JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M305076200 on June 10, 2003

J. Biol. Chem., Vol. 278, Issue 34, 32275-32283, August 22, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/34/32275    most recent
M305076200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tanioka, T.
Right arrow Articles by Tsujimoto, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tanioka, T.
Right arrow Articles by Tsujimoto, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Human Leukocyte-derived Arginine Aminopeptidase

THE THIRD MEMBER OF THE OXYTOCINASE SUBFAMILY OF AMINOPEPTIDASES*

Toshihiro Tanioka {ddagger}, Akira Hattori {ddagger} §, Shinako Masuda {ddagger}, Yoshihiro Nomura {ddagger} ¶, Hiroshi Nakayama ||, Shigehiko Mizutani ** and Masafumi Tsujimoto {ddagger}

From the {ddagger}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 Agriculture and Technology, Fuchu, Tokyo 183-0054, and **Department of Obstetrics and Gynecology, Nagoya University School of Medicine, Showa, Nagoya 466-8550, Japan

Received for publication, May 14, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Aminopeptidases hydrolyze N-terminal amino acids of proteins or peptide substrates. They are distributed widely in animal and plant tissues, as well as in bacteria and fungi, suggesting that they play important roles in various biological processes. They are essential for protein maturation, the activation, modulation, and degradation of bioactive peptides, and the determination of protein stability (1). In addition, several aminopeptidases are known as differentiation antigens and control cell proliferation and differentiation (2–5).

In our previous work, we cloned a cDNA for the placental leucine aminopeptidase (P-LAP)1/oxytocinase, a type II membrane-spanning protein that belongs to the M1 zinc metallopeptidase (gluzincin) family (6). Subsequently we cloned a cDNA encoding adipocyte-derived leucine aminopeptidase (A-LAP), which is also designated as puromycin-insensitive leucine-specific aminopeptidase or endoplasmic reticulum (ER)-aminopeptidase (ERAP)-1, as a highly homologous protein to P-LAP (7–9). Gluzincin aminopeptidases share the consensus HEXXH(X)18E zinc-binding motif essential for enzymatic activity (10). This growing family of mammalian zinc-containing aminopeptidase includes membrane-bound (P-LAP, aminopeptidase A, aminopeptidase N, and thyrotropin-releasing hormone degrading enzyme) (3, 4, 6, 11, 12), cytosolic (puromycin-sensitive aminopeptidase (PSA) and leukotriene A4 hydrolase) (13, 14), secretory (aminopeptidase B) (15), and ER resident (A-LAP/ERAP1) (9, 16) proteins. Mutational analyses revealed that essential amino acid residues are well conserved among members of the family (17–20).

Recent evidence facilitates new insights into the biological significance of the M1 family of aminopeptidases. It was reported that aminopeptidase A plays a role in the regulation of blood pressure by regulating the renin-angiotensin system in the brain (21). P-LAP/oxytocinase, which is also designated as insulin-regulated aminopeptidase, was shown to be the angiotensin IV receptor and may play a role in the memory retention and retrieval (22). Recently, we and others reported that A-LAP/ERAP1 is a final processing enzyme of the precursors of major histocompatibility complex (MHC) class I-presented antigenic peptides (9, 16). This enzyme was also shown to play roles in blood pressure regulation and angiogenesis (23, 24). It was also reported that PSA gene-deficient mice show dwarfism and display increased anxiety and analgesia (25). Furthermore, Osada et al. (26, 27) reported the roles of PSA in reproduction process. These results indicate that the mammalian aminopeptidases belonging to the M1 family of metallopeptidases play roles in the regulation of important biological processes.

Precursors to MHC class I-presented peptides with extra N-terminal residues are trimmed to mature epitopes in the ER. The peptides are first cleaved from endogenously synthesized proteins by proteasome or tripeptidyl peptidase II in the cytoplasm, transported into ER lumen, and then trimmed by aminopeptidase (28–30). Although A-LAP/ERAP1 was shown to be the trimming aminopeptidase in the ER, another enzyme is expected, because there remains trimming activity after removal of A-LAP/ERAP1 from ER lumenal protein fraction (9).

In an effort to elucidate the biological significance of the M1 family of aminopeptidases further, we have cloned in this study a novel member of this family, termed leukocyte-derived arginine (R) aminopeptidase (L-RAP) by searching a data base for homologous protein to P-LAP and A-LAP. Enzymatic analyses using synthetic substrates indicate that the enzyme has unique substrate specificity and preferentially cleaves N-terminal basic amino acids. Moreover, 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning and Sequencing of the Human L-RAP cDNA—The partial nucleotide sequences of cDNA encoding peptides with homology to P-LAP and A-LAP were found in the EST data base of GenBankTM by use of the TBLASTN program. The 337-base pair fragment from the EST clone (GenBankTM accession number AA361076 [GenBank] ) was used as a probe to screen a human leukocyte cDNA library (Invitrogen). The library were plated at a density of ~5 x 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 as described. DNA sequence was determined using a Taq dye terminator cycle sequencing kit and an Applied Biosystems model 377 DNA sequencer.

Expression of L-RAP in HeLa S3 Cells—The SalI-NotI fragment of the L-RAP-HA or L-RAP(s)-HA cDNA was inserted into the pSport-1 vector (Invitrogen). For transfection experiments, HeLa S3 cells plated in 24-well plates were grown to subconfluency in Dulbecco's modified Eagle's medium containing 10% calf serum. Then the cells were transfected with either pSport-1/L-RAP-HA or pSport-1/L-RAP(s)-HA employing LipofectAMINE and Plus reagent (Invitrogen) according to the manufacturer's instruction.

Production of Recombinant L-RAP—The cDNA encoding the signal sequence of bombyxin (i.e. MKILLATALMLSTVMWVSTA) followed by the open reading frame of L-RAP starting at Ala56 was ligated into the EcoRI sites of pFastbac1 vector. Recombinant baculovirus producing L-RAP was prepared using Bac-to-Bac system (Invitrogen) according to the manufacturer's instruction. Then High Five cells (Invitrogen) infected with the virus were cultured for 72 h in 3 liters of ExpressFive serum-free medium (Invitrogen) using a Cellmaster 1700 large scale culture system (Wakenyaku, Kyoto, Japan) supplied with 8 ppm of oxygen. Culture medium was collected by centrifugation.

Preparation of Antibody against L-RAP—Synthetic peptides corresponding to the C-terminal 12 amino acids of L-RAP including cysteine linker (i.e. LC12, CLPTLRTWLMVNT) and C-terminal 19 amino acids of L-RAP(s) (i.e. SC19, CHSDPKMTSNMVRIKRVTE) were coupled to keyhole limpet hemocyanin according to the method described previously and injected into rabbits for antibody production following standard procedures. Namely, the conjugates mixed with Freund's complete adjuvant were injected intraperitoneally (500 µg of peptide), followed by an injection every 7 days over a 3-week period of the conjugate (250 µg of peptide) with Freund's complete adjuvant.

Western Blot Analysis—Test samples were separated by SDS-PAGE on an 8% separating gel and transferred to polyvinylidene difluoride membranes (Pall). The membranes were blocked with Tris/HCl-buffered saline (NaCl/Tris) (pH 7.4) containing 0.1% Tween 20 (NaCl/Tris/Tween), 5% skimmed milk for 1 h at room temperature and then incubated in NaCl/Tris/Tween, 5% skimmed milk, and 2.5 µg/ml rabbit anti-L-RAP antibody for 2 h at room temperature. The filter was washed three times with NaCl/Tris/Tween and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Promega, Madison, WI), diluted to 1/20,000 in NaCl/Tris/Tween containing 5% skimmed milk. After washing the filter three times with NaCl/Tris/Tween, the blots were detected by an enhanced chemiluminescence method using an ECL plus Western blotting kit obtained from Amersham Biosciences. The results were visualized by fluorography using RX-U Fuji medical x-ray film.

Immunocytochemical Analysis—HeLa S3 cells grown on a cover glass were washed three times with PBS and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Cells were then permeabilized in PBS solution containing either 0.2% Triton X-100 or 25 µg/ml digitonin for 2 min. Coverslips were blocked for 1 h with PBS containing 3% bovine serum albumin (blocking buffer) and incubated for 1.5 h at room temperature with 5 µg/ml of affinity-purified rat anti-HA antibody and 5 µg/ml of mouse anti-KDEL monoclonal antibody in blocking buffer. The cells were then washed six times with PBS and incubated with 0.4 µg/ml of Alexa Fluor 488-labeled goat anti-rat IgG antibody and Alexa Fluor 594-labeled goat anti-mouse IgG antibody in blocking buffer for 1 h. After washing with PBS six times, cells were mounted in a drop of PermaFluor aqueous mounting medium (Immunon, Pittsburgh, PA) and viewed with a Leica TCS NT laser scanning microscope (Leica, Wetzlar, Germany).

Isolation of Lumenal Contents—To obtain cell-free extracts, Jurkat-T cells were suspended in SET buffer (250 mM sucrose, 1 mM EDTA, 20 mM Tris/HCl (pH 7.4) containing 100 µM phenylmethylsulfonyl fluoride), lysed at 4 °C in a Potter-Elvehjem glass Teflon-type tissue grinder and centrifuged for 10 min at 1,000 x g to remove nuclei and cell debris. The resulting lysate was centrifuged for 1 h at 100,000 x g at 4 °C to separate microsomal fraction from cytosolic fraction. The microsomal lumenal contents were separated by the method described previously with slight modification (9). In brief, the lumenal contents were released with 1 mg/ml of digitonin in SET buffer (pH 7.4) and then separated from the microsomal membranes by ultracentrifugation (400,000 x g,4 °C for 1 h). Membrane fraction was then resuspended in SDS sample buffer to obtain the membrane proteins.

Measurement of Aminopeptidase Activity—Aminopeptidase activity was determined with various fluorogenic substrates, aminoacyl-4-methylcoumaryl-7-amide (aminoacyl-MCAs). The reaction mixture containing 50 µM aminoacyl-MCA and enzyme preparation in 0.2 ml of 25 mM Tris/HCl buffer (pH 7.5) was incubated at 37 °C for 10 min. The reaction was terminated by adding 1 ml of 0.1 M sodium acetate buffer (pH 4.3) containing 0.1 M sodium monochloroacetate. The released 7-amino-4-methyl-coumarin was measured by spectrofluorophotometry (F-2000; Hitachi) at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Error bars indicate S.E. values from four individual experiments.

Cleavage of Peptides by L-RAP—Peptide (25 µM) were incubated with 2 µg/ml of L-RAP at 37 °C in 25 mM Tris/HCl buffer (pH 7.5). The reaction 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 x 100 mm) column (Senshu, Tokyo, Japan) using a Hewlett-Packard HP 1100 HPLC system (Agilent Technologies, Palo Alto, CA). Peptides were loaded onto the column equilibrated in 0.09% (v/v) trifluoroacetic acid. Elution of peptides were performed with a linear gradient of 0.09% (v/v) trifluoroacetic acid to 48% (v/v) acetonitrile 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 ion trap mass spectrometer (Finnigan, San Jose, CA).

Materials—S-Benzyl-Cys-, -Asp-, -Gln-, -Glu-, and -Gly-MCAs were purchased from BACHEM AG (Bubendorf, Switzerland). Arg-, Lys-, Ala-, Leu-, Phe-, and Met-MCAs were from Peptide Institute (Osaka, Japan). Recombinant human interferon (IFN)-{gamma} was obtained from PeproTech (Rocky Hill, NJ). Glycosidases were purchased from New England Biolabs (Beverly, MA). Protease inhibitors were all obtained from Sigma. Peptide hormones were all obtained from Peptide Institute. Antigenic peptides were synthesized and kindly provided by the RIKEN Brain Science Institute. The anti-HA monoclonal antibody was obtained from Roche Diagnostics. The monoclonal antibodies against ER retention signal KDEL of BiP and calnexin were purchased from StressGen Bioreagents (Victoria, British Columbia, Canada) and BD Biosciences, respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Molecular Cloning of cDNA for L-RAP—To identify sequences similar to the human P-LAP/oxytocinase gene, we searched public data bases of ESTs. Two EST clones (AA361076 [GenBank] and AA356078 [GenBank] ) derived from human T cell lymphoma were shown to encode novel peptides with significant amino acid homology to P-LAP. A full-length clone was then isolated from a human leukocyte cDNA library. Because the protein encoded by the cDNA was cloned from leukocyte cDNA library, we termed it L-RAP. We also cloned a cDNA encoding a truncated form of L-RAP and designated as L-RAP(s). 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 (GenBankTM accession number AF191545 [GenBank] ).

The cloned L-RAP cDNA is ~3.3 kb long. Fig. 1 shows the nucleotide sequence and the deduced amino acid sequence of the cDNA. The cDNA contains a full-length open reading frame, ending with a TAA stop codon at nucleotides 2881–2883. The first ATG triplet (starting at nucleotide 1) was putatively considered to be the initiation codon of the protein translation, because the 5'-untranslated region preceding this codon contains an in-frame stop codon. Taken together, the cDNA contains an open reading frame of 2880 bp flanked by a 150-bp 5'-untranslated region and a 303-bp 3'-untranslated region.



View larger version (55K):
[in this window]
[in a new window]
  		FIG. 1.
Nucleotide sequence of the cDNA and the deduced amino acid sequence of human L-RAP. Nucleotide residues are numbered from 5' to 3' with the first residue of the ATG codon encoding the putative initiating methionine. The deduced amino acid sequence is displayed below the nucleotide sequence as a one-letter code starting from the methionine. The N-terminal hydrophobic region is underlined, the potential glycosylation sites are circled, the HEXXH motif and conserved glutamic acid are boxed, and GAMEN motif is shadowed.

 

The predicted translation product of the L-RAP cDNA encodes a protein of 960 amino acids (including the initiator methionine) with a calculated molecular mass of 122,933 Da and contains nine potential N-glycosylation sites. Hydropathy analysis using the Kyte and Doolittle algorithm (31) showed that as in the case of P-LAP and A-LAP, the enzyme carries a significantly hydrophobic region near the N terminus that could function as an internal signal peptide and a membrane-spanning domain.

The deduced amino acid sequence contains typical consensus motifs of the zinc metallopeptidase family. There is an essential zinc-binding site (HEXXH) at amino acid residues 370–374 with a second glutamic acid separated by 18 amino acids. The sequence contains another sequence motif, GAMEN, at amino acid residues 334–338. These two motifs are found in several aminopeptidases and allow the classification of the L-RAP in the M1 family of metallopeptidases (17, 19).

Sequence Comparison and Phylogenetic Relationship—A computer search revealed that the amino acid sequence of L-RAP has apparent homology to P-LAP and A-LAP (Fig. 2A). Overall sequence identities of L-RAP to P-LAP and A-LAP were 43 and 49%, respectively, whereas other members of the M1 family of aminopeptidases show less (20–30%) homology to L-RAP. The identity between these three enzymes extends over their entire primary structures. Especially sequences around two conserved motifs (HEXXH(X)18E and GAMEN) show high degrees of homology. As for amino acid residues critical for the enzyme activity, all but one in L-RAP (i.e. His is replaced by Arg434) are conserved.



View larger version (72K):
[in this window]
[in a new window]
  		FIG. 2.
Similarity of L-RAP with other members of oxytocinase subfamily. A, alignment of L-RAP and L-RAP(s) proteins with other members of the oxytocinase subfamily. The deduced amino acid sequences of L-RAP and L-RAP(s) are aligned with the sequences of human P-LAP/insulin-regulated aminopeptidase and A-LAP/ERAP1. Gaps are inserted into the sequences for optimal alignment. Residues conserved among 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 between enzymes is proportional to the number of nucleotides and reflects evolutionary time since their divergence.

 

To gain better insights into the relationships among the M1 family of aminopeptidases, a phylogenetic tree was constructed by the method of Higgins and Sharp (32) based on the nucleotide sequence identities (Fig. 2B). According to this tree, it is apparent that L-RAP, P-LAP, and A-LAP belong to one distinctive group.

Tissue and Subcellular Localization of L-RAP—The tissue distribution of L-RAP was determined by Northern blot analysis using the full-length cDNA as a probe (Fig. 3). Expression of mRNA with size of 6.7 kb was observed in every tissue tested, suggesting its ubiquitous distribution. It is notable here that relatively high expression of L-RAP mRNA was observed in spleen and leukocytes.



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 3.
Northern blot analysis of poly(A)+ RNA from various human tissues. A human adult tissue Northern blot (BD Biosciences and Clontech) was probed with 32P-labeled cDNA.

 

To examine subcellular localization of L-RAP, L-RAP tagged with Influenza virus HA epitope at the C-terminal end (L-RAP-HA) was expressed in HeLa S3 cells (Fig. 4A). HeLa S3 cells transfected with constructs encoding L-RAP-HA were subjected to immunocytochemical analysis in the presence of 0.2% Triton X-100, which permeabilizes all cellular membranes non-selectively. A reticular, perinuclear staining pattern characteristic of the ER was observed. In addition, co-localization of L-RAP-HA with the ER retention signal sequence KDEL was clearly observed when merged. On the other hand, when the cells were treated with 5 µg/ml of digitonin to permeabilize plasma membrane selectively (33), neither L-RAP-HA nor KDEL immunoreactivity was detected. For comparison, we examined the localization of I{kappa}B, a cytosolic marker protein, and its immunoreactivity was detected after treatment with either Triton X-100 or digitonin (data not shown). Nearly the same results were obtained when the subcellular localization of L-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 located in the lumenal side.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 4.
Subcellular localization of L-RAP. A, HeLa S3 cells were transfected with L-RAP-HA and subjected to the immunocytochemical analysis employing antibodies against HA-tag and KDEL in the presence of 0.2% Triton X-100 or 5 µg/ml digitonin. B, localization of endogenous L-RAP in Jurkat-T cells. The cell lysate was first centrifuged at 100,000 x g to prepare the cytosolic and microsomal fractions. The lumenal contents were released from membrane fractions with digitonin and separated from microsomal membranes by further centrifugation at 400,000 x g. C, glycosidase treatment of endogenous L-RAP in Jurkat-T cells. Total cell lysate (10 µg) was denatured in 0.5% SDS and 1% 2-mercaptoethanol at 100 °C for 10 min and then the sample was treated either with peptide:N-glycosidase F (PNGase F) or endoglycosidase H (Endo H) (New England Biolabs) for 24 h at 37 °C and subjected to Western blot analysis.

 

We next examined the ER localization of endogenous L-RAP in Jurkat-T cells to determine whether the enzyme is a soluble or membrane-bound lumenal ER protein. As shown in Fig. 4B, both L-RAP and L-RAP(s) are fractionated mainly into the soluble fraction of lumenal contents. As expected, whereas BiP, which contains the KDEL sequence, was also obtained in the soluble fraction of lumenal contents, calnexin, ER membrane-bound protein, was detected in the microsomal membrane fraction. These results suggest that L-RAP and L-RAP(s) are soluble proteins located in the lumenal side of the ER. When examined the localization in HEK 293 cells, L-RAP is detected in the lumenal side of the ER (data not shown). As expected, L-RAP endogenously expressed in Jurkat-T cells are sensitive to endoglycosidase H and peptide:N-glycosidase F (Fig. 4C), indicating that the proteins contain high mannose-type sugar chains.

Characterization of Aminopeptidase Activity of L-RAP—To detect the aminopeptidase activity of L-RAP and L-RAP(s), HEK 293 cells were transiently transfected with either L-RAP or L-RAP(s) expression vector controlled under cytomegalovirus promoter. As in the case of A-LAP, both proteins were secreted into culture medium in this condition. As shown in Fig. 5A, aminopeptidase activity against Arg-MCA was clearly detected in the medium of L-RAP-expressing cells. Although a considerable amount of L-RAP(s) was detected in the medium of L-RAP(s)-expressing cells by Western blot analysis, little Arg-MCA degrading activity was detected. These results suggest that whereas L-RAP shows aminopeptidase activity, L-RAP(s) has little (if any) activity.



View larger version (25K):
[in this window]
[in a new window]
  		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 described under "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 µg/ml) was incubated with various aminoacyl-MCA substrates (25 µM) to assess peptidase activity. D, effects of various inhibitors on L-RAP activity. Aminopeptidase activity was measured using Arg-MCA as a substrate in the presence of various concentrations of inhibitors. o-PNT, 1,10-phenanthroline.

 

To further characterize the enzymatic properties of L-RAP, we established a large scale production system of the recombinant protein using a baculovirus system (Fig. 5B). L-RAP was purified to a single band of 115 kDa on SDS-PAGE from the culture medium. We then measured the relative hydrolytic activity of the enzyme toward various aminoacyl-MCAs. As shown 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 toward Arg-MCA were 3.18 µM, 2.30/s, and 7.25 x 105/M–1s–1, respectively. To other synthetic substrates tested, L-RAP had little activity. These results indicate that L-RAP preferentially hydrolyzes basic N-terminal amino acids.

The effects of various known inhibitors of aminopeptidases on the hydrolytic activity of L-RAP toward Arg-MCA are shown in Fig. 5D. Although amastatin was a potent inhibitor of the enzyme, bestatin was less active. Puromycin, a specific inhibitor of PSA, had little activity toward the enzyme. Of the chelating agents tested, 1,10-phenanthroline effectively inhibited the enzyme activity, but EDTA had no effect up to 1 mM. It is noteworthy here that the inhibitor profile of L-RAP is consistent with those of putative ER resident aminopeptidases, which trim the MHC class I-presented antigenic precursor peptides (34).

Next, we searched for L-RAP-mediated degradation of natural hormones to estimate the physiological role of the enzyme. Among hormones tested, the enzyme cleaved kallidin and angiotensin III (Fig. 6). No hydrolytic activity was observed toward oxytocin, vasopressin, and angiotensin II. These results indicate that the enzyme indeed exerts the aminopeptidase activity toward certain naturally occurring peptides, and its substrate specificity is different from P-LAP and A-LAP.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 6.
Cleavage of natural substrates by recombinant human L-RAP. A, cleavage of angiotensin III (Ang III). Angiotensin III (25 µM) was incubated with L-RAP (2 µg/ml) at 37 °C for the indicated times. B, conversion of kallidin to bradykinin. Kallidin (25 µM) was incubated with L-RAP (2 µg/ml) at 37 °C for the indicated times. After the reaction was terminated by adding 2.5% (v/v) formic acid, the generated peptides were separated and then their structures were determined as described under "Experimental Procedures."

 

Trimming of Precursors of Antigenic Peptides by L-RAP— Because L-RAP is a lumenal ER protein, we next examined whether L-RAP is a trimming enzyme of antigenic precursor peptides. For this purpose, we first examined the effects of IFN-{gamma} on the expression of L-RAP proteins. It is well known that IFN-{gamma} induces many components of the MHC class I-mediated antigen presentation pathway (28, 30). As shown in Fig. 7A, treatment of Jurkat-T and HEK 293 cells with IFN-{gamma} enhanced the L-RAP mRNA expression. On the other hand, the mRNA expression was barely detectable in U937 and HeLa S3 cells, and no induction was observed after the IFN-{gamma} treatment. Kinetic analysis indicated that in Jurkat-T cells, the expression level of the mRNA reached the highest 12 h after treatment and then declined to the basal level at 48 h (Fig. 7B). We also measured the expression level of L-RAP protein in Jurkat-T and HEK 293 cells by Western blot analysis using LC12 antibody (Fig. 7C). When the cells were treated with IFN-{gamma}, the expression of L-RAP protein was increased up to day 3.



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 7.
Effect of IFN-{gamma} on L-RAP expression in cultured cells. A, induction of L-RAP transcripts by IFN-{gamma}. Jurkat-T cells and HEK 293 cells were incubated with or without IFN-{gamma} (30 ng/ml) for 12 h at 37 °C. Expression level of L-RAP transcript was measured by Northern blot analysis. Each lane contained 6 µg of total RNA. B, kinetic analysis. Jurkat-T cells were incubated with IFN-{gamma} (30 ng/ml) for indicated times at 37 °C. Northern blot analysis of L-RAP and {beta}-actin transcripts was then performed. C, induction of L-RAP protein by IFN-{gamma}. Jurkat-T cells and HEK 293 cells were incubated with IFN-{gamma} (30 ng/ml) for indicated times at 37 °C. Expression level of L-RAP protein was measured by Western blot analysis. Each lane contained 10 µg of protein. D, measurement of L-RAP activity in the ER. After treatment of Jurkat-T cells with IFN-{gamma} for 2 days, ER lumenal proteins were extracted from microsomes of control or IFN-{gamma}-treated cells with digitonin. L-RAP was immunodepleted three times with anti-L-RAP LC12 antiserum, and residual Arg-MCA degrading activity was measured. SDS-PAGE analysis of L-RAP protein after immunodepletion is also shown. IP, immunoprecipitation.

 

To estimate the contribution of L-RAP to the Arg-MCA degrading activity in the ER, we examined the effects of loss of the enzyme (Fig. 7D). Although repeated treatment of the sample with anti-L-RAP LC12 antiserum, significant activity was still observed, suggesting that there is Arg-MCA degrading activity not attributable to L-RAP in the lumenal fraction. Besides this basal activity, Arg-MCA degrading activity that could be depleted by the antiserum was clearly observed, suggesting that about 30% of the activity is attributable to L-RAP. As expected, treatment of IFN-{gamma} caused the increase in the Arg-MCA degrading activity sensitive to the antiserum. An immunodepletion experiment indicated that IFN-{gamma} treatment caused about 2-fold increase in L-RAP activity. Residual activity after immunodepletion might reflect the basal activity upregulated by IFN-{gamma}. Alternatively there might be another Arg-MCA degrading activity in the lumenal fraction sensitive to IFN-{gamma}. Preimmune serum had no effect on the Arg-MCA degrading activity in the fractions (data not shown). Taken together, our results indicate that L-RAP can be up-regulated in several cells by IFN-{gamma}, and a significant portion of the Arg-MCA degrading activity is attributable to the enzyme in the ER.

We then incubated the recombinant enzyme with NH2-extended precursor of HIV-1 nef antigen (35). As shown in Fig. 8A, the enzyme efficiently removed N-terminal residues from NH2-extended antigen precursors (NYTPGPGVRY) to generate the final epitope, TPGPGVRY. When RU1 antigen precursor (TAVPYGSFKHV) was examined (36), final antigen epitope (VPYGSFKHV) was generated (data not shown). These results suggest that the enzyme reaction does not proceed if proline is located at the P2' site. We also examined another NH2-extended antigen precursor of melanoma-associated protein gp 100 antigen (FTITDQVPFSV) (37). As shown in Fig. 8B, transient accumulation of the final epitope of the antigen (ITDQVPFSV) was observed although the further degradation of the epitope occurred. These results suggest that like A-LAP/ERAP1, L-RAP can act as an antigen-trimming enzyme and accumulate MHC class-I-presented antigenic peptides in the ER lumen.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 8.
Trimming of precursors of antigenic peptides by L-RAP. A, generation of HIV-1 nef antigen by L-RAP. NH2-extended version of HIV-1 nef antigen (NYTPGPGVRY) (25 µM) was incubated with L-RAP (2 µg/ml) at 37 °C for the indicated times. B, generation of melanoma-associated protein gp 100 antigen by L-RAP. NH2-extended version of melanoma-associated protein gp 100 antigen (FTITDQVPFSV) (25 µM) was incubated with L-RAP (2 µg/ml) at 37 °C for the indicated times. After incubation, generated peptides were analyzed as described under "Experimental Procedures."

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have cloned a cDNA encoding a novel aminopeptidase, L-RAP. Like other M1 families of aminopeptidases, predicted L-RAP contains three domains: a 14-amino acid N-terminal domain, a 22-amino acid hydrophobic domain, and a 924-amino acid C-terminal domain. In the C-terminal domain, the cDNA predicts a protein that contains the HEXXH consensus sequence of zinc metallopeptidases with an additional glutamic acid residue 18 amino acids away, which constitute the active site of metallopeptidases. In addition, another consensus motif, GAMEN, which is considered to determine the exopeptidase specificity of gluzincin aminopeptidases, is located in N-terminal side of the HEXXH motif. These two motifs allow L-RAP to be classified into the M1 family of gluzincin aminopeptidases (10, 17, 19).

Eight mammalian aminopeptidases belonging to the M1 family are identified, and L-RAP is the ninth member of the family. Phylogenetic analysis indicates that P-LAP, A-LAP, and L-RAP form a distinct subfamily of the M1 family. Moreover, these three human genes are located contiguously around chromosome 5q15 (38). These results strongly suggest the latest diversion of these genes from a single ancestral gene, and therefore we propose here that P-LAP, A-LAP, and L-RAP should be classified into the oxytocinase subfamily of M1 aminopeptidases.

It has been recognized that the gluzincin aminopeptidases include membrane-bound, cytosolic, secretory, and ER resident proteins. Among them, the subcellular localization of aminopeptidases belonging to the oxytocinase subfamily is rather unique. P-LAP is located in some intracellular vesicles, and stimulus-dependent translocation of the enzyme into the plasma membrane was observed in several biological systems (39–43). It is generally believed that the physiological significance of P-LAP translocation is to enhance the cleavage of peptide hormone substrates at the cell surface (44). A-LAP/ERAP1 is the first aminopeptidase localized and functions as an antigen-trimming enzyme in the lumenal side of the ER (9, 16). Our results in this study indicate that L-RAP endogenously expressed in Jurkat-T cells and HEK 293 cells is also a soluble protein localized and functions in the ER lumen. Previous work shows that arginine degrading activity was clearly separated from leucine degrading activity in the ER lumenal protein prepared from rat liver (9). The unique localization of the enzymes may suggest the biological functions of the oxytocinase subfamily to be strictly regulated. Indeed, it is getting evident that the enzymes belonging to this subfamily play important roles in the maintenance of homeostasis such as blood pressure regulation, angiogenesis, and maintenance of memory (22–24).

When overexpressed in HEK 293 cells by the high expression vector controlled under cytomegalovirus promoter, L-RAP was detected in the medium. Overexpression of A-LAP in COS-7 cells also caused the secretion of the enzyme into the culture medium. It is plausible that when expressed highly, both A-LAP and L-RAP are secreted into the culture medium. Therefore it is tempting to speculate that some binding protein(s) such as a KDEL-containing protein or an ER membrane component may act as retention machinery for the enzymes. Most probably, saturation of this putative machinery may cause the secretion of the enzyme (45).

We characterized the enzymatic properties of L-RAP for the first time and found that the substrate specificity of L-RAP toward synthetic substrates is unique in its apparent preference for arginine. Another substrate cleaved by the enzyme is Lys-MCA. These results clearly indicate that the substrate specificity of L-RAP is rather restricted, and it preferentially cleaves N-terminal basic amino acids. Because the enzyme hydrolyzed Arg-MCA most preferentially, we termed it an arginine (R) aminopeptidase.

Searching for natural substrates, we found that the enzyme cleaves angiotensin III and kallidin. Considering that A-LAP, which cleaves angiotensin II and III and kallidin, regulates blood pressure (24, 46), it is tempting to speculate that L-RAP also plays a role in the regulation of blood pressure by inactivating angiotensin III and generating bradykinin. Putative ER retention machinery may play a role in the biological function of substrate peptides by regulating the secretion of the enzyme. However, it is necessary to identify natural substrates further to elucidate the physiological and/or pathological roles of the enzyme.

Although substrate specificity of L-RAP toward synthetic substrates is rather restricted, L-RAP could process several antigenic precursors of MHC class I-presented antigen having various N-terminal residues like A-LAP (9, 46). Several enzymes were purified either from cytoplasm or ER lumen as trimming enzymes. It now well recognized that the precursors to MHC class-I-presented peptides with extra N-terminal residues are trimmed to mature epitopes in the ER, and A-LAP/ERAP1 is the first enzyme responsible for the processing in the ER (9, 16). On the other hand, cytoplasmic enzymes such as PSA, lens leucine aminopeptidase, bleomycin hydrolase, and thimet oligopeptidase are thought to contribute to limit antigen presentation in vivo by destroying precursor peptides (34, 47–49).

L-RAP fulfills several criteria for the trimming enzyme of precursor peptides so far reported (34). 1) It is a metallopeptidase inhibited efficiently by 1,10-phenanthroline but not by EDTA. 2) It is located in the ER lumen. 3) Its expression in certain cells is enhanced by IFN-{gamma}. 4) It can trim certain antigenic precursors to MHC class I-presented antigen in vitro. 5) It hydrolyzes various N-terminal amino acids of peptide hormones and antigenic precursors except when proline is located at the P2' site. Taken together, these results strongly suggest that L-RAP can act as a trimming enzyme of precursors of antigenic peptides presented to MHC class I molecules in the ER. Broad specificity of L-RAP suits its role in cleaving a wide spectrum of peptides for MHC class I molecule. Because L-RAP is often expressed in cells lacking A-LAP/ERAP1 expression such as Jurkat-T cells, it is plausible that L-RAP and A-LAP/ERAP1 compensate each other for antigen presentation activity.

In summary, we have cloned a novel aminopeptidase, L-RAP, which belongs to the M1 family of metallopeptidases. The genetic and evolutionary analyses led us to propose the oxytocinase 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 circumstantial evidence that suggest the role of L-RAP as an antigentrimming enzyme. Considering the recent progress of the biological significance of the subfamily members, it is important to examine the in vivo role of L-RAP in future studies.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB109031 [GenBank] .

* 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 publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Laboratory of Cellular Biochemistry, RIKEN, The Institute of Physical and Chemical Research, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. Tel.: 81-48-467-9515; Fax: 81-48-462-4670; E-mail: ahattori{at}postman.riken.go.jp.

1 The abbreviations used are: 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, phosphate-buffered saline; HPLC, high pressure liquid chromatography; HEK, human embryonic kidney; HIV-1, human immunodeficiency virus, type 1. Back


   ACKNOWLEDGMENTS
 
We are grateful for N. Hirotani of Advanced Technology Development Center, RIKEN Brain Institute for synthesis of peptides used in this study.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Taylor, A. (1993) FASEB J. 7, 290–298[Abstract]
  2. Look, A. T., Ashmun, R. A., Shapiro, L. H., and Peiper, S. C. (1989) J. Clin. Invest. 83, 1299–1307[Medline] [Order article via Infotrieve]
  3. Nanus, D. M., Engelstein, D., Gastl, G. A., Gluck, L., Vidal, M. J., Morrison, M., Finstad, C. L., Bander, N. H., and Albino, A. P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7069–7073[Abstract/Free Full Text]
  4. Wu, Q., Lahti, J. M., Air, G. M., Burrows, P. D., and Cooper, M. D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 993–997[Abstract/Free Full Text]
  5. Shipp, M. A., and Look, A. T. (1993) Blood 82, 1052–1070[Free Full Text]
  6. Rogi, T., Tsujimoto, M., Nakazato, H., Mizutani, S., and Tomoda, Y. (1996) J. Biol. Chem. 271, 56–61[Abstract/Free Full Text]
  7. Hattori, A., Matsumoto, H., Mizutani, S., and Tsujimoto, M. (1999) J. Biochem. (Tokyo) 125, 931–938[Abstract/Free Full Text]
  8. Schomburg, L., Kollmus, H., Friedrichsen, S., and Bauer, K. (2000) Eur. J. Biochem. 267, 3198–3207[Medline] [Order article via Infotrieve]
  9. Saric, T., Chang, S.-C., Hattori, A., York, I. A., Markant, S., Rock, K. L., Tsujimoto, M., and Goldberg, A. L. (2002) Nat. Immunol. 3, 1169–1176[CrossRef][Medline] [Order article via Infotrieve]
  10. Hooper, N. M. (1994) FEBS Lett. 354, 1–6[CrossRef][Medline] [Order article via Infotrieve]
  11. Schauder, B., Schomburg, L., Kohrle, J., and Bauer, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9534–9538[Abstract/Free Full Text]
  12. Keller, S. R., Scott, H. M., Mastick, C. C., Aebersold, R., and Lienhard, G. E. (1995) J. Biol. Chem. 270, 23612–23618[Abstract/Free Full Text]
  13. Funk, C. D., Radmark, O., Fu, J. Y., Matsumoto, T., Jornvall, H., Shimizu, T., and Samuelsson, B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6677–6681[Abstract/Free Full Text]
  14. Constam, D. B., Tobler, A. R., Rensing-Ehl, A., Kemler, I., Hersh, L. B., and Fontana, A. (1995) J. Biol. Chem. 270, 26931–26939[Abstract/Free Full Text]
  15. Fukasawa, K. M., Fukasawa, K., Kanai, M., Fujii, S., and Harada, M. (1996) J. Biol. Chem. 271, 30731–30735[Abstract/Free Full Text]
  16. Serwold, T., Gonzalez, F., Kim, J., Jacob, R., and Shastri, N. (2002) Nature 419, 480–483[CrossRef][Medline] [Order article via Infotrieve]
  17. Vazeux, G., Iturrioz, X., Corvol, P., and Llorens-Cortes, C. (1998) Biochem. J. 334, 407–413
  18. Iturrioz, X., Rozenfeld, R., Michaud, A., Corvol, P., and Llorens-Cortes, C. (2001) Biochemistry 40, 14440–14448[CrossRef][Medline] [Order article via Infotrieve]
  19. Laustsen, P. G., Vang, S., and Kristensen, T. (2001) Eur. J. Biochem. 268, 98–104[Medline] [Order article via Infotrieve]
  20. Papadopoulos, T., Kelly, J. A., and Bauer, K. (2001) Biochemistry 40, 9347–9355[CrossRef][Medline] [Order article via Infotrieve]
  21. Reaux, A., Fournie-Zaluski, M. C., David, C., Zini, S., Roques, B., Corvol, P., and Llorens-Cortes, C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13415–13420[Abstract/Free Full Text]
  22. Albiston, A. L., McDowall, S. G., Matsacos, D., Sim, P., Clune, E., Mustafa, T., Lee, J., Mendelsohn, F. A. O., Simpson, R. J., Connolly, L. M., and Chai, S. Y. (2001) J. Biol. Chem. 276, 48623–48626[Abstract/Free Full Text]
  23. Miyashita, H., Yamazaki, T., Akada, T., Niizeki, O., Ogawa, M., Nishikawa, S., and Sato, Y. (2002) Blood 99, 3241–3249[Abstract/Free Full Text]
  24. Yamamoto, N., Nakayama, J., Yamakawa-Kobayashi, K., Hamaguchi, H., Miyazaki, R., and Arinami, T. (2002) Hum. Mutat. 19, 251–257[CrossRef][Medline] [Order article via Infotrieve]
  25. Osada, T., Ikegami, S., Takiguchi-Hayashi, K., Yamazaki, Y., Katoh-Fukui, Y., Higashinakagawa, T., Sakaki, Y., and Takeuchi, T. (1999) J. Neurosci. 19, 6068–6078[Abstract/Free Full Text]
  26. Osada, T., Watanabe, G., Kondo, S., Toyoda, M., Sakaki, Y., and Takeuchi, T. (2001) Mol. Endcrinol. 15, 960–971[Abstract/Free Full Text]
  27. Osada, T., Watanabe, G., Sakaki, Y., and Takeuchi, T. (2001) Mol. Endcrinol. 15, 882–893[Abstract/Free Full Text]
  28. Goldberg, A. L., Cascio, P., Saric, T., and Rock, K. L. (2002) Mol. Immunol. 39, 147–164[CrossRef][Medline] [Order article via Infotrieve]
  29. Kessler, B. M., Glas, R., and Ploegh, H. L. (2002) Mol. Immunol. 39, 171–179[CrossRef][Medline] [Order article via Infotrieve]
  30. Saveanu L., Fruci, D., and van Endert, P. M. (2002) Mol. Immunol. 39, 203–215[CrossRef][Medline] [Order article via Infotrieve]
  31. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105–132[CrossRef][Medline] [Order article via Infotrieve]
  32. Higgins, D. G., and Sharp, P. M. (1988) Gene 73, 237–244[CrossRef][Medline] [Order article via Infotrieve]
  33. Otto, J. C., and Smith, W. L. (1994) J. Biol. Chem. 269, 19868–19875[Abstract/Free Full Text]
  34. Serwold, T., Gaw, S., and Shastri, N. (2001) Nat. Immunol. 2, 644–651[CrossRef][Medline] [Order article via Infotrieve]
  35. Lucchiari-Hartz, M., van Endert, P. M., Lauvau, G., Maier, R., Meyerhans, A., Mann, D., Eichmann, K., and Nidermann, G. (2000) J. Exp. Med. 191, 239–252[Abstract/Free Full Text]
  36. Morel, S., Levy, F., Burlet-Schiltz, O., Brasseur, F., Probst-Kepper, M., Peitrequin, A.-L., Monsarrat, B., Van Velthoven, R., Cerottini, J.-C., Boon, T., Gairin, J. E., and Van den Eynde, B. J. (2000) Immunity 12, 107–117[CrossRef][Medline] [Order article via Infotrieve]
  37. Bakker, A. B., Schreurs, M. W., de Boer, A. J., Kawakami, Y., Rosenberg, S. A., Adema, G. J., and Figdor, C. G. (1994) J. Exp. Med. 179, 1005–1009[Abstract/Free Full Text]
  38. Hattori, A., Matsumoto, K., Mizutani, S., and Tsujimoto, M. (2001) J. Biochem. (Tokyo) 130, 235–241[Abstract/Free Full Text]
  39. Ross, S. A., Scott, H. M., Morris, N. J., Leung, W.-Y., Mao, F., Lienhard, G. E., and Keller, S. R. (1996) J. Biol. Chem. 271, 3328–3332[Abstract/Free Full Text]
  40. Nakamura, H., Itakura, A., Okamura, M., Ito, M., Iwase, A., Nakanishi, Y., Okada, M., Nagasaka, T., and Mizutani, S. (2000) Endocrinology 141, 4481–4485[Abstract/Free Full Text]
  41. Matsumoto, H., Nagasaka, T., Hattori, A., Rogi, T., Tsuruoka, N., Mizutani, S., and Tsujimoto, M. (2001) Eur. J. Biochem. 268, 3259–3266[Medline] [Order article via Infotrieve]
  42. Thoidis, G., and Kandror, K. V. (2001) Traffic 2, 577–587[CrossRef][Medline] [Order article via Infotrieve]
  43. Masuda, S., Hattori, A., Matsumoto, H., Miyazawa, S., Natori, Y., Mizutani, S., and Tsujimoto, M. (2003) Eur. J. Biochem. 270, 1988–1994[Medline] [Order article via Infotrieve]
  44. Keller, S. R. (2003) Front. Biosci. 8, S410–420
  45. Turano, C., Coppari, S., Altieri, F., and Ferraro, A. (2002) J. Cell. Physiol. 193, 154–163[CrossRef][Medline] [Order article via Infotrieve]
  46. Hattori, A., Kitatani, K., Matsumoto, H., Miyazawa, S., Rogi, T., Tsuruoka, N., Mizutani, S., Natori, Y., and Tsujimoto, M. (2000) J. Biochem. (Tokyo) 128, 755–762[Abstract/Free Full Text]
  47. Beninga, J., Rock, K. L., and Goldberg, A. L. (1998) J. Biol. Chem. 273, 18734–18742[Abstract/Free Full Text]
  48. Stoltze, L., Schirle, M., Schwarz, G., Schroter, C., Thompson, M. W., Hersh, L. B., Kalbacher, H., Stevanovic S., Rammensee, H.-G., and Schild, H. (2000) Nat. Immunol. 1, 413–418[CrossRef][Medline] [Order article via Infotrieve]
  49. Saric, T., Beninga, J., Graef, C. I., Akopian, T. N., Rock, K. L., and Goldberg, A. L. (2001) J. Biol. Chem. 276, 36474–36481[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Gen. Virol.Home page
C. Johnstone, S. Guil, M. A. Rico, B. Garcia-Barreno, D. Lopez, J. A. Melero, and M. Del Val
Relevance of viral context and diversity of antigen-processing routes for respiratory syncytial virus cytotoxic T-lymphocyte epitopes
J. Gen. Virol., September 1, 2008; 89(9): 2194 - 2203.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Goto, A. Hattori, S. Mizutani, and M. Tsujimoto
Asparatic Acid 221 Is Critical in the Calcium-induced Modulation of the Enzymatic Activity of Human Aminopeptidase A
J. Biol. Chem., December 21, 2007; 282(51): 37074 - 37081.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Maruyama, A. Hattori, Y. Goto, M. Ueda, M. Maeda, H. Fujiwara, and M. Tsujimoto
Laeverin/Aminopeptidase Q, a Novel Bestatin-sensitive Leucine Aminopeptidase Belonging to the M1 Family of Aminopeptidases
J. Biol. Chem., July 13, 2007; 282(28): 20088 - 20096.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. Pinyol, S. Bea, L. Pla, V. Ribrag, J. Bosq, A. Rosenwald, E. Campo, and P. Jares
Inactivation of RB1 in mantle-cell lymphoma detected by nonsense-mediated mRNA decay pathway inhibition and microarray analysis
Blood, June 15, 2007; 109(12): 5422 - 5429.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
C. Garcia-Rudaz, F. Luna, V. Tapia, B. Kerr, L. Colgin, F. Galimi, G. A. Dissen, N. D. Rawlings, and S. R. Ojeda
Fxna, a novel gene differentially expressed in the rat ovary at the time of folliculogenesis, is required for normal ovarian histogenesis
Development, March 1, 2007; 134(5): 945 - 957.
[Abstract] [Full Text] [PDF]

Home page