Dipeptidyl-peptidase IV (CD26)-role in the inactivation of regulatory peptides

Regulatory Peptides 85 (1999) 9–24 www.elsevier.com / locate / regpep

Review

Dipeptidyl-peptidase IV (CD26)-role in the inactivation of regulatory peptides Rolf Mentlein* ¨ Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany Anatomisches Institut der Universitat Received 12 July 1999

Abstract Dipeptidyl-peptidase IV (DPP IV/ CD26) has a dual function as a regulatory protease and as a binding protein. Its role in the inactivation of bioactive peptides was recognized 20 years ago due to its unique ability to liberate Xaa–Pro or Xaa–Ala dipeptides from the N-terminus of regulatory peptides, but further examples are now emerging from in vitro and vivo experiments. Despite the minimal N-terminal truncation by DPP IV, many mammalian regulatory peptides are inactivated — either totally or only differentially — for certain receptor subtypes. Important DPP IV substrates include neuropeptides like neuropeptide Y or endomorphin, circulating peptide hormones like peptide YY, growth hormone-releasing hormone, glucagon-like peptides(GLP)-1 and -2, gastric inhibitory polypeptide as well as paracrine chemokines like RANTES (regulated on activation normal T cell expressed and secreted), stromal cell-derived factor, eotaxin and macrophage-derived chemokine. Based on these findings the potential clinical uses of selective DPP IV inhibitors or DPP IV-resistant analogues, especially for the insulinotropic hormone GLP-1, have been tested to enhance insulin secretion and to improve glucose tolerance in diabetic animals. Thus, DPP IV appears to be a major physiological regulator for some regulatory peptides, neuropeptides, circulating hormones and chemokines.  1999 Elsevier Science B.V. All rights reserved. Keywords: Dipeptidyl-peptidase IV (EC 3.4.14.5); Neuropeptides; Hormones; Chemokines; Glucagon-like peptide-1; Growth hormone-releasing hormone

1. Discovery and classification of DPP IV The enzyme and binding protein dipeptidyl-peptidase IV (DPP IV, EC 3.4.14.5; CD26) was discovered by HopsuHavu and Glenner [40] in rat liver homogenates and commercial enzyme preparations as an activity liberating naphthylamine from Gly–Pro-2-naphthylamide, and initially termed glycylproline naphthylamidase. Since the amino Abbreviations: DPP IV, dipeptidyl-peptidase IV; GCP-2, granulocyte chemotactic protein-2; GIP, gastric inhibitory polypeptide or glucosedependent insulinotropic polypeptide; GLP, glucagon-like peptide; GRH, growth hormone-releasing hormone; HIV, human immunodeficiency virus; IP, interferon-g-inducible protein; MDC, macrophage-derived chemokine; MCP, monocyte chemotactic protein; NPY, neuropeptide Y; PHM, peptide histidine methionine; PYY, peptide YY; RANTES, regulated on activation normal T cell expressed and secreted; SDF, stromal cell-derived factor *Tel.: 149-431-880-2460; fax: 149-431-880-1557. E-mail address: [email protected] (R. Mentlein)

acid sequence Gly–Pro is frequently found in collagens, a possible metabolic significance in collagen metabolism was proposed. However, the enzyme is unable to cleave Pro–Pro or Pro–Hyp bonds which mostly follow the Gly– Pro sequence in collagens, and therefore the physiological functions of DPP IV remained obscure for many years. Nevertheless, its properties and distribution were thoroughly investigated, and the protein was rediscovered as a cellular marker and a binding protein several times. This review will focus on the role of DPP IV in the inactivation of bioactive peptides in mammals, neuropeptides, circulating hormones, cytokines and chemokines — a possible function that was recognized already 20 years ago due to the unique specificity of the peptidase [34,45]. Peptide bonds involving the cyclic amino acid proline do not only influence the conformation of peptide chains, but also restrict the attack by most proteases, even those with broad specificity [61,104,111]. Especially, sequences with proline in the N- or C-terminal penultimate position

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cannot be cleared by non-specialized exopeptidases. However, a number of peptidases exist which specifically or selectively attack proline bonds (Table 1). Mostly, these peptidases are not only involved in the cleavage of X–Pro or Pro–X bonds, but also in the degradation of corresponding alanyl bonds, although with reduced activity. For the inactivation of extracellular endocrine, paracrine or autocrine peptides only cell-surface ectoenzymes or secreted enzymes are of physiological importance. Among the proline / alanine-specific peptidases these are DPP IV (liberating N-terminal Xaa–Pro dipeptides), X–Pro aminopeptidase (liberating N-terminal amino acids from Xaa– Pro peptides), and membrane Pro–X–carboxypeptidase (liberating C-terminal residues from Pro–Xaa peptides). All these three peptidases are cell-surface ectoenzymes, but they are differently anchored in the plasma membrane. Moreover, in the case of DPP IV and X–Pro aminopeptidase (aminopeptidase P) soluble forms exist in body fluids which are either regarded as solubilized forms of the membrane counterparts or as specially coded soluble variants. The other prolyl- / alanyl-peptidases listed in Table 1 are either lysosomal or cytosolic enzymes and consequently thought to be involved mainly in intracellular protein turnover.

2. Distribution A physiological role in the inactivation of bioactive peptides has been postulated for all membrane-bound proline- / alanine-specific exopeptidases [66,104], but that of DPP IV has been investigated and documented best. In contrast to X–Pro aminopeptidase and Pro–X carboxypeptidase which showed a restricted distribution most vertebrate tissues contain DPP IV, but their activities vary widely (Fig. 1). In the kidney, where the enzyme is exceptionally concentrated, it is located primarily in the cortex and found in the brush-border and microvillus fractions. It can be immunostained in the glomeruli on podocytes in the region of the glomerular basement membrane and on proximal convoluted tubules [48]. Also, brush-border membranes of enterocytes in the intestine and microvilli of trophoblasts in the placenta are plentiful sources of DPP IV (for review [4,47,60]). In the liver, DPP IV is primarily located on hepatocytes at the plasma membranes around bile caniculi and on the bile duct epithelia. Also, on epithelial cells of the pancreatic duct DPP IV can be stained [36]. Thus, body compartments / fluids involved in nutrition and excretion (lumen of the intestine, bile, pancreatic fluid, urine) are

Table 1 Proline / alanine-specific mammalian endo- and exopeptidases a Protease

DPP IV EC 3.4.14.5 c DPP II EC 3.4.14.2 X–Pro aminopeptidase EC 3.4.11.9 Prolyl aminopeptidase EC 3.4.11.5 / 1 d Prolyl oligopeptidase EC 3.4.21.26 Membrane Pro–X carboxypeptidase EC 3.4.17.16 Lysosomal prolyl carboxypeptidase EC 3.4.16.2 X –Pro dipeptidase EC 3.4.13.9 Pro–X dipeptidase EC 3.4.13.8

Requirement of the cleaved peptides

Mr

Catalytic properties b

Cleavage site --

Residues accepted at j h

Chain length (residues)

Native

Subunit

Inhibitors

s–j- -h–

P,A

±P

3–80

220 000

110 000

s–j- -h–

A,P

Many

3–11

110 000

53 000

h- -j–s–

P

Many

3–40

270 000

90 000

j- -h–

Many

Many

2–30

300 000

54 000

–j- -h–

P, A

±P

4–30

81 000

81 000

–j- -h

P,A,G

±P

2–7

240 000

135 000

Dip-F Diprotin A Dip-F K–A–CH 2 Cl Phen Apstatin Phen Amastatin Dip-F Z–P-prolinal Phen

–j- -h

P

±P

>3

200 000

25 000

h- -j

P,Hyp

±P

2

108 000

54 000

j- -h

P,A

±P

2

300 000

Membrane m

pH Optimum

Soluble s

8.0

m (s)

4.5–6.0

s (m)

7–8

m (s)

7–8.5

s

7.5–8

s

8

m

Dip-F

4.5–5.5

s

EDTA Z-Pro Phen

7.5

s

8–9

s

a For details of the nomenclature see Enzyme Nomenclature available on the World Wide Web (http: / / www.chem.qmw.ac.uk / iubmb / enzyme / E34 / ), for details of sequences and structural relationships to other peptidases the MEROPS (http: / / www.bi.bbsrc.ac.uk / Merops / merops.htm) or PROSITE databases (http: / / www.expasy.ch / enzyme / ), and for information on the catalytic and molecular properties in links therein. b First line lists an example of an inhibitor selective for the enzyme class: Dip-F, diisopropyl fluorophosphate for serine-type peptidases; Phen, 1,10-phenanthroline, or EDTA for metallopeptidases. The second line lists a more specific inhibitor for the peptidase. c bacterial Xaa–Pro dipepeptidyl peptidase EC 3.4.14.11 catalyzes a similar reaction as the DPP IV EC 3.4.14.5 from animals, but belongs to another serine peptidase family. d Mammalian prolyl aminopeptidase is identical to EC 3.4.11.1 leucyl aminopeptidase [101].

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neuropeptides mainly in the cerebrospinal fluid. In the central nervous system DPP IV is found primarily in the circumventricular organs and on leptomeningeal cells [69]. Moreover, brain capillaries and some ependymal cells are strongly immunopositive for DPP IV [7]. However, in the embryonic brain DPP IV is abundantly present on neuronal cells [7]. In the peripheral nervous system DPP IV is located at the perineurium and Schwann cells [33]. Thus, DPP IV can be found at many epithelial and certain specialized mesodermal cells. The peptidase is often present at sites of physiological barriers (e.g. blood– brain barrier). Its location allows it to act on bioactive peptides in body fluids or communicating between immune cells.

3. Molecular and catalytic properties

Fig. 1. DPP IV activity in different rat tissues and purified or cultivated rat or human (h) cells. Activity was measured spectrophotometrically with Gly–Pro-4-nitroanilide as substrate (0.9 mM at pH 7.6 and 378C with tissues, or 0.5 mM at pH 8.5 and 378C with cells or serum) and related to g wet tissue / ml serum or to the number of cells determined by DNA quantification. Cultivated rat vascular smooth muscle cells, neurons, astroglial and oligodendroglial cells are devoid of DPP IV activity [64,65]. Please note that also a portion of DPP II activity (pH optimum 5.5) may contribute to the values in homogenates. Data for Wistar rat tissues from [53], for endothelial cells cultivated from human umbilical veins from [61], and for purified human peripheral lymphocytes from [67]; data for purified rat thymocytes and cultivated rat microglia cells are unpublished.

exposed to DPP IV. Therefore, in these tissues a simple digestive function of DPP IV in the final degradation of peptides produced by other endo- and exo-peptidases from nutritional proteins and their absorption has to be taken into account [12,35]. However, DPP IV is also in close contact with hormones circulating in the blood, since it is located on endothelial cells of the blood vessels [55] and, moreover, found as a soluble enzyme in the blood plasma. Among cells of the immune system DPP IV is expressed on activated T-helper lymphocytes [64] and subsets of macrophages [42]. In endocrine organs, DPP IV is strongly expressed at the capillary epithelia, but rarely in parenchymal cells except e.g. follicular epithelial cells of the thyroid gland or luteal cells [27,91]. In several tissues DPP IV is expressed in specialized fibroblasts, e.g. in the mammary gland [3], in the skin [89], or in the synovia [5]. In the adult nervous system, DPP IV has contact with

Human DPP IV solubilized from membranes by detergents is a glycoprotein with Mr of about 240 000 composed of two 120 000 subunits (e.g. the human placenta enzyme [87]). Due to the presence of sialic acids in the carbohydrate structure, DPP IV has an acidic isoelectric point. The cDNA codes for a polypeptide of 766 (rat 767) residues [70]. DPP IV is anchored in the plasma membrane by a 22 amino residues hydrophobic membranespanning domain (VLLG LLGAAALVTI ITVPVVLL) preceded by a short, intracellular hydrophilic sequence (MKTPWK) at the N-terminus (Fig. 2) and therefore classified as a type II integral membrane protein [39]. The remaining sequence of 738 residues contains nine potential N-linked glycosylation sites and is supposed to be located extracellularly. The rat sequence exhibits about 85% identity to the human sequence, lacks one residue and one glycosylation site [70]. The C-terminal regions (residues 625–752) of the human and rat sequences are completely conserved. The human DPP IV 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 b to 1.4 kb [1]. The 59-flanking domain contains neither a TATA box nor a CAAT box, but a 300-bp region extremely rich in C and G (72%) has potential binding sites for several transcription factors, such as NFkB, AP2 or Sp1 [1,8]. The DPP IV gene encodes two mRNAs sized about 4.2 and 2.8 kb which are expressed at different relative rates in individual tissues. DPP IV belongs to the serine class of proteases. A catalytic triad of serine, aspartic acid and histidine is found at the extracellular, C-terminal part of the molecule (Ser 631 , Asp 709 , His 741 in the mouse sequence [14]). Since the arrangement of the residues within this triade is inverted to the trypsin-like serine-proteases (His–Asp– Ser), DPP IV has been classified as a non-classical serine protease. The active site sequence GWSYG around the ] catalytic serine residue [77] corresponds to the consensus sequence GXSXG of serine proteases and is found in one ]

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R. Mentlein / Regulatory Peptides 85 (1999) 9 – 24

[60,87]; even E600 (diethyl-4-nitrophenyl phosphate, an inhibitor for serine esterases) is a covalent, slowly reacting inhibitor [87]. Metal ions such as Pb 21 , Hg 21 and Zn 21 are strongly inhibitory. Inhibitors of other classes of proteases such as EDTA, 1,10-phenanthroline (inhibitors of metalloproteases), N-ethylmaleimide, leupeptin (inhibitors for cysteine enzymes) or bestatin (inhibitor for many aminopeptidases) are ineffective. Specific DPP IV inhibitors acting at low concentrations are of special interest for physiological investigations and for potential clinical applications. The microbial peptides diprotin A (Ile–Pro–Ile) and diprotin B (Val–Pro–Leu) are competitive substrates that are slowly hydrolyzed and act as selective competitive inhibitors for DPP IV at micromolar concentrations [88]. In addition, several types of substrate analogues have been synthesized that are powerful and specific inhibitors at mM to pM concentrations, e.g. proline boronic acid dipeptide inhibitors (reversible), dipeptide phosphonates (irreversible), dipeptide 2cyanopyrrolidides or aminoacyl-pyrrolidides (both reversible; [20] for review). Natural peptides with N-terminal Xaa–Xaa–Pro sequence are more or less strong inhibitors for DPP IV, e.g. PYY(3–36) or the Tat protein of HIV-1 [109]. Mammalian DPP IV exhibits a relatively restricted substrate specificity (Fig. 3). In the P1 position almost exclusively proline and alanine are accepted. However, in a study with growth hormone-releasing hormone (GRH) analogues, those with other P1 residues, e.g. Ser, Val, Gly, Leu, were also cleaved although with low rates [9,59], and Fig. 2. Main structural features of human DPP IV (CD26). DPP IV is a type II transmembrane protein with six N-terminal amino acids in the cytoplasm and an extracellular domain with a short flexible segment, sugar- and cysteine-rich regions and a C-terminal catalytic region with a putative, serine-protease type catalytic centre (sequence GWSYG). Serine 630 forms a catalytic triade with asparate 708 and histidine 740 .

copy at positions 628–632 (human sequence, rat sequence positions 629–633). Any single substitution in this sequence results in loss of enzymatic activity and reactivity with diisopropyl fluorophosphate that covalently reacts with the active serine residue. Interestingly, a substrain of Fisher-344 rats lacks DPP IV activity, but produces DPP IV mRNA [108]. A missense mutation of the DPP IV gene in the catalytic site, namely the substitution of glycine(633) to arginine (active site GWSYR), appears to be responsible for the loss of activity ] [28]. Due to this deficiency for active DPP IV, this Fisher344 rat strain has become a valuable model to study the physiological functions of the peptidase. In accordance with the catalytic structure, DPP IV is inhibited more or less powerfully by serine enzymes inhibitors that covalently modify their active site. Diisopropyl fluorophosphate is a good inhibitor, whereas phenylmethane sulfonylfluoride is relatively ineffective

Fig. 3. Schematic representation of substrates cleaved by DPP IV. Dipeptides are liberated from the N-terminus of peptides with Pro or Ala in the P1 -position. Certain peptides with other small amino acids in P1 position are cleaved at low rates. In the P2 position, bulky, hydrophobic or basic amino acids with an obligate free amino group are preferred. Peptides with Pro or Hyp in the P19 position are not cleaved by DPP IV. Preferential residues for the P19 position are not known.

R. Mentlein / Regulatory Peptides 85 (1999) 9 – 24

macrophage-derived chemokine (MDC) is an example for a natural peptide from which Gly–Pro and Tyr–Gly are sequentially released by DPP IV [86]. Peptides or chromogenic substrates with Pro are far better hydrolyzed than the corresponding ones with Ala. In the P2 position any unsubstitued proteinogenic amino acid is accepted (including proline). Substrates with hydrophobic or basic residues are better hydrolyzed than those with acidic ones. The peptide bond between the P1 and P2 residues must be in the trans-position. Peptides with Pro or Hyp in the P19 -position are resistant to DPP IV hydrolysis, e.g. bradykinin (Arg–Pro–Pro– . . . ). The chain length of the peptides cleaved has not been systematically investigated. Peptides up to 80 residues appear to be good substrates, sequestration of larger proteins may depend on their tertiary structures [74]. Optimal cleavage rates are generally observed at pH values between 7.5 and 8.5. The intense studies of the kinetic properties and substrate requirements of DPP IV in the eighties has facilitated the later development of DPP IV-resistant analogues of peptide hormones. Apart from this action as a strict exopeptidase, endopeptidase activities on collagen or other extracellular matrix proteins have also been postulated occasionally for DPP IV [6]. At present it cannot, however, be excluded that these endopeptidase activities result from proteases closely related to DPP IV. Human seprase (also termed fibroblast activation protein alpha), an integral membrane gelatinase, has 68% homology in primary structure to human DPP IV, the membrane insertion and the carboxy terminus with the catalytic residues are highly homologous [30]. Moreover, recently a type II membrane protein has been cloned (NAALADase L, N-acetylated alpha-linked acidic dipeptidase-like) that is highly expressed in ovary and testis as well as in discrete brain regions. It has dipeptidase activity towards the neuropeptide N-acetyl-Laspartyl– L-glutamate as well as DPP IV activity [80]. In addition, a 82 000 variant of DPP IV (-b) has been purified from a T-cell line [43].

4. Cleavage of regulatory peptides by DPP IV The unique substrate specificity of DPP IV together with its localization as an ectoenzyme at the plasma membrane led to the assumption that this protease should either take part in the final catabolism of proline-rich peptides or have a regulatory role in the inactivation of bioactive peptides. Whereas initially the potency of DPP IV to cleave potential neuropeptide-, peptide hormone- or cytokine/ chemokine- substrates was investigated, later studies elucidated the role of DPP IV in body fluids or in cellular systems in vitro, and finally in vivo, often with the use of specific inhibitors or animals lacking DPP IV activity. Nearly all natural and synthetic bioactive peptide with N-terminal penultimate proline or alanine and up to 80 residues proved to be good substrates for DPP IV (Table

13

2). With the purified human enzyme Km values between 5 and 60 mM have been determined for both, natural Xaa– Pro as well as natural Xaa–Ala peptides with 7–42 residues [9,62,63,74]. These values are a bit lower than those for many chromogenic 4-nitranilide or 2-naphthylamide substrates (Km about 20–200 mM [74]). However, in vivo regulatory peptides act in the pico- or nanomolar range, and micromolar concentrations are never reached. At these low concentrations of substrates S the reaction rates v of the enzymes E are given by v 5 [E][S] k cat /Km [25]. Therefore, the rate (specificity) constant k cat / Km is a better basis for comparing the potency of a peptidase towards different peptides at physiological concentrations. As shown in Fig. 4, high rate constants (corresponding to high cleavage rates at low concentrations) are especially found for NPY, PYY or GRH, but also those of the other peptides listed are in the range for natural peptide substrates of other physiological relevant peptidases like neutral endopeptidase, angiotensin-converting enzyme or aminopeptidase N. Proteolytic cleavage of a biologically active peptide, especially a short truncation at the N-terminus, does not necessarily result in a modification of its biological activity like total or partial inactivation, or contrary-activation, or alteration of its bioavailability (e.g. binding properties to matrix or other proteins). As far known mammalian DPP IV is mainly involved in inactivation reactions. Processing of precursors to active peptides (activation) has been observed in lower vertebrates or invertebrates. For example, DPP IV-like (or DPP II-like, compare [68]) enzymes are necessary to process the pro-form of a sex pheromone to the mature a-mating factor in yeast, preprohormones of sea anemones to mature neuropeptides, promelittin to the venom in the honey-bee, cecropins to the mature antibacterial peptides in moths, the precursor of an antifreeze protein of the winter flounder, and precursors of the prohormone for caerulein in the frog Xenopus laevis (reviewed in [50,58]). However, DPP IV-like enzymes are also necessary for the inactivation of neuropeptides / peptide hormones in coelenterates, nematodes, insects, or molluscs (reviewed in [58]). In all cases it should be noted that DPP IV in lower vertebrates and in invertebrates differ from the mammalian enzyme in molecular properties and, more important, in specificity by e.g. accepting larger peptides as substrates. In the following text some most important physiological actions of DPP IV on regulatory peptides (Table 2) in mammals are discussed in detail.

5. Action of DPP IV on neuropeptides Substance P is a widespread neuropeptide that acts, e.g. as a transmitter of sensory information including noxious stimuli, as a potent contractor of smooth muscles, and as an immunoregulator. Three different G-protein-coupled receptor subtypes, NK 1 , NK 2 and NK 3 , are known that are

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Table 2 Mammalian regulatory peptides as substrates for DPP IV b Peptide

N-terminus

Residues no.

Cleavage a

Significance

References

Xaa–Pro peptides Tyr–melanostatin Endomorphin-2 Enterostatin b-Casomorphin Trypsinogen pro-peptide Bradykinin Substance P Corticotropin-like intermediate lobe peptide Gastrin-releasing peptide Neuropeptide Y Peptide YY Aprotinin RANTES

YP–LG–NH 2 YP–FF– NH 2 VP–DP–R YP–F . . . FP–T . . . RP–P . . . RP–KP–Q . . . RP–V . . .

4 4 5 7 8 9 11 22

11 11 11 111 1 Not 11 1

Inactivation Inactivation Inactivation Inactivation Questionable – Questionable Probably none

[74] [94] [11] [74] [74] [45] [34,45] [74]

VP–LP–A . . . YP–S . . . YP–I . . . RP–D . . . SP–Y . . .

27 36 36 58 68

1 111 111 1 1

[74] [62] [62] [74] [79,84]

GCP-2 SDF-1a SDF-1b MDC MCP-1 MCP-2 MCP-3 Eotaxin IP-10 Insulin-like growth factor-I Pro-colipase Interleukin-2 Interleukin-1b a 1 -Microglobulin Prolactin Trypsinogen Chorionic gonadotropin

GP–V . . . KP–V . . . KP–V . . . GP–YG–A . . . QP–DA– . . . QP–DS . . . QP–VG . . . GP–A . . . VP–L . . . GP–E . . . VP–DP–R . . . (pig) AP–T . . . AP–V . . . GP–VP–T . . . TP–V . . . (sheep) FP–T . . . (pig) AP–D . . . (a-chain)

[84] [79,96] [96] [86] [84] [79] [84] [79,100] [79] Mentlein (unpublished) [74] [74] [37] [74] [74] [74] [74]

Xaa–Ala peptides PHM GRH-(1–29) GRH-(1–44) GLP-1 GLP-2 Gastric inhibitory peptide

HA–E . . . YA–D . . . YA–D . . . HA–E . . . HA–E . . . YA–E . . .

[63] [9] [63] [63] [23] [63]

73 68 72 69 76 76 73 74 77 70 101 133 153 168 198 231 243

1 1 1 1 Not 1 Not 1 1 Not 1 Not Not Not 1 1 1

Probably none Inactivation at Y1-receptor Inactivation at Y1-receptor Probably none Inactivation at CCR1-, CCR-3 receptors, but not at CCR5 Probably none Inactivation at CXCR4-receptor Inactivation at CXCR4-receptor Inactivationn at CCR4-receptor – Inactivation – Inactivation at CCR3 receptor Unknown – Questionable – – – Probably none Probably none Probably none

27 29 44 30 34 42

11 11 11 11 11 11

Inactivation Inactivation Inactivation Inactivation Inactivation Inactivation

a

1, 1 1, 1 1 1 denote relative cleavage rates as concluded from the literature (not available for chemokines). Abbreviations: GCP-2, granulocyte chemotactic protein-2; GLP, glucagon-like peptide; GRH, growth hormone-releasing hormone, IP, interferon-ginducible protein; MDC, macrophage-derived chemokine; MCP, monocyte chemotactic protein; PHM, peptide histidine methionine; RANTES, regulated on activation normal T cell expressed and secreted; SDF, stromal cell-derived factor. b

stimulated also by the neurokinins A and B which share a common C-terminal pentapeptide sequence (Phe–Xaa– Gly–Leu–Met–NH 2 ) with substance P. Therefore, the sequential liberation of the dipeptides Arg–Pro and Lys– Pro from the N-terminus of the undecapeptide substance P [34,45] does not abolish its biological activity at the tachykinin receptors. However, the fragment substance P-(5–11) has a lower binding activity for the preferred substance P receptor, the NK 1 receptor [73]. Only effects for which the first four residues in substance P are essential [73], e.g. the degranulation of mast cells (probably non-

receptor-mediated by pertubation of the membrane), may be completely abolished by DPP IV cleavage. Substance P truncated by DPP IV can be further attacked by aminopeptidases. This sequential sequestration by DPP IV and subsequently by aminopeptidase N (EC 3.4.11.2, CD13) is the most important degradation pathway in human plasma, at the vascular endothelium, at fibroblasts and muscle myocytes [2,90,106,107]. However, many other potent substance P-degrading endopeptidases are known, especially the neutral endopeptidase (neprilysin, EC 3.4.24.11, CD10) [46].

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Fig. 4. Rate (specificity) constants k cat /Km for the cleavage of regulatory peptides by DPP IV purified from human placenta reflecting the relative potency to hydrolyze them at physiological (nanomolar) concentrations. Data taken from [9,62,63].

Bradykinin (R-PP-GFSPFR) is another example for regulatory peptide where DPP IV plays an additional role in the inactivation. Due to the Pro(3), it is not attacked initially by DPP IV, but after liberation of the N-terminal arginine by X–Pro aminopeptidase the following Pro–Pro dipeptide is released by DPP IV and the remaining peptide becomes susceptible to degradation by aminopeptidase N, which is an important degradation pathway in the perfused lung [83]. Also here, other neuropeptidases, especially neutral endopeptidase or angiotensin-converting enzyme are more generalized bradykinin-degrading enzymes [46]. Endomorphin-1 (YPWF-NH 2 ) and -2 (YPFF-NH 2 ) are endogenous opioid peptides with high affinity at m opioid receptors that produce potent analgesia in mice. DPP IV liberated the terminal Tyr–Pro with high activity from endomorphin-2 whereas an analogue with D–Pro in the penultimate position was resistant. This analogue was more potent and longer lasting to induce analgesia in vivo, moreover the DPP IV inhibitor Ala–pyrrolidonyl-2-nitrile itself elicited analgesia and potentiated the actions of endomorphin-2 [94]. These results suggests a role of DPP IV in endomorphin inactivation in vivo. b-Casomorphin, an opioid peptide produced by proteolytic digestion of b-casein from milk, is also an excellent substrate for DPP IV [74]. NPY and PYY are 36-amino acid regulatory peptides that share — together with pancreatic polypeptide — a high homology in primary and tertiary structure. NPY is an abundant neuropeptide in the central and peripheral nervous system; it is involved in the control of feeding, energy homeostatis and blood pressure. PYY is produced by endocrine cells of the small intestine and the colon, and released after a meal as a peptide hormone circulating in

the blood that inhibits several gastrointestinal functions, e.g. gastric acid release. The amino terminal sequence Tyr–Pro is conserved in NPY and PYY among all vertebrate species (as far as is known). A proline in the penultimate position is also found in pancreatic polypeptide (human sequence Ala–Pro–Leu– . . . ). NPY and PYY appear to be the best peptide substrates for DPP IV (Fig. 4) whereas pancreatic polypeptide is hardly cleaved [62]. All three peptides are resistant to other aminopeptidases except X–Pro aminopeptidase that liberates their N-terminal amino acid with considerably lower activity than DPP IV [62]. The potency of endopeptidases to cleave NPY or PYY is relatively low [57]. NPY, PYY and pancreatic polypeptide bind with different affinities to at least five receptor subtypes: the Y1 receptor has high affinities for full length NPY and PYY, the Y2 receptor binds and is stimulated by full length and N-terminally truncated NPY and PYY, the Y3 receptor shows high affinity for NPY and a low one for PYY, the Y4 receptor displays a high affinity for pancreatic polypeptide, and the Y5 receptor for all three peptides. As compared with the native peptides the N-terminally truncated ones, NPY(3–36) or PYY(3–36), have considerably reduced activities at the Y1 receptor, but not or scarcely reduced activities at the Y2 and Y5 receptors [29]. The Y1 receptor is located in the cerebral cortex and on vascular smooth muscle cells where it promotes vasoconstriction and cell proliferation, the Y2 receptor is often found presynaptically inhibiting e.g. the release of NPY and noradrenaline, and the Y5 receptor is abundant in the hypothalamus and involved in feeding behaviour. Considering these data DPP IV should convert NPY and PYY to agonists at the Y2 and Y5 receptors, but terminate

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their actions at the Y1 receptor subtype. Therefore, DPP IV has been already termed as NPY-converting enzyme and several physiological functions have been proposed, especially in the regulation of vascular smooth muscle contraction and angiogenesis [112]. NPY(3–36) has been identified as a considerable part (35%) of the total NPY extracted from porcine brain [32] and in the human cerebrospinal fluid [75]. It can also be isolated from tumor tissues [95]. PYY(3–36) is the major molecular form of PYY extracted from human or rabbit intestine [24] or circulating in the blood [31]. In accordance with these findings, NPY and PYY are very rapidly cleaved to the des(Tyr–Pro) derivatives by human serum and cultured endothelial cells from human umbilical veins [62]. However, in vitro or in vivo experiments with specific inhibitors or animals lacking DPP IV activity are needed prove these interesting roles for DPP IV as a regulator for biologically responses elicited by NPY and PYY. In conclusion, DPP IV has a major role in the differential or total inactivation of the neuropeptides NPY and endomorphin, and an additional one for the inactivation of substance P and bradykinin.

6. Action on circulating peptide hormones In Section 5 the involvement of DPP IV in the postsecretory processing of a peptide hormone with N-terminal Xaa–Pro sequence, PYY, was reviewed. Action of DPP IV on other Xaa–Pro peptide hormones circulating in the blood (Table 2) do not appear to be of biological importance. However, DPP IV plays a pivotal role for the inactivation of several Xaa–Ala peptide hormones that all belong to the GRH-glucagon family of peptide hormones. Within this family of 29–44 residue peptides considerable sequence homology is found mostly at the N-terminus. Peptides with N-terminal Tyr–Ala or His–Ala are cleaved and inactivated by DPP IV (GRH, GLP-1, GLP-2, GIP, PHM, PHI; Table 2), whereas those with N-terminal His– Ser [vasoactive intestinal peptide (VIP), secretin, glucagon, pituitary adenylate cyclase-activating peptide (PACAP)] are not attacked [63]. GRH is a 44-residue peptide of which biologically active, C-terminal truncated fragments are known. It is released from the hypothalamus and stimulates the release and the synthesis of growth hormone from / in the pituitary. In human plasma GRH(1–44)-NH 2 (or fragments) are rapidly metabolized to GRH(3–44)-NH 2 , further minor degradation products derive from endoproteolytic cleavage of the Arg(11)–Lys(12) or Lys(12)–Val(13) bonds [26]. All these proteolytic fragments are biologically inactive. By use of specific inhibitors, DPP IV and a trypsin-like endoprotease were identified as the proteases responsible for this processing. Following this initial report, DPP IV-mediated proteolysis was established as a major route of GRH degradation and inactivation also in rat [10],

porcine and bovine plasma in vitro and in cattle in vivo [51,52]. As consequence of these observations, peptide analogues of GRH(1–29) with improved stability to DPP IV were synthesized. Replacement of Tyr(1) by D-Tyr, NMeTyr or desNH 2 Tyr, of Ala(2) or of Asp(3) by the corresponding D-amino acids resulted in complete stability to human placenta DPP IV [9]. Surprisingly, replacement of the penultimate Ala by Ser, Thr, Gly, Val, or Leu did not abolish DPP IV cleavage, although these substitutions reduced the cleavage rates considerably [9,59]. Interestingly, replacement of Ala(2) by Pro increased the Vmax value only three-fold, but the Km value was doubled [9]. Thus, GRH with penultimate Ala or Pro are cleaved with about the same rate constants. GRH-derivatives that are resistant to DPP IV attack showed similar biological activity to induce growth hormone release in vitro (cultivated pituitary cells), but higher potency to increase serum growth hormone levels in vivo up to 60 min after injection (10 pmol / kg i.v. to Holstein steers) due to longer stability in vivo [51]. GLP-1, (formerly GLP-1(7–36)NH 2 ) and GLP-2 are 30- and 33-residue, respectively, gastrointestinal peptide hormones released postprandially from enteroglucagon cells (L-cells) in the small and large intestine, and are generated by an alternative post-translational processing of proglucagon (positions 78–108 and 126–159 of the human proglucagon precursor). GLP-1 stimulates insulin secretion in vitro and in vivo. It plays an important role in the regulation of postprandial insulin release and is the most important incretin (insulinotropic hormone) known so far (Fig. 5). In patients with type 2 diabetes, exogenous GLP-1 lowers postprandial plasma glucose excursions by stimulating insulin release and by inhibiting glucagon secretion. Therefore, the use of exogenous GLP-1 is considered a new possibility to treat patients with type 2 diabetes. However, the biological half-life of GLP-1 is very short. After subcutaneous administration, GLP-1 plasma levels return to basal concentrations after about 30 min. GLP-1 is also produced in the brain, and intracerebroventricular GLP-1 in rodent is a potent inhibitor of food and water intake. GLP-2 displays an intestinal growth factor activity in rodents, raising the possibility that GLP-2 may be therapeutically useful for enhancement of mucosal regeneration in patients with intestinal diseases [21]. The understanding of the inactivation of GLP-1 and GLP-2 is therefore of potential therapeutic interest enabling the synthesis of longer-acting analogues or the application of specific inhibitors that could prolong the effects the endogenous peptides. Especially, degradation resistant analogues of GLP-1 could be of potential value for the treatment of type 2 diabetes. Several studies in vitro and in vivo suggest that GLP-1 is primarily degraded at the N-terminus by the action of DPP IV resulting in biologically inactive des(His–Ala)– GLP-1 [63,16,18,19,49,81]. Incubation of GLP-1 with

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Fig. 5. Schematic drawing of a proposed role for DPP IV in the inactivation of incretins. GLP-1 and GIP are released postprandially from intestinal L- or K-cells, transported in the blood and stimulate insulin secretion from pancreatic b-cells. DPP IV at the surface of endothelial cells or soluble in the blood degrades both peptides at the N-terminus resulting in a rapid loss of hormonal activity.

human or rat plasma yielded this fragment as the main degradation product, and this conversion could be blocked by specific DPP IV inhibitors like diprotin a or Lyspyrrolidide [63,16,49]. Other GLP-1 degradation products were also detected, in particular after incubation with kidney membranes (cleavage between Ser(8) and Asp(9) [97]) or with neutral endopeptidase (at many sites [41]). However, after subcutaneous or intravenous administration of GLP-1 in healthy or type 2 diabetic humans more than 75% was recovered from the plasma as des(His–Ala)– GLP-1 [18]. After intravenous injection of 125 I-radiolabelled GLP-1 at picomolar concentrations into rats more than 50% was metabolized to des(His–Ala)–GLP-1 within 2 min, but not in DPP IV-negative rats [49]. Pharmacological inhibition of DPP IV with valine– pyrrolidide in anesthetized pigs, at a dose that reduced plasma DPP IV activity by more than 90%, increased the amount of intact GLP-1, both in the basal state and after infusion [15]. Moreover, DPP IV inhibition potentiated the insulinotropic effect of GLP-1 after intravenous infusion alone or with glucose. Oral administration of the DPP IV inhibitor Ile–thiazolidide to obese and lean Zucker rats reduced serum DPP IV activity by 65% and blocked the

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formation of des(His–Ala)–GLP-1 and des(His–Ala)–GIP [82]. In both phenotypes insulin secretion was enhanced by DPP IV inhibition, especially in the obese animals (150% over controls). By inhibitor treatment the glucose tolerance was improved in both phenotypes, and restored to nearnormal levels in obese animals. This was attributed to the glucose-lowering actions of increasing the circulating halflives of the endogenously released incretins GIP and, in particular, GLP-1. These studies suggest that manipulation of endogenously released GLP-1 (and GIP) by inhibition of DPP IV should be a valid therapeutic approach for lowering glucose levels in type 2 diabetes or other disorders involving glucose intolerance [38]. Another approach for the treatment of type 2 diabetes would be the pharmacological administration of DPP IVresistant analogues. As reviewed for GRH, GLP-1 stability to DPP IV should be enhanced by modifications of the N-terminal His(1), Ala(2) and Glu(3) by the corresponding D- or different amino acids. GLP-1 analogues with D-Ala(2), Gly(2), Ser(2), or Thr(2) had a longer half-life in vitro and in vivo, but retained only partially their biological activities [17,97]. In particular, D-Ala(2)– GLP-1 was completely stable to DPP IV cleavage and had biological potency comparable to GLP-1 on insulinoma cells in vitro, whereas the Gly(2)- and Ser(2)-derivatives — such as the corresponding GRH-analogues (see above) — were still degraded by DPP IV, though with low activity [97]. GLP-1 analogues with modifications of the N-terminal His(1) are often stable to DPP IV, but have considerably reduced activity except desamino-GLP-1 [97]. Despite their proteolytic stability, after subcutaneous injections these in vitro active analogues influenced plasma insulin concentrations in minipigs only for similar times (30 min) as GLP-1, however, the elevation of insulin levels was higher especially with desamino-GLP-1 and D-Ala(2)– GLP-1 [97]. In contrast, a single injection of 0.1 nmol Gly(2)–GLP-1 into diabetic mice fed with a high-fat diet corrected fasting hyperglycemia and glucose intolerance for several hours, whereas the activity of 1 nmol GLP-1 vanished within a few minutes of injection. These actions were correlated with increased insulin and decreased glucagon levels [13]. These data suggest that the effects of DPP IV-resistant analogues of GLP-1 on postprandial insulin levels are only partially improved over those of native GLP-1 under normal physiological conditions, but the effects may be more pronounced under diabetic or obese conditions. Like GLP-1, GLP-2 is also easily degraded to des(His– Ala)–GLP-2 by purified human DPP IV or with serum containing DPP IV activity [23]. When injected into normal rats, GLP-2 induces an increase in intestinal villus height and has a small trophic effect on small bowel weight. Administration of GLP-2 to DPP IV-deficient rats markedly increased the bioactivity of rat GLP-2 resulting in a significant increase in small bowel weight [23]. Moreover, in wild-type rats a DPP IV-resistant analogue r[Gly(2)]–

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GLP-2 produced a statistically significant increase in small bowel mass [23]. Similar results were obtained in mice with the corresponding human, DPP IV-resistant analogue h[Gly(2)]–GLP-2 [22]. These results show that inactivation of GLP-2 by DPP IV is of critical importance for its growth factor properties on the intestine and that DPP IV resistant analogues may be useful therapeutics to improve mucosal regeneration. Gastric inhibitory polypeptide or glucose-dependent insulinotropic polypeptide (GIP), a 42-amino-acid polypeptide, is secreted by duodenal K-cells after ingestion of fat and glucose. It inhibits the secretion of gastric acid and stimulates insulin release from pancreatic b-cells in the presence of elevated glucose levels. Thus, besides GLP-1, GIP also contributes to incretin activity of postprandial serum (Fig. 5). As with the other members of the GRHglucagon family with penultimate Ala, GIP is also rapidly degraded to GIP(3–42) by purified human DPP IV or serum with DPP IV activity [63]. This truncation of GIP in serum could be successfully inhibited by coincubation with specific DPP IV inhibitors like diprotin a or Lys– pyrrolidide [63,49]. GIP(3–42) was also detected after purification of GIP from pig intestine [44], and later it was shown that it lacked any biological activity [92]. As reported for GLP-1, GIP was also rapidly degraded in vivo to the truncated form (50% after 2 min) after injection of radioiodinated GIP into wild-type rats, but not in DPP IV-deficient ones [49]. Accordingly, after oral administration of a DPP IV inhibitor the half-life of GIP was prolonged in wild-type rats [79]. Thus, as for GLP-1 and GLP-2 DPP IV truncation is also the main degradation route for GIP in vivo. In conclusion, for several circulating hormones the role of DPP IV in their complete or receptor subtype-specific inactivation has been well documented. One releasing hormone (GRH) and several gastrointestinal hormones (GLP-1, GLP-2, GIP, PYY) are predominantly degraded by DPP IV located on endothelial cells or found as a soluble protease in the blood.

7. Action on chemokines Chemokines constitute a large group of small (8000– 10 000) secreted proteins that act as cell-type selective chemoattractants. They can be divided into four subfamilies, based on structural, functional and genetic criteria. The two most important subfamilies are the CXC and CC chemokines (a- and b-family), which differ in the spacing of the first two cysteine residues that form disulphide links with two other cysteines. In CXC chemokines these first cysteine residues are separated by one amino acid, in CC chemokines they are directly neighboured. Important members of the CXC chemokine subfamily are interleukin-8, platelet factor 4 and stromal cell-derived factor 1 (SDF-1). The CC-chemokine sub-

family includes macrophage inflammatory peptides 1a and 1b, and RANTES protein as well as GCP-2. CXC chemokines predominantly target neutrophils and, to a lesser extent, lymphocytes. CC chemokines mainly attract monocytes, but also lymphocytes, basophils, eosinophils, dendritic cells and NK cells. Modifications of the chemokines by post-translational proteolytic processing are frequent and regulate their activities towards the different receptors (CCR 1–9, CXCR 1–5,) that all belong to the G-protein-coupled seven transmembrane domain receptors. Several members of both chemokine subfamilies share a conserved Xaa–Pro or Xaa–Ala sequence at their Ntermini which conforms to the substrate specificity of DPP IV [104]. Indeed, DPP IV liberated the terminal Xaa–Pro dipeptides (compare Table 2) from full length RANTES, eotaxin, IP-10, MCP-2 (recombinant human DPP IV [79]; DPP IV purified from human prostasomes [84,100,103]), SDF-1a and SDF-1b (T-cell-bound DPP IV [96]; recombinant human DPP IV [78]; DPP IV purified from human prostasomes [85]), and GCP-2 (DPP IV purified from human prostasomes [84]). MCP-1 and MCP-3 are reported to be resistant to DPP IV-cleavage [79,84]. However, quantitative data on cleavage rates of chemokines are missing. From MDC not only the N-terminal dipeptide Gly–Pro but also the subsequent dipeptide Tyr–Gly is liberated [86]. This again shows that the substrate specificity of DPP IV is not only restricted to penultimate Pro and Ala; certain peptides with other small amino acids in this position [9,97] are also attacked although with considerably reduced activity. The N-terminal domain of chemokines (together with an exposed loop between the second and third Cys) is involved in receptor binding. Truncation can either activate or inactivate them, often only at certain receptor subtypes. A similar, receptor subtype selective effect of DPP IV truncation has been reviewed above for NPY and PYY. In the case of chemokines DPP IV mediated processing could thus provide a mechanism for the regulation of the target cell specificity and consequently for a differential cell recruitment. The CC chemokine RANTES is chemotactic for lymphocytes, monocytes, dendritic cells, eosinophils, basophils and NK cells. DPP IV truncation of RANTES resulted in the loss of its ability to increase cytosolic calcium concentrations and to induce chemotaxis in human monocytes in vitro, but did not reduce the calcium response in macrophages activated with macrophage colony-stimulating factor [79,84]. This differential activity was explained by altered specificity to chemokine receptor subtypes. RANTES(3–68) showed reduced activity relative to the full length peptide with cells expressing the CCR1 [79] or CCR3 [98] chemokine receptors, but retained [79] or even enhanced [93] the ability to stimulate the CCR5 receptors. Chemokine receptors, especially CCR5 and CXCR4, act as cofactors for HIV-1 entry into CD4 1 cells and their corresponding ligands can suppress HIV entry and thus

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replication. In accordance, DPP IV-truncated RANTES was able or even more potent to inhibit the cytopathic effects of HIV-1 [84,93]. Moreover, RANTES(3–68) acted as an antagonist for the chemotactic effect of full length RANTES, macrophage inflammatory protein-1a or b (MIP-1a / b) or MCP-3 on human monocytes [98]. DPP IV hydrolysis of RANTES appears to be relevant in several cellular systems, since RANTES(3–68) has been isolated from culture supernatants of stimulated human fibroblasts, skin samples, platelet preparations or sarcoma cells [76,84]. In conclusion, DPP IV converts RANTES to a chemokine with altered receptor subtype specificity (see Fig. 6). The CC chemokine eotaxin has been characterized as an important mediator in allergic reactions because it selectively attracts eosinophils, basophiles and TH 2 lymphocytes. The N-terminal Gly–Pro is efficiently cleaved from

Fig. 6. Schematic drawing of a proposed role for DPP IV in the inactivation of chemokines and possible physiological consequences. The chemokines eotaxin, RANTES or MCP-2 stimulate the receptor CCR3 that is expressed on eosinophil and basophil granulocytes as well as on TH 2 -lymphocytes. Beside chemotaxis, CCR3 mediates the degranulation of eosinophiles and basophiles as well as the synthesis of interleukins in TH 2 -lymphocytes which further stimulate the production of substances involved in killing of parasites or allergic reactions. Eotaxin, RANTES or MCP-2 are inactivated or even converted in CCR3 antagonists by the action of DPP IV located e.g. on endothelial cells or fibroblasts. Thus, DPP IV terminates these chemokine-induced reactions. In contrast to eotaxin, RANTES and MCP-2 stimulate further chemokine receptors which are expressed on other types of leukocytes; these actions are only partly terminated by DDP IV cleavage.

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eotaxin by DPP IV [79,100]. Eotaxin(3–74) showed reduced chemotactic activity for eosinophils in vitro and impaired binding and signalling properties through the CCR3 receptor [100]. Moreover, DPP IV truncated eotaxin exhibited antagonistic properties, since it desensitized calcium signalling and inhibited chemotaxis toward intact eotaxin. HIV-2 infection of CCR3-transfected cells was inhibited to the same extend by both eotaxin forms. Moreover, N-terminally truncated eotaxins (desGP, desGPA, desGPAS) have been purified from supernantants of cultured skin fibroblasts [71]. In conclusion, DPP IV terminates the chemotactic potency of eotaxin and thus limits the eotaxin-mediated inflammatory responses (Fig. 6). MDC, a CC chemokine, attracts monocytes, dendritic cells, activated lymphocytes and NK cells. DPP IV cleavage results in the formation of MDC(3–69) and MDC(5– 69) [86]. Compared with intact MDC(1–69) both DPP IV processed products had reduced chemotatic activity on lymphocytes and monocyte-derived dendritic cells [86,99]. They showed impaired binding to and calcium mobilization through the CCR4 receptor, the only identified MDC receptor so far. However, MDC(3–69) and MDC(5–69) retained their activities to attract and to bind to monocytes probably through a receptor subtype different from CCR4. Thus, it appears that MDC is another example for a chemokine that is only partially inactivated, namely to a certain receptor subtype. The CXC chemokine SDF-1, also termed pre-B-cell growth-stimulating factor (PBSF), is a chemoattractant for resting T-lymphocytes and monocytes. Two splice forms, SDF-1a and SDF-1b, arising from a single gene are known which differ only in four additional C-terminal residues in SDF-1b. Truncation of the N-terminal Lys–Pro by DPP IV from both peptides results in the loss of their chemotactic and antiviral activities in vitro [78,85,99]. DPP IV inactivates SDF-1a as a ligand for the CXCR4 receptor which is a lymphocyte chemotatic receptor as well as the main co-receptor for T-tropic HIV-1 strains. SDF1a(3–68) lost lymphocyte chemotactic and CXCR4-signalling properties [85]. However, in contrast to DPP IVtruncated RANTES, SDF-1a(3–68) had a diminished potency to inhibit HIV-1 infection [78,85]. Thus, SDF-1a is an example for a chemokine that is inactivated by DPP IV at its main receptor. In contrast, another CXC chemokine, GCP-2 was not reduced in its biological activity by DPP IV cleavage [84]. In conclusion, chemokines are a group of peptides in which a minimal N-terminal truncation by DPP IV may confer inactivation — totally or only differentially — for certain receptor subtypes. Moreover, DPP IV cleavage can convert several chemokine agonists into antagonists on distinct receptor subtypes. Chemokine cleavage by DPP IV is an emerging field in the last 2 years, and it can be expected that several more examples will be identified. Truncation by DPP IV and other proteases may be respon-

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sible for N-terminal heterogeneity of chemokines isolated from cell cultures [110]. As paracrine factors chemokines are targets for DPP IV on lymphocytes, macrophages, or fibroblasts. So far, the importance of N-terminal DPP IV processing for chemokines has been shown only in vitro with more or less purified enzyme preparations, but not yet in combined cellular systems. Especially, in vivo experiments that show the relevance for DPP IV as a differential terminator of chemotactic responses and inflammation are urgent and should be carried out by using DPP IV inhibitors or DPP IV negative animals.

8. Other enzymatic and binding functions of DPP IV Apart from its function as a regulatory protease, important other roles for DPP IV have been reported. As mentioned above, intestinal and renal DDP IV is involved in the final digestion of proline-containing oligopeptides and in the absorption of their fragments ([12] for review). DPP IV acts also as a binding and costimulatory protein. DPP IV binds to collagens, preferentially collagens I and III, via its cysteine rich domain, not by its catalytic site [56]. Binding to extracellular matrix proteins is probably a function for DPP IV isolated from liver as glycoprotein gp110 or bile caniculus domain-specific membrane glycoprotein (OX61 or HAM.4 antigen). Moreover, DPP IV has been shown to be identical with the cell surface marker CD26 [thymocyte-activating molecule (THAM) is the mouse homologue; 2B9 antigen] that is expressed on a fraction of resting T-cells at low density but strongly upregulated following T-cell activation (reviewed in [72,102]). CD26 / DPP IV can deliver a potent costimulatory activation signal into T-cells despite its small cytoplasmic region of only six amino acids. Signalling is dependent on its interaction with other molecules. It associates, presumably through its extracellular domain, with CD45, a protein tyrosine phosphatase, and with adenosine deaminase (ADA, EC 3.5.4.4), an enzyme involved in the salvage of purine nucleosides which catalyzes the deamination of adenosine and 29-deoxyadenosine to inosine and 29-deoxyinosine and whose deficiency causes severe combined immunodeficiency disease (SCID) in humans (DPP IV5adenosine deaminase complexing protein5ADAbp). Moreover, CD26 signalling is dependent on the expression of the T-cell receptor (TCR) complex. The dual function of DPP IV as a protease and a binding / stimulatory molecule is sustained by structural homologous proteins for which the one or other function has been reported. DPP IV belongs to the clan SC, family S9, subfamily B, of proteases to which other bacterial and eukaryote DPP IV belong as well as mammalian homologues with different peptidase activities, namely seprase / fibroblast activation protein alpha or the NAALADase (see above). In addition, structural homologues without pepti-

dase activities (and probably with binding activities?) have been cloned, the ‘dipeptidyl peptidase IV-like proteins (dipeptidyl aminopeptidase-related proteins)’ DPPX-L and DPPX-S [105] as well as brain-specific dipeptidyl peptidase-like protein BSPL [54] expressed predominately in brain and also in some peripheral tissues.

9. Conclusion and perspectives DPP IV/ CD26 has been discovered and investigated from two points of view: as a protease and as a binding / co-stimulatory protein. Its role as a regulatory protease and not merely a digestive enzyme for collagen or proline-rich peptide nutrients has been highlighted in recent years by in vitro and in vivo studies profiting from the development of specific inhibitors and DPP IV-deficient animals. Despite the only minimal truncation at the N-terminus of 30 to 80 residue peptides, DPP IV has a pivotal role in the inactivation of some circulating peptide hormones, especially those of the GRH-glucagon family with N-terminal Xaa–Ala, chemokines with N-terminal Xaa–Pro, and certain neuropeptides like NPY or endomorphins. Interestingly, apart from the extracellular conversion of NPY, PYY or chemokines to receptor subtype-selective agonists, no activating, prohormone-converting function for DPP IV (or a DPP IV-like enzyme) has yet been reported in mammals. Evidence for the importance of DPP IV in the inactivation of the insulinotropic peptides GLP-1 and GIP, as well as GLP-2 and GRH has been augmented from in vitro and in vivo studies which have resulted in the development of synthetic, DPP IV-resistant analogues of GLP-1 (and GRH). These analogues (or orally active DPP IV inhibitors) have potential therapeutical use for the treatment of type 2 diabetes, and corresponding uses can be anticipated for analogues of other hormones. The importance of DPP IV cleavage for certain chemokines and neuropeptides in vivo is far less clearly established and is a topic for future research. An interesting phenomenon is the generation of receptor subtype-selective agonists and antagonists by DPP IV cleavage. However, the inactivation of a regulatory peptide is not accomplished by DPP IV alone, and as in many other biological systems alternative routes exist which may limit the use of DPP IV resistant analogues or DPP IV inhibitors.

Acknowledgements My own experimental work in this field was supported by grants from the Deutsche Forschungsgemeinschaft. I thank the co-workers from my laboratory for their fruitful collaboration, especially my year long technicians Martina Burmester and Hella Rix-Matzen for their excellent, continuous work and their ever friendly help, and my

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teacher Eberhard Heymann for his steady encouragement and support. I am indebted Clemens Franke for drawing the figures, and to E. Brandt (Forschungsinstitut Borstel, ¨ Institute for Germany) and H.-U. Demuth (Hans Knoll Natural Product Research, Halle, Germany) for critical reading of the manuscript.

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