample buffer to obtain the membrane proteins.
Measurement of Aminopeptidase Activity—Aminopeptidase activitywas determined with various fluorogenic substrates, aminoacyl-4-methylcoumaryl-7-amide (aminoacyl-MCAs). The reaction mixture con-taining 50 �M aminoacyl-MCA and enzyme preparation in 0.2 ml of 25mM Tris/HCl buffer (pH 7.5) was incubated at 37 °C for 10 min. Thereaction was terminated by adding 1 ml of 0.1 M sodium acetate buffer(pH 4.3) containing 0.1 M sodium monochloroacetate. The released7-amino-4-methyl-coumarin was measured by spectrofluorophotometry(F-2000; Hitachi) at an excitation wavelength of 360 nm and an emis-sion wavelength of 460 nm. Error bars indicate S.E. values from fourindividual experiments.
Cleavage of Peptides by L-RAP—Peptide (25 �M) were incubated with2 �g/ml of L-RAP at 37 °C in 25 mM Tris/HCl buffer (pH 7.5). Thereaction was terminated by the addition of 2.5% (v/v) formic acid.Generated peptides were separated by reverse-phase HPLC on a DO-COSIL B (1 � 100 mm) column (Senshu, Tokyo, Japan) using aHewlett-Packard HP 1100 HPLC system (Agilent Technologies, PaloAlto, CA). Peptides were loaded onto the column equilibrated in 0.09%(v/v) trifluoroacetic acid. Elution of peptides were performed with alinear gradient of 0.09% (v/v) trifluoroacetic acid to 48% (v/v) acetoni-trile in 0.081% trifluoroacetic acid in 30 min at flow rate of 0.05 ml/min.The molecular masses of the peptides were determined by an LCQ iontrap mass spectrometer (Finnigan, San Jose, CA).
Materials—S-Benzyl-Cys-, -Asp-, -Gln-, -Glu-, and -Gly-MCAs werepurchased from BACHEM AG (Bubendorf, Switzerland). Arg-, Lys-,Ala-, Leu-, Phe-, and Met-MCAs were from Peptide Institute (Osaka,Japan). Recombinant human interferon (IFN)-� was obtained fromPeproTech (Rocky Hill, NJ). Glycosidases were purchased from NewEngland Biolabs (Beverly, MA). Protease inhibitors were all obtainedfrom Sigma. Peptide hormones were all obtained from Peptide Insti-tute. Antigenic peptides were synthesized and kindly provided by theRIKEN Brain Science Institute. The anti-HA monoclonal antibody wasobtained from Roche Diagnostics. The monoclonal antibodies againstER retention signal KDEL of BiP and calnexin were purchased fromStressGen Bioreagents (Victoria, British Columbia, Canada) and BDBiosciences, respectively.
RESULTS
Molecular Cloning of cDNA for L-RAP—To identify se-quences similar to the human P-LAP/oxytocinase gene, wesearched public data bases of ESTs. Two EST clones(AA361076 and AA356078) derived from human T cell lym-phoma were shown to encode novel peptides with significantamino acid homology to P-LAP. A full-length clone was thenisolated from a human leukocyte cDNA library. Because theprotein encoded by the cDNA was cloned from leukocyte cDNAlibrary, we termed it L-RAP. We also cloned a cDNA encodinga truncated form of L-RAP and designated as L-RAP(s). It
should be noted that a sequence in which eight nucleotides andone amino acid are different from L-RAP was submitted to thedata bank as mouse- and human-specific aminopeptidase (Gen-
BankTM accession number AF191545).The cloned L-RAP cDNA is �3.3 kb long. Fig. 1 shows the
nucleotide sequence and the deduced amino acid sequence of
FIG. 1. Nucleotide sequence of thecDNA and the deduced amino acidsequence of human L-RAP. Nucleotideresidues are numbered from 5� to 3� withthe first residue of the ATG codon encod-ing the putative initiating methionine.The deduced amino acid sequence is dis-played below the nucleotide sequence as aone-letter code starting from the methio-nine. The N-terminal hydrophobic regionis underlined, the potential glycosylationsites are circled, the HEXXH motif andconserved glutamic acid are boxed, andGAMEN motif is shadowed.
FIG. 2. Similarity of L-RAP with other members of oxytocinase subfamily. A, alignment of L-RAP and L-RAP(s) proteins with othermembers of the oxytocinase subfamily. The deduced amino acid sequences of L-RAP and L-RAP(s) are aligned with the sequences of humanP-LAP/insulin-regulated aminopeptidase and A-LAP/ERAP1. Gaps are inserted into the sequences for optimal alignment. Residues conservedamong the enzymes are shadowed. Asterisks indicate the essential residues for the hydrolytic activity of the enzymes so far reported. B,phylogenetic analysis of the nucleotide sequences of human aminopeptidases belonging to the M1 family of aminopeptidases. The distance betweenenzymes is proportional to the number of nucleotides and reflects evolutionary time since their divergence.
the cDNA. The cDNA contains a full-length open readingframe, ending with a TAA stop codon at nucleotides 2881–2883.The first ATG triplet (starting at nucleotide 1) was putativelyconsidered to be the initiation codon of the protein translation,because the 5�-untranslated region preceding this codon con-tains an in-frame stop codon. Taken together, the cDNA con-tains an open reading frame of 2880 bp flanked by a 150-bp5�-untranslated region and a 303-bp 3�-untranslated region.
The predicted translation product of the L-RAP cDNA en-codes a protein of 960 amino acids (including the initiatormethionine) with a calculated molecular mass of 122,933 Daand contains nine potential N-glycosylation sites. Hydropathyanalysis using the Kyte and Doolittle algorithm (31) showedthat as in the case of P-LAP and A-LAP, the enzyme carries asignificantly hydrophobic region near the N terminus thatcould function as an internal signal peptide and a membrane-spanning domain.
The deduced amino acid sequence contains typical consensusmotifs of the zinc metallopeptidase family. There is an essen-tial zinc-binding site (HEXXH) at amino acid residues 370–374with a second glutamic acid separated by 18 amino acids. Thesequence contains another sequence motif, GAMEN, at aminoacid residues 334–338. These two motifs are found in severalaminopeptidases and allow the classification of the L-RAP inthe M1 family of metallopeptidases (17, 19).
Sequence Comparison and Phylogenetic Relationship—Acomputer search revealed that the amino acid sequence ofL-RAP has apparent homology to P-LAP and A-LAP (Fig. 2A).Overall sequence identities of L-RAP to P-LAP and A-LAP were43 and 49%, respectively, whereas other members of the M1family of aminopeptidases show less (20–30%) homology toL-RAP. The identity between these three enzymes extends overtheir entire primary structures. Especially sequences aroundtwo conserved motifs (HEXXH(X)18E and GAMEN) show highdegrees of homology. As for amino acid residues critical for theenzyme activity, all but one in L-RAP (i.e. His is replaced byArg434) are conserved.
To gain better insights into the relationships among the M1family of aminopeptidases, a phylogenetic tree was constructedby the method of Higgins and Sharp (32) based on the nucleo-tide sequence identities (Fig. 2B). According to this tree, it isapparent that L-RAP, P-LAP, and A-LAP belong to one distinc-tive group.
Tissue and Subcellular Localization of L-RAP—The tissuedistribution of L-RAP was determined by Northern blot anal-ysis using the full-length cDNA as a probe (Fig. 3). Expressionof mRNA with size of 6.7 kb was observed in every tissuetested, suggesting its ubiquitous distribution. It is notable herethat relatively high expression of L-RAP mRNA was observedin spleen and leukocytes.
To examine subcellular localization of L-RAP, L-RAP taggedwith Influenza virus HA epitope at the C-terminal end (L-RAP-HA) was expressed in HeLa S3 cells (Fig. 4A). HeLa S3 cellstransfected with constructs encoding L-RAP-HA were sub-
jected to immunocytochemical analysis in the presence of 0.2%Triton X-100, which permeabilizes all cellular membranes non-selectively. A reticular, perinuclear staining pattern character-istic of the ER was observed. In addition, co-localization of
FIG. 3. Northern blot analysis of poly(A)� RNA from varioushuman tissues. A human adult tissue Northern blot (BD Biosciencesand Clontech) was probed with 32P-labeled cDNA.
FIG. 4. Subcellular localization of L-RAP. A, HeLa S3 cells weretransfected with L-RAP-HA and subjected to the immunocytochemical anal-ysis employing antibodies against HA-tag and KDEL in the presence of 0.2%Triton X-100 or 5 �g/ml digitonin. B, localization of endogenous L-RAP inJurkat-T cells. The cell lysate was first centrifuged at 100,000 � g to preparethe cytosolic and microsomal fractions. The lumenal contents were releasedfrom membrane fractions with digitonin and separated from microsomalmembranes by further centrifugation at 400,000 � g. C, glycosidase treat-ment of endogenous L-RAP in Jurkat-T cells. Total cell lysate (10 �g) wasdenatured in 0.5% SDS and 1% 2-mercaptoethanol at 100 °C for 10 min andthen the sample was treated either with peptide:N-glycosidase F (PNGaseF) or endoglycosidase H (Endo H) (New England Biolabs) for 24 h at 37 °Cand subjected to Western blot analysis.
L-RAP-HA with the ER retention signal sequence KDEL wasclearly observed when merged. On the other hand, when thecells were treated with 5 �g/ml of digitonin to permeabilizeplasma membrane selectively (33), neither L-RAP-HA norKDEL immunoreactivity was detected. For comparison, weexamined the localization of I�B, a cytosolic marker protein,and its immunoreactivity was detected after treatment witheither Triton X-100 or digitonin (data not shown). Nearly thesame results were obtained when the subcellular localization ofL-RAP(s)-HA was examined in HeLa S3 cells (data not shown).These results strongly suggest that both L-RAP and L-RAP(s)are localized in the ER, and their C-terminal ends are locatedin the lumenal side.
We next examined the ER localization of endogenous L-RAPin Jurkat-T cells to determine whether the enzyme is a solubleor membrane-bound lumenal ER protein. As shown in Fig. 4B,both L-RAP and L-RAP(s) are fractionated mainly into thesoluble fraction of lumenal contents. As expected, whereas BiP,which contains the KDEL sequence, was also obtained in thesoluble fraction of lumenal contents, calnexin, ER membrane-bound protein, was detected in the microsomal membrane frac-tion. These results suggest that L-RAP and L-RAP(s) are sol-uble proteins located in the lumenal side of the ER. When
examined the localization in HEK 293 cells, L-RAP is detectedin the lumenal side of the ER (data not shown). As expected,L-RAP endogenously expressed in Jurkat-T cells are sensitiveto endoglycosidase H and peptide:N-glycosidase F (Fig. 4C),indicating that the proteins contain high mannose-type sugarchains.
Characterization of Aminopeptidase Activity of L-RAP—Todetect the aminopeptidase activity of L-RAP and L-RAP(s),HEK 293 cells were transiently transfected with either L-RAPor L-RAP(s) expression vector controlled under cytomegalovi-rus promoter. As in the case of A-LAP, both proteins weresecreted into culture medium in this condition. As shown inFig. 5A, aminopeptidase activity against Arg-MCA was clearlydetected in the medium of L-RAP-expressing cells. Although aconsiderable amount of L-RAP(s) was detected in the mediumof L-RAP(s)-expressing cells by Western blot analysis, littleArg-MCA degrading activity was detected. These results sug-gest that whereas L-RAP shows aminopeptidase activity, L-RAP(s) has little (if any) activity.
To further characterize the enzymatic properties of L-RAP,we established a large scale production system of the recombi-nant protein using a baculovirus system (Fig. 5B). L-RAP waspurified to a single band of 115 kDa on SDS-PAGE from the
FIG. 5. Enzymatic characterization of recombinant human L-RAP. A, aminopeptidase activity of L-RAP using Arg-MCA as a substrate.HEK 293 cells were transfected with either L-RAP or L-RAP(s) and cultured for 48 h at 37 °C. Aminopeptidase activity was measured as describedunder “Experimental Procedures” after collection of supernatant. For comparison, supernatant of mock-transfected cells was also assayed. B,SDS-PAGE analysis of purified recombinant human L-RAP. C, substrate specificity of L-RAP toward synthetic substrates. Purified L-RAP (2�g/ml) was incubated with various aminoacyl-MCA substrates (25 �M) to assess peptidase activity. D, effects of various inhibitors on L-RAPactivity. Aminopeptidase activity was measured using Arg-MCA as a substrate in the presence of various concentrations of inhibitors. o-PNT,1,10-phenanthroline.
culture medium. We then measured the relative hydrolyticactivity of the enzyme toward various aminoacyl-MCAs. Asshown in Fig. 5C, Arg-MCA was hydrolyzed most efficiently,followed by Lys-MCA. The calculated Km, kcat, and kcat/Km
values from a Lineweaver-Burk plot of L-RAP activity towardArg-MCA were 3.18 �M, 2.30/s, and 7.25 � 105/M�1s�1, respec-tively. To other synthetic substrates tested, L-RAP had littleactivity. These results indicate that L-RAP preferentially hy-drolyzes basic N-terminal amino acids.
The effects of various known inhibitors of aminopeptidaseson the hydrolytic activity of L-RAP toward Arg-MCA are shownin Fig. 5D. Although amastatin was a potent inhibitor of theenzyme, bestatin was less active. Puromycin, a specific inhibi-tor of PSA, had little activity toward the enzyme. Of the che-lating agents tested, 1,10-phenanthroline effectively inhibitedthe enzyme activity, but EDTA had no effect up to 1 mM. It isnoteworthy here that the inhibitor profile of L-RAP is consist-ent with those of putative ER resident aminopeptidases, whichtrim the MHC class I-presented antigenic precursor peptides(34).
Next, we searched for L-RAP-mediated degradation of natu-ral hormones to estimate the physiological role of the enzyme.Among hormones tested, the enzyme cleaved kallidin and an-giotensin III (Fig. 6). No hydrolytic activity was observed to-ward oxytocin, vasopressin, and angiotensin II. These resultsindicate that the enzyme indeed exerts the aminopeptidaseactivity toward certain naturally occurring peptides, and itssubstrate specificity is different from P-LAP and A-LAP.
Trimming of Precursors of Antigenic Peptides by L-RAP—Because L-RAP is a lumenal ER protein, we next examinedwhether L-RAP is a trimming enzyme of antigenic precursorpeptides. For this purpose, we first examined the effects ofIFN-� on the expression of L-RAP proteins. It is well knownthat IFN-� induces many components of the MHC class I-me-diated antigen presentation pathway (28, 30). As shown in Fig.7A, treatment of Jurkat-T and HEK 293 cells with IFN-� en-hanced the L-RAP mRNA expression. On the other hand, themRNA expression was barely detectable in U937 and HeLa S3cells, and no induction was observed after the IFN-� treatment.Kinetic analysis indicated that in Jurkat-T cells, the expressionlevel of the mRNA reached the highest 12 h after treatmentand then declined to the basal level at 48 h (Fig. 7B). We alsomeasured the expression level of L-RAP protein in Jurkat-Tand HEK 293 cells by Western blot analysis using LC12 anti-body (Fig. 7C). When the cells were treated with IFN-�, theexpression of L-RAP protein was increased up to day 3.
To estimate the contribution of L-RAP to the Arg-MCA de-grading activity in the ER, we examined the effects of loss ofthe enzyme (Fig. 7D). Although repeated treatment of thesample with anti-L-RAP LC12 antiserum, significant activitywas still observed, suggesting that there is Arg-MCA degradingactivity not attributable to L-RAP in the lumenal fraction.Besides this basal activity, Arg-MCA degrading activity thatcould be depleted by the antiserum was clearly observed, sug-gesting that about 30% of the activity is attributable to L-RAP.As expected, treatment of IFN-� caused the increase in theArg-MCA degrading activity sensitive to the antiserum. Animmunodepletion experiment indicated that IFN-� treatmentcaused about 2-fold increase in L-RAP activity. Residual activ-ity after immunodepletion might reflect the basal activity up-regulated by IFN-�. Alternatively there might be another Arg-MCA degrading activity in the lumenal fraction sensitive toIFN-�. Preimmune serum had no effect on the Arg-MCA de-grading activity in the fractions (data not shown). Taken to-gether, our results indicate that L-RAP can be up-regulated
in several cells by IFN-�, and a significant portion of theArg-MCA degrading activity is attributable to the enzyme inthe ER.
We then incubated the recombinant enzyme with NH2-ex-tended precursor of HIV-1 nef antigen (35). As shown in Fig.8A, the enzyme efficiently removed N-terminal residues fromNH2-extended antigen precursors (NYTPGPGVRY) to generatethe final epitope, TPGPGVRY. When RU1 antigen precursor(TAVPYGSFKHV) was examined (36), final antigen epitope(VPYGSFKHV) was generated (data not shown). These resultssuggest that the enzyme reaction does not proceed if proline islocated at the P2� site. We also examined another NH2-ex-tended antigen precursor of melanoma-associated protein gp100 antigen (FTITDQVPFSV) (37). As shown in Fig. 8B, tran-sient accumulation of the final epitope of the antigen (IT-DQVPFSV) was observed although the further degradation ofthe epitope occurred. These results suggest that like A-LAP/ERAP1, L-RAP can act as an antigen-trimming enzyme andaccumulate MHC class-I-presented antigenic peptides in theER lumen.
DISCUSSION
In this study, we have cloned a cDNA encoding a novelaminopeptidase, L-RAP. Like other M1 families of aminopep-tidases, predicted L-RAP contains three domains: a 14-aminoacid N-terminal domain, a 22-amino acid hydrophobic domain,and a 924-amino acid C-terminal domain. In the C-terminaldomain, the cDNA predicts a protein that contains the HEXXH
FIG. 6. Cleavage of natural substrates by recombinant humanL-RAP. A, cleavage of angiotensin III (Ang III). Angiotensin III (25 �M)was incubated with L-RAP (2 �g/ml) at 37 °C for the indicated times. B,conversion of kallidin to bradykinin. Kallidin (25 �M) was incubatedwith L-RAP (2 �g/ml) at 37 °C for the indicated times. After the reactionwas terminated by adding 2.5% (v/v) formic acid, the generated peptideswere separated and then their structures were determined as describedunder “Experimental Procedures.”
consensus sequence of zinc metallopeptidases with an addi-tional glutamic acid residue 18 amino acids away, which con-stitute the active site of metallopeptidases. In addition, anotherconsensus motif, GAMEN, which is considered to determinethe exopeptidase specificity of gluzincin aminopeptidases, islocated in N-terminal side of the HEXXH motif. These twomotifs allow L-RAP to be classified into the M1 family of gluz-incin aminopeptidases (10, 17, 19).
Eight mammalian aminopeptidases belonging to the M1family are identified, and L-RAP is the ninth member of thefamily. Phylogenetic analysis indicates that P-LAP, A-LAP,and L-RAP form a distinct subfamily of the M1 family. More-over, these three human genes are located contiguously aroundchromosome 5q15 (38). These results strongly suggest the lat-est diversion of these genes from a single ancestral gene, andtherefore we propose here that P-LAP, A-LAP, and L-RAPshould be classified into the oxytocinase subfamily of M1aminopeptidases.
It has been recognized that the gluzincin aminopeptidasesinclude membrane-bound, cytosolic, secretory, and ER residentproteins. Among them, the subcellular localization of amin-opeptidases belonging to the oxytocinase subfamily is ratherunique. P-LAP is located in some intracellular vesicles, andstimulus-dependent translocation of the enzyme into theplasma membrane was observed in several biological systems(39–43). It is generally believed that the physiological signifi-cance of P-LAP translocation is to enhance the cleavage ofpeptide hormone substrates at the cell surface (44). A-LAP/ERAP1 is the first aminopeptidase localized and functions asan antigen-trimming enzyme in the lumenal side of the ER (9,16). Our results in this study indicate that L-RAP endog-enously expressed in Jurkat-T cells and HEK 293 cells is also a
soluble protein localized and functions in the ER lumen. Pre-vious work shows that arginine degrading activity was clearlyseparated from leucine degrading activity in the ER lumenalprotein prepared from rat liver (9). The unique localization ofthe enzymes may suggest the biological functions of the oxyto-cinase subfamily to be strictly regulated. Indeed, it is gettingevident that the enzymes belonging to this subfamily playimportant roles in the maintenance of homeostasis such asblood pressure regulation, angiogenesis, and maintenance ofmemory (22–24).
When overexpressed in HEK 293 cells by the high expressionvector controlled under cytomegalovirus promoter, L-RAP wasdetected in the medium. Overexpression of A-LAP in COS-7cells also caused the secretion of the enzyme into the culturemedium. It is plausible that when expressed highly, both A-LAP and L-RAP are secreted into the culture medium. There-fore it is tempting to speculate that some binding protein(s)such as a KDEL-containing protein or an ER membrane com-ponent may act as retention machinery for the enzymes. Mostprobably, saturation of this putative machinery may cause thesecretion of the enzyme (45).
We characterized the enzymatic properties of L-RAP for thefirst time and found that the substrate specificity of L-RAPtoward synthetic substrates is unique in its apparent prefer-ence for arginine. Another substrate cleaved by the enzyme isLys-MCA. These results clearly indicate that the substratespecificity of L-RAP is rather restricted, and it preferentiallycleaves N-terminal basic amino acids. Because the enzyme
FIG. 7. Effect of IFN-� on L-RAP expression in cultured cells. A,induction of L-RAP transcripts by IFN-�. Jurkat-T cells and HEK 293cells were incubated with or without IFN-� (30 ng/ml) for 12 h at 37 °C.Expression level of L-RAP transcript was measured by Northern blotanalysis. Each lane contained 6 �g of total RNA. B, kinetic analysis.Jurkat-T cells were incubated with IFN-� (30 ng/ml) for indicated timesat 37 °C. Northern blot analysis of L-RAP and �-actin transcripts wasthen performed. C, induction of L-RAP protein by IFN-�. Jurkat-T cellsand HEK 293 cells were incubated with IFN-� (30 ng/ml) for indicatedtimes at 37 °C. Expression level of L-RAP protein was measured byWestern blot analysis. Each lane contained 10 �g of protein. D, meas-urement of L-RAP activity in the ER. After treatment of Jurkat-T cellswith IFN-� for 2 days, ER lumenal proteins were extracted from micro-somes of control or IFN-�-treated cells with digitonin. L-RAP wasimmunodepleted three times with anti-L-RAP LC12 antiserum, andresidual Arg-MCA degrading activity was measured. SDS-PAGE anal-ysis of L-RAP protein after immunodepletion is also shown. IP,immunoprecipitation.
FIG. 8. Trimming of precursors of antigenic peptides by L-RAP. A, generation of HIV-1 nef antigen by L-RAP. NH2-extendedversion of HIV-1 nef antigen (NYTPGPGVRY) (25 �M) was incubatedwith L-RAP (2 �g/ml) at 37 °C for the indicated times. B, generation ofmelanoma-associated protein gp 100 antigen by L-RAP. NH2-extendedversion of melanoma-associated protein gp 100 antigen (FTIT-DQVPFSV) (25 �M) was incubated with L-RAP (2 �g/ml) at 37 °C for theindicated times. After incubation, generated peptides were analyzed asdescribed under “Experimental Procedures.”
hydrolyzed Arg-MCA most preferentially, we termed it an ar-ginine (R) aminopeptidase.
Searching for natural substrates, we found that the enzymecleaves angiotensin III and kallidin. Considering that A-LAP,which cleaves angiotensin II and III and kallidin, regulates bloodpressure (24, 46), it is tempting to speculate that L-RAP alsoplays a role in the regulation of blood pressure by inactivatingangiotensin III and generating bradykinin. Putative ER reten-tion machinery may play a role in the biological function ofsubstrate peptides by regulating the secretion of the enzyme.However, it is necessary to identify natural substrates further toelucidate the physiological and/or pathological roles of theenzyme.
Although substrate specificity of L-RAP toward synthetic sub-strates is rather restricted, L-RAP could process several anti-genic precursors of MHC class I-presented antigen having vari-ous N-terminal residues like A-LAP (9, 46). Several enzymeswere purified either from cytoplasm or ER lumen as trimmingenzymes. It now well recognized that the precursors to MHCclass-I-presented peptides with extra N-terminal residues aretrimmed to mature epitopes in the ER, and A-LAP/ERAP1 is thefirst enzyme responsible for the processing in the ER (9, 16). Onthe other hand, cytoplasmic enzymes such as PSA, lens leucineaminopeptidase, bleomycin hydrolase, and thimet oligopeptidaseare thought to contribute to limit antigen presentation in vivo bydestroying precursor peptides (34, 47–49).
L-RAP fulfills several criteria for the trimming enzyme ofprecursor peptides so far reported (34). 1) It is a metallopeptidaseinhibited efficiently by 1,10-phenanthroline but not by EDTA. 2)It is located in the ER lumen. 3) Its expression in certain cells isenhanced by IFN-�. 4) It can trim certain antigenic precursors toMHC class I-presented antigen in vitro. 5) It hydrolyzes variousN-terminal amino acids of peptide hormones and antigenic pre-cursors except when proline is located at the P2� site. Takentogether, these results strongly suggest that L-RAP can act as atrimming enzyme of precursors of antigenic peptides presented toMHC class I molecules in the ER. Broad specificity of L-RAPsuits its role in cleaving a wide spectrum of peptides for MHCclass I molecule. Because L-RAP is often expressed in cells lack-ing A-LAP/ERAP1 expression such as Jurkat-T cells, it is plau-sible that L-RAP and A-LAP/ERAP1 compensate each other forantigen presentation activity.
In summary, we have cloned a novel aminopeptidase, L-RAP,which belongs to the M1 family of metallopeptidases. The ge-netic and evolutionary analyses led us to propose the oxytoci-nase subfamily of M1 aminopeptidases, which include P-LAP/oxytocinase/insulin-regulated aminopeptidase, A-LAP/ERAP1,and L-RAP. In addition, we have presented a variety of circum-stantial evidence that suggest the role of L-RAP as an antigen-trimming enzyme. Considering the recent progress of the bio-logical significance of the subfamily members, it is important toexamine the in vivo role of L-RAP in future studies.
Acknowledgments—We are grateful for N. Hirotani of AdvancedTechnology Development Center, RIKEN Brain Institute for synthesisof peptides used in this study.
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