©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Form of Dipeptidylpeptidase IV Found in Human Serum
ISOLATION, CHARACTERIZATION, AND COMPARISON WITH T LYMPHOCYTE MEMBRANE DIPEPTIDYLPEPTIDASE IV (CD26) (*)

Jonathan S. Duke-Cohan (1)(§) (2), Chikao Morimoto (1) (3), Joshua A. Rocker (1), Stuart F. Schlossman (1) (3)

From the (1) Division of Tumor Immunology, Dana Farber Cancer Institute, and Departments of (2) Pathology and (3) Medicine, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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,() 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) .

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.


MATERIALS AND METHODS

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 NaHPO7HO, 0.31 mM KHPO, 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 NaHPO 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.


RESULTS

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).




DISCUSSION

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 -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) .

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.() 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) .

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.

  
Table: Purification of CD26/DPPIV from 100 ml of normal human serum


  
Table: N-terminal sequences determined for serum DPPIV after proteolytic digestion

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).



FOOTNOTES

*
This work was supported by National Institutes of Health Grants AI23360-08, AI12069, CA55601, and CA34183. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Division of Tumor Immunology, Dana Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3816; Fax: 617-632-4569.

The abbreviations used are: DPPIV, dipeptidyl peptidase IV; ADA-1, adenosine deaminase type I; ADA-CP, adenosine deaminase complexing protein; ConA, concanavalin A; DFP, diisopropylfluorophosphate; DPPII, dipeptidyl peptidase II; NANase II, recombinant neuraminidase II; PBL, peripheral blood leukocytes; PBS, phosphate-buffered saline; pNA, p-nitroanilide; PNGase F, recombinant peptide:N-glycosidase F; rsCD26, recombinant soluble CD26; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis.

Dr. J. Kameoka; diluted 1:20 in TTBS.

J. S. Duke-Cohan, C. Morimoto, and S. F. Schlossman, unpublished data.


ACKNOWLEDGEMENTS

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.


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