Vesicular Localization and Characterization of a Novel Post-Proline-Cleaving Aminodipeptidase, Quiescent Cell Proline Dipeptidase This information is current as of September 26, 2021. Murali Chiravuri, Fernando Agarraberes, Suzanne L. Mathieu, Henry Lee and Brigitte T. Huber J Immunol 2000; 165:5695-5702; ; doi: 10.4049/jimmunol.165.10.5695 http://www.jimmunol.org/content/165/10/5695 Downloaded from References This article cites 28 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/165/10/5695.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Vesicular Localization and Characterization of a Novel Post-Proline-Cleaving Aminodipeptidase, Quiescent Cell Proline Dipeptidase1 Murali Chiravuri,* Fernando Agarraberes,† Suzanne L. Mathieu,* Henry Lee,* and Brigitte T. Huber2* A large number of chemokines, cytokines, and signal peptides share a highly conserved X-Pro motif on the N-terminus. The cleavage of this N-terminal X-Pro dipeptide results in functional alterations of chemokines such as RANTES, stroma-derived factor-1, and macrophage-derived chemokine. Until recently, CD26/DPPIV was the only known protease with the ability to cleave N-terminal X-Pro motifs at neutral pH. We have isolated and cloned a novel serine protease, quiescent cell proline dipeptidase (QPP), with substrate specificity similar to that of CD26/DPPIV. In this paper we show that QPP, like CD26/DPPIV, is synthesized Downloaded from with a propeptide and undergoes N-glycosylation. Interestingly, this glycosylation is required for QPP enzymatic activity, but not for its localization. Unlike the cell surface molecule, CD26/DPPIV, QPP is targeted to intracellular vesicles that are distinct from lysosomes. Proteinase K treatment of intact vesicles indicates that QPP is located within the vesicles. These vesicles appear to have a secretory component, as QPP is secreted in a functionally active form in response to calcium release. The presence of QPP in the vesicular compartment suggests that molecules bearing the N-terminal X-Pro motif can be cleaved at multiple sites within and outside the cell. These results expand the potential site(s) and scope of a process that appears to be an important mechanism of http://www.jimmunol.org/ post-translational regulation. The Journal of Immunology, 2000, 165: 5695–5702. roteolytic cleavage of proteins can have profound effects C-terminal amino acid by prolylcarboxypeptidase (PCP; angio- on their stability and function (1–3). There is a striking tensinase C) (7, 8). P conservation of X-Pro motifs on the N termini of many Few proteolytic enzymes that cleave peptide bonds containing chemokines, cytokines, and other signal peptides, suggesting a se- proline have been identified. These include exopeptidases such as lective pressure for this motif in the evolution of these molecules CD26/DPPIV and PCP (4). We have isolated and cloned a serine (4). For many of these factors, the significance of the X-Pro motif protease, quiescent cell proline dipeptidase (QPP), which has sub- has yet to be ascertained. Recently, however, the work of several strate specificity very similar to that of CD26/DPPIV (9). Both by guest on September 26, 2021 groups has shown that the N-terminal X-Pro motif of the chemo- these enzymes cleave N-terminal dipeptides when the penultimate 3 kines RANTES, stroma-derived factor-1␣ (SDF1␣), SDF1␤, amino acid is a proline or, less preferably, an alanine residue (9– eotaxin, and macrophage-derived chemokine (MDC) is cleaved by 11). Recently, we observed that highly specific aminodipeptidase dipeptidyl peptidase IV (DPPIV/CD26), resulting in an alteration inhibitors cause cell death in quiescent lymphocytes, but not in of their biological function (2, 3, 5, 6). Thus, it appears that the activated lymphocytes (12). QPP is the likely target of these in- N-terminal X-Pro motif on signal molecules may serve as a site for hibitors, suggesting that this protease plays an important role in post-translational modification and regulation. The cleavage of resting cells (12). However, unlike CD26/DPPIV, which is found proline-containing peptide bonds on the C terminus can also affect on the cell surface (10, 11), QPP was isolated from intracellular the function of molecules. Angiotensin, for example, undergoes a fractions (9). Although QPP and CD26/DPPIV share substrate loss of vasoactive function following post-proline cleavage of the specificity, there is no significant sequence homology between these two proteases, suggesting an evolutionary convergence of *Department of Pathology, Program in Immunology, and †Department of Physiology, functional activity. QPP does, however, share significant sequence Sackler School of Graduate Biomedical Sciences, Tufts University School of Medi- cine, Boston, MA 02111 homology with the lysosomal serine protease PCP (42% sequence Received for publication March 15, 2000. Accepted for publication August 22, 2000. identity) (7, 9). It is interesting to note that the three post-proline- The costs of publication of this article were defrayed in part by the payment of page cleaving proteases, CD26/DPPIV, PCP, and QPP, all have the charges. This article must therefore be hereby marked advertisement in accordance same sequential ordering of the active site residues, Ser-Asp-His with 18 U.S.C. Section 1734 solely to indicate this fact. (4, 9), and are functionally active as homodimers (7, 9, 11). 1 This work was supported by National Institutes of Health Research Grants AI36696 In this work we show that similar to its functional homologue, and AI43469 (to B.T.H.). CD26/DPPIV, QPP is synthesized with a cleaved pro-peptide. 2 Address correspondence and reprint requests to Dr. Brigitte T. Huber, Department of Pathology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, QPP undergoes N-linked glycosylation at the Golgi apparatus, MA 02111. E-mail address: [email protected] which is required for its proteolytic activity. Unlike CD26/DPPIV, 3 Abbreviations used in this paper: SDF, stroma-derived factor; QPP, quiescent cell QPP localizes to a post-Golgi vesicular compartment. QPP-con- proline dipeptidase; DPPIV, dipeptidyl peptidase IV; DPPII, dipeptidyl peptidase II; taining vesicles have similar density to lysosomes, but further frac- PCP, prolylcarboxypeptidase; GFP, green fluorescent protein; M6P, mannose-6-phos- phate; LAMP1, lysosome-associated membrane protein-1; MDC, macrophage-de- tionation shows that QPP-containing vesicles form a distinct com- rived chemokine, TGN, trans-Golgi network; zGGL-AMC, z-glycine-glycine- partment, and unlike mannose-6-phosphate (M6P)-dependent leucine-aminomethylcoumarin; Ala-Pro-AFC, alanine-proline-aminofluorocoumarin; HA, hemagglutinin; PNS, postnuclear supernatant; ER, endoplasmic reticulum; Glc- lysosomal targeting (13), QPP localizes to vesicles by a M6P- NAc, N-acetylglucosamine; PNGase F, peptide-N-glycosidase F. independent mechanism. QPP is localized in the vesicular lumen, Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 5696 CHARACTERIZATION OF A CD26/DPPIV-LIKE PROTEASE as it is not susceptible to proteinase K digestion of intact vesicles. Following this the samples were brought up to 1% Nonidet P-40 and 0.05 Functionally active QPP can also be detected in cell culture su- M sodium phosphate. PNGase F (500 U) was added and incubated for the pernatant, and its secretion can be induced by cell activation and indicated times at 37°C. The reaction was terminated by boiling the sample in 2ϫ reducing loading buffer for 5 min, followed by SDS-PAGE and calcium mobilization, suggesting that there is a secretory compo- immunoblot analysis. The controls were treated in the same manner except nent to the QPP-containing vesicular compartment. The study of for the addition of PNGase F. post-translational modification of N-terminal X-Pro-containing signal molecules has been a rapidly expanding field in the last few Confocal microscopy years. The discovery of a novel post-proline-cleaving aminodipep- 293T human fibroblasts were transfected with QPP-GFP or QPP-Myc, tidase in a distinct compartment from CD26/DPPIV broadens the plated on coverslips, pretreated with fibronectin (100 ␮g/ml), and blocked scope of post-proline-cleavage as a regulatory mechanism. with 1% BSA in PBS. These cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X in PBS. The primary Ab was incubated for1hat4°C, followed by three washes and incubation of the secondary Materials and Methods Ab, where applicable, for1hat4°C. The samples were washed and vi- Materials sualized by confocal microscopy. Anti-Myc Ab was purchased from PharMingen (San Diego, CA), and anti- Biochemical organelle fractionation hemagglutinin (anti-HA) Ab was purchased from BabCo (Berkeley, CA). The anti-lysosome-associated membrane protein-1 (anti-LAMP1) mAb The isolation of subcellular organelles on discontinuous density gradients H4A3 was obtained from the Developmental Studies Hybridoma Bank was performed as previously described (14). In addition, a continuous gra- ϫ 8 (Iowa City, IA), and the anti-calnexin mAb was purchased from StressGen dient fractionation was performed. Briefly, 1 10 cells were lysed by (Victoria, Canada). Rab11 and adaptin-␣ Abs were purchased from Trans- cavitation or Dounce homogenization (Kontes, Vineland, NJ) in 0.25 M duction Laboratories (Lexington, KY).