DPPIV

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European Journal of Cell Biology 82, 53 ± 73 (2003, February) ¥ ¹ Urban & Fischer Verlag ¥ Jena http://www.urbanfischer.de/journals/ejcb

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The multifunctional or moonlighting protein CD26/DPPIV Emil Boonacker, Cornelis J. F. Van Noorden1) Academic Medical Center, University of Amsterdam, Department of Cell Biology and Histology, Amsterdam/The Netherlands Received July 25, 2002 Received in revised version November 20, 2002 Accepted November 21, 2002

Signal transduction ± immunity ± cancer ± apoptosis ± adhesion ± protease ± biocomplexity CD26/DPPIV can be considered a moonlighting protein because it is a multifunctional protein that exerts its different functions depending on cell type and intra- or extracellular conditions in which it is expressed. In the present review, we summarize all its known functions in relation to physiological and pathophysiological conditions. The protein is a proteolytic enzyme, receptor, costimulatory protein, and is involved in adhesion and apoptosis. The CD26/DPPIV protein plays a major role in immune response. Abnormal expression is found in the case of autoimmune diseases, HIV-related diseases and cancer. Natural substrates for CD26/DPPIV are involved in immunomodulation, psycho/neuronal modulation and physiological processes in general. Therefore, targeting of CD26/ DPPIV and especially its proteolytic activity has many therapeutic potentials. On the other hand, there are homologous proteins with overlapping proteolytic activity, which thus may prevent specific modulation of CD26/DPPIV. In conclusion, CD26/DPPIV is a protein present both in various cellular compartments and extracellularly where it exerts different functions and thus is a true moonlighting protein.

Introduction Since its description in 1966 (Hopsu-Havu and Glenner, 1966), dipeptidyl peptidase IV (DPPIV; EC 3.4.14.5) has been considered to be a unique peptidase that cleaves dipeptides from peptides and proteins containing proline in the penultimate position. It can also cleave dipeptides with alanine in that position. However, proteolysis is only one of the multiple functions that this protein executes. DPPIV, also known as CD26, is involved as well in signal transduction and can bind to 1)

Professor Dr. C. J .F. Van Noorden, Department of Cell Biology and Histology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam/The Netherlands, e-mail: [email protected], Fax: ‡ 31 2 06974156.

a variety of proteins. Due to its multifunctional character and its widespread expression, the exact functions of CD26/DPPIV in vivo have not yet been elucidated. Multifunctional proteins are also called moonlighting proteins (Jeffery, 1999; Ejiri, 2002), and functions of such proteins can vary on the basis of their intracellular or extracellular localization, cell type, oligomeric or polymeric state, and concentrations of ligand, cofactor or product. Numerous cell types express CD26/DPPIV, both intracellularly and extracellularly, thus making it look like an essential moonlighting protein in cell biology. However, Fischer rats that lack CD26/DPPIV have only minor physiological defects, are viable and show no difference in growth rate as compared with control rats (Tiruppathi et al., 1993). Therefore, the functions of CD26/DPPIV are probably not unique and other proteins must be able to perform similar tasks. In the present review, we discuss the multiple cellular processes in which CD26/DPPIV is thought to be involved. Special attention is given to its functioning in the complex microenvironment of the living cell which allows its moonlighting properties to be exerted. Moonlighting of proteins as well as posttranslational modifications of proteins provide an extra dimension to gene expression in the ultimate functioning of cells. Investigations focussed on moonlighting and posttranslational modifications of proteins are only in their infancy yet but are essential for the understanding how a cell functions. Many reviews on specific aspects of CD26/DPPIV have been published recently (Fleischer, 1995; De Meester et al., 1999; Kahne et al., 1999; Hildebrandt et al., 2000; Langner and Ansorge, 2000, 2002; Gorrell et al., 2001) and are referred to for more detailed information on specific aspects of CD26/DPPIV.

Moonlighting properties of CD26/DPPIV The T cell activation antigen, CD26 or DPPIV (CD26/DPPIV), is a 110-kDa multifunctional moonlighting cell surface protein that is expressed by many cell types. In humans, it is expressed constitutively on epithelial cells of liver, intestine, kidney and in

0171-9335/03/82/02-53 $15.00/0

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E. Boonacker, C. J. F. Van Noorden

a soluble form as sCD26/DPPIV in the circulation. The expression of CD26/DPPIV on T and B cells is regulated (Fox et al., 1984; Srivastava and Bhargava, 1986; Fleischer, 1987; Ulmer et al., 1992; Ansorge et al., 1995; Buhling et al., 1995; Micouin and Bauvois, 1997; Bauvois et al., 2000). CD26/DPPIV has at least 5 functions. The protein functions as (1) serine protease, (2) receptor, (3) costimulatory protein, (4) adhesion molecule for collagen and fibronectin, and (5) is involved in apoptosis. Moonlighting of CD26/DPPIV provides cells with a tool that can be used for multiple purposes. CD26/DPPIV can be considered a housekeeping protein. Hong et al. (1989) have studied expression of CD26/DPPIV mRNA in tissues by means of immunoprecipitation and Northern blot analysis. They detected only one type of CD26/ DPPIV mRNA. However, Kahne et al. (1996) showed a broad molecular heterogeneity of the CD26/DPPIV protein by means of isoelectric focussing. These findings indicate that heterogeneity of the molecular forms of CD26/DPPIV is due to posttranslational modifications. The human CD26/DPPIV gene is located on the long arm of chromosome 2 (2q24.3) and spans approximately 70 kb. It contains 26 exons, ranging in size from 45 bp to 1.4 kb (Abbott et al., 1994). The 5'-flanking domain contains neither a TATA box nor a CAAT box, commonly found in housekeeping genes, but a 300-bp sequence that is extremely rich in C and G which has potential binding sites for several transcription factors, such as NFkB, AP2 or Sp1 (Bohm et al., 1995). These binding sites are features of promotors of genes lacking tissue-specific expression and are also characteristic of a promotor of a housekeeping gene. However, the housekeeping nature of the protein is conflicting with tissue-specific transcription of the CD26/DPPIV gene (Bohm et al., 1995), because CD26/DPPIV is expressed constitutively in liver, kidney and intestine, whereas its expression is regulated in T and B cells (Boonacker et al., 2002). Bauvois et al. (2000) described that CD26/DPPIV expression is activated by interferons and retinoic acid in chronic B lymphocytic leukemia cells via the signaling pathway involving Stat1a and the GAS response element (TTCnnnGAA) of the CD26/DPPIV promotor.

Proteolytic function of CD26/DPPIV Membrane-bound proteases are found in a wide variety on many cell types. Their expression is usually finely regulated, depending on specific functions of the cell and their engagement in defined physiological pathways. Protein turnover, ontogeny, inflammation, tissue remodeling, cell migration and invasion are some of the many physiological and pathological events in which membrane-bound proteases play a crucial role. They act both as effectors and as regulatory molecules by activation of proforms or degradation of biomolecules by cleavage (Riemann et al., 2001). CD26/DPPIV was originally believed to be the only membrane-bound protease specific for proline as the penultimate residue at the amino terminus of a peptide chain and to cleave off the terminal dipeptide (Sedo and Malik, 2001). The presence of proline residues gives unique structural features to peptide chains, because it is the only cyclic amino acid substantially affecting the susceptibility of proximal peptide bonds to cleavage (Yaron and Naider, 1993; Demuth and Heins, 1995).

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Many biologically active peptides contain this unique evolutionary-conserved proline residue as a regulatory element in proteolytic processing. Therefore, proline-specific proteases play an important role in modulating activity of biologically active peptides such as neuropeptides, hormones, cytokines and chemokines (Yaron and Naider, 1993; Ansorge et al., 1995). Although the natural substrate(s) for CD26/DPPIV remain(s) to be identified, an almost endless list of possible substrates have been proposed, such as substance P, b-casomorphin, kentsin, somatoliberin, RANTES, IL-2, fibrin a-chain, stromal derived factor-1a (SDF-1a), eotaxin, and glucagon1±29 (Tables I ± III). Lambeir et al. (2001b) provided insight in the selectivity of CD26/DPPIV for specific chemokines, by ranking the chemokines as substrates on the basis of their kcat/Km, which determines the half-life of the substrate at a given enzyme concentration (Table IV). In general, short peptides such as morphiceptin are considered to be effective substrates for CD26/DPPIV. In contrast, none of the cytokines with higher molecular weights with proline in the penultimate position have been identified as a CD26/DPPIV substrate, whereas smaller fragments of these cytokines up to 24 amino acids long, containing an N-terminal sequence that is similar to that of IL1b, IL-2 or murine IL-6, are cleaved. The rate of CD26/DPPIVcatalyzed proteolysis is inversely related with the chain length of the protein to be cleaved (Hoffmann et al., 1993; Lambeir et al., 2001b). Substrate recognition by CD26/DPPIV is defined not only by the size of the substrate, but also by its amino acid sequence. The formalism of Schechter and Berger (1968) provides a classification system for amino acids in a peptide on the basis of their position in that peptide in relation to the cleavage site; amino acids preceding the scissile bond in the direction of the N-terminus are called P1, P2, etc, whereas residues into the direction of the C-terminus following the scissile bond are called P1', P2', etc. Proteins containing proline in P1 usually have large hydrophobic/aromatic side chains. Proteins containing alanine in the P1 position have often aspartate, glutamate, or glutamine in the P1' position. The P2' position is then often occupied by a small polar amino acid (glycine, serine, or alanine). A variety of amino acids with small polar, long aliphatic and positively-charged side chains are often found in the P3' position. A clear pattern has not yet been found for the P4', P5' and P6' position of CD26/DPPIV substrates, except for a prevalence of threonine at P5'. It indicates that substrate recognition by CD26/DPPIV does not extend much further than the P3' position. The 3-dimensional structure of CD26/DPPIV has not been elucidated yet on the basis of crystal structures (Ansorge et al., 2000), but computing was used to resolve parts of its structure (Brandt, 1997; Gorrell et al., 2000). Recently, the 3-dimensional structure of the pig serine protease prolyl oligopeptidase has been elucidated and appeared to be homologous to CD26/ DPPIV (Fulop et al., 2001). Serine proteases like CD26/DPPIV and prolyl oligopeptidase have the amino acid sequence serineaspartate-histidine in their catalytic site. This is the reverse sequence found in classical serine proteases that are members of the chymotrypsin and subtilisin families (David et al., 1993; Hooper et al., 2001). CD26/DPPIV and its structural homologues prolyl oligopeptidase and proline imidopeptidase (Table V) contain a similar peptide sequence that forms the last 200 C-terminal residues in their extracellular domain with an a/bhydrolase fold which contains the catalytic triad serineaspartate-histidine. The histidine in the catalytic triad of serine

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Tab. I. Possible substrates of CD26/DPPIV that are involved in physiological processes. Substrate

Reference

a1-Microglobulin Aprotinin Bradykinin Chorionic gonadotropin Corticotropin-like intermediate lobe peptide Enterostatin Fibrinogen a-chain Gastric inhibitory polypeptide Gastric releasing peptide Growth hormone-releasing factor (GHRF) Glucagon-like peptide-1 (GLP-1) Glucagon-like peptide-2 (GLP-2) Glucagon Glucose-dependent insulinotropic polypeptide Peptide histidine methionine (PHM) Insulin-like growth factor-1 Morphiceptin Monocyte chemotactic protein-1 Monocyte chemotactic protein-2 Monocyte chemotactic protein-3 Procalcitonin Pro-colipase Prolactin Substance P Trypsinogen Tyr-melanostatin Vasoactive intestinal peptide (VIP)

(Nausch et al., 1990) (Nausch et al., 1990) (Kato et al., 1978) (Nausch et al., 1990) (Nausch et al., 1990) (Bouras et al., 1995) (Heymann and Mentlein, 1984) (Mentlein et al., 1993) (Nausch et al., 1990) (Frohman et al., 1986, 1989; Bongers et al., 1992; Martin et al., 1993) (Mentlein et al., 1993; Kieffer et al., 1995; Pauly et al., 1996) (Drucker et al., 1997b) (Pospisilik et al., 2001) (Kieffer et al., 1995; Pauly et al., 1996) (Mentlein et al., 1993) (Mentlein, 1999) (Tiruppathi et al., 1990, 1993) (Proost et al., 1998a, b, c) (Oravecz et al., 1997) (Proost et al., 1998a) (Wrenger et al., 2000) (Heymann et al., 1986; Nausch et al., 1990) (Nausch et al., 1990) (Heymann and Mentlein, 1978; Kato et al., 1978) (Nausch et al., 1990) (Shane et al., 1999) (Lambeir et al., 2001a)

Tab. II. Possible substrates of CD26/DPPIV involved in inflammatory processes.

Tab. III. Possible substrates of CD26/DPPIV involved in neurological and psychomodulatory processes.

Substrate

Reference

Substrate

Reference

Eotaxin IP-10 I-TAC Kentsin LD78b Lymphotoxin MDC67 MDC69 Mig Monocyte chemotactic protein RANTES

(Struyf et al., 1999) (Oravecz et al., 1997) (Lambeir et al., 2001b) (Bueno et al., 1986) (Lambeir et al., 2001b) (Ansorge et al., 1991) (Lambeir et al., 2001b) (Lambeir et al., 2001b) (Lambeir et al., 2001b) (Van Coillie et al., 1998) (Oravecz et al., 1997; Iwata et al., 1999) (Proost et al., 1998b) (Shioda et al., 1998) (Bauvois et al., 1992)

b-Casomorphin

(Heymann and Mentlein, 1986; Nausch et al., 1990) (Shane et al., 1999) (Bueno et al., 1986) (Mentlein et al., 1993) (Mentlein et al., 1993) (Heymann and Mentlein, 1978)

SDF-1a SDF-1b TNF-a

proteases plays at least two roles in the hydrolysis of peptide bonds. First, it acts as proton acceptor in the formation of an acyl-enzyme intermediate and second, it acts as proton donor in the subsequent deacylation step (Kraut, 1977). The central tunnel of an unusual seven-blade b-propeller domain covers the catalytic cleft formed by the a/b-hydrolase fold. Both the hydrolase and propeller domain of these proteases are part of the surface of the inner cavity in which catalysis occurs (Abbott et al., 1999). The propeller domain is the reason why these proteases are in fact oligopeptidases because they exclude large peptide sequences from occupying the catalytic pocket. In this fashion, the propeller can protect larger peptides and proteins from proteolysis. The entrance to the catalytic pocket lies in the center of the lower face of the b propeller as a pore-closing domain. It has been suggested that the lining of the opening of this narrow pore allows for specific substrate entry due to charged flexible side chains and that this

Endomorphin-1 and -2 Kentsin NPP Y Peptide YY Substance P

Tab. IV. Natural substrates of CD26/DPPIV on the basis of the order of kcat/Km according to Lambeir et al. (2001b). 6

Peptide

kcat/Km in 10

M

SDF-1a MDC69 I-TAC MDC67 IP-10 Mig Eotaxin RANTES LD78b

52 41 1.2  0.1 0.5  0.1 0.5  1 > 0.4  0.2 0.08  0.01 0.04  0.01 0.003  0.002

1

s

1

may be a substrate-mediated event (Fulop et al., 1998). These findings indicate that the catalytic triad of CD26/DPPIV is not so specific at all for proline and for alanine and adjacent amino acids, but rather the molecular environment of the active site in the enzyme molecule defines for a large part substrate specificity. Substrate-assisted catalysis (SAC) is one of the mechanisms by which substrate specificity is acquired. SAC has been demonstrated for serine proteases as well. When, for example, the serine amino acid in the catalytic triad is replaced by site-

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Tab. V. CD26/DPPIV structure and/or activity homologues (DASH). Modified after (Sedo and Malik, 2001). Protein CD26/DPPIV ˆ FAPb ˆ ADA binding-protein

(E.C.3.4.14.5)

DPPII (QPP)

(E.C.3.4.14.2)

DPPIII

(E.C.3.4.14.4)

Activity

Reference

Release of an N-terminal dipeptide, Xaa-Xbb-/-Xcc, from a polypeptide, preferentially when Xbb is Pro, provided Xcc is neither Pro nor hydroxyproline. (Membranebound serine peptidase) Release of an N-terminal dipeptide, Xaa Xbb / Xcc, preferentially when Xbb is Ala or Pro. Substrates are oligopeptides, preferentially tripeptides. (Lysosomal serine-type peptidase at acidic pH) Release of an N-terminal dipeptide from a peptide comprising four or more residues, with broad specificity. Also acts on dipeptidyl 2-naphthylamides. (Cytosolic peptidase)

(Tiruppathi et al., 1993; Rawlings and Barrett, 2000)

DPPIV-b DPP-7 DPPVIII (DPP4-related protein) DPPT-L (mahogany protein, attractin) Dipeptidyl aminopeptidase-like protein (DPPX long/ short, brain specific dipeptidyl peptidase-like protein (BSPL, DPPVI) FAPa (separase) Human immunodeficiency virion1 protease Lysosomal prolyl carboxypeptidase (PCP/angiotensinase C)

(E.C.3.4.23.16) (E.C.3.4.16.2)

Membrane Pro-X carboxypeptidase Glutamate carboxypeptidase II ˆ PSMA ˆ NAALADase I (N-acetyl a-linked acidic dipeptidase I) NAALADase II NAALADase L (Ileal 100 protein) Membrane dipeptidase

(E.C.3.4.17.16)

(E.C.3.4.13.19)

Xaa-Pro-dipeptidyl peptidase

(E.C.3.4.14.11)

X-Pro dipeptidase

(E.C.3.4.13.9)

Prolyl oligopeptidase

(E.C.3.4.21.26)

(E.C.3.4.17.21)

Specific for a P1 residue that is hydrophobic, and P1' variable, but often Pro Cleavage of a Pro / Xaa bond to release a C-terminal amino acid Release of a C-terminal residue other than proline, by preferential cleavage of prolyl bond. (No activity) Release of an unsubstituted C-terminal glutamyl residue, typically from Ac Asp Glu or folylpoly-gamma-glutamates. (No activity) Hydrolysis of dipeptides. Hydrophobic dipeptides are cleaved preferentially, including prolyl amino acids. (Membrane-bound) Cleaves Xaa Pro / bonds to release unblocked Nterminal dipeptides from substrates including Ala Pro / P-nitro-anilide and (sequentially) Tyr Pro / Phe Pro / Gly Pro / Ile Hydrolysis of Xaa / Pro dipeptides; also acts on aminoacyl-hydroxyproline analogues. No action on Pro / Pro. (Cytosolic) Hydrolysis of Pro / Xaa  Ala / Xaa in oligopeptides. (Generally cytosolic)

directed mutagenesis, a functional group in the substrate can substitute for the missing catalytic residue. Similarly, replacement of the catalytic histidine by alanine in a subtilisin protease reduced the catalytic efficiency by a million-fold. When substrates contain a histidine, they are hydrolyzed much faster than homologous substrates containing alanine or glutamine in the same position. In contrast, the wild-type enzyme hydrolyzes all substrates with similar catalytic efficiency (Carter and Wells, 1987). Substrate specificity is also determined by complex formation with other proteins that modulate the accessibility of the active site. Therefore, different isoforms of CD26/DPPIV may have different substrate specificities (Malik et al., 2000). These aspects of substrate specificity of CD26/DPPIV make it very difficult to predict which natural substrates are processed by which isoforms of CD26/DPPIV. For instance, glucagon1±29 is a 29 amino acid long peptide hormone that is released from pancreatic a-cells to induce increased blood glucose levels during fasting by stimulating

(Chiravuri et al., 1999; Underwood et al., 1999; Fukasawa et al., 2001) (Fukasawa et al., 1999)

(Blanco et al., 1997) (Banbula et al., 2001) (de Lecea et al., 1994; Abbott et al., 2000) (Duke-Cohan et al., 1996, 1998; Tang et al., 2000) (Wada et al., 1992; Yokotani et al., 1993; Hough et al., 1998) (Garin-Chesa et al., 1990; Levy et al., 1999; Park et al., 1999) (Dunn, 1998) (Yang et al., 1968; Walter et al., 1980; Rawlings and Barrett, 1996) (Dehm and Nordwig, 1970) (Rawlings and Barrett, 1997) (Sedo and Malik, 2001) (Pangalos et al., 1999) (Campbell et al., 1966) (Meyer-Barton et al., 1993)

(Yoshimoto et al., 1983) (Abbott et al., 2000; Gorrell et al., 2001)

hepatic glycogenolysis and gluconeogenesis. The circulating half-life of glucagon1±29 is approximately 5 ± 6 min in dogs and humans. Recently, evidence has been presented that CD26/ DPPIV is partly responsible for the inactivation of glucagon (Hinke et al., 2000). It is still controversial, which tissues are responsible for clearance of glucagon from the circulation. It is likely that the kidney plays a dominant role, because CD26/ DPPIV is present on the apical surface of epithelial cells in renal proximal tubules where it can degrade glucagon1±29 present in primary urine. However, it is not understood why glucagon1±29 is not degraded in the circulation (Hendriks and Benraad, 1981; Marki, 1983), as CD26/DPPIV is present on the surface of T cells and B cells and as freely circulating sCD26/DPPIV. This would implicate that only certain isoforms of CD26/DPPIV are responsible for degradation of glucagon1±29 in vivo, as various isoforms are expressed in different cell types (Kahne et al., 1996). Kasahara et al. (1984) observed multiple isoforms of sCD26/DPPIV in human serum after cellulose-acetate electrophoresis. For some of these molecular isoforms, the origin could

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Tab. VI. Levels of soluble CD26/DPPIV in plasma in relation with physiological or pathophysiological processes (›, upregulated; ï, downregulated). Modified after (Gorrell et al., 2000). Serum DPPIV

Reference

ï ï › › › › › › ï ï ï ï › ï ï › › › ï ï ï ï ï › ï › ï ï ï ï ï ï ï

(Maes et al., 1999) (Durinx et al., 2001) (Korom et al., 1997) (Hildebrandt et al., 1999) (Katoh et al., 2000) (Kubota et al., 1994) (Perner et al., 1999) (Haacke et al., 1986) (Cordero et al., 2000) (Rose et al., 2002) (Polgar, 1992; Maes et al., 2001a, b) (Korosi et al., 2001) (Steinbrecher et al., 2001) (Maes et al., 1998b) (Cordero et al., 2000) (Nishikawa et al., 1995) (Firneisz et al., 2001) (Hanski et al., 1986) (Ivanov et al., 1992; McCaughan et al., 1993) (Subramanyam et al., 1993; Hosono et al., 1999) (Lefebvre et al., 2002) (Hildebrandt et al., 2001a) (Schmitz et al., 1996) (Lakatos et al., 2000) (Fukasawa et al., 1982; Uematsu et al., 1998) (Gotoh et al., 1988) (Polgar, 1992; Hildebrandt et al., 2001b) (Katoh et al., 2000; Bock et al., 2001) (Cordero et al., 2001; Cuchacovich et al., 2001) (Maes et al., 1996) (Van Der Velden et al., 1999) (Stancikova et al., 1992; Cuchacovich et al., 2001) (Maes et al., 1998a; Durinx et al., 2001)

be determined, such as liver, spleen or kidney. However, the origin of sCD26/DPPIV is still a matter of debate. Shedding or secretion of CD26/DPPIV from different cell types can be responsible for the existence of soluble isoforms of CD26/ DPPIV. A correlation between levels of sCD26/DPPIV and specific physiological or pathophysiological processes is shown in Table VI. This observation makes the issue of in vivo processing of molecules such as glucagon1±29 by the various isoforms of CD26/DPPIV even more complex. It has been suggested that CD26/DPPIV is also able to degrade collagen, thereby facilitating trafficking of cells through the extracellular matrix. CD26/DPPIV has been reported to be one of the mediators of lymphocyte migration in the thymus from the cortical region to the medulla during maturation (Savino et al., 1993). Furthermore, gelatin zymography demonstrated gelatinase activity of immunopurified CD26/DPPIV. Gelatinase activity is only possible when the enzyme has endopeptidase activity in addition to its exopeptidase activity. Indeed, it has been reported that rat CD26/DPPIV exhibits weak endopeptidase activity and is able to cleave denaturated fibrillar collagen, but it cannot degrade native collagen (Bermpohl et al., 1998). The weak endopeptidase activity indicates that CD26/DPPIV and its homologue fibroblast activation protein (FAP-a; Table V) are members of a new subfamily of gelatinolytic integral membrane serine proteases (Pineiro-Sanchez et al., 1997). These proteinases are all expressed as homodimers, and this seems to be a condition for full expression of endoproteinase activity as activity seems to be dependent on subunit association (Pineiro-Sanchez et al.,

1997). However, heterodimer complexes of FAP-a and CD26/DPPIV have been described as well (Rettig et al., 1993, 1994; Scanlan et al., 1994). It is unknown yet what the effect of heterodimerization is on protease activity.

Abstinence in alcoholics Ageing Allograft rejection, kidney transplantation Anorexia nervosa, bulimia nervosa Atopic dermatitis Autoimmune disease Biliary atresia Cancer of bile duct or pancreas Colorectal carcinoma Crohn×s disease (CD26 higher on T cells) Depression/anxiety Diabetes mellitus Encephalitis Fibromyalgia Gastrointestinal cancer Graves× disease Hepatic dysfunction, hepatitis C Hepatoma Hepatoma/melanoma cell line (more acidic isoforms) HIV infection Hypertension Inflammatory bowel disease (CD26 higher on T cells) Immunosuppression Liver cirrhosis Oral squamous cell carcinoma Osteoporosis Pregnancy Psoriasis Rheumatoid arthritis (CD26 higher on T cells) Schizophrenia Smoking Systemic lupus erythematosus (hypersialylated isoform) Women versus men

Receptor function of CD26/DPPIV The highly invasive human prostate tumor cell line 1-LN synthesizes and secretes large amounts of plasminogen activators and matrix metalloproteinases (MMPs). It was demonstrated in these cells that the receptor for plasminogen type 2 (Pg 2) is CD26/DPPIV. Pg 2 has six glycoforms that differ in their sialic acid content. Only the highly sialylated Pg 2g, Pg 2d and Pg 2e glycoforms bind to CD26/DPPIV (Gonzalez-Gronow et al., 2001). In human rheumatoid synovial fibroblasts, Pg binding to the Pg 2 receptor and activation of Pg by urokinasetype plasminogen activator induce a significant rise in the cytosolic free Ca2‡ concentration (Gonzalez-Gronow et al., 1993) via interactions of Pg with CD26/DPPIV associated with the integrin aIIbb3 on the cell surface (Gonzalez-Gronow et al., 1994, 1998). Lactose is a sugar which interferes with the binding of Neu5Ac (a2 ± 3) or (a2 ± 6) residues to sialic acidbinding proteins and inhibits the intracellular Ca2‡ flux by blocking binding of Pg 2g, Pg 2d and Pg 2e glycoforms to CD26/ DPPIV on the surface of fibroblasts (Gonzalez-Gronow et al., 1998), suggesting that binding occurs via carbohydrate chains. A similar inhibition of the intracellular Ca2‡ flux was observed

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when cells were preincubated with the anti-CD26/DPPIV mAb 236.3. Triggering of CD26/DPPIV can induce MMP-9 expression, and when this occurs in cancer cells, it possibly facilitates metastasis (Gonzalez-Gronow et al., 2001). Pg 2e alone is also able to stimulate expression of proMMP-9 (Gonzalez-Gronow et al., 2001). On the other hand, CD26/DPPIV also plays a role in differentiation of cells, thereby blocking the cells from conversion into a malignant phenotype. When a Ser ! Ala mutation was inserted in the catalytic triad, CD26/DPPIV did not suppress tumorigenicity or anchorage-independent growth of cancer cells, nor did it reverse the block in differentiation, showing that DPPIV activity is required in suppressing the malignant phenotype (Wesley et al., 1999). As a receptor, CD26/DPPIV is able to bind adenosine deaminase (ADA) (Kameoka et al., 1993). ADA is a 41-kDa enzyme expressed in all mammalian cells that catalyzes deamination of adenosine and 2'-deoxyadenosine to inosine and 2'-deoxyinosine, respectively. Although the location of this enzyme is mainly cytosolic, it is also expressed on the surface of B cells and T cells, and this expression is increased upon cell activation. CD26/DPPIV is directly associated with ADA and is identical with the so-called ADA-binding protein (Fig. 1). ADA plays a major role in the development and function of lymphoid tissues. ADA deficiency causes both defects in the development of the immune response and haematologic malignancies (Blackburn et al., 1998). In humans, ADA deficiency is inherited as an autosomal recessive condition and causes severe combined immunodeficiency due to the sensitivity of T cells and B cells to adenosine and deoxyadenosine, which downregulates T cell and B cell activation. Adenosine and deoxyadenosine are the substrates that are degraded by ADA. Thus, the ADA-CD26/DPPIV complex is able to reduce local concentrations of adenosine (Dong et al., 1996). Furthermore, binding of ADA to CD26/DPPIV on the surface of T cells induces cell proliferation (Martin et al., 1995). HIV gp120 protein inhibits binding between ADA and CD26/ DPPIV, and thus inhibits adenosine breakdown. In this way, it may contribute to the pathogenesis of HIV-related diseases (Valenzuela et al., 1997), but the fact that adults with defective ADA-CD26/DPPIV binding are healthy suggests that binding is not essential (Richard et al., 2000). Gp120 interacts with CD26/DPPIV but gp120-mediated disruption of the ADACD26/DPPIV complex is a consequence of interactions between gp120 and a domain on the CD26/DPPIV protein different from the ADA-binding site (Franco et al., 1998). CD26/DPPIV colocalizes with CXCR4 (Herrera et al., 2001), which is the receptor for one of the natural substrates of CD26/DPPIV, SDF-1a (Fig. 1). SDF-1a binding to CXCR4 induces chemotaxis and antiviral activity of Th2 cells, but not Th1 cells. CXCR4 and CD26/DPPIV on membranes of T (CD4‡) and B (CD4 ) cell lines co-immunoprecipitate. Treatment of cells with SDF-1a induces CD26/DPPIV to be cointernalized with CXCR4 (Fig. 2). SDF-1a-mediated downregulation of plasma membrane-bound CD26/DPPIV by internalization is not blocked by pertussis toxin indicating that internalization is not mediated by Gi proteins. Internalization of CXCR4 receptors does not occur in cells that express mutant CXCR4 receptors that cannot be internalized, whereas CD26/DPPIV internalization is not hampered, indicating that internalization of CD26/DPPIV and CXCR4 follows different routes (Herrera et al., 2001). Longer periods of treatment with SDF-1a result in a homogeneous distribution of CXCR4

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underneath the plasma membrane and a clustered localization of CD26/DPPIV in intracellular vesicles. This confirms that internalization of the two proteins follows different routes. Codistribution and cointernalization also occur in peripheral blood lymphocytes. Since CD26/DPPIV is a cell surface ectopeptidase that has the capacity to cleave SDF-1a, the CXCR4-CD26/DPPIV complex is likely a functional unit in which CD26/DPPIV may directly modulate SDF-1a-induced chemotaxis and antiviral activity of lymphocytes (Fig. 3). The physical association of CXCR4 and CD26/DPPIV, directly or as part of a supramolecular structure, suggests a role of CD26/ DPPIV in the pathophysiology of viral infection. Furthermore, comodulation of CXCR4 and CD26/DPPIV by SDF-1a suggests that the ADA-CD26/DPPIV and the CXCR4-CD26/DPPIVADA complexes are important for the functioning of lymphocytes (Fig. 3) (Herrera et al., 2001). Immunohistochemical analysis indicated that crosslinking of CD26/DPPIV with a monoclonal antibody induces not only capping and internalization of the molecule, but also its colocalization with the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR), as this protein binds to CD26/DPPIV via M6P residues in the glycosylation domain of CD26/DPPIV (Ikushima et al., 2000). Colocalization was found in cytoplasmic vesicles close to the cell surface after crosslinking of CD26/DPPIV (Fig. 2). Addition of exogenous M6P inhibited not only the internalization of CD26/DPPIV induced by anti-CD26/DPPIV antibody crosslinking but also T cell proliferation induced by CD3 and CD26/DPPIV costimulation. Apparently, M6P/IGFIIR binding to CD26/DPPIV is necessary for the costimulatory function of CD26/DPPIV. T cell proliferation induced by anti-CD3 and PHA is not inhibited by M6P (Ikushima et al., 2000). CD26/DPPIV on the apical membrane of hepatocytes (Kreisel et al., 1993) and intestinal epithelial cells (Matter et al., 1990) is continuously internalized and reexpressed. Internalization of CD26/DPPIV is independent of DPPIV activity. Although internalization has been described for CD26/ DPPIV to be modulated by M6P/IGFIIR on T cells (Ikushima et al., 2000), it is not yet clear which carbohydrate moiety is responsible for this interaction. However, treatment of CD26/ DPPIV with either a glycosidase or a phosphatase completely abolished this binding capacity, demonstrating that both glycosylation and phosphorylation of CD26/DPPIV are required for binding with M6P/IGFIIR. It was found that only PHA-activated T cells express M6P-CD26/DPPIV, suggesting that activation induces mannose-6-phosphorylation of CD26/ DPPIV (Ikushima et al., 2000). These data suggest that binding of CD26/DPPIV to M6P/IGFIIR is mediated by M6P residues on the CD26/DPPIV carbohydrate moiety. The function of this recycling in hepatocytes and intestinal epithelial cells seems to be repair of the oligosaccharide chains attached to the glycoprotein, or in the regulation of terminal glycosylation, e.g. in response to physiological stimuli (Kreisel et al., 1993). Recycling of ectoenzymes has been demonstrated in only a few cases, such as for 5'-nucleotidase in a hepatoma cell line (Van den Bosch et al., 1988). Other membrane components such as the transferrin receptor (Snider and Rogers, 1985) and the M6P/IGFIIR also exhibit recycling and resialylation of their oligosaccharides. In this setting, it is striking to see that all molecules associated with T cell activation, such as CD3/TCR complex (Moller et al., 1990; Morel et al., 1992; Herrera et al., 2001; Hwang and Sprent, 2001; Dietrich et al., 2002; Menne et al., 2002), CXCR4 (Herrera et al., 2001) and all the

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Fig. 1. Schematic representation of molecules associated with CD26/ DPPIV. From top to bottom: adenosine A1a receptor (A1aR) and its ligand adenosine, adenosine deaminase (ADA), CD45, the CD26/ DPPIV dimer, its transporter M6P/IGFIIR, one of the natural substrates of CD26/DPPIV, SDF-1a, that is inactivated (in red frame) by CD26/DPPIV, the receptor of SDF-1a, CXCR4, RANTES which is another natural substrate of CD26/DPPIV but is still bioactive after proteolysis (in green frame), and the receptor of RANTES, CCR5, the TCR/CD3 complex and intracellular Fyn59 and ZAP70 which are

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associated with TCR, the CD4 complex associated with intracellular Lck59 and phospholipase C (PLC) which liberates IP3 and consequently induces an increase in intracellular Ca‡‡. Activation of T cells by CD26 crosslinking results in increased activity of phosphotyrosine kinases via CD45, ZAP70, Lck59 and Fyn59, phosphorylation of CD3 and Ca2‡ influx via PLC which are similar effects as upon stimulation of the TCR/CD3 pathway. Modified after (Gorrell et al., 2000).

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Fig. 2. Schematic representation of traffic of plasma membranebound proteins in cells. All proteins can be internalized and recycled. IL-4 stimulation elevates M6P/IGFIIR expression and enhances mannose glycoconjugate uptake and endocytosis and downregulates CD26/DPPIV expression on the plasma membrane of Th2 cells. IFN-g stimulation decreases M6P/IGFIIR expression and thus diminishes internalization of CD26/DPPIV in Th1 cells and an elevated particle

sorting towards the lysosomal compartment. Ligand binding by M6P/ IGFIIR and subsequent internalization and accumulation in the endolysosomal compartment induces transcription activity. Early endosome (EE), sorting endosome (SE), late endosome (LE), endolysosome (EL) and lysosome (L). Membrane-bound proteins are represented by the same symbols as in Figure 1.

molecules associated with CD26/DPPIV are internalized and recycled in T lymphocytes as is shown in the schematic representation in Figure 2. Further studies on the exact cellular distribution patterns of CD26/DPPIV and all the molecules that it is associated with in

living cells during T cell activation will provide further insight in functions of CD26/DPPIV, and possibly novel mechanisms in which costimulatory molecules are involved in the T cell signaling process.

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Fig. 3. Schematic representation of Th1 and Th2 actions towards sites of inflammation and preferential attraction of Th1 cells by chemokines that are bioactive after CD26/DPPIV proteolysis, IP-10 and RANTES (in green frames). Chemokines that are inactivated by CD26/DPPIV, SDF-1a, eotaxin and MDC (in red frames) attract Th2 cells. The

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chemokines are released by dendritic cells, epithelial cells, stromal cells and endothelial cells at the site of inflammation. Neutrophils and macrophages are preferentially recruited to the site of inflammation by Th1 cells, whereas basophils and eosinophils are preferentially recruited by Th2 cells.

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Costimulatory function of CD26/ DPPIV Crosslinking of CD26/DPPIV and CD3 with immobilized mAbs induces T cell activation and IL-2 production (Morimoto and Schlossman, 1998; Boonacker et al., 2002). Treatment of T cells with anti-CD26/DPPIV antibodies leads to a decreased surface expression of CD26/DPPIV due to internalization (Fig. 2), and this modulation of CD26/DPPIV results in an elevated proliferative response to anti-CD3 or anti-CD2 stimulation (Dang et al., 1990a). T cell activation leads to redistribution of proteins in lipid rafts (Montixi et al., 1998; Xavier et al., 1998; Horejsi et al., 1999; Janes et al., 1999). This redistribution in rafts results in association of the T cell receptor (TCR/CD3) with signal-transduction molecules, indicating a specific role of rafts in T cell activation. Costimulatory molecules such as CD2, CD5, CD9, and CD44 are also raft associated (Yashiro-Ohtani et al., 2000), and strengthen TCRmediated signaling when coligated with CD3 by inducing aggregation of rafts as well as enhancing association of TCR/ CD3 and rafts. Crosslinking of CD26/DPPIV induces tyrosine phosphorylation of a panel of cellular proteins, which are similar to those that are phosphorylated after TCR/CD3 stimulation (Hegen et al., 1997). Moreover, inhibition by M6P was demonstrated for anti-CD3 and anti-CD26 costimulation, but not by G6P or M1P. The inhibitory effect was not observed in cells stimulated by anti-CD3 and PHA (Ikushima et al., 2000). Stimulation with a combination of anti-CD3 and anti-CD26/DPPIV antibodies induces substantially more IL-2 production by CD26(DPPIV‡)-transfected Jurkat cells (Jurkat cells are T cells that lack CD26/DPPIV) than when the mutant lacking enzyme activity ± CD26(DPPIV )-transfected Jurkat cells ± is stimulated. However, mutant CD26(DPPIV )-transfected cells produce significantly more IL-2 than control Jurkat cells lacking CD26/DPPIV expression (Tanaka et al., 1993). IL-2 has to be processed by CD26/DPPIV to become fully activated. As a costimulatory molecule, CD26/DPPIV is also able to induce cell proliferation via interactions with CD45 (Fig. 1). CD45 is a tyrosine phosphatase, which in its inactive state forms a dimer (Desai et al., 1993). It has been demonstrated that CD26/DPPIV is present in lipid rafts, and that crosslinking with anti-CD26/DPPIV antibodies increases the recruitment of CD26/DPPIVand CD45 molecules to these rafts (Ishii et al., 2001). This indicates that CD26/DPPIV and CD45 are associated. Recruitment results in increased tyrosine phosphorylation of receptor signaling molecules such as cCbl, p56Lck, ZAP-70, Erk1/2, and CD3z (Hegen et al., 1997; Ishii et al., 2001), suggesting that CD26/DPPIV induces monomerisation of CD45 and stimulates TCR signaling (Braun et al., 1998). However, the costimulatory function of CD26/DPPIV remains incompletely understood, because proteolytic activity of CD26/DPPIV seems to play an important but not an essential role in the costimulatory function of CD26/DPPIV (Tanaka et al., 1993).

Adhesion of CD26/DPPIV to collagen and fibronectin Indications of interactions of CD26/DPPIV as an adhesion molecule with proteins of the extracellular matrix were

obtained by experiments that show that the small peptide Gly-Pro-Ala which is recognized by CD26/DPPIV as substrate interferes with spreading of rat hepatocytes on a matrix containing fibronectin and collagen (Hanski et al., 1985). Similar results were obtained with substrates and inhibitors of CD26/DPPIV, but not with tripeptides that were not substrates for CD26/DPPIV. In agreement with this observation, it was found that peripheral T cells can migrate through a monolayer of endothelial cells on a collagen gel when high levels of CD26/ DPPIV are expressed (Masuyama et al., 1992). Moreover, an antibody against CD26/DPPIV delayed fibronectin-mediated adhesion of rat hepatocytes to denaturated collagen (Hanski et al., 1988). Nitrocellulose binding assays using 125I-labelled DPPIV that was purified to homogeneity from rat hepatocytes revealed a direct interaction of DPPIVand fibronectin. Binding to fibronectin occurs at a site in the CD26/DPPIV molecule that is distinct from its exopeptidase substrate recognition site because competitive peptide inhibitors and phenylmethylsulphonyl fluoride enhanced fibronectin binding, possibly as a result of an altered conformation of DPPIV (Piazza et al., 1989). CD26/DPPIV acts as collagen receptor on murine fibroblasts (Bauvois, 1988), and on human T cells as a collagen receptor that induces cell activation (Dang et al., 1990b). The binding site for collagen was found to be present in the cysteinerich region of the molecule (Loster et al., 1995).

Involvement of CD26/DPPIV in apoptosis Jurkat T cells transfected with CD26(DPPIV‡) appeared to be less apoptotic than cells transfected with CD26(DPPIV ). The higher rates of apoptosis of the CD26(DPPIV ) transfectants was explained by the finding that CD95 (Fas/Apo-1) was upregulated in mutants without DPPIV activity in comparison with transfectants with DPPIVactivity (Morimoto et al., 1994). CD95 is a member of the nerve growth factor/tumor necrosis factor receptor family that mediates apoptosis (Trauth et al., 1989). In a human hepatoma cell line, CD26/DPPIV functions in the absence of most receptors expressed on T cells. Surprisingly, these hepatoma cells underwent apoptosis after stimulation with an immobilized anti-CD26/DPPIV mAb. This effect was not limited to this cell line, because CD26/DPPIV stimulation of another hepatoma cell line, HepG2, also induced apoptosis. This effect was transduced by a tyrosine kinase, because phosphatase inhibition enhanced apoptosis (Gaetaniello et al., 1998). This is in line with the fact that tyrosine phosphorylation can be demonstrated after crosslinking of CD26/DPPIV, but in contrast with the finding that CD26(DPPIV‡)-transfected Jurkat cells are protected from apoptosis after HIV infection, appararently because CD95 was not upregulated in the hepatoma cells (Morimoto et al., 1994). A possible explanation is that the opposite functions of CD26/DPPIV depend on its moonlighting properties and thus the molecule either exerts its different functions in different cell types, or different isoforms exert unique functions in different cellular contexts. The remarkable difference between the role of CD26/DPPIV in lymphocytes and hepatoma cells in relation to apoptosis is an example that the protein exerts its specific functions depending on the context of multifunctional molec-

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ular machineries and thus that CD26/DPPIV is a moonlighting protein.

The role of CD26/DPPIV in immune responses CD26/DPPIV is expressed on T lymphocytes, B lymphocytes and NK cells (Ansorge et al., 1995; Mattern et al., 1995). CD26positive T helper cells are for 76% CD4‡ and for 16% CD8‡ (Mattern et al., 1995). Expression of CD26/DPPIV on T helper cells is tightly regulated upon activation (Boonacker et al., 2002). Detailed analysis of subsets of human CD4‡ T helper cells indicates that CD26/DPPIV expression is more restricted than that of most other accessory proteins since it is expressed only on CD4‡ memory T helper (CD45RO‡/CD29‡) cells. This unique population is the only cell type that can recall antigens, can induce immunoglobulin synthesis in B cells and activate MHC-restricted cytotoxic T cells (Morimoto et al., 1989; Dang et al., 1990a). CD4‡ cells lacking CD26/DPPIV cannot be triggered to elicit helper functions but can respond to mitogens and alloantigens. CD26/DPPIV is not a stable surface marker; T cell activation induces increased expression of CD26/DPPIV on the T cell surface in the initial stages of proliferation and it reaches a maximum after three days. Then, expression decreases again and cells stop to proliferate after 11 days of culture (Ansorge et al., 1995; Mattern et al., 1995). The increase in CD26/DPPIV expression upon stimulation is more profound on Th1 cells than on Th2 cells, so that CD26/DPPIV expression is many-fold higher on Th1 cells during activation and proliferation, but Th1 and Th2 cells show similar DPPIV activity due to posttranslational alterations in Km and Vmax values of DPPIV (Boonacker et al., 2002). Kahne et al. (1996) demonstrated differences in intracellular localization patterns of isoforms of DPPIV. These patterns were found to change significantly upon mitogenic stimulation. Some isoforms were found only in soluble fractions of resting T cells, whereas after PHA-stimulation they were present in membrane fractions. These isoforms on the plasma membrane either originate from the cytosol and are translocated or represent a newly synthesized form of the enzyme. CD26/DPPIV isoforms of haematogenous sources are strongly heterogeneous. Mitogenic stimulation of human mononuclear cells leads to changes in both isoelectric points and cellular localization patterns of distinct isoforms of CD26/ DPPIV. When comparing staining patterns of DPPIV activity and immunolocalization patterns of CD26/DPPIV isoforms, strong differences were found. Lysates from healthy individuals also showed strong differences in activity patterns after isoelectric focussing (Kahne et al., 1996). Noteworthy is that these isoforms of CD26/DPPIV are recognized by all known antiCD26/DPPIV mAbs. A direct quantitative relationship between the amount of antibody binding and DPPIV activity was not found. It can be concluded on the basis of these phenomena that the CD26 family consists of isoforms exhibiting DPPIV activity as well as isoforms without detectable activity (Klobusicka and Babusikova, 1999a). However, it is not completely clear whether CD26/DPPIV was responsible for all bands that showed substrate conversion, because substrate conversion could also be performed by homologous proteases, such as attractin, DPPII, DPPIV-b, or DPPVIII (Table V).

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Since CD26/DPPIV participates in various functions of the immune system, variations in expression of this molecule and its DPPIVactivity may be the basis of the roles of CD26/DPPIV in regulating T cell development in particular and the immune response in general. Because IFN-g, CD26/DPPIV, CCR5, CXC4, and CXCR3 (the latter 3 are the receptors of IP-10, Mig and I-TAC which are all processed by CD26/DPPIV; Table I) are Th1 hallmarks (Bonecchi et al., 1998; Loetscher et al., 1998), processing of chemokines may constitute an important mechanism to reduce attraction of leukocytes in a Th1 response (Fig. 3). It is crucial to dampen the immune response after influx of sufficient immune cells in an inflamed site. At the moment that CD26/DPPIV-mediated cleavage of chemokines has reduced the bioactivity of chemokines, expression of proteolytically less active isoforms of CD26/DPPIV makes sense. This is in accordance with the finding that CD26/DPPIV expression on Th2 cells is lower than on Th1 cells, whereas DPPIVactivity on Th1 cells and Th2 cells is similar (Boonacker et al., 2002). Moreover, adult and neonate T cell populations differ in their DPPIV activity with higher activity on neonatal cells. The shaping of the T cell repertoire is more dynamic and faster during the neonatal period and has been linked with a difference in CD26/DPPIV expression (Vissinga et al., 1987).

Consequences of abnormal expression of CD26/DPPIV Many diseases are related with altered plasma levels of sCD26/ DPPIV (Table VI).

CD26/DPPIV and cancer Malignant cells often show altered CD26/DPPIV expression or even do not express CD26/DPPIV (Wesley et al., 1999). For example, loss of CD26/DPPIV occurs during malignant transformation of melanocytes when the cells become independent of exogenous growth factors for survival (Albino et al., 1992; Morrison et al., 1993). Tetracycline-inducible expression of DPPIV/CD26 in malignant human melanoma cells that were transfected with the CD26/DPPIV gene induced a profound switch of phenotype of the cells to nonmalignancy. CD26/ DPPIV re-expression led to inhibition of tumorgenicity and anchorage-independent growth, reversal of the block in differentiation, and a re-acquired dependence on exogenous growth factors for survival. Suppression of tumorgenicity and reversal of the block in differentiation were dependent on DPPIV activity (Wesley et al., 1999). However, other studies using melanoma cells expressing mutant CD26/DPPIV lacking either extracellular serine protease activity or the six amino acid long cytoplasmic tail demonstrated that DPPIV activity and the cytoplasmic tail are not required for decreased metastatic potential (Pethiyagoda et al., 2000). Surprisingly, dependence on exogenous growth factors was not related with DPPIV activity. Re-expression of either wild-type CD26/ DPPIV or mutant CD26(DPPIV ) rescued expression of a second cell surface serine protease, FAP-a, which can form a heterodimer with CD26/DPPIV. This observation suggests that FAP-a also plays a role in regulating growth of melanocytes (Wesley et al., 1999) and demonstrates the complex roles that these multifunctional proteases play. Downregulation of CD26/ DPPIV seems to play an important role in the early development of melanomas. Because CD26/DPPIV can inactivate

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circulating growth hormone-releasing factor (GHRF) (Frohman et al., 1989), downregulation of CD26/DPPIV may facilitate tumor growth by prolonging the presence of GHRF in the circulation, resulting in higher levels of the growth hormone. Differentiation of gliomas is related with CD26/DPPIV activity and its subcellular distribution pattern. Low activity is found in poorly differentiated gliomas and vice versa. These differences may be related with changes in pI-isoforms of CD26/DPPIV. When comparing differentiated slowly proliferating glioma cells with poorly differentiated rapidly proliferating glioma cells, DPPIVactivity of a number of pI-isoforms was distinctly higher in differentiated glioma cells. The higher DPPIV activity in differentiated glioma cells was particularly related with membrane-associated isoforms (Sedo et al., 1998). Stromal cells in benign prostate tumors express different cellsurface peptidases than normal prostatic stromal cells and epithelial cells. For example, expression of aminopeptidase A (APA) is increased in tumor stroma, whereas aminopeptidase N (APN) is absent in most cancer cells (Bogenrieder et al., 1997). CD26/DPPIV levels are also decreased in metastatic prostate tumors. Binding of CD26/DPPIV to fibronectin and collagen may alter an aggressive or metastatic phenotype to a more quiescent phenotype. Along this line, Cheng et al. (1998) have demonstrated that adhesion of CD26/DPPIV to fibronectin is involved in metastasis of breast cancer in lung. Remarkably, CD26/DPPIV was expressed on lung endothelial cells whereas fibronectin was present on the surface of the metastatic cancer cells. Attachment of circulating cancer cells to endothelial cells via adhesion molecules is considered to be responsible for organspecific metastasis. Rat R3230AC-MET cancer cells and RPC-2 prostate cancer cells that metastasize to the lung bind membrane vesicles isolated from the lung vasculature. In contrast, vesicles that are derived from the vasculature of organs to which these cancer cells do not metastasize, do not bind. Purification of the endothelial cell adhesion molecule from rat lung extracts and the use of a mAb that inhibits selective adhesion revealed that the protein was CD26/DPPIV (Johnson et al., 1993). CD26/DPPIV has been studied in T-acute lymphoblastic leukemia (T-ALL). Noteworthy is the discrepancy between the high expression of CD26/DPPIV but moderate DPPIV activity on T lymphoblasts in the majority of T-ALL cases (Klobusicka and Babusikova, 1999a, b). Degradation of extracellular matrix by proteinases has been implicated during invasion and metastasis (Werb, 1997; Van Noorden et al., 1998). Interactions between plasminogen and CD26/DPPIV have been reported to initiate signal transduction that regulates expression of MMP-9 in prostate cancer cells (Gonzalez-Gronow et al., 2001). Therefore, the role of CD26/ DPPIV does not seem to be unequivocal in carcinogenesis. On the one hand, CD26/DPPIV expression in cancer cells seems to downregulate cancer progression because it induces differentiation. On the other hand, CD26/DPPIVactivity seems to play a direct role in invasion and metastasis as well.

CD26/DPPIV and autoimmune diseases In T cell-mediated experimental autoimmune encephalomyelitis (EAE), a critical role for CD26/DPPIV in the modulation of effector functions of CD4‡ T lymphocytes has been demonstrated (Natarajan and Bright, 2002). Signs of EAE were partially suppressed by in vivo administration of the specific CD26/DPPIV inhibitor I40 both in a preventive way and in a

therapeutic way (Steinbrecher et al., 2001). The protective mechanism of CD26/DPPIV inhibition can be explained by its modulation of T cell effector function. The CD26/DPPIV inhibitors I40 and I49 also suppressed secretion of TNF-a and to a lesser extent that of IFN-g, cytokines that upregulate the immune response. Earlier reports show that T cell proliferation and secretion of IL-2, IL-6, and IL-10 in mouse spleen and thymocytes is suppressed by in vivo administration of CD26/ DPPIV inhibitors (Reinhold et al., 1997a). Likewise, proliferation and secretion of various cytokines including TNF-a and IFN-g were suppressed in human T cells (Reinhold et al., 1997b, 1998). These findings are in line with the fact that EAE is mediated by CD4‡ Th1 cells which typically secrete TNF-a, IFN-g and lymphotoxin. Moreover, it was found that the immunosuppressive TGF-b1 cytokine was upregulated in spinal cord tissue and plasma of mice that were treated with the DPPIV inhibitor I40 (Reinhold et al., 1997a, b; Steinbrecher et al., 2001). Expression of CD26 molecules on the surface of T cells from peripheral blood and cerebrospinal fluid of patients with progressive multiple sclerosis (Hafler et al., 1985) and Graves× disease is elevated (Eguchi et al., 1989). In conclusion, it can be stated that autoimmune diseases are related with elevated CD26/DPPIV expression on T cells.

CD26/DPPIV and AIDS CD26/DPPIV on human T cells consists of a set of isoforms as has been demonstrated by isoelectric focussing studies (Torimoto et al., 1992; Ansorge et al., 1995; Mattern et al., 1995; Kahne et al., 1996; Smith et al., 1998). DPPIV activity is found only in basic isoforms of CD26/DPPIV, but not in acidic isoforms. A shift to acidic isoforms has been observed during HIV infection (Smith et al., 1998). CD4‡ cells of patients infected with HIV have an intrinsic defect in their ability to recognize and respond to antigens before a reduction of the total number of CD4‡ cells occurs in these patients (Lane et al., 1985; Van Noesel et al., 1990; Schnittman et al., 1990). The memory function to recall antigens is a property of CD4‡ T cells expressing CD26/DPPIV. It is the only type of T helper cell that is known to proliferate in response to soluble antigens. They also activate both MHC-restricted cytotoxic T cells to kill virallyinfected target cells and B cells to secrete immunoglobulins (Morimoto et al., 1989). In this respect, it is striking that there is a selective decrease in CD26/DPPIV-positive T cells in HIV-1infected individuals prior to a general reduction in the number of CD4‡ cells (Blazquez et al., 1992). These findings indicate the importance of the immuno-modulating role of CD26/DPPIV which is supported by the finding that antigen response in HIV1-infected individuals can be restored by the addition of sCD26/ DPPIV in vitro (Schmitz et al., 1996). Furthermore, it has been shown that DPPIV protease activity of plasma sCD26/DPPIV was both decreased in HIV-1-infected individuals and inversely correlated with HIV-1 RNA levels in cells. In contrast, cleavage of SDF-1 by CD26/DPPIV reduces its anti-HIV activity and thus the presence of CD26/DPPIV may facilitate entry of the virus into cells (Ohtsuki et al., 1998; Proost et al., 1998c). Antigen-specific memory CD4‡ T cells infected with HIV-1 showed significantly higher CXCR4 and HIV-1 expression in Th0/2-oriented responses (with low CD26/ DPPIVexpression) in comparison with Th1-oriented responses (with high CD26/DPPIV expression). Similarly, in naive CD4‡ T cells activated in the presence of IL-4 or IL-12 and infected with the same HIV-1 virus, IL-4 upregulated CXCR4 and HIV1 expression whereas IL-12 downregulated both (Fig. 3). The

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downregulatory effect of IL-12 on CXCR4 expression was found to be dependent on its capacity to induce IFN-g production. These observations can account for the higher risk of progression of HIV-related diseases in HIV-1-infected individuals undergoing a Th0/2-oriented immune response with low levels of CD26/DPPIV (Galli et al., 1998). Treatment with neuraminidase significantly reduced the amount of acidic isoforms of CD26/DPPIV on T cells of HIVinfected individuals and re-established a more basic pattern that resembled the pattern of non-HIV-infected individuals (Smith et al., 1998). Generally, sialic acids stabilize plasma membranes by their negative charge, but also serve as an immunobarrier, for instance between mother and fetus, thus blocking the formation of antibodies against fetal tissues (Schauer, 1988). Sialylation has regulating and modifying effects in cellular processes by masking recognition sites on molecules and inhibiting their receptor functions (Palabrica et al., 1992). Smith et al. (1998) suggested an allosteric-like mechanism in which highly charged basic short-chain peptides which are present on HIV particles bind noncovalently to sialic acids of the CD26/DPPIV glycoprotein, resulting in inhibition of its CD26/DPPIV activity by partly blocking access of substrates to the active site. Possible pathological effects of hypersialylation of CD26/ DPPIV are the following: (1) modification of the modality of CD26/DPPIV as an adhesion molecule, (2) impairment of CD26/DPPIV as receptor to respond to signals for cell activation, (3) alterations in the affinity of DPPIV for its natural substrates, and (4) conformational changes of CD26/ DPPIV that may directly or indirectly lead to premature apoptosis. Concentrated domains of sialic acids linked to CD26/ DPPIV by extended N-linked oligosaccharides as occurs for example during aging and HIV-1 infection (Smith et al., 1998) are more effective as a negative charge than limited domains of negatively-charged amino acids in the protein moiety for binding viral proteins or peptides. Similarly, it was shown that sialylation plays a key role in the apical targeting of a secreted CD26/DPPIV isoform. So, hypersialylation of CD26/DPPIV is both a natural and a pathological phenomenon. Increased sialylation occurs in principle with all proteins on the plasma membrane of cells during aging (Abdul-Salam et al., 2000). Thus a lower degree of sialylation is found in children as compared with adults. Therefore, it is striking that older HIVinfected individuals develop HIV-related diseases more rapidly than younger HIV-infected persons, and die more rapidly after infection (Fletcher et al., 1992; Adler et al., 1997). HIV-1infected cells from older individuals do not appear to be more susceptible to immune-mediated destruction, but the more rapid progression of HIV-related diseases appears to be due to the inability of older persons to replace functional T cells that have been destroyed. This is related to the protease activity and costimulatory function of CD26/DPPIV. Inhibition of CD26/ DPPIV protease activity by sialylation fits in this concept, since IL-2 processing that is necessary for proliferation of T cells is inhibited when CD26/DPPIV activity is blocked. Jurkat cells transfected with CD26(DPPIV ) are more susceptible to HIV1 infection than cells transfected with wild-type CD26/DPPIV (Morimoto et al., 1994), whereas replication of HIV-1 virus is reduced in CD26(DPPIV‡) transfectants as compared to wildtype Jurkat cells. Moreover, the costimulatory effect of CD26/ DPPIV can be downregulated by sialylation because interactions of CD26/DPPIV with other proteins (e.g. ADA and CD45) are reduced.

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Therapeutic potentials of CD26/ DPPIV targeting The exact picture of all biological functions of CD26/DPPIV is difficult to compose due to its moonlighting properties. However, evidence exists for therapeutic potentials of DPPIV inhibitors, especially because processing of biologically active peptides by DPPIV activity modulates their metabolism. Tables I, II and III list biologically active peptides that can be processed by CD26/DPPIV. GHRF is degraded by DPPIV and administration of a DPPIV inhibitor together with GHRF may be useful to treat children with growth hormone deficiency to prolong the availability of GHRF (Bongers et al., 1992). DPPIV is involved in the degradation of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) in vitro and in vivo. GIP and GLP-1 are considered to be the most important insulin-releasing hormones that are part of the enteroinsular axis. The term enteroinsular axis refers to the signaling pathway between gut and pancreatic islets that amplify the insulin response to induce absorbance of glucose. N-terminal truncation by DPPIV abolishes insulinotropic activity of GIP and GLP-1 and this hydrolysis is the primary mechanism of their inactivation in vivo. Inhibition of DPPIV with orally administered Ile-thiazolidine enhances insulin secretion and improves glucose tolerance in response to an oral challenge in lean and obese Zucker rats. These rats exhibit characteristics of non-insulin-dependent diabetes mellitus (Pederson et al., 1998). This phenomenon was attributed to a block in the inactivation of GIP and GLP-1 by DPPIV, resulting in signal amplification of the enteroinsular axis. Therefore, it is believed that inhibitors of DPPIValone or in combination with GLP-1 can be used to lower glucose levels in diabetes (Drucker, 1998). Likewise, GLP-2 can also be inactivated by DPPIV. GLP-2 displays intestinal growth factor activity in rodents. Administration of a modified form of GLP-2, [Gly]2-GLP-2, which is resistant to DPPIV hydrolysis in mice, increases their small bowel weight, predominantly due to a significant increase in villous height (Brubaker et al., 1997; Drucker et al., 1997a, b). Therefore, DPPIV inhibitors may be useful to increase the intestinotropic properties of GLP-2 on mucosal regeneration in patients with intestinal disease (Drucker, 1998). Binding of plasminogen to CD26/DPPIV initiates a signal transduction mechanism which regulates expression of MMP-9 by prostate cancer cells (Gonzalez-Gronow et al., 2001). Therefore, inhibitors of DPPIV may block metastasis of cancer. Alkyldiamine-induced arthritis in rats, a model that shares several pathological features with rheumatoid arthritis, was suppressed by a series of DPPIV inhibitors in a dose-dependent manner (Tanaka et al., 1997). T cells with high levels of CD26/ DPPIV preferentially migrate into the rheumatoid synovium to induce inflammation and tissue destruction (Mizokami et al., 1996). Again, DPPIV inhibitors may be useful as immunosuppressants for the treatment of autoimmune diseases such as rheumatoid arthritis and for the prevention of rejection of transplants, as DPPIV can alter the balance of chemokine activity towards stimulation and attraction of Th1 cells. CD26/ DPPIV is preferentially expressed on Th1 cells and cleaves RANTES, eotaxin, MDC, SDF-1a, and SDF-1b. The cleavage products of these chemokines trigger Th1-specific but not Th2-

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specific chemokine receptors (Oravecz et al., 1997; Proost et al., 1998a, 1999; Shioda et al., 1998; Struyf et al., 1998, 1999). In general, it can be concluded that DPPIV inhibitors can be used as therapeutic agents in cases when bioactive molecules are processed at a too high rate by DPPIV, or when these molecules are available at lower amounts than necessary. Therefore, DPPIV inhibitors can be used to prolong the availability of bioactive molecules. On the other hand, synthesis of DPPIV-resistant analogues of bioactive molecules can also be a strategy for therapy. In the therapeutic strategies mentioned above, DPPIV activity is inhibited, but stimulation of DPPIV activity may also be used in therapy. For instance, treatment with sCD26/ DPPIV may help to restore the antigen response of, for example, HIV-1-infected individuals and to delay progression of HIV-1-related diseases (Hosono et al., 1999). However, one has to realize that processing of regulatory peptides is not accomplished by CD26/DPPIV alone, and like in many other biological systems alternative routes exist which may limit the use of DPPIV inhibitors or DPPIV-resistant analogues of bioactive peptides. Although DPPIV inhibitors have been shown to modulate numerous cell functions, the existence of CD26/DPPIV homologues (Table V) that lack DPPIV activity but still are biologically active clearly indicates that DPPIV activity is not crucial in all functions of CD26/DPPIV and/or structurehomologues (DASH). Moreover, enzymatically active members of the DASH family probably execute some of their functions independently of their proteolytic activity as well (Hegen et al., 1993; Steeg et al., 1995). Although a number of these DASH proteins have been identified, functional roles of the individual proteins are not known yet. Their broad distribution patterns, subcellular localization patterns and substrate preferences argue for specific physiological regulation mechanisms and functions of each particular family member. On the other hand, co-expression of CD26/DPPIV and DPPIV-b (Jacotot et al., 1996), CD26/DPPIVand attractin (DPPT-L) (Duke-Cohan et al., 1996), CD26/DPPIVand DPPII (Hagihara et al., 1987), and CD26/DPPIV and FAP-a (Wesley et al., 1999) are evidence for their cooperation. Therefore, it should be realized that in the case of specific inhibition of CD26/DPPIV activity functional substitution may interfere with therapy. Such phenomena could be the reason why Japanese Fischer 334 rats are displaying only pathologies of the digestive apparatus, despite a complete lack of CD26/ DPPIV expression (Tiruppathi et al., 1993). Moreover, the existence of possible DASH crosstalk is indirectly supported by the upregulation of endogenous FAP-a by recombinant expression of CD26/DPPIV in melanoma cells (Wesley et al., 1999). In conclusion, there is growing evidence suggesting that the contextual repertoire of DASH proteins, with partially overlapping functional and molecular characteristics, seems to be an important dynamically titrated feature of the cell phenotype. Eventually, from a functional point of view, an individual DASH molecule itself does not need to be highly specific, it depends on the microenvironment of the molecule what function and what specificity are exerted. In other words, specificity of the proteins can be provided by cells and substrates in the immediate environment (Monsky et al., 1994; Duke-Cohan et al., 1996; Jacotot et al., 1996; Chiravuri et al., 1999; Mentlein, 1999). Most of the molecules belonging to this group fulfill multiple functions relying on their moon-

lighting properties and thus on the site and context of their expression in living cells.

Conclusions The role of CD26/DPPIV in many processes seems logical. Expression of CD26/DPPIV in renal, intestinal and liver epithelial cells is mainly involved in the degradation/catabolism of proteins, but its functional role in the immune response is still largely unknown. To elucidate the function of CD26/DPPIV in the immune system, it is necessary to find a good model in which it is possible to monitor this extraordinary protein. Especially, a model is desired in which it is possible to study redistribution and oligosaccharide-reprocessing of CD26/DPPIV during T cell activation and subsequent interactions with other molecules to help to elucidate the functions of CD26/DPPIV in the immune response. High numbers of CD26/DPPIVpositive cells in inflamed tissues are related with autoimmune diseases (Hafler et al., 1985; Nakao et al., 1989) whereas low numbers of CD26/DPPIV-positive cells indicate immunodeficiency. However, the main function(s) of CD26/DPPIV on T cells still remain(s) to be elucidated. Therefore, a better understanding of the functions of the various isoforms of the multifunctional CD26/DPPIV protein and other proline-specific peptidases in the specific cellular compartments and in extracellular fluids as sCD26/DPPIV is necessary. Acknowledgements. The careful preparation of the manuscript by Ms. T. M. S. Pierik and the figures by Mr. J. Peeterse are gratefully acknowledged.

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