Distribution of Adipocyte-derived Leucine Aminopeptidase (A-LAP)/ER-aminopeptidase (ERAP)-1 in Human Uterine Endometrium
Departments of Obstetrics and Gynecology (DS,HA,AI,SM) and Laboratory Medicine (TN), Nagoya University Graduate School of Medicine, Nagoya, Japan, and Laboratory of Cellular Biochemistry (AH,MT), RIKEN, Wako, Japan
Correspondence to: H. Ando, MD, PhD, Dept. of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: ando{at}med.nagoya-u.ac.jp
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Summary |
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(J Histochem Cytochem 52:11691175, 2004)
Key Words: adipocyte-derived leucine aminopeptidase aminopeptidase endometrium endoplasmic reticulum implantation puromycin-insensitive leucyl-specific aminopeptidase (PILSAP)
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Introduction |
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In our previous work, we cloned a cDNA encoding placental leucine aminopeptidase (P-LAP)/oxytocinase, a type II membrane-spanning protein that belongs to the M1 zinc-metallopeptidase (gluzincin) family (Rogi et al. 1996). We then cloned a cDNA for the adipocyte-derived leucine aminopeptidase (A-LAP), which was also designated as endoplasmic reticulum (ER)-aminopeptidase (ERAP)-1 or puromycin-insensitive leucyl-specific aminopeptidase (PILSAP), as a highly homologous protein to P-LAP (Hattori et al. 1999
; Schomburg et al. 2000
; Saric et al. 2002
). A-LAP trims certain precursors to major histocompatibility complex (MHC) class I-presented antigenic peptides (Serwold et al. 2002
). The proteolytic activity of A-LAP is controlled by the size rather than the NH2 terminus of the peptide substrate (York et al. 2002
). Gluzincin aminopeptidases share the consensus His-(X)18-Glu motif essential for enzymatic activity (Hooper 1994
). This growing family of mammalian zinc-containing membrane-bound aminopeptidases includes P-LAP, A-LAP, thyrotropin-releasing hormone-degrading enzyme, aminopeptidase A, and aminopeptidase N (Olsen et al. 1988
; Wu et al. 1990
; Nanus et al. 1993
; Schauder et al. 1994
; Rogi et al. 1996
; Hattori et al. 1999
). In addition, we have recently cloned a cDNA encoding a novel aminopeptidase, leukocyte-derived arginine (R) aminopeptidase (L-RAP) (Tanioka et al. 2003
). We have reported that, like A-LAP, L-RAP is an aminopeptidase normally retained in the ER and trims certain precursors to MHC class I-presented antigenic peptides (Tanioka et al. 2003
). Moreover, three human genes of P-LAP, A-LAP, and L-RAP are located contiguously around chromosome 5q15 (Horio et al. 1999
; Hattori et al. 2001
; Tanioka et al. 2003
). These results strongly support the latest divergence of three genes from a single ancestral gene, and therefore we have proposed that these three aminopeptidases should be classified in the oxytocinase subfamily of M1 aminopeptidases (Tanioka et al. 2003
).
Human endometrium undergoes dramatic morphological and functional changes during the menstrual cycle. In an effort to elucidate the biological significance of the M1 family of aminopeptidases in human reproductive processes, including implantation and menstruation, we have recently reported the expression and localization of P-LAP and aminopeptidase A in the endometrium (Ando et al. 2002; Toda et al. 2002
). The endometrium is the maternal frontier to the implanted embryo, a semi-allograft, which is initially recognized by the uterine leukocytes. Furthermore, the endometrium may be susceptible to foreign organisms via the vagina. We studied the distribution and intracellular localization of A-LAP in human endometrium. We speculated that A-LAP and P-LAP, both of which belong to the same subfamily of aminopeptidases, might show some similarities and differences in their distribution in human cyclic endometrium.
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Materials and Methods |
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Tissue Collection for Immunohistochemistry
We retrieved endometrial biopsy specimens from the pathology files at Nagoya University Hospital, Nagoya, Japan as described previously (Ando et al. 2002). Patient clinical charts were reviewed and cases were selected on the basis of a history of regular menstrual cycles and no use of any intrauterine device or hormone therapy for at least 6 months before the biopsy. Histological slides of the endometrium were reviewed and cases were further selected on the basis of consistent histological findings. As a result of these reviews, biopsy specimens from 39 patients, aged 3443 years, were available for examination. Endometrial dating criteria were used to assess the phase of the menstrual cycle (Noyes et al. 1950
). The results of this histological classification were as follows: 14 proliferative phase, eight early secretory phase, eight mid-secretory phase, nine late secretory phase. Endometrial dating was strictly defined histologically by an expert pathologist in this technique (TN). All biopsy samples had been proved to be histologically benign. The use of pathology specimens for this research was approved by the institutional review board. These samples, which had been fixed in 10% formalin and embedded in paraffin, were used for IHC.
Tissue Collection for Endometrial Cell Culture
Fresh endometrial tissues in the late proliferative phase were collected by curettage from six women with regular menstrual cycles, aged 3446 years, during hysterectomy for leiomyoma at Nagoya University Hospital. Written informed consent was obtained from the women before surgery. Individual curetted samples were quickly washed with cold PBS to remove blood and secretions. Samples were immediately moved to our laboratory in an ice bucket. PBS was removed by brief centrifugation and then the samples were used for cell culture after glands and stroma were separated as described below.
Endometrial Cell Culture
Endometrial stromal cells (ESCs) and endometrial glandular epithelial cells (EECs) were separated essentially as described previously (Satyaswaroop et al. 1979) with only slight modification (Ando et al. 2002
). Briefly, endometrial tissue was minced into small pieces (
1 mm3) and these pieces were filtered through a cell strainer consisting of 100-µm pore size nylon mesh (Becton Dickinson; Franklin Lakes, NJ) to remove blood cells. Then the minced tissue was incubated with stirring at 37C for 20 min in PBS, 0.5% collagenase (Wako; Osaka, Japan), and DNase (0.1 mg/ml; Sigma, St Louis, MO). For separating EECs, the incubation time was extended to 30 min. The dispersed ESCs were separated from endometrial glands and undigested tissues by filtration through a cell strainer consisting of 70-µm pore size nylon mesh (Becton Dickinson). The glands were obtained by the backflash of the nylon mesh with PBS. To ensure purity, the glands were sorted microscopically. Then the glands were incubated with stirring at 37C for 5 min in PBS containing 0.25% trypsin (Sigma). The separated ESCs and EECs were washed and pelleted by centrifugation (400 x g, 10 min) and then suspended (106 cells/ml) in RPMI 1640 (Sigma) containing FCS (10% v/v; Life Technologies, Gaithersburg, MD), 100 IU/ml penicillin, and 100 µg/ml streptomycin. For Western blotting, ESCs and EECs were plated onto 10-cm Falcon dishes (Becton Dickinson) and 10-cm collagen-coated dishes (Sumitomo Bakelite; Tokyo, Japan), respectively, for 48 hr at 37C in a humidified atmosphere of 5% CO2 in air. For immunofluorescent cytochemistry, cells were grown on the Chamber-Tek chamber slide (Miles; Naperville, IL) for 48 hr at 37C in a humidified atmosphere of 5% CO2 in air. The purity of ESCs and EECs was
99% and
94%, respectively.
Western Blotting Analysis
The frozen tissue samples or the harvested cell samples were homogenized using a motor-driven Teflon pestle for 10 min on ice in PBS extraction buffer containing 1% Triton X-100 and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 10 µg/ml leupeptin). Tissue extracts were obtained as supernatants after centrifugation at 15,000 x g for 30 min at 4C and were stored at 80C. Protein concentrations were determined using a protein assay kit (Bio-Rad Laboratories; Hercules, CA). Immunoblotting analysis was carried out according to the method reported previously (Ando et al. 2002). In brief, 10 µg of protein was equally diluted in a sample buffer and denatured at 95C for 5 min. Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked with 5% skim milk and then immunoblotted with antiA-LAP antibody (1:1000 dilution). Rabbit polyclonal IgG antibody was raised against recombinant human A-LAP expressed in Chinese hamster ovary cells (Hattori et al. 2000
). It is highly specific and it does not crossreact with recombinant human P-LAP expressed in Chinese hamster ovary cells (Matsumoto et al. 2000
). After washing with PBS, membranes were treated with an ECL blotting detection system (Amersham Pharmacia Biotech; Piscataway, NJ). Then the membranes were dehybridized and then immunoblotted with antiß-actin antibody. Densitometric analysis was applied for statistical comparison (Image Master 1D; Amersham Biosciences, Piscataway, NJ). Densitometric data from three independent experiments were expressed as the mean ± SD.
Immunohistochemistry
Formalin-fixed, paraffin-embedded tissue sections were cut to a thickness of 3 µm. Deparaffinized sections in 0.01 M citrate buffer were treated three times for 5 min at 90C at 750 W in an H2500 microwave oven (M and M; Tokyo, Japan) for heat-induced epitope retrieval. Endogenous peroxidase activity was blocked by incubation with 0.5% (w/v) H2O2 in methanol for 10 min, and nonspecific immunoglobulin binding was blocked by incubation with 10% normal goat serum in PBS for 10 min. IHC staining was carried out based on the labeled streptavidinbiotin (LSAB) method. A Ventana Basic DAB Detection Kit (Ventana Medical Systems; Tucson, AZ), yielding a brown product from diaminobenzidine (DAB)/copper sulfate, was used to detect A-LAP. Staining procedures were done automatically using a Ventana BenchMark IHC Staining System (Ventana Medical Systems) according to the manufacturer's instructions. This system is a completely automatic computerized staining system that yields stable and reproducible data. The primary antibody against A-LAP was diluted 1:100 in PBS. In negative control sections, the primary antibody was replaced with rabbit IgG. Human kidney sections were used as a positive staining control. The slides were counterstained with hematoxylin before mounting. Stained sections were observed under an Olympus (Tokyo, Japan) BH2 microscope and photographed using a CCD Color Camera CS600 (Olympus). The staining intensity was classified as follows: strong, moderate, weak, very weak, and negative. Multiple sections obtained from the same tissue block were stained repeatedly over a 2-month period.
Immunofluorescent Microscopy
EECs and ESCs on chamber slides after fixation with 3.7% formaldehyde in PBS were treated with 0.2% Triton X-100 in PBS for 10 min and soaked in PBS containing 1% BSA (BSA-PBS) and 5% skim milk for 30 min. Cells were then incubated with anti-ER retention signal KDEL Antibody (StressGen Bioreagents; Victoria, Canada) and antiA-LAP antibody, diluted with BSA-PBS (1:200 dilution) for 1 hr, washed three times for 5 min with BSA-PBS, and incubated with secondary antibodies, rhodamine-conjugated anti-goat IgG (Jackson ImmunoResearch Laboratories; West Grove, PA) and FITC-conjugated anti-mouse IgG (Santa Cruz Biotechnology; Santa Cruz, CA) for 1 hr. Cells were examined using an confocal microscope (Radiance; Bio-Rad Laboratories).
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Results |
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Discussion |
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Although tissue distribution of A-LAP is broad (Schomburg et al. 2000; Serwold et al. 2002
), its localization in each organ has not been studied as extensively as that of P-LAP (Nagasaka et al. 1997
). A heterograft recognition system may be activated in the cyclic endometrium because the endometrium is susceptible to foreign organisms, such as bacteria and fungi. 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 the ER lumen, and then trimmed by A-LAP. Therefore, our data on the distribution of A-LAP may contribute to elucidation of the immune system in the cyclic endometrium.
Intracellular localization of A-LAP was homogenous throughout the menstrual cycle. In contrast to the intracellular localization change of P-LAP along with the apocrine secretion of endometrial glands (Toda et al. 2002), the intracellular localization of endometrial A-LAP was unchanged. Therefore, A-LAP was still localized when P-LAP disappeared after apocrine secretion was finished in the late secretory phase. Enzymatically, aminopeptidases hydrolyze N-terminal amino acids of peptide substrates with their own specificity. P-LAP specifically degrades oxytocin, vasopressin, and angiotensin III (Tsujimoto et al. 1992
), while A-LAP degrades angiotensin II and kallidin (Hattori et al. 2000
). Among the bioactive peptides, it appears that angiotensin II may play important roles in such processes as angiogenesis, vasoconstriction, and trophoblast invasion in the cyclic endometrium (Li and Ahmed 1996
; Ando et al. 2002
; Xia et al. 2002
). The spatiotemporal localization of angiotensin II is highly regulated by the local reninangiotensin system and by angiotensin-degrading enzymes (Vinson et al., 1997
; Ando et al., 2002
). It is unclear at present how A-LAP as an ER aminopeptidase may contribute to the degradation of angiotensin II in the endometrium. Recently, our group identified A-LAP in human invasive trophoblastic cells (Ino et al. 2003
). It is noteworthy that A-LAP was present at a detectable level only in the endometrial tissue of ectopic pregnancy at 68 weeks. Further studies are necessary to clarify the distribution and function of A-LAP in the endometrium and invasive trophoblasts during the establishment of pregnancy.
In conclusion, we showed the presence and distribution of A-LAP in human cyclic endometrium. Belonging to the same subfamily of aminopeptidases, A-LAP and P-LAP share similarities in endometrial distribution. However, each leucine aminopeptidase has its unique intracellular localization in glandular epithelial cells. Further understanding of the function of A-LAP in the endometrium appears likely to yield insights into the cyclic changes during the normal menstrual cycle, although extensive work will be required to prove the involvement of this enzyme in various physiological changes of the endometrium.
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Acknowledgments |
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We are grateful for the technical assistance of Hiroko Sato in the immunohistochemical analysis.
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Footnotes |
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Literature Cited |
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Ando H, Nagasaka T, Nomura M, Tsukahara S, Kotani Y, Toda S, Murata Y, et al. (2002) Premenstrual disappearance of aminopeptidase A in endometrial stromal cells around endometrial spiral arteries/arterioles during the decidual change. J Clin Endocrinol Metab 87:23032309
Hattori A, Kitatani K, Matsumoto H, Miyazawa S, Rogi T, Tsuruoka N, Mizutani S, et al. (2000) Characterization of recombinant human adipocyte-derived leucine aminopeptidase expressed in Chinese hamster ovary cells. J Biochem (Tokyo) 128:755762[Abstract]
Hattori A, Matsumoto H, Mizutani S, Tsujimoto M (1999) Molecular cloning of adipocyte-derived leucine aminopeptidase highly related to placental leucine aminopeptidase/oxytocinase. J Biochem (Tokyo) 125:931938[Abstract]
Hattori A, Matsumoto K, Mizutani S, Tsujimoto M (2001) Genomic organization of the human adipocyte-derived leucine aminopeptidase gene and its relationship to the placental leucine aminopeptidase/oxytocinase gene. J Biochem (Tokyo) 130:235241[Abstract]
Hooper NM (1994) Families of zinc metalloproteases. FEBS Lett 354:16[CrossRef][Medline]
Horio J, Nomura S, Okada M, Katsumata Y, Nakanishi Y, Kumano Y, Takami S, et al. (1999) Structural organization of the 5'-end and chromosomal assignment of human placental leucine aminopeptidase/insulin-regulated membrane aminopeptidase gene. Biochem Biophys Res Commun 262:269274[CrossRef][Medline]
Imai K, Maeda M, Fujiwara H, Okamoto N, Kariya M, Emi N, Takakura K, et al. (1992) Human endometrial stromal cells and decidual cells express cluster of differentiation (CD) 13 antigen/aminopeptidase N and CD10 antigen/neutral endopeptidase. Biol Reprod 46:328334[Abstract]
Ino K, Kikkawa F, Suzuki T, Kajiyama H, Shibata K, Nomura S, Itakura A, et al. (2003) Expression of placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in human normal and malignant invasive trophoblastic cells. Lab Invest 83:17991809[CrossRef][Medline]
Li X-F, Ahmed A (1996) Dual role of angiotensin II in the human endometrium. Hum Reprod 11(suppl2):95108[Abstract]
Look AT, Ashmun RA, Shapiro LH, Peiper SC (1989) Human myeloid plasma membrane glycoprotein CD13 (gp150) is identical to aminopeptidase N. J Clin Invest 83:12991307[Medline]
Matsumoto H, Rogi T, Yamashiro K, Kodama S, Tsuruoka N, Hattori A, Takio K, et al. (2000) Characterization of a recombinant soluble form of human placental leucine aminopeptidase/oxytocinase expressed in Chinese hamster ovary cells. Eur J Biochem 267:4652
Nagasaka T, Nomura S, Okamura M, Tsujimoto M, Nakazato H, Oiso Y, Nakashima N, et al. (1997) Immunohistochemical localization of placental leucine aminopeptidase/oxytocinase in normal human placental, fetal and adult tissues. Reprod Fertil Dev 9:747753[Medline]
Nanus DM, Engelstein D, Gastl GA, Gluck L, Vidal MJ, Morrison M, Finstad CL, et al. (1993) Molecular cloning of the human kidney differentiation antigen gp160: human aminopeptidase A. Proc Natl Acad Sci USA 90:70697073[Abstract]
Noyes RW, Hertig AT, Rock J (1950) Dating the endometrial biopsy. Fertil Steril 1:325[Medline]
Olsen J, Cowell GM, Konigshofer E, Danielsen EM, Moller J, Laustsen L, Hansen OC, et al. (1988) Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA. FEBS Lett 238:307314[CrossRef][Medline]
Rogi T, Tsujimoto M, Nakazato H, Mizutani S, Tomoda Y (1996) Human placental leucine aminopeptidase/oxytocinase. A new member of type II membrane-spanning zinc metallopeptidase family. J Biol Chem 271:5661
Saric T, Chang SC, Hattori A, York IA, Markant S, Rock KL, Tsujimoto M, et al. (2002) An IFN-gamma-induced aminopeptidase in the ER, ERAP1, trims precursors to MHC class I-presented peptides. Nature Immunol 3:11691176[CrossRef][Medline]
Satyaswaroop PG, Bressler RS, de la Pena MM, Gurpide E (1979) Isolation and culture of human endometrial glands. J Clin Endocrinol Metab 48:639641[Abstract]
Schauder B, Schomburg L, Kohrle J, Bauer K (1994) Cloning of a cDNA encoding an ectoenzyme that degrades thyrotropin-releasing hormone. Proc Natl Acad Sci USA 91:95349538
Schomburg L, Kollmus H, Friedrichsen S, Bauer K (2000) Molecular characterization of a puromycin-insensitive leucyl-specific aminopeptidase, PILS-AP. Eur J Biochem 267:31983207
Serwold T, Gonzalez F, Kim J, Jacob R, Shastri N (2002) ERAAP customizes peptides for MHC class I molecules in the endoplasmic reticulum. Nature 419:480483[CrossRef][Medline]
Tanioka T, Hattori A, Masuda S, Nomura Y, Nakayama H, Mizutani S, Tsujimoto M (2003) Human leukocyte-derived arginine aminopeptidase: the third member of the oxytocinase subfamily of aminopeptidases. J Biol Chem 278:3227532283
Taylor A (1993) Aminopeptidases: structure and function. FASEB J 7:290298
Toda S, Ando H, Nagasaka T, Tsukahara S, Nomura M, Kotani Y, Nomura S, et al. (2002) Existence of placental leucine aminopeptidase/oxytocinase/insulin-regulated membrane aminopeptidase in human endometrial epithelial cells. J Clin Endocrinol Metab 87:13841389
Tsujimoto M, Mizutani S, Adachi H, Kimura M, Nakazato H, Tomoda Y (1992) Identification of human placental leucine aminopeptidase as oxytocinase. Arch Biochem Biophys 292:388392[Medline]
Vinson GP, Saridogan E, Puddefoot JR, Djahanbakhch O (1997) Tissue renin-angiotensin system and reproduction. Hum Reprod 12:651662[Abstract]
Wu Q, Lahti JM, Air GM, Burrows PD, Cooper MD (1990) Molecular cloning of the murine BP-1/6C3 antigen: a member of the zinc-dependent metallopeptidase family. Proc Natl Acad Sci USA 87:993997[Abstract]
York IA, Chang SC, Saric T, Keys JA, Favreau JM, Goldberg AL, Rock KL (2002) The ER aminopeptidase ERAP1 enhances or limits antigen presentation by trimming epitopes to 89 residues. Nature Immunol 3:11771184[CrossRef][Medline]
Xia Y, Wen HY, Kellems RE (2002) Angiotensin II inhibits human trophoblast invasion through AT1 receptor activation. J Biol Chem 277:2460124608