From the
Human CD26, a Type II membrane glycoprotein with intrinsic
dipeptidylpeptidase IV (DPPIV) activity and ability to bind adenosine
deaminase type I (ADA-1), is expressed on epithelial cells
constitutively, but on T lymphocytes its expression is regulated. A
soluble form of CD26/DPPIV has been described in plasma and related to
immunological status, but it has been defined by the presence of DPPIV
activity rather than by isolation. Using nondenaturing chromatographic
techniques followed by nondenaturing native preparative
electrophoresis, we obtained a homogeneous preparation of soluble serum
DPPIV and compared it with a recombinant soluble CD26/DPPIV (rsCD26).
We show that serum DPPIV is a monomer of 175 kDa in contrast to rsCD26
of 105-110 kDa, that it exists as a trimer, and that it is
probably a serine proteinase. Deglycosylation removed N-linked
sugar from both serum DPPIV and rsCD26; no O-linked
glycosylation was observed, revealing a protein core of 130 kDa for
serum DPPIV. The large serum form expresses functional DPPIV activity
with substrate and inhibitor specificities and pH activity profile
similar to those of rsCD26. Epitope analysis showed that monoclonal
antibodies against five epitopes expressed by rsCD26 also bound, but
more weakly, with serum DPPIV. Analysis of peptides after limiting
proteolysis and N-terminal sequences reveals no homology with rsCD26
but some identity with other peptidases. Unlike rsCD26, the serum form
does not bind ADA-1 and has no ADA-1 already associated with it.
Similarly to rsCD26, serum DPPIV is a potent T cell costimulator. We
conclude that the serum form of DPPIV is unique and is not a breakdown
product of membrane CD26. The conservation of DPPIV activity and five
epitopes specific to rsCD26 suggest, however, a significant structural
similarity.
Initially identified as a 105-kDa T cell activation antigen
defined by the monoclonal antibody Ta1
(1) , the CD26 antigen was
subsequently shown to delineate the T cell subset responding to recall
antigens
(2, 3) . Although the antigen is expressed in
the liver, kidney, and intestine
(4) , only in the T cell are the
levels of membrane CD26 under tight cellular regulation with expression
up-regulated upon cell activation. CD26 has been shown to have
dipeptidylpeptidase IV activity (DPPIV,
CD26 not only marks the activated state but
is itself involved in the signal transducing process; cross-linking of
CD3 and CD26 results in enhanced T cell activation in the absence of
antigen-presenting cells (10). It is unlikely that CD26 is directly
involved in transducing the activation signal across the T cell
membrane, since it has only a very short cytoplasmic region of 6 amino
acids
(11) . The protein tyrosine phosphatase, CD45RO, has been
shown to associate with CD26 and may provide a putative mechanism for
the costimulation
(12) . Other associations include the strong
binding of adenosine deaminase type I (ADA-1) to CD26
(13) , this
may be of particular importance since ADA activity helps regulate the
early stages of signal transduction in T lymphocytes
(14) . We
have confirmed that the costimulatory potential of CD26 occurs
extracellularly by showing that a soluble recombinant CD26 (rsCD26)
representing the extracellular domain can enhance the T cell-mediated
reaction to recall antigens
(15) . Reinforcing the proposal that
soluble CD26 is costimulating, we found that in the absence of recall
antigen, the rsCD26 has no effect upon the proliferative response. In
the course of this latter study, we demonstrated that a natural form of
DPPIV/CD26 could be identified in normal human serum, and that the
levels of this naturally occurring soluble DPPIV influenced the level
of reactivity of T cells to recall antigens.
In this report, we
describe the properties of DPPIV/CD26 isolated from the serum and show
that while it shares similar enzymatic and antigenic properties with
the membrane form, in several biochemical aspects there are distinct
differences. In particular, the soluble form has a molecular mass of
175 kDa, and it does not bind ADA-1. Nevertheless, it retains the
ability to costimulate the T lymphocyte response to the recall antigen,
tetanus toxoid. Furthermore, N-terminal sequencing after tryptic
digestion suggested structural disparity between membrane CD26 and
soluble serum DPPIV. Accordingly, we suggest that although 105-kDa
membrane type CD26 may be found in the serum in small amounts, the
majority of serum DPPIV activity is provided by a novel peptidase
structurally distinct from CD26/DPPIV.
In this report we identify and describe the properties of a
novel form of DPPIV, which is soluble in serum and distinct to the
membrane form. The presence of soluble CD26 has been described
previously in detergent-free cell extracts
(23) , and its
presence has been inferred in serum from the identification of
significant circulating DPPIV activity or by reactivity with defined
anti-CD26 antibodies
(15, 24, 25, 26) .
We have shown previously that serum soluble CD26/DPPIV may play an
important role in regulating the immune response to recall antigens
in vivo(15) and wished to confirm the identity of the
serum form with the membrane form. Nevertheless, to clear serum of
DPPIV activity using the high affinity anti-CD26 antibody 1F7 required
large amounts of antibody, suggesting that there may be structural
differences between the membrane and soluble forms.
We thus set
about purifying the DPPIV activity from human serum. Using both
commercially available frozen serum as well as freshly isolated plasma,
we developed a nondenaturing procedure for purifying the soluble DPPIV
activity using techniques that should have maintained functional
integrity. Although we had a large panel of antibodies available, we
did not use these for affinity purification, since we found that in
every case the harsh elution conditions required (glycine-HCl, pH
1-2, or 3 M KSCN) resulted in a reduction in the DPPIV
specific activity of at least 1 order of magnitude. In every
purification, the DPPIV activity and anti-CD26 reactivity was
associated with a homogeneous band of 175 kDa obtained by preparative
native gel electrophoresis. In two preparations (out of seven total),
we were also able to copurify some lower molecular mass DPPIV activity
corresponding to the 105-kDa form of CD26, but quantitation by laser
densitometry confirmed that this was always less than 6% of the total
preparation, and separated away at the preparative native
electrophoresis stage.
Soluble DPPIV has been identified in
pathological and normal human urine with a molecular mass of around
280-400 kDa determined by gel filtration
(27, 28) ,
although it is unlikely that the 400-kDa form represents a dimer of the
serum form, since its size would not normally transfer by glomerular
filtration. Kidney and epithelial-associated DPPIV/CD26 is a membrane
dimer in the range 230-270 kDa
(29) with reduced subunits
of CD26 having a molecular mass of 105-115 kDa, from which it is
difficult to deduce any relationship with the serum form described
here. One previous study purified an ADA-1 complexing protein (ADA-CP)
of 105 kDa from human plasma
(30) , and their yield coincided
with the small amounts of 105-kDa soluble CD26 we could isolate from
some plasma samples (5-10 µg/100 ml serum). Nevertheless,
their procedure utilized a polyclonal antibody developed against
ADA-CP, and purification was monitored by ADA activity, which would
miss the larger non-ADA-binding form of DPPIV.
This raises the issue
of the origin of large 175-kDa DPPIV in the serum. Having shown that
the material in its reduced and deglycosylated form is larger than the
membrane form, it cannot result from shedding or proteolytic digestion
of membrane CD26/DPPIV. Several mRNA species have been reported for
human CD26
(11) , although the evidence to date suggests that
human DPPIV/CD26 is produced from a single location on chromosome
2
(31) , perhaps allowing alternative splicing as a possible
origin of serum CD26. One further possibility is that initiation occurs
at two distinct promoter sites, as has already been shown for another
membrane ectoenzyme, rat
We may safely exclude
the possibility that the DPPIV activity is the result of contaminating
105-kDa soluble CD26 or other aminopeptidases that may release the
substrate chromophore by sequential degradation. The levels of
aminopeptidase P in serum are essentially zero
(25) , while
aminopeptidase M (CD13) activity could not be detected (by hydrolysis
of Leu-pNA). Furthermore, the substrate specificities, inhibitor
kinetics, and pH optimum of serum DPPIV were very similar to those of
rsCD26.
One of the differences between the serum and membrane forms
was in the relative expression of epitopes detected by the monoclonal
anti-CD26 antibodies. Although these epitopes were expressed by the
large 175-kDa DPPIV, the antibodies bound more strongly to rsCD26. This
made itself apparent particularly in a previous report by our group,
where very high concentrations of 1F7 antibody (40 µg/ml) were
required to clear DPPIV activity and CD26 antigenic activity from the
serum
(15) . Nevertheless, the eventual complete clearance of
DPPIV activity by this antibody, albeit at high concentrations, also
implies that all the DPPIV activity in serum could be attributed to
1F7-reactive antigen and implies structural similarities between all
functional DPPIV moieties in serum. In support, we have now produced a
polyclonal rabbit antibody against purified serum DPPIV, which binds
strongly to Jurkat cells (CD26-negative) transfected with cDNA encoding
the 105-kDa CD26/DPPIV.
An additional
difference between serum and recombinant CD26 concerns the inability of
serum DPPIV to bind ADA. This is not as straightforward as initially
presented since human ADA exists as two forms, a low molecular mass
(40-kDa) Type 1 form and a high molecular mass (100-kDa) Type 2
form
(37) . ADA-1 accounts for almost all intracellular and
membrane-associated ADA, while ADA-2 is predominantly found circulating
in the serum. Recent reports show that ADA-1 interacts strongly with
the membrane form of CD26
(13, 22) and that CD26 is
equivalent to what was previously reported as ADA-CP. ADA-2 is not well
characterized, but its levels vary significantly with immune activity,
particularly during opportunistic or systemic bacterial
infections
(38, 39, 40) . There exists the
possibility that serum DPPIV interacts with serum ADA-2 rather than
with ADA-1, but this remains to be tested. Nevertheless, ADA-1 does not
appear to play a role in the T cell costimulatory activity mediated by
serum DPPIV since the non-ADA-1-binding serum DPPIV is a potent
costimulator of recall antigen-driven T lymphocyte responses in
vitro.
Although we have speculated as to the genetic origin of
serum DPPIV, we do not yet know its cellular origin. This direction of
investigation will be aided considerably by the isolation of a cDNA
encoding the serum form of DPPIV, an aspect that is currently under
investigation. The intriguing similarities and differences between the
serum form and membrane form suggest a common origin. The clear ability
of both DPPIV/CD26 forms to enhance immune function supports the
proposal that DPPIV/CD26 is an important regulator of the in vivo immune response, both as a membrane antigen
(10) and as a
soluble serum protein
(15) . An important question that remains
to be determined is whether both forms are present in the T lymphocyte
and, if so, whether both are regulated by the same pathway after T cell
activation, or whether they are under separate and distinct controls.
Dashes (-)
indicate N-terminal or C-terminal extensions; a single dash (-)
indicates that a gap has been inserted for alignment. An x indicates that an amino acid could not be reliably identified at
that position. Similarity was determined by conservative replacement
using the FASTA program (Genetics Computer Group, Madison, WI).
We thank W. S. Lane and R. Robinson for all the help
with N-terminal sequencing and H. Saito, A. Yaron, K. V. S. Prasad, M.
Hegen, J. Kameoka, K. Tachibana, and T. Sato for helpful advice.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
EC
3.4.14.5) in its extracellular domain
(5, 6) , and the
costimulatory potential appears to be partially dependent upon this
enzyme activity (7), which can cleave N-terminal dipeptides with
proline, and less effectively, alanine in the penultimate
position
(8) . Although a substrate of relevance to T cell
activation has not yet been identified, other substrates, including the
neuropeptide substance P, may be processed in vivo by
DPPIV/CD26
(9) .
Recombinant Soluble CD26
The full extracellular
domain of T cell membrane-expressed CD26 (rsCD26) was obtained as a
soluble secreted product from transfected CHO cells as described
previously (15). The transfected CHO cells were grown in serum-free
CHO-SFM medium to confluence (Life Technologies Inc.). Sodium azide
(0.1%, w/v, final) was added to the rsCD26-containing conditioned
medium, which was passed at 1 ml/min over a 38 1.5-cm
concanavalin A (ConA)-agarose column (Sigma) equilibrated in 2x
phosphate-buffered saline (2
PBS; 30.8 mM NaCl, 0.54
mM Na
HPO
7H
O, 0.31
mM KH
PO
, pH 6.8) at room temperature
(as were all subsequent column procedures). After washing with 5 column
volumes of 2
PBS, bound glycoprotein was eluted with 2
PBS containing 200 mM
-methylmannoside (Sigma) and
dialyzed extensively against 50 mM sodium acetate buffer, pH
4.65. After equilibrating a 8.5
2.5-cm s-Sepharose (Pharmacia
Biotech Inc.) ion exchange column in 50 mM sodium acetate, pH
4.65 (equilibration buffer), the DPPIV-active ConA eluate was then
loaded at 1 ml/min. The column was then washed with 10 column volumes
of equilibration buffer and eluted with a 0-1 M NaCl
gradient in equilibration buffer. The rsCD26 elutes as a clean peak at
50 mM NaCl, and all eluted DPPIV activity was associated with
this peak.
DPPIV Activity
The primary substrate used for
determining DPPIV activity in all assays was
Gly-Pro-p-nitroanilide (Gly-Pro-pNA; Sigma), which is
hydrolyzed by DPPIV to release pNA absorbing strongly at 405 nm.
Kinetic assays were performed using varying amounts of substrate in
order to obtain estimates of the K and
V
using standard Michaelis-Menten kinetics. For
screening assays of DPPIV activity, 150 µl of substrate was used at
1 mg/ml (2 mM final concentration) in phosphate buffer (pH
7.6, 100 mM) in 96-well flat-bottomed microtiter plates
together with 10 µl of appropriately diluted sample. For samples
obtained after elution from the s-Sepharose column, the pH was raised
to pH 7.6 by addition of 25 µl of 1 M Tris-HCl, pH 7.6, to
the reaction mixture. One unit of enzyme activity was defined as the
amount of enzyme that cleaved 1 µmol of Gly-Pro-pNA/min at pH 7.6
and 20 °C. Inhibition of the hydrolysis of Gly-Pro-pNA was
determined in the presence of the specific DPPIV inhibitor diprotin A
(Bachem, King of Prussia, PA). To test for the presence of non-DPPIV
peptide hydrolase activity, the following substrates were used (all
obtained from Bachem): Leu-pNA, Ala-pNA, Arg-pNA, succinyl-Gly-Pro-pNA,
Gly-Arg-pNA, and Val-Ala-pNA (a weak DPPIV substrate). The pH optimum
was determined substituting phosphate buffer (100 mM, pH
4.5-9) in place of standard incubation buffer.
Serum DPPIV
Serum DPPIV was prepared by isolating
from 100 ml of pooled normal human male AB serum the fraction of
protein precipitated by 50-70% saturated ammonium sulfate. After
extensive dialysis against 50 mM acetate buffer, pH 4.65, the
material was passed onto, and eluted from, a s-Sepharose column in an
identical manner to that described above for rsCD26. The DPPIV-positive
fractions were pooled and dialyzed against 2 PBS prior to
binding and elution from a ConA-agarose column as described above for
rsCD26. The ConA eluate was dialyzed against acetate equilibration
buffer and reapplied to the s-Sepharose column, which was then washed
with 10 volumes of equilibration buffer. The DPPIV activity was eluted
by a 0-0.5 M NaCl gradient in equilibration buffer, and
all DPPIV activity was found in the 10-50 mM NaCl eluate
(determined by conductance). The eluted material was then concentrated
to 1-2 ml using 10-kDa cut-off centrifugal concentrators
(Centriprep 10; Amicon, Beverley, MA) and was then applied to a
preparative native 7.5% acrylamide gel buffered with Tris-glycine.
After electrophoresis, the gel was cut horizontally into 1-mm strips
and the >30-kDa material was extracted from the gel slices by
electroelution (model 422; Bio-Rad). The eluted material expressing
DPPIV activity was characterized by running on a 7.5% SDS-PAGE gel
under reducing conditions followed by silver staining (Amersham Corp.).
The molecular mass of serum DPPIV was also determined by its
application (50 µg in 100 µl of PBS) on to a 100
1.5-cm
Superdex 200 column (Pharmacia) equilibrated in PBS after calibration
using blue dextran (for void volume), thyroglobulin (669 kDa),
apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), and bovine
serum albumin (66 kDa), all obtained from Sigma. Flow rate was 0.4
ml/min, protein elution was monitored by optical density at 280 nm, and
the DPPIV-containing fractions were localized by ability to hydrolyze
Gly-Pro-pNA using the standard conditions described above.
Enzymatic Deglycosylation
Sequential
deglycosylations were performed using recombinant
N-acetylneuraminidase II (NANase II; 10 milliunits/10 µg
of substrate protein), O-glycosidase (2 milliunits/10 µg
of substrate protein), and peptide:N-glycosidase F (PNGase F;
5 milliunits/10 µg of substrate protein) using buffers and reagents
supplied (Glyko, Novato, CA). Briefly, the NANase II (removing
(2-3)-,
(2-6)-, and
(2-8)-linked
NeuAc) and O-glycosidase digestions were performed for 1 h at
37 °C in 50 mM sodium phosphate, pH 6.0. The pH of the
samples was then raised by addition of Na
HPO
to
0.125 M final, pH 8.0, followed by boiling in the presence of
SDS and
-mercaptoethanol for 5 min to denature the protein.
Nonidet P-40 (2.5 µl/40-µl sample) was added prior to
incubation with PNGase F for 3 h at 37 °C, which removed
N-linked tri- and tetraantennary complex-type oligosaccharide
chains, polysialic acids, and high mannose and hybrid oligosaccharide
chains. The samples were again incubated with SDS-PAGE reducing sample
buffer at 100 °C prior to electrophoresis on 10% acrylamide gels.
To check for complete deglycosylation, the enzyme-treated proteins were
stained for carbohydrate moieties in 10% SDS-PAGE gels by oxidation
with periodic acid followed by staining with Schiff's reagent
(Sigma).
Epitope Analysis
1 µg of serum DPPIV or rsCD26
was incubated in 100 µl of PBS containing 0.05% Tween 20 (Sigma;
PBS-Tween) together with 5 µg of purified control or anti-CD26
antibodies for 1 h at 4 °C with rotation. Control murine monoclonal
antibodies used were W6/32
(anti--microglobulin/histocompatibility leukocyte
antigen Class I, IgG
; Ref. 16) and UCHL1 (anti-CD45RO,
IgG
; Ref. 17). The anti-CD26 murine monoclonal IgG
antibodies used were 1F7 (IgG
) and 5F8 (IgG
)
described previously
(2, 18) and 10F8A, 9C11, 4G8, and
2F9 (all IgG
), which were produced in our laboratory after
immunization of mice with CD26-transfected 300.19 cells, a murine pre-B
cell lymphoma line. All antibodies were purified by binding to and
elution from Protein A-Sepharose (Pharmacia). By epitope analysis using
Surface Plasmon Resonance (Biacore, Pharmacia Biosensor AB), 5F8, 2F9,
4G8, 9C11, and 1F7 detected five unique epitopes while 10F8A was
blocked by 1F7 and 2F9. Anti-mouse IgG-agarose beads (Sigma; 50 µl)
were added to each sample and incubated for an additional 16 h at 4
°C with rotation, followed by five washes of the beads with
PBS-Tween to remove unbound CD26 and antibody. Twenty µl of the
washed beads were then incubated in duplicate together with Gly-Pro-pNA
(2 mM final concentration). The enzyme activity, determined as
hydrolysis and release of pNA measured at 405 nm, of the
antibody-immobilized CD26/DPPIV provided a relative measure of the
affinity of the particular antibody for CD26.
Peptide and Sequence Analysis
For N-terminal
sequencing, 50 µg (280 pmol) of serum DPPIV was
electrophoresed on a 7.5% SDS-PAGE gel, electrotransferred to a
polyvinylidene difluoride membrane (0.45 µm; Bio-Rad), and stained
with 0.1% Ponceau S in 5% acetic acid (Sigma). After cutting out the
stained band at 175-180 kDa, protein was eluted from a small
piece of membrane for amino acid analysis to estimate the amount of
transferred protein. The protein was then subjected to tryptic
digestion on the polyvinylidene difluoride membrane, and the products
were resolved by narrow-bore reverse phase high performance liquid
chromatography as described
(19) . Peaks suitable for N-terminal
sequencing were selected, checked for molecular mass by matrix-assisted
laser desorption mass spectroscopy, and sequence determined using a
model 477A protein sequencer linked to a model 120A PTH-amino acid
analyzer (Applied Biosystems, Foster City, CA). All sequence analysis
was performed using the GCG Unix package (Genetics Computer Group,
Madison, WI).
Affinity Labeling of Enzyme Active Site
To
determine whether serum DPPIV is a serine protease similar to rsCD26,
the active sites of both proteins (5 µg in 20 µl of 100
mM Tris-HCl, pH 8.0) were labeled with 25 µCi of
[H]diisopropylfluorophosphate (DFP, 1 mCi/ml;
DuPont NEN) for 24 h at 37 °C, after which the proteins were
separated by SDS-PAGE (7.5%). The gel was fixed in 25% isopropanol, 10%
acetic acid, and 2
10-mm slices of the lanes containing
radiolabeled sample were cut out, homogenized in 1.5-ml polypropylene
tubes, and the slurries were incubated with 1 ml of scintillation
mixture (Atomlight; DuPont NEN) for 5 days with occasional shaking
until all material had entered into solution. Further scintillation
fluid (2 ml) was added, and the samples were counted in a
-scintillation counter.
CD26 Binding to Adenosine Deaminase
(ADA-1)
Purified serum DPPIV or rsCD26 (50 µg/ml in 20
mM acetate buffer, pH 4.5) was immobilized on the
carboxyl-methyl dextran surface of a CM5 sensor chip by
N-hydroxysuccinimide/carbodiimide-mediated amine coupling
(Biacore, Pharmacia Biosensor). The ability of bovine adenosine
deaminase (ADA-1, 10 µg/ml in 10 mM HEPES, pH 7.4, 100
mM NaCl, 0.05% Tween 20) to bind to the CD26-coated surface
was determined by measuring surface plasmon resonance
(20) . In a
separate technique, and to simultaneously determine whether adenosine
deaminase was already bound to serum DPPIV, samples of serum DPPIV and
rsCD26 (1 µg/ml) were incubated for 30 min at 4 °C in the
presence or absence of ADA-1 (10 µg/ml in Tris-buffered saline, pH
7.4, containing 0.1% Tween; TTBS), followed by an incubation with a
polyclonal rabbit anti-human ADA(
)
or normal
rabbit serum control. Protein A-Sepharose (Sigma; 50 µl) was added
and the samples incubated overnight while rotating at 4 °C. After 4
washes in TTBS, the Protein A-Sepharose beads were tested for DPPIV
activity by incubation with Gly-Pro-pNA (2 mM final
concentration).
Proliferation Assays
Peripheral blood leukocytes
were isolated from donors tested previously for positive proliferative
responses to tetanus toxoid, where such responses could be further
enhanced by addition of rsCD26. PBL were resuspended at 5
10
in 200 µl of RPMI 1640 containing 10% autologous
serum depleted of endogenous CD26 by batch incubation with anti-CD26
antibodies as described previously
(15) , with or without tetanus
toxoid (Connaught Laboratories, Swiftwater, PA; 10 µg/ml) and serum
DPPIV or rsCD26 at the indicated concentrations and incubated in
round-bottomed 96-well microtiter plates for 7 days at 37 °C in a
humidified 7.5% CO
atmosphere. Each well was pulsed with 1
µCi of tritiated thymidine (DuPont NEN) and was incubated for an
additional 24 h before harvesting and counting incorporated thymidine
using a
-scintillation counter.
Purification of Serum DPPIV
DPPIV from serum was
isolated by sequential purification using ion exchange chromatography
(s-Sepharose), ConA affinity chromatography, and a second separation on
s-Sepharose. Approximate levels of purification and yields are
presented in . The chromatographic procedures yielded a
preparation, which displayed three protein bands by SDS-PAGE under
reducing conditions. None of the bands co-migrated with the 105-kDa
rsCD26. The preparation was further purified by eluting the three bands
from a nondenaturing native preparative Tris-glycine gel. All DPPIV
enzyme activity was associated with a single high molecular mass band
on the native gel, which consisted predominantly of a 175-180-kDa
moiety by non-reducing and reducing SDS-PAGE (Fig. 1, A and B). Under native conditions, the serum DPPIV activity
migrated differently than rsCD26 (Fig. 1C). This
preparation was further analyzed by matrix-assisted laser desorption
mass spectrometry
(21) , which revealed a strong peak at
approximately 175 kDa, while rsCD26 produced a strong signal at
101-105 kDa. In addition, gel filtration chromatography on
Superdex 200 revealed that in its native state, the serum DPPIV
migrates between thyroglobulin (669 kDa) and apoferritin (443 kDa) as a
large complex of 570 kDa. Since the material applied to the gel
filtration column was not appreciably contaminated by any other
protein, the serum DPPIV would appear to exist as a trimer. Due to
availability, the DPPIV was purified from human serum rather than
plasma. Nevertheless, purification of DPPIV from 50 ml of plasma of 7
normal donors revealed an identical 175-180-kDa band. In two
instances, the 105-kDa form copurified with the 175-kDa form, but was
less than 6% of the total protein preparation by laser densitometry.
Figure 1:
Panel A, analysis by SDS-PAGE
(6%) under non-reducing conditions of rsCD26 (lane1)
and soluble serum DPPIV (lane2). PanelB, analysis by SDS-PAGE (10%) under reducing conditions
of rsCD26 (lane1) and soluble serum DPPIV (lane2). Panel C, analysis by Tris-glycine native
PAGE (7.5%) of rsCD26 (lane1) and soluble serum
DPPIV (lane2).
Biochemical Characterization of Serum
CD26/DPPIV
The membrane form of CD26 is heavily glycosylated
(approximately 16% of the molecular mass; Ref. 18), so we examined the
pattern of glycosylation of the serum form to determine whether it
resulted from hyperglycosylation of the identical peptide backbone
found in the membrane form. Accordingly, samples of both rsCD26 and
serum DPPIV were sequentially digested by neuraminidase (NANase II), by
O-glycosidase, and then by PNGase F (removing NeuAc,
O-linked, and all N-linked oligosaccharides and
polysialic acids). As shown in Fig. 2, the pattern of digestion
is similar for rsCD26 and serum DPPIV, demonstrating that both have
significant N-linked glycosylation but little if any
O-glycosylation. Nevertheless, the deglycosylated backbone of
serum DPPIV remains larger than that of rsCD26 (approximately 130 kDa
in comparison with 90 kDa, the latter figure agreeing well with the 89
kDa predicted from the amino acid sequence), demonstrating that the
higher molecular mass of the serum form is not due to
hyperglycosylation. Staining for glycoprotein revealed that enzymatic
deglycosylation resulted in removal of all carbohydrate residues
detectable by periodic acid-Schiff's reagent (Fig. 2,
lanes 9-12).
Figure 2:
Analysis of glycosylation of rsCD26
(lanes 1-4) and soluble serum DPPIV (lanes
5-8) by sequential enzymatic deglycosylation and separated
on a 10% SDS-PAGE gel under reducing conditions followed by silver
staining. Lanes1 and 5, untreated sample.
Lanes2 and 6, removal of terminal NeuAc
with NANase II. Lanes3 and 7, NANase II and
removal of O-linked sugars by O-glycosidase.
Lanes4 and 8, NANase II,
O-glycosidase, and removal of N-linked sugars by
PNGase F. Lanes9 and 10, periodic
acid-Schiff staining for glycoprotein of the same rsCD26 samples as in
lanes 1 and 4; lanes11 and
12, glycoprotein staining of the same serum DPPIV samples as
in lanes5 and 8.
Enzymatic Properties of Serum CD26
The DPPIV
enzyme activities of the rsCD26 and serum DPPIV were analyzed by
Michaelis-Menten kinetics by assessing kinetics of hydrolysis of
substrate at different concentrations. Analysis using the
Lineweaver-Burke transformation yielded an identical
K of 0.28 mM for Gly-Pro-pNA for
both recombinant and serum DPPIV and the values for V
of 1.52 units/nmol rsCD26 and 0.65 units/nmol serum DPPIV were in
the same range, using Gly-Pro-pNA as substrate. Inhibition by the
specific DPPIV inhibitor diprotin A was similar for both serum DPPIV
and rsCD26, with an IC
of 0.125 mM for rsCD26,
and 0.25 mM for serum DPPIV. To further exclude the
possibility that we had purified irrelevant aminopeptidases that,
working in concert or by sequential hydrolysis, could release the pNA
chromophore from substrate, we tested a number of substrates that could
be hydrolyzed by non-DPPIV aminopeptidases. One candidate for
sequential hydrolysis is aminopeptidase M (EC 3.4.11.2), but no
hydrolysis of Ala-pNA, Arg-pNA or Leu-pNA was observed, and activity by
aminopolypeptidase (EC 3.4.11.14) may be similarly excluded. The
substrate succinyl-Gly-Pro-pNA was also not hydrolyzed. The only
substrate apart from the dipeptide chromatogen Gly-Pro-pNA that could
be hydrolyzed was Val-Ala-pNA, which was cleaved by both the serum
DPPIV and rsCD26 (relative to rate of hydrolysis of Gly-Pro-pNA,
hydrolysis of Val-Ala-pNA was 0.098 and 0.079, for rsCD26 and serum
DPPIV, respectively). Although DPPIV primarily hydrolyzes dipeptides
with a proline in the penultimate position, it is well documented that
alanine in the penultimate position may also render a dipeptide
sequence susceptible to cleavage by DPPIV
(8) . The Gly-Pro-pNA
substrate may also be a substrate for dipeptidylpeptidase II (DPPII),
as may Val-Ala-pNA, but this enzyme has an acidic pH
optimum
(8) . We determined the pH optima for both the rsCD26 and
the serum DPPIV and showed them to be identical at pH8.5, while there
was almost a complete inhibition of activity at acidic pH 4.5-5,
suggesting that we were not purifying a DPPII-like molecule.
Epitope Analysis
In order to compare the antigenic
sites of rsCD26 and serum DPPIV, we used a panel of monoclonal
anti-CD26 antibodies developed in our laboratory that had been shown to
detect five distinct antigenic epitopes of CD26 by cytofluorimetry and
by cross-blocking using surface plasmon resonance. Both serum DPPIV and
rsCD26 were incubated with the murine monoclonal anti-CD26 antibodies
(1F7, 5F8, 9C11, 2F9, and 4G8, which detect distinct epitopes; and
10F8A, which detects an epitope shared by the 1F7 and 2F9 determinants)
and with murine monoclonal control antibodies (W6/32 and UCHL1) after
which the antibody-antigen complexes were removed from unbound material
by binding to anti-mouse IgG-Sepharose beads. Complexed CD26 was
determined by DPPIV activity, which was proportional to the amount of
CD26/DPPIV immobilized (Fig. 3). The complete panel of anti-CD26
antibodies can bind to both serum DPPIV and rsCD26, and despite the
binding being greater to the recombinant form, the implication is that
the two proteins share some structural motifs.
Figure 3:
Analysis of epitope expression by rsCD26
(light stipple) and serum DPPIV (heavystipple). Units of enzyme activity represent the DPPIV
activity of the CD26/DPPIV bound to CD26-specific antibodies
immobilized on anti-mouse Ig-Sepharose beads. UCHL1 (anti-CD45RO) and
W6/32 (anti-histocompatibility leukocyte antigen Class I) are control
antibodies.
N-terminal Sequencing of Tryptic Peptides of Serum
DPPIV
N-terminal sequences of 10 peptides derived after tryptic
digestion of reduced and alkylated serum DPPIV were determined
(). Of these, none revealed a complete identity with any
sequence within available computerized data bases. Nevertheless, six of
the peptides revealed significant homology and similarity (using
conservative substitutions) to a metalloendopeptidase (leishmanolysin),
a serine endopeptidase (myeloblastin), a protein classified originally
as a proteolytic plasminogen activator (neutral proteinase, of which
the protease activity is now questionable), urokinase plasminogen
activator, and chymotrypsin-like serine protease precursor.
Serum DPPIV Is a Serine Proteinase
To determine
the functional classification of serum DPPIV and compare it with
rsCD26, which is known to be a serine proteinase, we incubated the
serum DPPIV with [H]DFP, which reacts with
serines in the active site of serine proteinases. As shown in
Fig. 4
, the serum DPPIV could be labeled with DFP and formed a
sharp peak around 175-180 kDa, while the rsCD26 was more broadly
spread over the region of 105-115 kDa, which might be attributed
to heterogeneous glycosylation
(18) .
Figure 4:
[H]DFP binding to
rsCD26 (
) and serum DPPIV (
). Each lane
was cut into 2-mm slices, each of which was dissolved in scintillation
fluid and counted.. The abcissa represents distance (cm)
traveled in the gel and is correlated with the indicated molecular mass
markers, which were run on the same gel. The ordinate (dpm)
represents the amount of [
H]DFP-labeled
protein.
Binding of ADA-1 to CD26
rsCD26 has been shown
previously to be identical to the membrane protein that binds
ADA-1
(13, 22) . We used two techniques to determine
whether serum DPPIV binds ADA-1. The first technique utilized surface
plasmon resonance to measure the interaction of ADA-1 with serum or
recombinant CD26 immobilized on a carboxymethyl dextran surface. It is
clear that the rsCD26 has a high affinity for ADA-1 but that this is
not so for serum DPPIV (Fig. 5A), which shows a binding
profile similar to that of the control. To confirm this, we utilized a
technique whereby rabbit anti-ADA antibodies were incubated with rsCD26
or serum DPPIV, and the immune complexes formed were removed with
Protein A-Sepharose beads. Binding of ADA-1 to CD26 was then confirmed
by detection of DPPIV activity associated with the extensively washed
beads. In the absence of exogenously added ADA-1
(Fig. 5B, -ADA), neither the rsCD26 nor
the serum CD26 had any associated ADA-1 prior to the experiment. Upon
addition of exogenous ADA (Fig. 5B, +ADA),
only the rsCD26 bound to the ADA, and no evidence of ADA-binding to
serum DPPIV was observed. Although the rsCD26 DPPIV activity developed
within 10 min of adding substrate, the samples were left for 24 h
after, which time the samples incubated with serum CD26 had still not
developed a signal above background, showing that the lack of ADA
binding was absolute, and not a relative difference. It is also of
interest to note that deglycosylated rsCD26 is still able to bind ADA-1
as well as the untreated form, suggesting that the ADA binding site on
rsCD26 is not in the heavily glycosylated region proximal to the
membrane.
Figure 5:
Panel A, analysis of ADA-binding
of rsCD26 and serum DPPIV detected by surface plasmon resonance. ADA
(10 µg/ml in 10 mM HEPES, 100 mM NaCl, 0.05%
Tween 20) was passed over a control carboxymethyl dextran surface
(lowesttrace), over serum DPPIV (middletrace), and over rsCD26 (uppertrace),
both immobilized on a carboxymethyl dextran surface. One thousand
resonance units (ordinate) represents approximately 1
ng/mm of binding protein. Panel B, analysis of ADA
binding of rsCD26 and serum DPPIV by immobilization with anti-ADA.
rsCD26 and serum DPPIV, incubated with or without ADA, were allowed to
interact with rabbit anti-ADA or normal rabbit serum
(Control), followed by removal of immune complexes with
Protein A-Sepharose beads. DPPIV/CD26 complexed to the beads was
determined by DPPIV enzyme activity. The lightstippledbars represent untreated, and the heavystippledbars represent deglycosylated, rsCD26
and serum DPPIV.
T Cell Costimulatory Activity of CD26
We have
shown previously that rsCD26 is able to enhance the responses of
peripheral blood leukocytes (PBL) to the recall antigen, tetanus
toxoid
(15) . Accordingly, we tested whether serum DPPIV could
also enhance the responses of PBL to tetanus toxoid. As shown in
Fig. 6
, the serum DPPIV was as capable as rsCD26 at costimulating
the response to tetanus toxoid, with the peak response in the range
0.5-1 µg/ml. As we have routinely observed, concentrations of
rsCD26 higher than 1 µg/ml tend to be inhibitory, and a similar
phenomenon was observed for serum DPPIV. It is important to note here
that all autologous plasma samples used as culture supplements during
the incubation with recall antigen were precleared of endogenous
CD26/DPPIV activity by pretreatment of the plasma samples with
anti-CD26 antibody and removal of immune complexes with anti-mouse IgG
magnetic beads.
Figure 6:
T cell costimulatory activity developed by
soluble CD26/DPPIV. rsCD26 () and serum DPPIV (
)
were incubated with PBL and tetanus toxoid in medium supplemented with
soluble CD26-depleted autologous plasma. Proliferation was assayed by
measurement of cell-incorporated [
H]thymidine at
7 days. Verticalbars represent S.E. In the absence
of tetanus toxoid, neither form of CD26/DPPIV had any effect upon the
base response of 2351 ± 108 cpm (data not
shown).
-glutamyl transpeptidase
(32) .
There is also support for a distinct separate gene since DPP-X has been
observed, which shows great homology to DPPIV but does not exhibit any
dipeptidylpeptidase IV activity itself
(33) . Further evidence
that there may be disparate genes coding for CD26-like molecules is
provided by the recent report that fibroblast activation protein
is a type II membrane glycoprotein with remarkably similar sequence and
organizational structure to membrane CD26 and, in fact, can form a
heterodimer with membrane CD26
(34) .
(
)
The N-terminal
sequences, however, suggest little sequence similarity between rsCD26
and serum DPPIV. Some caution must be exercised here since we may be
comparing only those regions sensitive to proteolytic digestion; we
have preliminary data that both proteins appear to have large
protease-resistant regions. One recent report favors the notion that
the DPPIV-related enzymes have arisen by divergent evolution from an
/
-hydrolase ancestor, where distinct secondary and tertiary
structures may be retained despite complete differences in sequence
(35). In contrast, the disparity of sequence and conservation of
structure between the eukaryotic serine proteases, cysteine proteases,
subtilisins, and the
/
hydrolase fold enzymes provides a
strong argument for convergent evolution
(36) .
Table:
Purification of CD26/DPPIV from 100 ml of normal
human serum
Table:
N-terminal sequences
determined for serum DPPIV after proteolytic digestion
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.