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Department of Pathology, Program in Immunology, and
Department of Physiology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA 02111
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(SDF1),3 SDF1
,
eotaxin, and macrophage-derived chemokine (MDC) is cleaved by
dipeptidyl peptidase IV (DPPIV/CD26), resulting in an alteration of
their biological function (2, 3, 5, 6). Thus, it appears
that the N-terminal X-Pro motif on signal molecules may serve as a site
for post-translational modification and regulation. The cleavage of
proline-containing peptide bonds on the C terminus can also affect the
function of molecules. Angiotensin, for example, undergoes a loss of
vasoactive function following post-proline cleavage of the C-terminal
amino acid by prolylcarboxypeptidase (PCP; angiotensinase C) (7, 8). Few proteolytic enzymes that cleave peptide bonds containing proline have been identified. These include exopeptidases such as CD26/DPPIV and PCP (4). We have isolated and cloned a serine protease, quiescent cell proline dipeptidase (QPP), which has substrate specificity very similar to that of CD26/DPPIV (9). Both these enzymes cleave N-terminal dipeptides when the penultimate amino acid is a proline or, less preferably, an alanine residue (9, 10, 11). Recently, we observed that highly specific aminodipeptidase inhibitors cause cell death in quiescent lymphocytes, but not in activated lymphocytes (12). QPP is the likely target of these inhibitors, suggesting that this protease plays an important role in resting cells (12). However, unlike CD26/DPPIV, which is found on the cell surface (10, 11), QPP was isolated from intracellular fractions (9). Although QPP and CD26/DPPIV share substrate specificity, there is no significant sequence homology between these two proteases, suggesting an evolutionary convergence of functional activity. QPP does, however, share significant sequence homology with the lysosomal serine protease PCP (42% sequence identity) (7, 9). It is interesting to note that the three post-proline-cleaving proteases, CD26/DPPIV, PCP, and QPP, all have the same sequential ordering of the active site residues, Ser-Asp-His (4, 9), and are functionally active as homodimers (7, 9, 11).
In this work we show that similar to its functional homologue, CD26/DPPIV, QPP is synthesized with a cleaved pro-peptide. QPP undergoes N-linked glycosylation at the Golgi apparatus, which is required for its proteolytic activity. Unlike CD26/DPPIV, QPP localizes to a post-Golgi vesicular compartment. QPP-containing vesicles have similar density to lysosomes, but further fractionation shows that QPP-containing vesicles form a distinct compartment, and unlike mannose-6-phosphate (M6P)-dependent lysosomal targeting (13), QPP localizes to vesicles by a M6P-independent mechanism. QPP is localized in the vesicular lumen, as it is not susceptible to proteinase K digestion of intact vesicles. Functionally active QPP can also be detected in cell culture supernatant, and its secretion can be induced by cell activation and calcium mobilization, suggesting that there is a secretory component to the QPP-containing vesicular compartment. The study of post-translational modification of N-terminal X-Pro-containing signal molecules has been a rapidly expanding field in the last few years. The discovery of a novel post-proline-cleaving aminodipeptidase in a distinct compartment from CD26/DPPIV broadens the scope of post-proline-cleavage as a regulatory mechanism.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-Myc Ab was purchased from PharMingen (San Diego, CA), and
anti-hemagglutinin (anti-HA) Ab was purchased from BabCo
(Berkeley, CA). The anti-lysosome-associated membrane protein-1
(anti-LAMP1) mAb H4A3 was obtained from the Developmental Studies
Hybridoma Bank (Iowa City, IA), and the anti-calnexin mAb was
purchased from StressGen (Victoria, Canada). Rab11 and adaptin- Abs
were purchased from Transduction Laboratories (Lexington, KY). The
reporter substrates alanine-proline-aminofluorocoumarin (Ala-Pro-AFC)
and z-glycine-glycine-leucine-aminomethylcoumarin (zGGL-AMC)
were purchased from Enzyme Systems Products (Dublin, CA). Tunicamycin,
ionomycin, and cyclohexamide were purchased form Sigma (St. Louis, MO),
and peptide-N-glycosidase F (PNGase F) was purchased from
New England Biolabs (Beverly, MA).
Constructs and transfections
To make the QPP-green fluorescent protein (GFP) construct, enhanced GFP was amplified from the cloning vector pEGFP-N1, obtained form Clontech (Palo Alto, CA), and cloned into the pCI-neo expression vector from Promega (Madison, WI). The sense primer incorporated five glycine codons upstream of GFP. QPP was amplified with primers containing restriction site linkers and cloned in frame upstream of enhanced GFP. The QPP-Myc fusion was generated by PCR using the high fidelity DeepVent polymerase (New England Biolabs), with an antisense primer that coded for 10 aa comprising the c-Myc epitope tag (EQLLISEEDL). Likewise, a QPP-HA fusion was generated using an antisense primer that coded for 10 aa comprising the HA epitope tag (YPYDVPDYA). All constructs were cloned into the pCI-neo expression vector. Transfections were performed using the calcium phosphate method.
Western blot analysis
Cells (1–2 x 107) were resuspended in lysis buffer (20 mM HEPES, 1.5 mM MgCl2, 2 mM EDTA, 10 mM KCl, 0.1% Nonidet P-40, 5 µg/ml antipain, and 5 µg/ml leupeptin) for 30 min at 4°C. Lysed cells were centrifuged at 3,000 rpm on a microcentrifuge for 10 min, and the pellet was discarded. The postnuclear supernatant was subjected to a 30,000 x g centrifugation for 30 min. The protein concentration was measured using the bicinchoninic acid protein estimation kit (Pierce, IL). For the immunoblot assay of supernatants, the supernatants of QPP-transfected or vector-transfected (control) cells were centrifuged at 300 x g to remove all cells and subjected to SDS-PAGE analysis.
Enzyme assays
QPP and proteasome enzymatic activities were measured using the
fluorogenic substrates Ala-Pro-AFC (20 µM in 50 mM HEPES buffer) and
zGGL-AMC (100 µM in 50 mM HEPES), respectively, on a Fluoromax
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 was 4-methylumbelliferyl--D-glucosaminide. Twenty
microliters of the samples obtained from the discontinuous gradient
were incubated with 0.5 ml of the substrate solution (1 mM
4-methylumbelliferyl--D-glucosaminide and 0.1 M sodium
acetate, pH 4.5) for 5 min. For the continuous gradient fractions, 10
µl of the samples were incubated for 20 min. The reaction was stopped
with 1.25 ml of stop solution (0.5 M glycine and 0.5 M
Na2CO3). The samples were
read using an excitation wavelength of 364 nm and an emission
wavelength of 448 nm.
PNGase F assays
Fifty micrograms of lysate from control or QPP-transfected cells was boiled for 10 min under denaturing conditions (0.5% SDS and 1% 2-ME). Following this the samples were brought up to 1% Nonidet P-40 and 0.05 M sodium phosphate. PNGase F (500 U) was added and incubated for the indicated times at 37°C. The reaction was terminated by boiling the sample in 2x reducing loading buffer for 5 min, followed by SDS-PAGE and immunoblot analysis. The controls were treated in the same manner except for the addition of PNGase F.
Confocal microscopy
293T human fibroblasts were transfected with QPP-GFP or QPP-Myc, plated on coverslips, pretreated with fibronectin (100 µg/ml), and blocked 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 for 1 h at 4°C, followed by three washes and incubation of the secondary Ab, where applicable, for 1 h at 4°C. The samples were washed and visualized by confocal microscopy.
Biochemical organelle fractionation
The isolation of subcellular organelles on discontinuous density gradients was performed as previously described (14). In addition, a continuous gradient fractionation was performed. Briefly, 1 x 108 cells were lysed by cavitation or Dounce homogenization (Kontes, Vineland, NJ) in 0.25 M sucrose, and the postnuclear supernatant (PNS) was taken from a low speed centrifugation. For discontinuous fractionation, a metrizamide/Percoll (Accurate Chemical & Scientific Corp., Westbury, NY) gradient was set up, using 35% metrizamide, 17% metrizamide, and 6% Percoll, and the PNS was layered over it. Following ultracentrifugation at 60,000 x g the four interface layers were harvested and analyzed. The orientation of the gradient is as follows: lightest fraction = fraction I, most dense fraction = fraction IV. For the continuous gradient fine fractionation of subcellular 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 linear gradient of 5–20% metrizamide, 1 ml of 6% Percoll (Pharmacia, Uppsala, Sweden), and 2.5 ml of PNS. The gradients were centrifuged at 160,000 x g for 2 h at 4°C, using a SW41 swinging rotor (Beckman, Fullerton, CA). Gradients were fractionated in 250-µl aliquots. These fractions were analyzed as described.
Proteinase K treatment
The QPP-containing vesicular fraction was obtained by discontinuous gradient fractionation and either were left untreated or were treated with proteinase K. Fractions were incubated in chymostatin (30 µM) for 10 min on ice. These vesicles either were left unpermeabilized or were permeabilized with 1% Triton X. Proteinase K (10 µg/ml) and CaCl2 (1 mM) were added to the samples for 20 min at 0°C. The reaction was terminated by the addition of AEBSF (100 µM).
QPP purification
QPP was purified from Jurkat cells as previously described (9). Briefly, Jurkat S-110 supernatant was dialyzed against 4 l of 50 mM acetic acid, titrated to pH 4.5 with NaOH. The protein sample was clarified by centrifugation at 1000 x g for 10 min at 4°C. The clarified supernatant was concentrated on a Centricon 50 membrane to about 10 ml. The concentrated sample was loaded onto a 3 ml of HiTrap SP Sepharose column and equilibrated with 50 mM acetate, pH 4.5 (start buffer). The column was 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 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 onto a Superose 12 gel filtration column and equilibrated with 50 mM acetate, pH 4.5, and 150 mM NaCl. Active fractions were pooled and used as a purified preparation of the activity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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QPP was initially purified from the cytoplasmic fraction of Jurkat
T cells (9). To determine the cellular localization of
QPP, confocal microscopy was performed on cells expressing a QPP-GFP
fusion protein. 293T human fibroblasts transfected with this chimeric
construct showed a cytoplasmic distribution (Fig. 1
A). Unlike CD26/DPPIV, QPP
does not localize to the cell membrane. This was confirmed by
biochemical means; namely, surface biotinylation of QPP expressing
cells, followed by immunoprecipitation, did not yield any biotinylated
QPP molecules (M. Chiravuri et al., unpublished observations). To
ensure that the distribution of QPP-GFP was not altered by the presence
of the GFP portion of the construct, immunohistochemical analysis was
performed with a QPP-Myc construct (Fig. 1B). Fig. 1C shows that QPP-GFP and QPP-Myc colocalize, demonstrating
that the nature of the tag did not affect QPP localization.
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Proteins are routed to the secretory pathway by signal peptides
that insert the protein into the endoplasmic reticulum (ER) membrane.
Primary sequence analysis of QPP using a Kyte and Doolittle
hydrophobicity 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. 1
D 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.
QPP undergoes N-linked glycosylation
SDS-PAGE analysis shows that QPP migrates as a 58-kDa species,
even though the predicted m.w. of QPP based on its primary sequence is
53 kDa. Similar to the lysosomal protease PCP (7), QPP has
six potential N-glycosylation sites, and glycosylation at
these sites could cause the observed increased in molecular mass.
Tunicamycin is an inhibitor of N-acetylglucosamine (GlcNAc)
transferase that blocks assembly of the GlcNAc-dolichol complex and
thus prevents glycosylation of asparagine residues (16).
QPP-transfected fibroblasts were treated with tunicamycin (5 µg/ml)
for various periods of time. Fig. 2A shows that the upper 58-kDa
band gradually disappeared with increased time of tunicamycin
treatment. We also treated QPP with PNGase F, an enzyme that cleaves
between the innermost GlcNAc and asparagine residues of high mannose,
hybrid and complex carbohydrates from N-linked glycoproteins
(17). PNGase F treatment also caused the disappearance of
the 58-kDa band (Fig. 2B). These results indicate that the
58-kDa band represents a processed glycosylated form of QPP, while the
53-kDa band is the unprocessed form.
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C, following the inhibition
of new protein synthesis, the ratio of the 53-kDa form of QPP to the
58-kDa form decreases. These results indicate that the 58-kDa species
is the mature molecule.
To determine the functional significance of the glycosylation, we
analyzed the effect of glycosylation on QPP enzyme activity. Cells
transfected with QPP were treated with tunicamycin for various time
periods, and the ability of QPP to cleave the reporter substrate
Ala-Pro-AFC was measured. QPP activity was significantly decreased
following tunicamycin treatment, indicating that the 58-kDa species is
the active form of QPP, while the unglycosylated form lacks QPP
activity (Fig. 2D). On the other hand, tunicamycin treatment
did not alter the localization of QPP (see Fig. 6 below). As a control,
the cleaving activity of the proteasome was measured in each of the
samples. The proteasome is a cytosolic molecular complex
(18) that does not undergo a significant decrease in
enzyme activity following tunicamycin treatment. Consistent with this,
no change in proteasome activity was seen in the tunicamycin-treated
samples.
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To localize QPP we performed biochemical organelle fractionation
using both discontinuous and continuous metrizamide/Percoll density
gradients. This was conducted on human fibroblasts transfected with a
QPP-Myc construct as well as on untransfected Jurkat T cells. The
transfected fibroblasts had identical phenotype and growth
characteristics as untransfected cells. QPP-Myc fibroblasts were lysed
by cavitation, and these lysates were fractionated. Four fractions were
obtained from the discontinuous gradients and were analyzed using
organelle-specific markers. As a cytosolic marker we measured the
chymotrypsin activity of the proteasome complex, using the reporter
substrate zGGL-AMC (19, 20). The lysosomal marker used was
-hexosaminidase activity (14), while LAMP1 was used as
a marker for late endosomes and lysosomes (21). The ER
marker used was calnexin (22), while the
trans-Golgi network (TGN) marker was Rab11
(23). To detect QPP we performed Western blot analysis and
measured its activity with the QPP reporter substrate Ala-Pro-AFC.
The analysis of the four fractions is shown in Fig. 3. The least dense fraction (I; soluble)
had the most proteasome activity (Fig. 3A), but contained
very little QPP (Fig. 3, F and G). Fractions
enriched in Rab11 (II; Fig. 3B) and calnexin (IV; Fig. 3C) also showed little QPP activity (Fig. 3F).
Lysosomes and late endosomes were identified in fraction III by the
presence of LAMP1 (Fig. 3D), and this fraction also had
maximal activity of lysosomal -hexosaminidase activity (Fig. 3E). The fraction containing lysosomes (III) had the
majority of QPP enzymatic activity (Fig. 3F). This was
confirmed using Western blot analysis (Fig. 3G).
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). The proteasome activity localizes to
the soluble fraction (I; Fig. 4A), and the majority of Rab11
migrates in fraction II (Fig. 4B), while calnexin localizes
to the fraction IV (Fig. 4C). As in the transfected
fibroblasts, LAMP1 was highly enriched in fraction III, although some
was also found on the less dense fraction II. Most of the
-hexosaminidase activity was found in fraction III (Fig. 4, D and E). The presence of membrane-bound LAMP1
but low -hexosaminidase activity in fraction II might indicate that
a fraction of the lysosomes was broken during Dounce homogenization,
causing the release of soluble proteins such as -hexosaminidase and
decreasing the density of the lysosomes. The majority of QPP
Ala-Pro-AFC cleaving activity was found in fraction III (Fig. 4F). These results suggest that QPP localizes to vesicles
that are either part of the lysosomal compartment or to vesicles that
have similar density to lysosomes.
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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 to alternate vesicles
that migrate with the lysosomes under the discontinuous gradient
fractionation conditions.
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D). These fractions were also 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 ruptured
vesicles, perhaps due to the increased centrifugation time 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 correlates 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 corresponding 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
localization of QPP, fibroblasts treated or untreated with tunicamycin
were subjected to organelle purification using discontinuous gradients.
As shown in Fig. 6A,
unglycosylated QPP was still found in the same fraction (III) and did
not show increased accumulation in the Golgi apparatus. This shows that
glycosylation and the M6P receptor pathway are not required for the
routing of QPP, suggesting a nonlysosomal vesicular localization.
However, M6P-independent localization of proteins to lysosomes has also
been described (26).
To confirm that QPP is located within vesicles, QPP-HA fibroblasts were
fractionated using discontinuous gradients, and intact vesicles from
fraction III were treated with proteinase K. This fraction contained
lysosomes/late endosomes and QPP-containing vesicles (Fig. 6B, lane 1). When the intact vesicles were first
permeabilized 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. Jurkat cells in
PBS with 1 mM CaCl2 were treated with ionomycin
for 15 min at 37°C, and QPP activity in the supernatant was measured.
As Fig. 7 shows, ionomycin treatment of
Jurkat cells caused increased the secretion of QPP. As a control for
lysed cells, the activity of the cytosolic proteasome complex was
measured in the supernatant. The proteasome activity remained similar
for both cell populations. These results suggest that QPP secretion can
be triggered by calcium mobilization.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although there are many potential substrates with N-terminal X-Pro motifs, few known enzymes have the requisite substrate specificity to cleave these motifs (4). Until recently, only the cell surface molecule CD26/DPPIV was known to have the correct substrate specificity at physiologic pH to be able to cleave the X-Pro motif from these molecules. Like CD26/DPPIV, QPP cleaves N-terminal dipeptides when the penultimate amino acid is a proline and, to a lesser extent, an alanine (4, 9, 10, 11). The discovery of a CD26/DPPIV-like activity intracellularly expands the potential realm of post-proline-cleavage and regulation.
QPP was initially isolated and cloned from Jurkat T cells (9). Although functionally homologous to CD26/DPPIV, QPP bears no sequence homology with the former enzyme. It does, however, share homology with the carboxypeptidase, PCP (7, 9). Similarly to CD26/DPPIV and PCP, QPP is targeted to the ER, undergoes cleavage of a propeptide, and is N-glycosylated. The glycosylation of QPP is necessary for its enzymatic activity, either to assume its native three-dimensional conformation or for substrate binding. However, the absence of glycosylation does not perturb the intracellular localization of QPP. While many of the reported experiments were conducted on QPP-transfected fibroblasts, overexpressing of QPP did not alter the phenotype or growth characteristics of the cells.
Unlike CD26/DPPIV, QPP does not localize to the cell surface, but to intracellular vesicles distinct from the TGN. This was confirmed by the fact that surface biotinylation did not yield any biotinylated QPP molecules, as tested biochemically with immunoprecipitations (our unpublished observations). In a discontinuous gradient, QPP-containing vesicles comigrated with the lysosomes; however, in a continuos gradient we were able to show that QPP has a different distribution from the lysosomal marker LAMP1. QPP-containing vesicles are downstream from the TGN and are distinct from Rab11-containing vesicles and clathrin-coated endocytic vesicles. Interestingly, QPP-containing vesicles have a similar 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, 28, 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 signaling (H. Lee et al., manuscript in preparation). Such secretion behavior would allow the cell to release the aminodipeptidase activity in response to stimulation. Given that extracellular signal molecules play a pivotal role during cellular activation, the release of aminodipeptidase activity may play an important modulatory role. It remains to be seen whether QPP and CD26/DPPIV work in concert or through distinct pathways.
We recently found that highly specific inhibitors of post-proline-cleaving aminodipeptidases trigger a caspase-dependent apoptotic 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 homeostasis. Interestingly, the target of these inhibitors appeared to be QPP rather than CD26/DPPIV, indicating that there may be some separation of function between these two enzymes that share substrate specificity, but have distinct subcellular localizations. Although QPP was isolated from lymphocytes, Northern blot analysis indicates that QPP is expressed in multiple tissues (M. Chiravuri et al., unpublished observations), suggesting a diverse role for this gene product.
Post-prolyl aminodipeptidase-mediated post-translational regulation of signal molecules has been a rapidly expanding field of study in the last few years. The discovery of alternate CD26/DPPIV-like activities in distinct subcellular locations contributes to the understanding of what appears to be a complex regulatory mechanism at the post-translational level.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Brigitte T. Huber, Department of Pathology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111.
3 Abbreviations used in this paper: SDF, stroma-derived factor; QPP, quiescent cell proline dipeptidase; DPPIV, dipeptidyl peptidase IV; DPPII, dipeptidyl peptidase II; PCP, prolylcarboxypeptidase; GFP, green fluorescent protein; M6P, mannose-6-phosphate; LAMP1, lysosome-associated membrane protein-1; MDC, macrophage-derived chemokine, TGN, trans-Golgi network; zGGL-AMC, z-glycine-glycine-leucine-aminomethylcoumarin; Ala-Pro-AFC, alanine-proline-aminofluorocoumarin; HA, hemagglutinin; PNS, postnuclear supernatant; ER, endoplasmic reticulum; GlcNAc, N-acetylglucosamine; PNGase F, peptide-N-glycosidase F.
Received for publication March 15, 2000. Accepted for publication August 22, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(SDF-1) and SDF-1 are abolished by CD26/dipeptidyl peptidase IV-mediated cleavage. Proc. Natl. Acad. Sci. USA 95:6331.
stimulation. J. Immunol. 159:5896.[Abstract]
and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. J. Biol. Chem. 266:19599.2-adrenergic receptor/-arrestin complex recruits the clathrin adaptor AP-2 during endocytosis. Proc. Natl. Acad. Sci. USA 96:3712.This article has been cited by other articles:
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  J. P. Goetze Biochemistry of Pro-B-Type Natriuretic Peptide-Derived Peptides: The Endocrine Heart Revisited Clin. Chem., September 1, 2004; 50(9): 1503 - 1510. [Abstract] [Full Text] [PDF]
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) and proteasome activity (
)
in cells treated with tunicamycin for the indicated times.
