Dipeptidase Aminodipeptidase, Quiescent Cell Proline of a Novel

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

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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-phosphate; 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,

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). The reporter substrates alanine-pro- sucrose, and the postnuclear supernatant (PNS) was taken from a low speed Downloaded from line-aminofluorocoumarin (Ala-Pro-AFC) and z-glycine-glycine-leucine- centrifugation. For discontinuous fractionation, a metrizamide/Percoll (Ac- aminomethylcoumarin (zGGL-AMC) were purchased from Enzyme curate Chemical & Scientific Corp., Westbury, NY) gradient was set up, Systems Products (Dublin, CA). Tunicamycin, ionomycin, and cyclohex- using 35% metrizamide, 17% metrizamide, and 6% Percoll, and the PNS ϫ amide were purchased form Sigma (St. Louis, MO), and peptide-N-glyco- was layered over it. Following ultracentrifugation at 60,000 g the four sidase F (PNGase F) was purchased from New England Biolabs interface layers were harvested and analyzed. The orientation of the gra- ϭ ϭ (Beverly, MA). dient is as follows: lightest fraction fraction I, most dense fraction fraction IV. For the continuous gradient fine fractionation of subcellular http://www.jimmunol.org/ Constructs and transfections organelles, a metrizamide/Percoll gradient was set up as follows: 1 ml of 35% metrizamide was placed on the bottom of the tube, followed by a To make the QPP-green fluorescent protein (GFP) construct, enhanced linear gradient of 5–20% metrizamide, 1 ml of 6% Percoll (Pharmacia, GFP was amplified from the cloning vector pEGFP-N1, obtained form Uppsala, Sweden), and 2.5 ml of PNS. The gradients were centrifuged at Clontech (Palo Alto, CA), and cloned into the pCI-neo expression vector 160,000 ϫ g for2hat4°C, using a SW41 swinging rotor (Beckman, from Promega (Madison, WI). The sense primer incorporated five glycine Fullerton, CA). Gradients were fractionated in 250-␮l aliquots. These frac- codons upstream of GFP. QPP was amplified with primers containing re- tions were analyzed as described. striction site linkers and cloned in frame upstream of enhanced GFP. The QPP-Myc fusion was generated by PCR using the high fidelity DeepVent Proteinase K treatment polymerase (New England Biolabs), with an antisense primer that coded The QPP-containing vesicular fraction was obtained by discontinuous gra-

for 10 aa comprising the c-Myc epitope tag (EQLLISEEDL). Likewise, a by guest on September 26, 2021 QPP-HA fusion was generated using an antisense primer that coded for 10 dient fractionation and either were left untreated or were treated with pro- aa comprising the HA epitope tag (YPYDVPDYA). All constructs were teinase K. Fractions were incubated in chymostatin (30 ␮M) for 10 min on cloned into the pCI-neo expression vector. Transfections were performed ice. These vesicles either were left unpermeabilized or were permeabilized ␮ using the calcium phosphate method. with 1% Triton X. Proteinase K (10 g/ml) and CaCl2 (1 mM) were added to the samples for 20 min at 0°C. The reaction was terminated by the Western blot analysis addition of AEBSF (100 ␮M). Cells (1–2 ϫ 107) were resuspended in lysis buffer (20 mM HEPES, 1.5 QPP purification ␮ mM MgCl2, 2 mM EDTA, 10 mM KCl, 0.1% Nonidet P-40, 5 g/ml antipain, and 5 ␮g/ml leupeptin) for 30 min at 4°C. Lysed cells were QPP was purified from Jurkat cells as previously described (9). Briefly, centrifuged at 3,000 rpm on a microcentrifuge for 10 min, and the pellet Jurkat S-110 supernatant was dialyzed against 4 l of 50 mM acetic acid, was discarded. The postnuclear supernatant was subjected to a 30,000 ϫ g titrated to pH 4.5 with NaOH. The protein sample was clarified by cen- centrifugation for 30 min. The protein concentration was measured using trifugation at 1000 ϫ g for 10 min at 4°C. The clarified supernatant was the bicinchoninic acid protein estimation kit (Pierce, IL). For the immu- concentrated on a Centricon 50 membrane to about 10 ml. The concen- noblot assay of supernatants, the supernatants of QPP-transfected or vec- trated sample was loaded ontoa3mlofHiTrap SP Sepharose column and tor-transfected (control) cells were centrifuged at 300 ϫ g to remove all equilibrated with 50 mM acetate, pH 4.5 (start buffer). The column was cells and subjected to SDS-PAGE analysis. washed with 10 column volumes of start buffer and eluted with a linear 0–300 mM NaCl gradient in start buffer. Active fractions were pooled and Enzyme assays concentrated to about 1 ml on a Centricon 50 membrane, then to about 0.2 ml on a Microcon 30 membrane. The concentrated material was loaded QPP and proteasome enzymatic activities were measured using the fluoro- onto a Superose 12 gel filtration column and equilibrated with 50 mM genic substrates Ala-Pro-AFC (20 ␮M in 50 mM HEPES buffer) and acetate, pH 4.5, and 150 mM NaCl. Active fractions were pooled and used zGGL-AMC (100 ␮M in 50 mM HEPES), respectively, on a Fluoromax as a purified preparation of the activity. fluorescence plate reader (Molecular Devices, Menlo Park, CA; AFC: excitation 390 nm; emission 510 nm; AMC: excitation, 460 nm; emission, 510 nm). For the ␤-hexosaminidase assay, the fluorogenic substrate used Results was 4-methylumbelliferyl-␤-D-glucosaminide. Twenty microliters of the QPP is an intracellular cytoplasmic protease samples obtained from the discontinuous gradient were incubated with 0.5 ml of the substrate solution (1 mM 4-methylumbelliferyl-␤-D-glucosamin- QPP was initially purified from the cytoplasmic fraction of Jurkat ide and 0.1 M sodium acetate, pH 4.5) for 5 min. For the continuous T cells (9). To determine the cellular localization of QPP, confocal gradient fractions, 10 ␮l of the samples were incubated for 20 min. The microscopy was performed on cells expressing a QPP-GFP fusion reaction was stopped with 1.25 ml of stop solution (0.5 M glycine and 0.5 protein. 293T human fibroblasts transfected with this chimeric MNa2CO3). The samples were read using an excitation wavelength of 364 nm and an emission wavelength of 448 nm. construct showed a cytoplasmic distribution (Fig. 1A). Unlike CD26/DPPIV, QPP does not localize to the cell membrane. This PNGase F assays was confirmed by biochemical means; namely, surface biotinyla- Fifty micrograms of lysate from control or QPP-transfected cells was tion of QPP expressing cells, followed by immunoprecipitation, boiled for 10 min under denaturing conditions (0.5% SDS and 1% 2-ME). did not yield any biotinylated QPP molecules (M. Chiravuri et al., The Journal of Immunology 5697

Primary sequence analysis of QPP using a Kyte and Doolittle hy- drophobicity plot (15) indicates a hydrophobic sequence in the N-terminus of QPP, aa 1–21. To verify this, QPP was puriﬁed from Jurkat cells and subjected to N-terminal sequencing. Fig. 1D shows a comparison of the amino acid sequence of full-length QPP deduced from its cDNA (9) and the N-terminal sequence of mature QPP puriﬁed from Jurkat cells. It can be seen that a 29-aa peptide is cleaved from newly synthesized QPP. The cleaved sequence may be either a signal peptide alone or may include a signal peptide and a short propeptide.

FIGURE 1. Localization of QPP by confocal microscopy in 293T hu- QPP undergoes N-linked glycosylation man fibroblasts and N-terminal sequencing of mature QPP. Human 293T SDS-PAGE analysis shows that QPP migrates as a 58-kDa species, human fibroblasts were transfected with a QPP-GFP (green; A) and a QPP- even though the predicted m.w. of QPP based on its primary se- Myc (red; B) construct. These cells were permeabilized and analyzed with quence is 53 kDa. Similar to the lysosomal protease PCP (7), QPP an anti-Myc Ab. C, Merged computer images, where colocalization regis- ters as yellow/orange. D, QPP is synthesized with a propeptide that is has six potential N-glycosylation sites, and glycosylation at these cleaved. Purified QPP was subjected to N-terminal sequencing, and this sites could cause the observed increased in molecular mass. Tu- sequence was compared with the full-length sequence of QPP. nicamycin is an inhibitor of N-acetylglucosamine (GlcNAc) trans- Downloaded from ferase that blocks assembly of the GlcNAc-dolichol complex and thus prevents glycosylation of asparagine residues (16). QPP- unpublished observations). To ensure that the distribution of QPP- transfected fibroblasts were treated with tunicamycin (5 ␮g/ml) for GFP was not altered by the presence of the GFP portion of the various periods of time. Fig. 2A shows that the upper 58-kDa band construct, immunohistochemical analysis was performed with a gradually disappeared with increased time of tunicamycin treat- QPP-Myc construct (Fig. 1B). Fig. 1C shows that QPP-GFP and ment. We also treated QPP with PNGase F, an enzyme that cleaves http://www.jimmunol.org/ QPP-Myc colocalize, demonstrating that the nature of the tag did between the innermost GlcNAc and asparagine residues of high not affect QPP localization. mannose, hybrid and complex carbohydrates from N-linked glycoproteins (17). PNGase F treatment also caused the disappearance QPP is synthesized with a cleaved propeptide of the 58-kDa band (Fig. 2B). These results indicate that the 58- Proteins are routed to the secretory pathway by signal peptides that kDa band represents a processed glycosylated form of QPP, while insert the protein into the endoplasmic reticulum (ER) membrane. the 53-kDa band is the unprocessed form. by guest on September 26, 2021

FIGURE 2. QPP is N-glycosylated, and N-glycosylation is required for QPP enzymatic activity. Immunoblot analysis was performed with anti-Myc Ab. A, QPP-Myc (transfected) ﬁbroblasts treated with tunicamycin (5 ␮g/ml) for the indicated times. B, Anti-Myc Western blot of QPP-Myc, either untreated or treated with PNGase F. C, QPP-Myc ﬁbroblasts treated with cyclohexamide (15 ␮g/ml) for the indicated times. D, Analysis of QPP enzymatic activity (f) and proteasome activity (Ⅺ) in cells treated with tunicamycin for the indicated times. 5698 CHARACTERIZATION OF A CD26/DPPIV-LIKE PROTEASE

To further confirm this post-translational modification, we ana- QPP localizes to vesicles with similar density to lysosomes lyzed the effect of blocking new protein synthesis with cyclohex- To localize QPP we performed biochemical organelle fractionation amide. Cells were transfected with QPP and treated for varying using both discontinuous and continuous metrizamide/Percoll den- lengths of time with cyclohexamide. As shown in Fig. 2C, follow- sity gradients. This was conducted on human fibroblasts trans- ing the inhibition of new protein synthesis, the ratio of the 53-kDa fected with a QPP-Myc construct as well as on untransfected Ju- form of QPP to the 58-kDa form decreases. These results indicate rkat T cells. The transfected fibroblasts had identical phenotype that the 58-kDa species is the mature molecule. and growth characteristics as untransfected cells. QPP-Myc fibro- To determine the functional significance of the glycosylation, blasts were lysed by cavitation, and these lysates were fraction- we analyzed the effect of glycosylation on QPP enzyme activity. Cells transfected with QPP were treated with tunicamycin for var- ated. Four fractions were obtained from the discontinuous gradi- ious time periods, and the ability of QPP to cleave the reporter ents and were analyzed using organelle-specific markers. As a substrate Ala-Pro-AFC was measured. QPP activity was signifi- cytosolic marker we measured the chymotrypsin activity of the cantly decreased following tunicamycin treatment, indicating that proteasome complex, using the reporter substrate zGGL-AMC (19, ␤ the 58-kDa species is the active form of QPP, while the unglyco- 20). The lysosomal marker used was -hexosaminidase activity sylated form lacks QPP activity (Fig. 2D). On the other hand, (14), while LAMP1 was used as a marker for late endosomes and tunicamycin treatment did not alter the localization of QPP (see lysosomes (21). The ER marker used was calnexin (22), while the Fig. 6 below). As a control, the cleaving activity of the proteasome trans-Golgi network (TGN) marker was Rab11 (23). To detect was measured in each of the samples. The proteasome is a cyto- QPP we performed Western blot analysis and measured its activity solic molecular complex (18) that does not undergo a significant with the QPP reporter substrate Ala-Pro-AFC. Downloaded from decrease in enzyme activity following tunicamycin treatment. The analysis of the four fractions is shown in Fig. 3. The least Consistent with this, no change in proteasome activity was seen in dense fraction (I; soluble) had the most proteasome activity (Fig. the tunicamycin-treated samples. 3A), but contained very little QPP (Fig. 3, F and G). Fractions http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Subcellular organelle fractionations of QPP-Myc 293T human fibroblasts using discontinuous gradients. Four fractions were obtained and assayed for proteasome activity as a cytosolic marker (A), immunoblot analysis of the secretory vesicle marker Rab11 (B), immunoblot analysis of the ER marker calnexin (C), immunoblot analysis of the lysosome/late endosome marker LAMP1 (D), enzyme assay of lysosomal ␤-hexosaminidase activity (E), QPP enzymatic activity (F), and immunoblot analysis with an anti-Myc Ab (G). The Journal of Immunology 5699 enriched in Rab11 (II; Fig. 3B) and calnexin (IV; Fig. 3C) also in the transfected fibroblasts, LAMP1 was highly enriched in frac- showed little QPP activity (Fig. 3F). Lysosomes and late endo- tion III, although some was also found on the less dense fraction somes were identified in fraction III by the presence of LAMP1 II. Most of the ␤-hexosaminidase activity was found in fraction III (Fig. 3D), and this fraction also had maximal activity of lysosomal (Fig. 4, D and E). The presence of membrane-bound LAMP1 but ␤-hexosaminidase activity (Fig. 3E). The fraction containing ly- low ␤-hexosaminidase activity in fraction II might indicate that a sosomes (III) had the majority of QPP enzymatic activity (Fig. fraction of the lysosomes was broken during Dounce homogeni- 3F). This was confirmed using Western blot analysis (Fig. 3G). zation, causing the release of soluble proteins such as ␤-hex- QPP was first isolated from Jurkat cells where it is constitutively osaminidase and decreasing the density of the lysosomes. The ma- expressed. Therefore, we also analyzed QPP distribution within jority of QPP Ala-Pro-AFC cleaving activity was found in fraction these cells. Jurkat cells were lysed by Dounce homogenization, III (Fig. 4F). These results suggest that QPP localizes to vesicles and organelle fractionation was performed as described above. The that are either part of the lysosomal compartment or to vesicles that same cell fractionation was conducted on untransfected Jurkat have similar density to lysosomes. cells (Fig. 4). The proteasome activity localizes to the soluble fraction (I; Fig. 4A), and the majority of Rab11 migrates in fraction II QPP is routed to a nonlysosomal vesicular compartment (Fig. 4B), while calnexin localizes to the fraction IV (Fig. 4C). As To further define the localization of QPP within the lysosome- containing fraction (III), we performed colocalization experiments with QPP and the late endosomal and lysosomal marker, LAMP1 (21). QPP-GFP and LAMP1 did not colocalize, as determined by

confocal microscopy (Fig. 5, A–C), suggesting that QPP is routed Downloaded from to alternate vesicles that migrate with the lysosomes under the discontinuous gradient fractionation conditions. A higher resolution was obtained by performing continuous gradient separations. QPP-expressing ﬁbroblasts were separated into 51 fractions and probed for the late endosomal and lysosomal

marker, LAMP1, and QPP (Fig. 5D). These fractions were also http://www.jimmunol.org/ probed for adaptin-␣, a component of adaptor protein 2 that is found in clathrin-coated vesicles from the plasma membrane (24, 25), and Rab11, a small GTP-binding protein that associates with the TGN, some secretory vesicles, and recycling endosomes (23). Fig. 5D shows that the distribution pattern of QPP is different from that of LAMP1. Most QPP is found in relatively more dense vesicles, with its peak at fractions 45 and 47. The appearance of QPP in the least dense soluble fractions is presumably from rup- tured vesicles, perhaps due to the increased centrifugation time by guest on September 26, 2021 required for this fractionation method. The LAMP1 expression pattern has two peaks: one at fraction 25, possibly representing the late endosomes, and the other at fraction 41, representing the lysosomes, according to the ␤-hexosaminidase marker (Fig. 5E). The distribution pattern of Rab11 is significantly different from that of QPP, as it localizes to the less dense vesicles, in agreement with the data from the discontinuous gradients (Fig. 3B). Adaptin-␣ also shows a different distribution than QPP, and their respective fractions of maximum expression are clearly different. Analyses of the fractions for QPP and ␤-hexosaminidase enzymatic activities show a distribution in accordance with the Western blot analysis (Fig. 5E). The QPP activity reaches a maximum in fraction 47 and is slightly elevated in the first few fractions, where the soluble QPP would be found. The ␤-hexosaminidase enzymatic activity corre- lates with the Western blot profile of lysosomal LAMP1 distribution, with maximum activity at fraction 41 and low activity throughout the endosomal range. It should be noted that the elevated QPP activity in the least dense fractions has no correspond- ing increase in ␤-hexosaminidase activity, further supporting the idea that, while the intact QPP-containing vesicles and the lysosomes have similar densities, as was seen with the discontinuous gradients, they are not the same vesicles. Many lysosomal proteins, particularly lysosomal matrix proteins, are targeted to the lysosome through the M6P receptor pathway (13). To determine the effect of glycosylation on the local- FIGURE 4. Subcellular organelle fractionation of untransfected CD26Ϫ ization of QPP, fibroblasts treated or untreated with tunicamycin Jurkat T cells. The fractions were analyzed for proteasome activity (A), were subjected to organelle purification using discontinuous gra- Rab11 expression (B), expression of the ER marker calnexin (C), immu- dients. As shown in Fig. 6A, unglycosylated QPP was still found noblot analysis of LAMP1 (D), lysosomal ␤-hexosaminidase activity, and in the same fraction (III) and did not show increased accumulation QPP enzymatic activity (F). in the Golgi apparatus. This shows that glycosylation and the M6P 5700 CHARACTERIZATION OF A CD26/DPPIV-LIKE PROTEASE

FIGURE 5. QPP localizes to nonlysosomal vesicles. A, Confo- cal microscopy of QPP-GFP (green) ﬁbroblasts. These cells were permeabilized and probed with the anti-LAMP1 (red) Ab (B) and a merged computer image (C) was made. D, Cellular fractionation of QPP-HA ﬁbroblasts on a continuous density gradient. The gradient was prepared as described in Materials and Methods. Immunoblot analysis was performed of LAMP1, adaptin-␣, QPP, and Rab 11, respectively. E, Analysis and comparison of QPP and ␤-hexosaminidase enzymatic activities in the various fractions. Downloaded from http://www.jimmunol.org/

receptor pathway are not required for the routing of QPP, suggest- Discussion by guest on September 26, 2021 ing a nonlysosomal vesicular localization. However, M6P-inde- A large number of signal proteins from diverse families such as pendent localization of proteins to lysosomes has also been de- cytokines, chemokines, and neuropeptides show a remarkable con- scribed (26). servation of an X-Pro motif on the N-terminus (4). Over the last To confirm that QPP is located within vesicles, QPP-HA fibro- few years, it has become apparent that a number of these signal blasts were fractionated using discontinuous gradients, and intact peptides are shorn of their X-Pro motifs by CD26/DPPIV with vesicles from fraction III were treated with proteinase K. This profound consequences to their biological function. These include fraction contained lysosomes/late endosomes and QPP-containing the chemokines RANTES, SDF1, MDC, and eotaxin (2, 3, 5, 6). vesicles (Fig. 6B, lane 1). When the intact vesicles were first per- The ubiquity of the X-Pro motif on signal molecules suggests that meabilized with detergent and then treated with proteinase K, QPP was degraded (Fig. 6B, lane 2). However, in the absence of detergent, no degradation of QPP by proteinase K was seen (Fig. 6B, lane 3), indicating that QPP is located on the inside of the vesicles.

QPP is secreted in an active form Given that QPP localizes to a post-Golgi vesicular fraction distinct from lysosomes, we analyzed the secretion behavior of QPP. To determine whether QPP is secreted, we tested the supernatants of QPP-transfected fibroblasts and vector-transfected fibroblasts for QPP and observed that QPP was secreted in a functionally active form (data not shown). Calcium-dependent exocytosis is a well described phenomenon (27, 28). QPP release from Jurkat cells was monitored after the addition of the calcium ionophore, ionomycin. FIGURE 6. QPP localizes in an M6P-independent manner and is found Jurkat cells in PBS with 1 mM CaCl were treated with ionomycin within the vesicles. A, QPP-Myc transfected fibroblasts were treated with 2 ␮ for 15 min at 37°C, and QPP activity in the supernatant was mea- tunicamycin (5 g/ml) for 24 h and then subjected to organelle fractionation using discontinuous gradients. The four fractions were analyzed by sured. As Fig. 7 shows, ionomycin treatment of Jurkat cells caused anti-Myc immunoblot analysis. B, Proteinase K treatment of QPP-contain- increased the secretion of QPP. As a control for lysed cells, the ing vesicles. Fibroblasts transfected with QPP-HA were subjected to or- activity of the cytosolic proteasome complex was measured in the ganelle fractionation. The fraction containing lysosomes and QPP-express- supernatant. The proteasome activity remained similar for both cell ing vesicles (III) was 1) left untreated, 2) treated with Triton X and populations. These results suggest that QPP secretion can be trig- proteinase K, or 3) treated with proteinase K alone. These fractions were gered by calcium mobilization. analyzed using the anti-HA Ab. The Journal of Immunology 5701

ilar density as lysosomes, but under more stringent fractionation can be seen to be more dense than lysosomes. Also, as seen with confocal microscopy analysis, QPP does not colocalize with LAMP1, and unlike most soluble lysosomal proteins, QPP localizes in a M6P-independent manner. However, a glycosylation-independent mechanism of routing to lysosomes has also been described in the literature (26). Within its vesicular compartment, QPP may come into contact with substrate molecules that are in the process of being synthesized or secreted. QPP does not appear to localize to clathrin-coated endocytic vesicles, but this does not rule out contact with endocytosed substrate molecules. Calcium-dependent exocytosis of vesicular proteins is a well- described phenomenon (27–29). QPP secretion occurs via a calcium-dependent mechanism and can be induced by the calcium ionophore ionomycin, although secreted QPP represents only a minor part of the total QPP activity. Preliminary experiments indicate that QPP is also secreted in response to TCR-mediated sig- naling (H. Lee et al., manuscript in preparation). Such secretion

behavior would allow the cell to release the aminodipeptidase ac- Downloaded from tivity in response to stimulation. Given that extracellular signal molecules play a pivotal role during cellular activation, the release FIGURE 7. QPP secretion is regulated in Jurkat cells. Jurkat cells in of aminodipeptidase activity may play an important modulatory ␮ PBS with 1 mM CaCl2 were treated with either PBS or 10 M ionomycin role. It remains to be seen whether QPP and CD26/DPPIV work in for 15 min at 37°C. The supernatant was then analyzed for QPP activity concert or through distinct pathways. (f). The amount of enzyme released is expressed as a percentage of the We recently found that highly specific inhibitors of post-proline- http://www.jimmunol.org/ total activity in the cells. As a control the chymotrypsin activity of the cleaving aminodipeptidases trigger a caspase-dependent apoptotic proteasome complex was analyzed (Ⅺ). pathway in quiescent lymphocytes, but not activated lymphocytes (12). These results reinforce the idea that post-proline-cleaving aminodipeptidases play an important role in lymphocyte ho- this motif may be part of an important post-translational regulatory meostasis. Interestingly, the target of these inhibitors appeared to mechanism in a variety of signal pathways in multiple tissues. be QPP rather than CD26/DPPIV, indicating that there may be Although there are many potential substrates with N-terminal some separation of function between these two enzymes that share X-Pro motifs, few known enzymes have the requisite substrate substrate specificity, but have distinct subcellular localizations. Al- specificity to cleave these motifs (4). Until recently, only the cell though QPP was isolated from lymphocytes, Northern blot analy- by guest on September 26, 2021 surface molecule CD26/DPPIV was known to have the correct sis indicates that QPP is expressed in multiple tissues (M. Chira- substrate specificity at physiologic pH to be able to cleave the vuri et al., unpublished observations), suggesting a diverse role for X-Pro motif from these molecules. Like CD26/DPPIV, QPP this gene product. cleaves N-terminal dipeptides when the penultimate amino acid is Post-prolyl aminodipeptidase-mediated post-translational regu- a proline and, to a lesser extent, an alanine (4, 9–11). The discov- lation of signal molecules has been a rapidly expanding field of ery of a CD26/DPPIV-like activity intracellularly expands the po- study in the last few years. The discovery of alternate CD26/DP- tential realm of post-proline-cleavage and regulation. PIV-like activities in distinct subcellular locations contributes to QPP was initially isolated and cloned from Jurkat T cells (9). the understanding of what appears to be a complex regulatory Although functionally homologous to CD26/DPPIV, QPP bears no mechanism at the post-translational level. sequence homology with the former enzyme. It does, however, share homology with the carboxypeptidase, PCP (7, 9). Similarly Acknowledgments to CD26/DPPIV and PCP, QPP is targeted to the ER, undergoes We thank Dr. J. Fred Dice for helpful discussions and support. We also cleavage of a propeptide, and is N-glycosylated. The glycosylation thank Nicole D’Avirro, Shurjo Sen, and Sreya Urs for critical reading of of QPP is necessary for its enzymatic activity, either to assume its the manuscript. native three-dimensional conformation or for substrate binding. However, the absence of glycosylation does not perturb the intra- References cellular localization of QPP. While many of the reported experiments 1. Varshavsky, A. 1996. The N-end rule: functions, mysteries, uses. Proc. Natl. Acad. Sci. USA 93:12142. were conducted on QPP-transfected fibroblasts, overexpressing of 2. Shioda, T., H. Kato, Y. Ohnishi, K. Tashiro, M. Ikegawa, E. E. Nakayama, H. Hu, QPP did not alter the phenotype or growth characteristics of the cells. A. Kato, Y. Sakai, H. Liu, et al. 1998. Anti-HIV-1 and chemotactic activities of Unlike CD26/DPPIV, QPP does not localize to the cell surface, human stromal cell-derived factor 1␣ (SDF-1␣) and SDF-1␤ are abolished by CD26/dipeptidyl peptidase IV-mediated cleavage. Proc. Natl. Acad. Sci. USA but to intracellular vesicles distinct from the TGN. This was con- 95:6331. firmed by the fact that surface biotinylation did not yield any bi- 3. Oravecz, T., M. Pall, G. Roderiquez, M. D. Gorrell, M. Ditto, N. Y. Nguyen, otinylated QPP molecules, as tested biochemically with immuno- R. Boykins, E. Unsworth, and M. A. Norcross. 1997. Regulation of the receptor specificity and function of the chemokine RANTES (regulated on activation, precipitations (our unpublished observations). In a discontinuous normal T cell expressed and secreted) by dipeptidyl peptidase IV (CD26)-medi- gradient, QPP-containing vesicles comigrated with the lysosomes; ated cleavage. J. Exp. Med. 186:1865. however, in a continuos gradient we were able to show that QPP 4. Vanhoof, G., F. Goossens, I. De Meester, D. Hendriks, and S. Scharpe. 1995. Proline motifs in peptides and their biological processing. FASEB J. 9:736. has a different distribution from the lysosomal marker LAMP1. 5. Struyf, S., P. Proost, D. Schols, E. De Clercq, G. Opdenakker, J. P. Lenaerts, QPP-containing vesicles are downstream from the TGN and are M. Detheux, M. Parmentier, I. De Meester, S. Scharpe, and J. Van Damme. 1999. CD26/dipeptidyl-peptidase IV down-regulates the eosinophil chemotactic po- distinct from Rab11-containing vesicles and clathrin-coated endo- tency, but not the anti-HIV activity of human eotaxin by affecting its interaction cytic vesicles. Interestingly, QPP-containing vesicles have a sim- with CC chemokine receptor 3. J. Immunol. 162:4903. 5702 CHARACTERIZATION OF A CD26/DPPIV-LIKE PROTEASE

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