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From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Deletion of the Zinc-Binding Motif of CD13/Aminopeptidase N Molecules Results in Loss of Epitopes That Mediate Binding of Inhibitory Antibodies By Richard A. Ashmun, Linda H. Shapiro, and A. Thomas Look The myeloid cell-surface glycoprotein CD13/ aminopeptidase N (APN; EC 3.4.11.2) contains a pentapeptide (HExxH) in its extracellular domain that is characteristic of many zincdependent metalloproteinases. This region contains residues important for zinc binding and constitutes part of the catalytic domain of several metalloproteases. We deleted an internal fragment of 117 base pairs (bp) from the human CD13/APN cDNA, resulting in an in-frame deletion that included the sequences coding for this pentapeptide motif. The mutant cDNA was subcloned into a retroviral expression vector, and polypeptidesencoded by the altered cDNA were expressed in transfected murine NIH-3T3 fibroblasts. The mutant CD13/APN molecules lacked enzymatic activity, and their intracellular processing to the cell surface was retarded by comparison with normal CD13/APN polypeptides. The mutant molecules also lacked epitopes required for binding of four of 19 CD13-specific monoclonal antibodies (MoAbs) tested in flow cytometricassays. Each of the four MoAbs also inhibitedthe enzymatic activity of wild-type APN molecules, suggesting that these antibodies may inhibit aminopeptidase activity by interferingwith the enzyme’s zinc-coordinating properties. Cells engineered to express mutant CD13/ APN polypeptides at the cell surface provide a tool for defining the physiologic role of this enzyme on normal and malignant myeloid cells and marrow stromal cells. o 1992 by The American Society of Hematology. T metallopr~teinases.~~ We have now created a deletion mutant of APN that lacks a 39-amino acid segment in the extracellular domain, including the critical pentapeptide motif. Presented here are findings that indicate the location of epitopes that may be important in the zinc-coordinating activities of CD13/APN. HE CELL-SURFACE glycoprotein CD13 (also known as gp150) was first identified on leukocytes of the myeloid series and was later shown to be identical to aminopeptidase N (APN; EC 3.4.11.2),1,2 a prominent membrane-bound metallopeptidase of epithelial cells that form the brush borders of the small intestine and renal tubules. CD13/APN exemplifies a group of cell-surface peptidases that have been found on discrete subsets of hematopoietic cells. While CD13/APN expression is restricted to myeloid progenitors and mature monocytes, macrophages, and granulocytes within the hematopoietic system, other membrane-bound peptidases show activity on granulocytes and lymphoid progenitors (CDlO/neutral endopeptidase; EC 3.4.24.11),3-5 activated T lymphocytes (CD26/dipeptidyl peptidase IV, EC 3.4.14.5),6-s and early B-lymphocyte progenitors (murine BP1/6C3, identical to aminopeptidase A, EC 3.4.11.7).9-” We previously demonstrated that the APN activity of CD13 molecules on myeloid cells can be inhibited with two of a panel of 11 monoclonal antibodies (MoAbs) that specifically bind epitopes of human CD13.12 APN has an absolute requirement for zinc for its enzymatic activity, and its extracellular domain includes a pentapeptide sequence (HExxH, at amino acid positions 388 to 392) that mediates zinc binding and catalytic activity in a series of related From the Departments of Hematology-Oncology and Tumor Cell Biology, St Jude Children’s Research Hospital, Memphis, TN; and the Division of Hematology-Oncology, Department of Pediatrics, The University of Tennessee, Memphis, College of Medicine, Memphis, TN. Submitted October 21, 1991; accepted February 13, 1992. Supported in part by National Institutes of Health Grant No. ROI -CA42804, Leukemia Program Project Grant No. POI-CA20180, Cancer Center Research Grant No. P3O-CA21765, and the American Lebanese Syrian Associated Charities (ALSAC). Address reprint requests to Richard A . Ashmun, PhD, Department of Hematology-Oncology, St Jude Children’s Research Hospital, PO Box 318, Memphis, TN38IOI. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section I734 solely to indicate this fact. 0 I992 by The American Society of Hematology. 0006-4971/92/7912-0019$3.00/0 3344 MATERIALS AND METHODS Construction and expression of a mutant APN cDNA. The full-length human CD13/APN cDNA subcloned in a pBluescript plasmid (Stratagene, La Jolla, CA)’ was digested with the BstEII restriction endonuclease, which excised an internal 117-base pair (bp) restriction fragment from the cDNA, but did not cut elsewhere in the cDNA or in the vector. The deleted fragment included the coding sequences for the extracellular zinc-binding motif (HExxH, at amino acid positions 388 to 392; see Fig 1). The digested cDNA molecules were separated from the small insert fragment by electrophoresis and gel elution, religated, and used to transform XL1 blue bacteria (Stratagene). Digested DNA lacking the deleted sequences was religated correctly to maintain the CD13/APN open-reading frame, as confirmed by sequence analysis using the dideoxynucleotide chain termination method14 as applied to double-stranded DNA templates (Stratagene). Mutant cDNA molecules were excised from the pBluescript plasmid by digestion with Sal1 andXbaI, which cut within the polylinker of the plasmid and in the 3’ noncoding sequences of the cDNA, respectively. The insert DNA was recovered by electrophoresis and gel elution and blunt-end-ligated into the unique BamHI site of the retroviral expression vector ~ Z I P ~ ~ O S V ( X provided ) - ~ , ’ ~ by Dr Richard A. Mulligan (Whitehead Institute, Boston, MA). Colonies containing recombinant plasmids were identified by hybridization, and a retroviral construct with the mutant CD13/APN cDNA cloned in the correct orientation was isolated and selected on the basis of restriction endonuclease mapping. This construct was transfected into NIH-3T3 cells by calcium phosphate precipitation techniques as previously described.I6 The cells were then cultured in complete medium containing 800 pg/mL G418 (Geneticin; Sigma Chemical, St Louis, MO) to select for transfectants expressing the neomycin resistance gene contained in the vector. Stable G418-resistant transformants were isolated after 2 to 3 weeks. We had previously generated NIH-3T3 transfectants that expressed high levels of normal human APN,’JZ thus providing a basis for comparison with the deletion mutant. The predicted amino acid sequences of normal and deletion mutant APN molecules are illustrated in Fig 1. Flow cytomeh’c analysis of MoAb binding to transfectants expressing mutant APN polypeptides. NIH-3T3 transfectants expressing Blood, Vol79, No 12 (June 15). 1992: pp 3344-3349 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. CD13/AMINOPEPTIDASE N DELETION MUTANT 1 I Cytoplasmic Transmembrane Segment Domain I Extracellular Domain 3345 I 7 Normal CD13 COOH NHz I ., ,,. I , I ,, 928 Deletion Mutant CD13 NH, i i i COOH Fig 1. Schematic of the predicted structure of normal and mutant CD13/APN molecules. The structure of the protein encoded by the CD13/APN deletion mutant cDNA (928 amino acids, bottom) was predicted to lack 39 amino acids as compared with the normal CD13/APN molecules (967 amino acids, top). The deleted residues (shaded region) included those residues encoded by the portion of CD13/APN cDNA between twoBstEll sites, and includedthe extracellular pentapeptide sequence HELAH, which is associated with catalytic activity and zinc coordination. normal CD13/APN (NIHKD13wt) or the deletion mutant (NIH/ CD13del) were analyzed by flow cytometry as previously described.16Briefly, cells were incubated at 4°C for 30 minutes in a titered excess of either a CD13-specific MoAb or an isotypematched control mouse myeloma protein. After being washed with cold staining medium (Dulbecco's modified Eagle's medium, supplemented with 1.0% fetal calf serum, L-glutamine, 10 mmol/L HEPES, antibiotics, and 2 mmol/L sodium azide), cells were incubated for 30 minutes with fluoresceinated affinity-purified goat antiserum to mouse immunoglobulin (Coulter, Hialeah, FL). After further washing in cold staining medium, the cells were resuspended in staining medium containing 0.25 mmol/L propidium iodide and analyzed with a Coulter EPICS 753 flow cytometer. Nineteen different CD13/APN-specific MoAbs were used in this analysis. They were gifts from the following investigators: SJ-1D1, Dr J. Mirro, St. Jude Children's Research Hospital, Memphis, TN; MCS-2, Dr K. Sagawa, Kurume University, Fukuoka, Japan; WM15 and WM47, Drs E. Favaloro and K. F. Bradstock, ICPMR, Westmead Hospital, NSW, Australia; F23, Dr C.L. Finstad, Memorial Sloan-Kettering Cancer Center, New York, Ny,Mou28 and Mou48, Dr R. Winchester, Hospital for Joint Diseases, New York, Ny, CLB/Mon/Gran/2, Dr P.M. Lansdorp, British Columbia Cancer Research Center, Vancouver, BC, and Dr A.E.G. van dem Borne, Central Laboratory of the Netherlands, Amsterdam; RMAG6, Drs P.J. O'Connell and A.J. d'Apice, Royal Melbourne Hospital, Victoria, Australia; 22A5, Dr M.A. Horton, St. Bartholomew's Hospital, London, U K MY7, Dr J.D. Griffin, DanaFarber Cancer Institute, Boston, MA; 3D8, Dr A.E. Koch, Northwestern University Medical School, Chicago, IL; 46All and 43E6, Dr H.-J. Buhring, Medizinische Klinik 11, Tubingen, Germany; U71 and U81, Dr D. Bourel, Centre Regional de Tranfusion Sanguine, Rennes, France; MY32, Dr C.I. Civin, Johns Hopkins Oncology Center, Baltimore, MD; 72a, Dr R.F. Todd, University of Michigan Medical Center, Ann Arbor, MI; and TUK1, Dr B. Uchanska-Ziegler, Institut fur Experimentelle Immunologie der Philipps Universitat Marburg, Marburg, Germany. Analysis of peptidase activiy. Cell-surface APN activity was measured with a sensitive spectrophotometric assay, as previously described.12 Briefly, intact NIH/CD13wt cells were incubated at 37°C in isotonic buffer containing 6 mmol/L alanine-p-nitroanilide (Sigma), a substrate for CD13/APN. Samples were periodically removed and chilled to arrest enzymatic activity. After centrifugation at PC, cell-free supernatants were assayed for optical density at 405 nm to detect the presence of freep-nitroaniline liberated by cleavage of the substrate by CD13/APN. All measurements were made in triplicate. This assay specifically detects APN activity, as demonstrated by the ability of the aminopeptidase-specific inhibitors bestatin and actinonin to block all catalytic activity.I2To test for inhibition of cell-surface APN activity by CD13/APN-specific MoAbs, NIH/CD13wt cells were preincubated for 1 hour at 37°C in the presence of saturating concentrations of individual CD13/ APN-specific MoAbs, followed by addition of substrate and measurement of cell-surface APN activity by the above method. Immunoprecipitation of mutant CD13IAPN molecules. NIH/ CD13wt cells, NIH/CD13del cells, or parental NIH-3T3 cells were metabolically labeled with [35S]methioninefor 1 hour, incubated an additional hour in medium containing unlabeled methionine, lysed, and immunoprecipitated with either CD13/APN-specific MoAb (WM15 or MY7) or isotype-matched control mouse myeloma protein. Protein-A-Sepharose CL-4B (Pharmacia Fine Chemicals, Piscataway, NJ) coated with goat antibody to mouse immunoglobulin was used as the immunoabsorbent. Immunoprecipitates were heat-denatured and analyzed on polyacrylamide gels containing sodium dodecyl sulfate (SDS), as previously described.16 For kinetic studies, parallel cultures of NIH/CD13wt or NIH/ CD13del cells were labeled for 15 minutes with [35S]methionine, followed by incubation in medium containing an excess of cold methionine. At various times of chase, cells were lysed and immunoprecipitated with MoAb MY7, and the immunoprecipitates were analyzed as described above. RESULTS Inhibitory MoAbs do not recognize mutant CD13IAPN molecules. NIH/CD13wt transfectants and NIH/CD13del transfectants were stained with a panel of 19 MoAbs specific for defined extracellular epitopes of CD13/APN, and then were analyzed by flow cytometry. All of the MoAbs recognized epitopes of normal CD13/APN molecules (Fig 2A and C), whereas four of the 19 (F23, WM15, U71, and U81) did not react with the deletion mutant (Fig 2F), suggesting that the epitopes normally recognized by these antibodies were either abolished or modified by the deletion of 39 amino acids from the extracellular domain. Although 15 of the MoAbs did recognize epitopes on the CD13/APN mutant (Fig 2B and D), the extent of binding was generally less than one tenth of that with the NIH/ CD13wt transfectants. Sixteen of the 19 MoAbs, including the four that failed to recognize the deletion mutant, were tested for their ability to inhibit cell-surface A€" activity. A subset of seven MoAbs significantly inhibited the activity of the peptidase, whereas the other nine had little or no effect. The seven inhibitory MoAbs were in two groups: (1) four MoAbs that were highly inhibitory (WM15, F23, U71, and USl), and were the same MoAbs that failed to recognize the mutant CD13/AF"; and (2) three MoAbs with intermediate inhibitory effects (3D8, MY7, and CLB/Mon/Gran/2), which did bind to the CD13/APN mutant (Fig 3). All nine of the noninhibitory MoAbs recognized epitopes on the CD13/ AI" mutant by flow cytometric assays. Mutant CDISIAPN molecules lack enzymatic activity. APN activity was measured on the surface of intact parental NIH-3T3 cells, NIH/CD13wt transfectants, and NIH/ From www.bloodjournal.org by guest on June 16, 2017. For personal use only. ASHMUN, SHAPIRO, AND LOOK 3346 NIH3T3 / Normal CD13 120 r NIH3T3 I Mutant CD13 "I B 2 s 100 .. B BO E p .c 2 I] 60 40 m 0" 50 1 W n 500 3 : F E l , IA m.. 250- 0 E, P 20 D 1 10 100 1 10 100 Log Fluorescence Intensity Fig 2. Flow cytometric analysis of normal and deletion mutant CD13/APN on NIH-3T3 transfectants. NIH/CD13wt or NIH/CD13del transfectants were stained with a panel of CD13/APN-specific MoAbs; binding was compared with that of an isotype-matched control mouse myeloma protein (dotted lines). All 19 MoAbs recognized the normal CD13/APN on NIH/CD13wt cells, as shown for MoAbs Mou28 (A), MY7 (C), and WM15 (E). Fifteen of the 19 MoAbs also bound t o the CDl3/APN deletion mutant on NIH/CD13del cells, as shown for Mou28 (B) and MY7 (D). However, four MoAbs (WM15, F23. U71, and U81) failed to bind t o the deletion mutant, as shown for WM15 (F). CD13del transfectants (Fig 4). The mutant cells lacked detectable APN activity, indicating that the CD13/APN molecules expressed on their cell surface were enzymatically inactive. NIH/CD13wt cells, by contrast, displayed high levels of APN activity, more than 15 times that of parental NIH-3T3 cells, whose aminopeptidase activity is characteristically 10w.l~ Immunoprecipitated mutant CD13IAPN molecules display alteredprocessing. CD13/AI" molecules were immunoprecipitated from NIH/CD13wt cells and from NIH/CD13del cells with either MY7 or WM15 MoAbs. The MY7 antibody recognized both normal and mutant CD13/APN molecules in flow cytometric assays (Fig 2), and immunoprecipitated both normal and mutant molecules (Fig 5, lanes 3 and 6). By contrast, WM15 recognized normal but not mutant CD13/APN molecules in flow cytometric assays (Fig 2); it also immunoprecipitated normal CD13/APN molecules (Fig 5, lane 2), but not the mutant polypeptides (Fig 5, lane 5). The majority of the normal CD13/APN molecules were present as 150-Kd species, previously shown to be the mature cell-surface form of this enzyme; a minority were present as 130-Kd species, previously shown to be the transient intracellular precursor.lb The CD13/APN deletion mutant also appeared as two species, with electro- MAb Binding\/ Normal CD13 Deletion Mutant CD13 + - + + + + Fig 3. Inhibition of cell-surface APN activity by CDl3/APN-specific MoAbs. Cell-surface APN activity on intact NIH/CD13wt cells was measured after the binding of each of 16 CDlB/APN-specific MoAbs, as described in Materials and Methods. The measured activity is expressedas a percentage of control activity measured in the absence of any MoAb. The four MoAbs (WM15, F23, U71, and U81) that failed t o recognize mutant CD13/APN molecules corresponded t o the most inhibitory for cell-surface CD13/APN a c t i v i i . Three MoAbs displaying intermediate inhibition of APN (3D8, MY7, and CLB/Mon/Gran/2) recognized both native and deletion mutant enzyme molecules, as did the nine MoAbs that were noninhibitory. phoretic mobilities consistent with slightly smaller molecular masses ( 145 Kd and 125 Kd, respectively) as predicted by the modified cDNA sequence. As opposed to the normal CD13/APN molecules, most of the mutants were present as smaller 125-Kd species. Pulse-chase analysis demonstrated processing of normal CD13/APN molecules to mature, 150-Kd cell-surface molecules within 2 hours (Fig 6, left), whereas the deletion mutant remained incompletely processed as intracellular precursor molecules even after 8 - +0.0 0.5 Time (hr) 1.o Fig 4. Measurement of the enzymatic activity of mutant CD13/ APN molecules. Cell-surface APN activity was measured on NIH/ CDl3wt cells (0). NIH/CD13del cells (O),or parental NIH3T3 cells (A). Enzymatic activity on NIH/CD13del cells was indistinguishable from that on parental NIH3T3 cells, indicating that the deletion mutant lacked enzymatic activity. From www.bloodjournal.org by guest on June 16, 2017. For personal use only. CD13/AMINOPEPTIDASE N DELETION MUTANT NIH3T3 + Normal CD13 -0, c, c 0 0 s 3 h NIH3T3 + Mutant CD13 - 0,, c c s NIH3T3 0 b h 3347 c, C 0 0 E3 >-I h 1 2 3 4 5 6 7 8 9 - 200 - 116 - 97 Fig 5. Characterizationof normaland mutant CD13/APN glycoproteins. Cells were metabolically labeled with ["Slmethionine for 1 hour, followed by incubation for an additional hour in complete medium; detergent lysates were immunoprecipitated,and immune complexes were analyzed in polyacrylamide gels containing SDS. CD13/APN molecules were immunoprecipitatedfrom NIH/CD13wt cells (lanes 1 to 3). NIHICD13del cells (lanes 4 to 6). or parental NIH3T3 cells (lanes 7 to 9). using MoAbs MY7 (lanes 3.6.9). WM15 (lanes 2,5,8), or isotype-matched control myeloma protein (lanes 1.4.7). Normal CD13/APN molecules were precipitated with either MY7 or WMlS, and were present predominantlyas the mature, 150 kD cell surface form (left arrow, lanes 2 and 3). Mutant CD13/APN molecules were precipitatedonly by MY7, and were present predominantly as the lower-molecular weight precursor form (right arrow, lane 6). hours (Fig 6, right), with only a small percentage of the molecules reaching the cell surface. DISCUSSION We have produced a deletion mutant of CD13/APN that lacks a part of the extracellular domain, including a putative zinc-binding motif. Many extracellular epitopes were preserved in the altered CD13/APN molecule, as 15 of 19 CD13/APN-specific MoAbs bound to the mutant glycoproteins. However, the four highly inhibitory MoAbs that failed to bind must recognize epitopes in the vicinity of the critical pentapeptide sequence associated with zinc coordination and catalytic activity. These MoAbs may exert their inhibitory action by interfering with zinc coordination of the native enzyme, by blocking a residue important in catalytic activity, or both. The antigenic determinants of these four MoAbs may also include residues elsewhere on the molecule that would normally be placed in juxtaposition with the deleted amino acids on the normal CD13/APN protein." By contrast, the three MoAbs with intermediate inhibitory effects (3D8, MY7, and CLB/Mon/Gran/2) may recognize epitopes lying near but outside of the deleted segment. Flow cytometric profiles for these three MoAbs were similar to those for the nine noninhibitory MoAbs (Fig 2A-D), indicating that the epitopes recognized by these MoAbs were fully preserved on the deletion mutant molecules. Steric effects of binding in close proximity to the native enzyme's active site could explain moderate, but incomplete, inhibition of APN activity by this subset of MoAbs. Comparison of the deleted CD13/APN sequences with sequences of other metalloproteases suggests that they are pivotal in forming a tridentate pocket for zinc in the APN extracellular domain. The pentapeptide sequence containing histidines 388 and 392 (HExxH) is characteristic of zinc-binding metalloproteases.'3.'XStudies of CDlO/neutral endopeptidase have also demonstrated the importance of this extracellular region in enzymatic a~tivity.'~.~" Point mutations of either of the two analogous histidine residues within its similar pentapeptide sequence (HExxH, at amino acid positions 583 to 587) resulted in loss of zinc binding and catalytic activity, whereas a point mutation of a glutamic acid residue contained within the pentapeptide sequence eliminated catalytic activity, apparently without affecting zinc binding.I9 These findings suggest that while zinc coordination is closely associated with the histidine residues present within this critical region, catalytic activity also depends on the presence of other important residues in the enzyme's active site. Dimerization of APN molecules, which occurs intracellularly during biosynthesis, while the molecules are still in the transient precursor form, may be necessary for the transport of CD13/APN molecules through the Golgi, and is sensitive to even small alterations in molecular structure, as is ultimate processing of the protein to the cell surface.2'.22 In our study, the deletion of a 39-amino acid segment resulted in markedly diminished processing and intracellular retention of mutant CD13/APN molecules, suggesting that the critical dimerization and final processing of these molecules were significantly affected by this deletion. This raises the possibility that levels of normal CD13/APN could be modulated on the surface of a cell by engineering coexpression of mutant CD13/APN, leading to intracellular retention of heterodimers of normal and mutant molecules. Experiments to test this idea are under way. APN is a widely distributed ectoenzyme found on a spectrum of tissues, including the brush border epithelial cells of small intestine, renal proximal tubules, and placenta. In the small intestine, APN is found on the apical surface of epithelial cells, where it cleaves N-terminal amino acids from small oligopeptides as part of protein From www.bloodjournal.org by guest on June 16, 2017. For personal use only. ASHMUN, SHAPIRO, AND LOOK 3348 NIH3T3 + Mutant CD13 NIH3T3 + Normal CD13 I Chase 0 0.51 2 Time (hr) I 4 6 800.51 2 4 6 8’ - 200 F - 116 - 97 Fig 6. Biochemical analysis of normal and mutant CD13/APN molecules. Parallel cultures of either NIH/CD13wt cells or NIH/CD13/del cells were labeled with PsSJmethioninefor 15 minutes, and then incubated in medium containing unlabeled methionine for the indicated chase times. Detergent lysates were immunoprecipitated with t h e MoAb MY7, and immune complexes were analyzed in polyacrylamide gels containing SDS. Normal CD13/APN molecules were present in t h e transient precursor 130-Kd form, and were chased to the mature, 150-Kd cell-surfaceform by 2 hours (left). Deletion mutant CD13/APN molecules were detected as 125-Kd precursor forms predominantly, with only a small percentage appearing as t h e processed mature cell surface form of approximately 145 Kd (right). dige~tion.~ This . ~ ~enzyme has also been implicated as having a regulatory role in the inactivation of biologically active oligopeptides, best exemplified by its participation in the degradation of opioid dipeptides on synaptic membranes of the central nervous ~ y s t e m . ~ ~The - ~ ’ precise function of APN on the surface of myeloid cells is still unknown, although some evidence suggests a regulatory r0le,2~-~* in which the enzyme either removes key residues from active peptides or converts inactive peptides to active forms. The lineage-specific pattern of expression of APN within the hematopoietic system suggests that the enzyme’s role may be central to myeloid cell function. Mutational analysis to delineate structural motifs of CD13/APN polypeptides essential for enzymatic activity and binding of inhibitory MoAbs, as described in this report, should prove useful in delineating the role of APN on myeloid cells. ACKNOWLEDGMENT We thank Kevin Coleman, Elizabeth Mann, Edward Wingfield, and Sam Lucas for excellent technical assistance, and John Gilbert for editorial review. We thank the investigators listed in the Methods for their gifts of MoAbs. REFERENCES 1. Look AT, Ashmun RA, Shapiro LH, Peiper SC: Human myeloid plasma membrane glycoprotein CD13 (gp150) is identical to aminopeptidase N. J Clin Invest 83:1299, 1989 2. Olsen J, Cowell GM, Konigshofer E, Danielsen EM, Moller J, Laustsen L, Hansen OC, Welinder KG, Engberg J, Hunziker W, Spiess M, Sjostrom H, Noren 0:Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA. FEBS Lett 238:307,1988 3. Letarte M, Vera S, Tran R, Addis JBL, Onizuka RJ, Quackenbush EJ, Jongeneel CV, Mclnnes RR: Common acute lymphoblastic leukemia antigen is identical to neutral endopeptidase. J Exp Med 168:1247,1988 4. Jongeneel CV, Quackenbush El,Ronco P, Verroust P, Carrel S, Letarte M: Common acute lymphoblastic leukemia antigen expressed on leukemia and melanoma cell lines has neutral endopeptidase activity. J Clin Invest 83:713, 1989 5. Shipp MA, Vijayaraghavan J, Schmidt EV, Masteller EL, d’Adamio L, Hersh LB, Reinherz E L Common acute lymphoblastic leukemia antigen (CALLA) is active neutral endopeptidase 24.1 1 (“enkephalinase”): Direct evidence by cDNA transfection analysis. Proc Natl Acad Sci USA 86297,1989 6. Vivier I, Marguet D, Naquet P, Bonicel J, Black D, Li CX, Bernard A-M, Gorvel J-P, Pierres M: Evidence that thymocyteactivating molecule is mouse CD26 (dipeptidyl peptidase IV). J lmmunol 147:447,1991 7. Hegen M, Niedobiteck G, Klein CE, Stein H, Fleischer B: The T cell triggering molecule Tp103 is associated with dipeptidyl aminopeptidase IV activity. J Immunol 144:2908,1990 8. Ulmer AJ, Mattern T, Feller AC, Heymann E, Flad HD: CD26 antigen is a surface dipeptidyl peptidase IV (DPPIV) as characterized by monoclonal antibodies clone Tii-19-4-7 and 4ELlC7. Scand J Immunol31:429,1990 9. Wu Q, Tidmarsh GF, Welch PA, Pierce JH, Weissman IL, Cooper MD: The early B-lineage antigen BP-1 and the transformation-associated antigen 6C3 are on the same molecule. J Immunol 143:3303,1989 10. Wu Q, Lahti JM, Air GM, Burrows PD, Cooper MD: Molecular cloning of the murine BP-116C3 antigen: A member of the zinc-dependent metallopeptidase family. Proc Natl Acad Sci USA 87:993,1990 11. Wu Q, Li L, Cooper MD, Pierres M, Gorvel J P Aminopeptidase A activity of the murine B-lymphocyte differentiation antigen BP-l/6C3. Proc Natl Acad Sci USA 88:676,1991 12. Ashmun RA, Look A T Metalloprotease activity of CD13/ aminopeptidase N on the surface of human myeloid cells. Blood 75:462, 1990 13. Vallee BL, Auld DS: Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 2 9 5647,1990 14. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463, 1977 15. Cepko CL, Roberts BE, Mulligan R C Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell 371053, 1984 16. Look AT, Peiper SC, Rebentisch MB, Ashmun RA, Roussel From www.bloodjournal.org by guest on June 16, 2017. For personal use only. CD13/AMINOPEPTIDASE N DELETION MUTANT MF, Rettenmier CW, Sherr CJ: Transfer and expression of the gene encoding a human myeloid membrane antigen (gp150).J Clin Invest 75569,1985 17. Laver WG, Air GM, Webster RG, Smith-Gill SJ: Epitopes on protein antigens: Misconceptions and realities. Cell 61553, 1990 18. Vallee BL, Auld DS: Short and long spacer sequences and other structural features of zinc binding sites in zinc enzymes. FEBS Lett 257:138,1989 19. Devault A, Sales V, Nault C, Beaumont A, Roques B, Crine P, Boileau G: Exploration of the catalytic site of endopeptidase 24.11 by site-directed mutagenesis. FEBS Lett 23154,1988 20. Devault A, Nault C, Zollinger M, Fournie-Zaluski M-C, Roques BR, Crine P, Boileau G: Expression of neutral endopeptidase (enkephalinase) in heterologous COS-1 cells. J Biol Chem 263:4033,1988 21. Danielsen EM: Perturbation of intestinal microvillar enzyme biosynthesis by amino acid analogs. J Biol Chem 265:14566, 1990 22. Danielsen EM: Biosynthesis of intestinal microvillar proteins. Dimerization of aminopeptidase N and lactase-phlorizin hydrolase. Biochemistry29:305,1990 23. Noren 0, Sjostrom H, Danielsen EM, Cowell GM, Skovbjerg H The enzymes of the enterocyte plasma membrane, in Desnuelle P (ed): Molecular and Cellular Basis of Digestion. Amsterdam, The Netherlands, Elsevier/North-Holland, 1986, p 335 24. Semenza G: Anchoring and biosynthesis of stalked brush 3349 border membrane proteins: Glycosidasesand peptidases of enterocytes and renal tubuli. A n n Rev Cell Biol2255,1986 25. Matsas R, Stephenson SL, Hryszko J, Kenny AJ, Turner AJ: The metabolism of neuropeptides. Phase separation of synaptic membrane preparations with Triton X-114 reveals the presence of aminopeptidaseN. Biochem J 231:445,1985 26. Turner AJ, Hooper NM, Kenny AJ: Metabolismof neuropeptides, in Kenny AJ, Turner AJ (eds): Mammalian Ectoenzymes. New York, NY,Elsevier Science, 1987, p 211 27. Turner AJ, Matsas R, Kenny AJ: Commentary: Are there neuropeptide-specific peptidases? Biochem Pharmacol 34:1347, 1985 28. Saito M, Aoyagi T, Umezawa H, Nagai Y: Bestatin, a new specific inhibitor of aminopeptidases, enhances activation of small lymphocytes by concanavalin A. Biochem Biophys Res Commun 76526,1977 29. Schorlemmer HU, Bosslet K, Sedlacek HH: Ability of the immunomodulating dipeptide bestatin to activate cytotoxic mononuclear phagocytes. Cancer Res 43:4148,1983 30. Talmadge JE, Lenz BF, Pennington R, Long C, Phillips H, Schneider M, Tribble H Immunomodulatory and therapeutic properties of bestatin in mice. Cancer Res 46:4505,1986 31. Jarstrand C, Blomgren H Increased granulocyte phagocytosis after oral administration of bestatin, a new immunomodulator.J Clin Lab Immunol7115,1982 32. Muller WEG, Zahn RK, Arendes J, Munsch N, Umezawa H: Activation of DNA metabolism in T-cells by bestatin. Biochem Pharmacol28:3131,1979 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 1992 79: 3344-3349 Deletion of the zinc-binding motif of CD13/aminopeptidase N molecules results in loss of epitopes that mediate binding of inhibitory antibodies RA Ashmun, LH Shapiro and AT Look Updated information and services can be found at: http://www.bloodjournal.org/content/79/12/3344.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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