Clinical Science (2005) 108, 1–16 (Printed in Great Britain)
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`Dipeptidyl peptidase IV and related enzymes
`in cell biology and liver disorders
`
`Mark D. GORRELL
`A.W. Morrow Gastroenterology and Liver Centre at Royal Prince Alfred Hospital, Centenary Institute of Cancer Medicine and
`Cell Biology and Discipline of Medicine, The University of Sydney, Sydney, New South Wales, Australia
`
`A B S T R A C T
`
`DP (dipeptidyl peptidase) IV is the archetypal member of its six-member gene family. Four members
`of this family, DPIV, FAP (fibroblast activation protein), DP8 and DP9, have a rare substrate speci-
`ficity, hydrolysis of a prolyl bond two residues from the N-terminus. The ubiquitous DPIV glyco-
`protein has proved interesting in the fields of immunology, endocrinology, haematology and
`endothelial cell and cancer biology and DPIV has become a novel target for Type II diabetes
`therapy. The crystal structure shows that the soluble form of DPIV comprises two domains, an
`α/β-hydrolase domain and an eight-blade β-propeller domain. The propeller domain contains the
`ADA (adenosine deaminase) binding site, a dimerization site, antibody epitopes and two open-
`ings for substrate access to the internal active site. FAP is structurally very similar to DPIV, but FAP
`protein expression is largely confined to diseased and damaged tissue, notably the tissue remodel-
`ling interface in chronically injured liver. DPIV has a variety of peptide substrates, the best studied
`being GLP-1 (glucagon-like peptide-1), NPY (neuropeptide Y) and CXCL12. The DPIV family has
`roles in bone marrow mobilization. The functional interactions of DPIV and FAP with extra-
`cellular matrix confer roles for these proteins in cancer biology. DP8 and DP9 are widely distri-
`buted and indirectly implicated in immune function. The DPL (DP-like) glycoproteins that lack
`peptidase activity, DPL1 and DPL2, are brain-expressed potassium channel modulators. Thus the
`six members of the DPIV gene family exhibit diverse biological roles.
`
`INTRODUCTION
`
`Few proteinases are capable of cleaving the post-proline
`bond and very few can cleave a prolyl bond two pos-
`itions from the N-terminus. The latter small subset of
`serine proteinases, the post-proline dipeptidyl aminopep-
`tidases, consists of the four enzymes of the DP (di-
`peptidyl peptidase) IV gene family, DPIV, FAP (fibro-
`
`blast activation protein), DP8 and DP9 [1], and DP-II
`(E.C. 3.4.14.2) [2]. DPIV (E.C. 3.4.14.5) is a ubiquitous,
`multifunctional homodimeric glycoprotein with roles in
`nutrition, metabolism, the immune and endocrine sys-
`tems, bone marrow mobilization, cancer growth and cell
`adhesion. DPIV ligands include ADA (adenosine de-
`+
`+
`/H
`ion exchanger 3 [4] and
`aminase) [3], kidney Na
`fibronectin [5]. Important DPIV substrates include at
`
`Key words: adenosine deaminase, CD26, diabetes, dipeptidyl peptidase IV, fibroblast activation protein, post-proline amino-
`peptidase.
`Abbreviations: ADA, adenosine deaminase; DP, dipeptidyl peptidase; DPL, DP-like; ECM, extracellular matrix; FAP, fibroblast
`activation protein; G-CSF, granulocyte colony-stimulating factor; GIP, glucose-dependent insulinotropic peptide; GKO, gene
`knockout; GLP, glucagon-like peptide; GRP, gastrin-releasing peptide; HSC, hepatic stellate cell; IFN, interferon; IL, interleukin;
`MAb, monoclonal antibody; MMP, matrix metalloproteinase; NPY, neuropeptide Y; PACAP, pituitary adenylate cyclase-activating
`peptide; POP, prolyl oligopeptidase; PPAR, peroxisome proliferator-activated receptor; sCD26, soluble CD26; SREBP, sterol
`regulatory element-binding protein; srhCD26, soluble recombinant human CD26; uPA, urokinase plasminogen activator; VIP,
`vasoactive intestinal peptide.
`Correspondence: Dr Mark D. Gorrell (email M.Gorrell@centenary.usyd.edu.au).
`
`C(cid:1) 2005 The Biochemical Society
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`Petitioner GE Healthcare – Ex. 1041, p. 1
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`

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`2
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`M. D. Gorrell
`
`Table 1 Some natural substrates of DPIV
`MDC, macrophage-derived chemokine; Mig, monokine induced by IFN γ ;
`IP10,
`IFN γ -induced protein;
`IFN-inducible T-cell α chemoattractant;
`IGF-1,
`I-TAC,
`insulin-like growth factor-1; hCGα, human chorionic gonadotrophin α chain;
`GHRF, growth hormone releasing factor; LHα leutinizing hormone α chain.
`
`Substrate
`
`GLP-1
`GLP-2
`GIP
`GRP
`Substance P
`NPY
`Peptide YY(1–36)
`PACAP38
`CCL5/RANTES
`CCL11/eotaxin
`CCL22/MDC
`CXCL9/Mig
`CXCL10/IP10
`CXCL11/I-TAC
`CXCL12/SDF-1
`IGF-1
`Prolactin
`hCGα
`GHRF
`LHα
`Thyrotropin α
`Peptide histidine methionine
`Enkephalins
`Vasostatin-1
`
`N-terminus
`
`His-Ala-Glu-
`His-Ala-Asp-
`Tyr-Ala-Asp-
`Val-Pro-Leu-
`Arg-Pro-Lys-
`Tyr-Pro-Ser-
`Tyr-Pro-Ile-
`His-Ser-Asp-
`Ser-Pro-Tyr-
`Gly-Pro-Gly-
`Gly-Pro-Tyr-
`Thr Pro-Val-
`Val-Pro-Leu-
`Phe-Pro Met-
`Lys-Pro-Val-
`Gly-Pro-Glu-
`Thr-Pro-Val-
`Ala-Pro-Asp-
`Tyr-Ala-Glu-
`Phe-Pro-Asn-
`Phe-Pro-Asp-
`His-Ala-Asp-
`Tyr-Pro-Val-
`Leu-Pro-Val-
`
`Reference
`
`[19]
`[20]
`[19]
`[21,22]
`[21]
`[23]
`[23]
`[22,24]
`[25]
`[25]
`[25]
`[26]
`[25,26]
`[26]
`[27]
`[28]
`[21]
`[21]
`[19,24]
`[28]
`[28]
`[19,24]
`[29]
`[30]
`
`least nine chemokines, NPY (neuropeptide Y), pep-
`tide YY, GLP (glucagon-like peptide)-1, GLP-2 and
`GIP (glucose-dependent insulinotropic peptide; Table 1).
`DPIV inhibitors are in clinical trials as a new therapy
`for non-insulin dependent diabetes mellitus (Type II di-
`abetes) [6,7]. Therapeutic benefit is derived from reduced
`inactivation of GLP-1 and GIP by DPIV-mediated cleav-
`age, thus stimulating greater insulin production. Further-
`more, on a high-fat diet, the DPIV GKO (gene knock-
`out) mouse has reduced appetite and increased energy
`expenditure compared with wild-type animals [8], sug-
`gesting that DPIV-selective inhibitors may be useful as
`anti-obesity agents that might combat liver steatosis.
`Lymphocytes and endothelial and epithelial cells express
`DPIV (for review, see [9]). In addition to the integral
`membrane form, a soluble form of DPIV occurs in serum
`[10].
`DPIV has a post-proline dipeptidyl aminopeptidase
`activity preferentially cleaving Xaa-Pro or Xaa-Ala di-
`peptides (where Xaa is any amino acid) from the N-ter-
`minus of polypeptides. The POP (prolyl oligopeptidase;
`EC 3.4.21.26) family, a group of aminopeptidases and
`
`C(cid:1) 2005 The Biochemical Society
`
`COLOUR
`
`Figure 1 Schematic presentation of the proteins of the
`DPIV family and of POP
`The arrangement of structural domains is depicted. Approximate positions of
`N-glycosylation sites, cysteine residues and some residues required for enzyme
`activity and ADA or antibody binding are depicted. Not to scale.
`
`endopeptidases able to hydrolyse the post-proline bond,
`includes the DPIV gene family. The DPIV gene family
`is distinguished by a pair of glutamate residues that are
`distant from the catalytic serine in the primary struc-
`ture (Figure 1) [1], but within the catalytic pocket in
`the tertiary structure [11]. These glutamate residues, at
`positions 205 and 206 in DPIV, are essential for DP
`activity [12,13]. The DPIV gene family has six members,
`including FAP, DP8, DP9 and the two non-enzymes
`DPL1 and DPL2 (Table 2).
`This review complements recent reviews [14–18] in
`discussing the structure, activities and roles of the DPIV
`gene family in T-cell function, chemoattraction of leuco-
`cytes, cancer, angiogenesis, fibrinolysis, haematopoiesis
`and energy metabolism.
`
`IN VIVO EXPRESSION OF DPIV/CD26
`
`DPIV is expressed in all organs, by capillary endothelial
`cells and activated lymphocytes and on apical surfaces
`of epithelial, including acinar, cells. In humans, DPIV is
`present in the gastrointestinal tract, biliary tract, exocrine
`pancreas, kidney, thymus, lymph node, uterus, placenta,
`prostate, adrenal, parotid, the sweat, salivary and mam-
`mary glands and endothelia of all organs examined,
`including liver, spleen, lungs and brain (reviewed pre-
`viously, see [9,14]). DPIV is a 110 kDa glycoprotein
`
`Petitioner GE Healthcare – Ex. 1041, p. 2
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`

`

`Table 2 Enzymes of the human DPIV family
`Key: (cid:1), yes;×, no; ?, not known.
`Characteristic
`
`Hydrolysis of H-Gly-Pro
`Hydrolysis of H-Ala-Pro
`Hydrolysis of H-Arg-Pro
`Cleavage of chemokines
`Gelatinase activity (collagen I)
`Dimeric form
`Binding to adenosine deaminase
`mRNA expression in normal adult tissues
`Protein expression in normal adult
`Protein expression by activated fibroblasts
`Expression by fetal mesenchymal cells
`Expression by activated hepatic stellate cells
`Expression by lymphocytes
`
`DP8
`(cid:1)
`(cid:1)
`Poor
`?
`×
`×
`×
`Ubiquitous
`?
`?
`?
`?
`(cid:1)
`
`that is catalytically active only as a dimer. CD26 cell-
`surface expression on T-cells increases 5–10- fold follow-
`ing antigenic or mitogenic stimulation.
`Human DPIV overexpression in mice produces fewer
`thymocytes and peripheral blood leucocytes
`from
`+
`thymocytes
`2 months of age, more single positive CD8
`+
`+
`and CD4
`peripheral blood
`and more apoptotic CD8
`lymphocytes [31].
`Many epithelial tumours and cancer cell lines express
`DPIV, but DPIV expression is down-regulated or absent
`in tumour cells [1]. However, solid tumours contain
`DPIV in the stromal fibroblasts [1,32].
`
`FAP
`
`FAP has 52 % amino acid identity with DPIV, but FAP
`and DPIV differ in expression patterns and substrate
`specificities (Table 2). FAP has a collagen type I-specific
`gelatinase activity [33,34]. In contrast, we have detected
`no gelatinase activity from recombinant human DPIV
`in zymograms of transfected CHO (Chinese-hamster
`ovary) cells or of purified protein [33] or in gelatinase
`assays of transfected monkey fibroblastic [14] or human
`epithelial [35] cell lines. Like DPIV, catalysis depends
`upon dimerization [33,36]. Interestingly, DPIV and FAP
`form heterodimers [37]. A soluble form of FAP has
`been isolated from normal bovine and human serum but,
`curiously, despite the abundance of serum DPIV, serum
`FAP is homodimeric [38,39].
`Controlling gelatinases is vital for organ structure.
`Unlike MMPs (matrix metalloproteinases), which have
`a proenzyme form, the gelatinase activity of FAP is con-
`stitutive. FAP is normally restricted to a subset of glu-
`cagon: producing α-cells in pancreatic islets [32]. FAP
`is strongly expressed by activated HSCs (hepatic stellate
`cells), notably near lipid accumulation, called steatosis,
`
`Dipeptidyl peptidase IV gene family
`
`3
`
`DPIV
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`×(cid:1)
`(cid:1)
`(cid:1)
`Ubiquitous
`Ubiquitous
`(cid:1)
`(cid:1)
`×
`(cid:1)
`
`FAP
`
`poor
`(cid:1)
`(cid:1)
`?
`
`(cid:1)
`×
`Ubiquitous
`Serum, pancreas
`(cid:1)
`(cid:1)
`(cid:1)
`×
`
`DP9
`(cid:1)
`(cid:1)
`Poor
`?
`×
`×
`×
`Ubiquitous
`?
`?
`?
`?
`(cid:1)
`
`COLOUR
`
`Figure 2 Schematic representation of FAP and DPIV in
`cirrhotic liver
`In cirrhotic nodules, FAP is expressed by myofibroblasts (mf) in the septum and by
`HSCs following activation in the portal–parenchymal interface. DPIV is expressed
`on activated T-cells, capillary endothelium and the bile canalicular surface of
`hepatocytes. Not to scale.
`
`in liver and by mesenchymal cells in other sites of
`tissue remodelling such as stromal fibroblasts of epithelial
`tumours and healing wounds and embryonic mesen-
`chymal cells [32–34,40,41]. The FAP GKO mouse has
`a normal phenotype for body weight, organ weights,
`histological examination of major organs and haemato-
`logical analysis [42].
`The HSC has an important role in the pathogenesis
`of cirrhosis. Following liver injury, HSCs undergo activ-
`ation and transdifferentiation to become myofibroblasts.
`Significant functional changes accompany this pheno-
`typic change, including alterations in ECM (extracellular
`matrix) production and expression of various MMPs and
`their inhibitors. FAP is expressed by myofibroblasts
`and a subset of activated human HSCs at the tissue–re-
`modelling interface, which is the PPI (portal–paren-
`chymal
`interface), of cirrhotic liver [33] (Figure 2).
`
`C(cid:1) 2005 The Biochemical Society
`
`Petitioner GE Healthcare – Ex. 1041, p. 3
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`

`

`4
`
`M. D. Gorrell
`
`Table 3 Physical attributes of human DPIV-related proteins
`(cid:1), yes;×, no; ?, not known.
`∗
`The long form is a splice variant.
`Attribute
`DPIV
`
`DP8
`
`FAP
`
`Synonyms
`GenBank® accession
`
`CD26, ADAbp
`M80536
`
`References
`Gene location
`Human
`Mouse
`Number of exons
`Gene size (kb)
`Transmembrane domain
`Monomer mobility
`Number of amino acids
`
`[57]
`
`2q24.2
`2
`26
`81.8
`(cid:1)
`110 kDa
`766
`
`Seprase
`U09278
`
`[37]
`
`2q24.3
`2
`26
`72.8
`(cid:1)
`95 kDa
`760
`
`–
`AF221634
`NM 130434
`[45,48]
`
`15q22.32
`9
`20
`71
`×
`100 kDa
`882
`
`DP9
`
`–
`AY374518
`NM 139159
`[13,48,49]
`
`19q13.3
`17
`19
`47.3
`×
`110 kDa
`863
`
`∗
`DP9 long
`
`–
`AF542510
`AF452102
`[35]
`
`22
`48.6
`×
`?
`971
`
`DPL1
`
`DPL2
`
`POP
`
`DPP6, DPPX
`M96859
`M96860
`[52]
`
`7q36.2
`5
`26
`911
`(cid:1)
`97 kDa
`∗
`, 803
`865
`
`PEP, PREP
`AB020018
`
`DPP10
`AY387785
`NM 1004360
`[48,50,51]
`
`2q14.1
`1
`26
`1402
`(cid:1)
`97 kDa
`∗
`796
`
`6q21
`10
`15
`40.2
`×
`80 kDa
`712
`
`FAP-positive cells are present in early stages of liver
`injury, and FAP immunostaining intensity strongly cor-
`relates with the histological severity of fibrosis in chronic
`liver disease [40].
`Conferring FAP expression upon a human epithelial
`cell line increases tumorigenicity in mice, but has the
`opposite effect on a melanoma cell line [43,44]. The bio-
`logical importance of these observations is unknown
`because tumour cells do not naturally express FAP
`in vivo.
`
`DP8 AND DP9
`
`The discovery of DP8 and DP9, which are ubiquitously
`expressed enzymes with DPIV-like peptidase activity
`[35,45], means that previous studies using DPIV inhi-
`bitors to infer functions of DPIV will require reinter-
`pretation where the inhibitor is found to also inhibit
`DP8 or DP9 [46]. However, little is known about the
`expression or functional significance of DP8 or DP9.
`DP8 and DP9 are both soluble proteins localized in the
`cytoplasm. Both are DPs that are active as monomers
`and hydrolyse H-Ala-Pro- and H-Gly-Pro-derived sub-
`strates, although less efficiently than DPIV. Neither
`DP8 nor DP9 exhibit gelatinase activity and no natural
`substrates are known.
`
`DP8
`DP8 has 26 % amino acid identity with the protein
`sequences of DPIV and FAP and is a dipeptidyl amino-
`peptidase, hydrolysing the prolyl bond after a penulti-
`mate proline [45]. However, some biochemical charac-
`teristics of DP8 are similar to the endopeptidase POP
`(Table 3). Like DP8, POP is a soluble cytoplasmic protein,
`is active as a monomer and lacks N-linked and O-linked
`
`C(cid:1) 2005 The Biochemical Society
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`glycosylation sites. Like DPIV, DP8 mRNA expression
`is ubiquitous. DP8 mRNA levels are elevated in both
`activated and transformed lymphocytes. DPIV traverses
`the TGN (trans-Golgi network), which is in a secretion
`pathway, enters secretory vesicles then moves to the cell
`surface [47]. Despite finding DP8 in Golgi as well as
`elsewhere in cytoplasm, we have not found evidence
`of secretion of DP8 by transfected COS or 293T cells
`[35].
`
`DP9
`DP9 is the closest relative to DP8, having 61 % amino acid
`identity. DP9 has two forms, having open reading frames
`of 2589 bp and 2913 bp. A ubiquitous predominant DP9
`mRNA transcript at 4.4 kb represents the short form and
`a less abundant 5.0 kb transcript present predominantly
`in muscle represents the long form (Table 3) [35,48,49].
`DP9 has only two potential N-linked glycosylation sites.
`Paradoxically for a cytoplasmic protein, DP9 contains an
`RGD (Arg-Gly-Asp) potential cell attachment sequence,
`which is the best characterized integrin-binding motif,
`near its N-terminus. In contrast, the RGD motif in mouse
`DPIV has a different location, on propeller blade four
`where ADA binds to human DPIV. We obtain DP9 sizes
`on SDS/PAGE of 110 kDa and 95 kDa [35], whereas
`others report a single band at about 95–100 kDa [48,49].
`DP9 has a predicted polypeptide size of 98 263 Da, so
`we propose that intact fully glycosylated DP9 runs at
`110 kDa.
`Northern blot analysis on normal tissues shows DP9
`mRNA expression predominantly in muscle, liver and
`leucocytes. However, in silico examination of 255 human
`DP9 ESTs (expressed sequence tags; UniGene Cluster
`Hs.237617) indicate that DP9 mRNA expression is most
`abundant in leucocytic cell lines and diseased and tumour-
`bearing tissues including melanoma [35].
`
`Petitioner GE Healthcare – Ex. 1041, p. 4
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`

`Dipeptidyl peptidase IV gene family
`
`5
`
`Table 4 In vivo immunological and haematopoietic effects of DPIV inhibitors
`∗
`GKO mouse was not inhibitor treated.
`
`Outcome
`
`Name of inhibitor
`
`Type of inhibitor
`
`Wild-type mouse
`
`DPIV GKO mouse
`
`Reference
`
`Pro-Pro Diphenyl phosphonate ester
`Ala-Pro-nitrobenzoylhydroxylamine
`Lyz-[Z-(NO2)]-thiazolidide
`Isoleucyl-thiazolidide
`Val-boro-Pro
`Val-boro-Pro
`Diprotin A (Ile-Pro-Ile)
`Diprotin A or valine-pyrrolidide
`
`Irreversible, competitive
`Irreversible, suicide
`Reversible, competitive
`Reversible, competitive
`Irreversible
`Irreversible
`Reversible, competitive
`Reversible, competitive
`
`Prolonged cardiac allograft survival
`Suppression of experimental arthritis
`Suppression of experimental arthritis
`Exacerbates NPY + Con A-stimulated paw inflammation
`Tumour Regression
`Accelerates recovery from neutropenia
`Impaired G-CSF-induced haematopoiesis
`Increased bone marrow transplant efficiency
`
`Not done
`Same as wild type
`Same as wild type
`Not done
`Same as wild type
`Same as wild type
`∗
`GKO same as inhibitor
`∗
`GKO same as inhibitor
`
`[63]
`[58]
`[58]
`[65]
`[66]
`[61]
`[67]
`[64]
`
`THE NON-ENZYME DPIV GENE FAMILY
`MEMBERS: DPL1 AND DPL2
`
`Like DPL1, DPL2 associates with and modulates A-type
`potassium channels [56].
`
`Two enzymatically inactive proteins closely related to
`DPIV lack DPIV catalytic activity due to mutations of the
`catalytic serine residue and its neighbouring tryptophan
`residue, giving a surrounding sequence of Gly-Lys-Asp-
`Tyr-Gly-Gly instead of the motif Gly-Trp-Ser-Tyr-Gly-
`Gly. Since we cloned the second human DPIV paralogue
`that lacks the catalytic serine, we use the names DPL (DP-
`like) 1 and 2 for these proteins to simplify the nomen-
`clature [1,50]. As restoring the enzyme activity of DPL1
`or DPL2 would very likely require both the serine and
`tryptopan residues, their biological activities are probably
`exerted via binding interactions. It is very unlikely that
`provision only of a serine residue could rescue an enzyme
`activity in DPL2, as has been hypothesized [51].
`DPL1 was previously called DPPX or DPP6 [52]. Ex-
`pression of neuronal DPL1 increases in response to
`kainic acid injection into the hippocampus, suggesting
`possible involvement in CNS (central nervous system)
`plasticity [53]. DPL1 has a crucial role in the traffick-
`ing, membrane targeting and function of A-type potas-
`sium channels in somatodendritic compartments of
`neurons, which are important in neuronal function and
`in dysfunction, such as Parkinson’s disease [54]. Despite
`the absence of DP activity, DPL1 exerts an important
`developmental function. The mouse rump white mu-
`tation, which lacks expression of the DPL1 gene, is em-
`bryonic lethal in homozygotes and causes a pigmentation
`defect in heterozygotes [55].
`DPL2 has been cloned by others and called DPP10
`[48,51,56]. Like DPL1, DPL2 is alternatively spliced. The
`DPL2, DPIV and FAP genes are all on chromosome 2,
`and DPL2 is more closely related to DPIV and FAP than
`is DPL1. Intronic portions of the DPL2 gene link to
`asthma [51]. DPL2 mRNA expression occurs in brain,
`adrenal gland and pancreas [48,50]. This is similar to the
`expression pattern of the long form of DPL1 [52,53].
`
`INHIBITOR DATA INDICATE IMPORTANCE
`OF DPIV-RELATED DPs
`
`It is likely that many DPIV inhibitors are selective for
`the DPIV family rather than DPIV itself [48]. Therefore
`some potential functions of DP8 and DP9 may be
`inferred from studies in which a function has been at-
`tributed to DPIV by observing diminution of that func-
`tion in cells or animals treated with a DPIV inhibitor.
`In several paradigms, DPIV inhibitor treatment elicits
`similar responses in cells and animals irrespective of pos-
`session or lack of DPIV expression (Table 4). For example,
`DPIV inhibitors suppress collagen-induced and alkyl-
`diamine-induced arthritis to similar extents in DPIV-
`deficient German Fischer344 and wild-type rats [58].
`Moreover, T-cell proliferation in vitro [59] and immune
`responses in vivo [58] are diminished in the presence
`of DPIV inhibitors; however, the DPIV-deficient rat has
`normal immune responses [60]. These data imply that the
`peptidase activities of DP8 and/or DP9 have important
`functions in activated lymphocytes. FAP is excluded from
`consideration because it is not present in leucocytes.
`The DPIV/DP-II inhibitor Val-boro-Pro stimulates
`haematopoiesis to similar extents in DPIV GKO and
`wild-type mice [61], indicating that bone marrow ex-
`presses important DPIV-related enzymes. In animal
`models, DPIV inhibitors suppress antibody production
`[62] and prolong the survival of heart transplants [63]. It is
`possible that these phenomena also relate to functions of
`DP8 and/or DP9, rather than DPIV. Therapeutic DPIV
`inhibitors should either avoid inhibition of DP8 and
`DP9, or the effects of inhibiting DP8 and DP9 should
`be understood.
`Recently, the non-selective DPIV inhibitors valine-
`pyrrolidide and diprotin A have been shown to greatly
`
`C(cid:1) 2005 The Biochemical Society
`
`Petitioner GE Healthcare – Ex. 1041, p. 5
`
`

`

`6
`
`M. D. Gorrell
`
`COLOUR
`
`Figure 3 Secondary, tertiary and quaternary structure of DPIV
`(A) The DPIV homodimer as a ribbon diagram with each monomer coloured blue to red, carbohydrates are depicted in ball-and-stick representation and positions
`of important amino acids as spheres. The orange and yellow residues Leu294 and Val341 are essential for ADA binding, whereas the red positively charged residues
`Arg343 and Lys441 are important in epitopes of antibodies that inhibit ADA binding [86]. Glu205 and Glu206, coloured blue, are essential for enzyme activity [12].
`Both domains contribute to the dimerization interface, the β-propeller contributing two β-strands that protrude from blade 4. Substrate access to the catalytic site
`occurs via the side openings that face each other and are between the eight-blade β-propeller (bottom) and α/β hydrolase (top) domains. The transmembrane
`
`C(cid:1) 2005 The Biochemical Society
`
`Petitioner GE Healthcare – Ex. 1041, p. 6
`
`

`

`Dipeptidyl peptidase IV gene family
`
`7
`
`enhance the efficiency of bone marrow cell transplant-
`ation [64]. The administration of DPIV inhibitor to the
`−/−
`mouse in this model would be interesting.
`DPIV
`
`(Figure 3). A single amino acid point mutation near the
`C-terminus, His750 → Glu, is sufficient to prevent dimer-
`ization [79].
`
`THE THREE-DIMENSIONAL STRUCTURE
`OF DPIV
`
`The seven DPIV crystal structures recently reported
`reflect a sudden global interest in the pharmaceutical
`design of DPIV inhibitors [11,72–77]. The DPIV glyco-
`protein is a dimer (Figure 3). Each monomer subunit
`consists of two domains, an α/β-hydrolase domain (re-
`sidues 39–51 and 501–766) and an eight-blade β-propeller
`domain (residues 59–497), that enclose a large cavity of
`(1 A˚ = 0.1 nm) in diameter. Access to
`approx. 30–45 A˚
`this cavity is provided by a large side opening of ap-
`prox. 15 A˚ [76]. However, only elongated peptides, or
`unfolded or partly unfolded protein fragments, can reach
`the small pocket within this cavity that contains the
`active site. DPIV contains nine N-linked glycosylation
`sites that lie predominantly on the propeller domain near
`the dimerization interface [75] and perhaps shield this
`trypsin-resistant extracellular protein from proteolysis.
`
`The active site and catalytic mechanism
`The residues forming the catalytic triad are Ser630, Asp708
`and His740. In addition, Tyr547 in the hydrolase domain is
`essential for catalytic activity and in the crystal structure
`appears to stabilize the tetrahedral oxyanion intermediate
`form of a substrate [78]. Two glutamate residues in the
`catalytic pocket, Glu205 and Glu206 (Figure 3A), align
`the substrate peptide by forming salt bridges to its N-ter-
`minus, leaving room for only two amino acids before the
`peptide reaches the active serine residue, thus explain-
`ing its dipeptide-cleaving activity. Furthermore, in the
`substrate second position only amino acids with smaller
`side chains such as proline, alanine and glycine can fit
`into the narrow hydrophobic pocket. Thus the crystal
`structures have helped to explain the substrate specificity
`of DPIV and the mutation data showing that Glu205 and
`Glu206 are essential for catalysis [12,13].
`An intriguing aspect of DPIV biochemistry is the de-
`pendence of peptidase activity upon homodimerization.
`Dimerization requires the hydrolase domain [13] and
`a protrusion from the fourth blade of the β-propeller
`
`The unusual propeller of DPIV
`β-Propellers have four to eight blades formed by a
`repeated subunit containing at least 30 and generally
`50 amino acids in a β-sheet of four anti-parallel strands.
`Propellers commonly act as scaffolding for protein–pro-
`tein interactions [80,81]. The points of contact with
`ligand and antibodies are formed by loops contributed
`by adjacent propeller blades such that binding epitopes
`depend upon tertiary structure. DPIV has all of these
`characteristics.
`The structure of DPIV is unique among leucocyte
`surface molecules. Other leucocyte surface antigens that
`include a β-propeller domain are CD100 [82] and integrin
`α-chain [83], which have seven-blade propellers. As
`DPIV is a type II protein, the propeller domain points
`its lower face towards the extracellular milieu (see Fig-
`ure 3A). The eight-blade β-propeller domain of DPIV is
`more disordered than other propellers.
`
`The ADA binding site on DPIV
`ADA (EC 3.5.4.4) is a soluble globular 43 kDa enzyme
`present in all mammalian tissues. ADA catalyses the
`irreversible deamination of adenosine to inosine and of
`(cid:4)
`(cid:4)
`-deoxyadenosine to 2
`-deoxyinosine. ADA derived
`2
`from rabbits, cattle and humans binds to human, but not
`mouse, DPIV. ADA binds human DPIV with a KA of
`4–20 nM. Both monomeric and dimeric DPIV bind
`ADA [13,77]. Localizing ADA to the cell surface by its
`binding to CD26 probably reduces inhibition of T-cell
`proliferation by extracellular adenosine.
`Three charged residues on ADA, Glu139, Arg142 and
`Asp143 [84,85], have been identified by point mutation as
`important for ADA–DPIV binding. The crystal structure
`of DPIV with ADA shows that the ADA binding is
`located, as predicted from the model [86], on the outer
`edges of the fourth and fifth blades on the lower side
`near the lower face of the β-propeller domain of DPIV.
`Only one salt bridge binds ADA to DPIV (Figure 3C).
`Most of the involved residues on DPIV are hydrophobic
`and most of the 13 involved residues on ADA, which
`are all polar, are charged [77]. Generally, protein–protein
`binding primarily involves hydrophobic surfaces with
`
`domain (residues 7–28) is above the molecule in this view of the extracellular portion, residues 39–766, of human DPIV. The Figure was prepared using the atomic
`co-ordinates having Protein Data Bank code 1N1M [11], MOLSCRIPT and Canvas v. 8.06. (B) A surface representation of the DPIV monomer with the negatively charged
`surface coloured red and positively charged surface coloured blue. Viewed side-on (on left) and towards the propeller lower face (on right). An inhibitor is coloured
`yellow to indicate the position of the catalytic pocket. Reproduced from [11] with permission. (C) In these surface representations of DPIV and its ligand, ADA amino
`acids in the binding interface are labelled. Positively and negatively charged residues are blue and red, and non-polar and polar uncharged residues are yellow and
`orange respectively. The helices 1 and 2 of ADA are labelled α1 and α2. The DPIV monomer is oriented similarly to the monomer at the right in (A). In each image,
`c(cid:1) (2004) American Society for Biochemistry
`a large arrow points towards the central opening of the propeller lower face. Reproduced from [77] with permission.
`and Molecular Biology.
`
`C(cid:1) 2005 The Biochemical Society
`
`Petitioner GE Healthcare – Ex. 1041, p. 7
`
`

`

`8
`
`M. D. Gorrell
`
`some salt bridges that are mostly peripheral in the binding
`interface. Thus the amino acid composition of the ADA–
`DPIV binding site is unusual, perhaps due to the short
`evolutionary time that it has undergone selective pressure.
`ADA binding to human DPIV is blocked by certain
`anti-DPIV MAb (monoclonal antibodies) that define a
`similar epitope. MAb that block ADA binding rely upon
`Val341 and Thr440-Lys441 of DPIV for binding [86]. The
`DPIV structure shows that these amino acids are on one
`side of the propeller and distant from both of the open-
`ings, which explains the lack of interference from either
`ADA or MAb with the catalytic activity of DPIV
`or ADA. This location of ADA binding also positions
`ADA away from the cell surface and perhaps increases
`accessibility to ligands such as A1-adenosine receptor [87]
`and plasminogen-2 [85].
`
`Protein structure in the DPIV family
`Relating the structures of the DPIV family members to
`their substrate specificities will be valuable in designing
`selective inhibitors. Arg125 in propeller blade 2 contacts
`inhibitors and substrates [11,72–77]. Arg125 is conserved
`in DPIV in all species from bacteria to human, but is
`absent from DP8 and DP9. The sequence motif of the
`Glu205-containing α-helix that limits DPIV to cleavage
`after the second amino acid of substrates [11] is conserved
`in all six proteins of the DPIV gene family in all species
`[1]. The structures of FAP, DP8, DP9, DPL1 and DPL2
`can now be predicted using DPIV as a template, but
`solving their structures will provide precise information
`concerning mechanisms of substrate specificity and pro-
`tein–protein interaction in this gene family.
`
`SOLUBLE DPIV
`
`The naturally occurring dimeric soluble form of DPIV
`exists in extracellular fluids, including serum, seminal
`fluid, saliva and bile. The richest natural sources of soluble
`DPIV are seminal fluid [88,89] and kidney [75].
`Altered serum DPIV levels occur in many diseases
`[14,90]. ADA binding assays are specific for CD26 and
`show that at least 90 % of serum DPIV activity on H-Gly-
`Pro is derived from CD26 [10]. The origin(s) of the
`remaining DPIV activity is unknown, but could include
`FAP [39]. The naturally occurring soluble form of CD26
`starts at Ser39 [10] and is derived from cell-surface
`CD26 [35]. The mechanism of shedding sCD26 (sol-
`uble CD26) from the cell surface is unknown, but is
`thought to be proteolytic [47].
`The level of DPIV in serum from healthy adults is
`−1 · ml−1, which cor-
`
`about 22 nmol p-nitroanilide · min
`responds to approx. 7 µg/ml. It may derive from all
`DPIV-expressing cells that contact blood, generally endo-
`thelial cells and lymphocytes. Serum DPIV levels
`generally decrease in disease unless liver injury or ex-
`
`C(cid:1) 2005 The Biochemical Society
`
`tensive lymphocyte proliferation is involved [14]. Serum
`CD26 measurement has no diagnostic value by itself.
`However, it may be useful in combination with other
`disease makers such as with α-l-fucosidase for colon
`cancer [91]. Perhaps the function of serum DPIV is to
`inactivate and thereby prevent systemic effects from bio-
`active peptides following their local production.
`
`Functions of soluble DPIV
`The functions of sCD26 have been studied only in
`relation to T-cell proliferation. Exogenous srhCD26 (re-
`combinant human sCD26) is not itself mitogenic, but
`is able to enhance proliferation of activated peripheral
`blood lymphocytes [59,92]. Concentrations of srhCD26
`greater than the optimum (0.5 µg/ml) diminish this effect
`[92]. We have obtained concordant data using uninfected
`subjects and Herpes simplex virus as the recall antigen [9].
`Thus sCD26 exerts regulatory effects on in vitro T-cell
`memory responses, generally decreasing strong responses
`and increasing weak responses.
`sCD26 is taken up via binding to mannose 6-phosphate
`+
`monocytes, leading to an increase in
`receptor by CD14
`their antigen-presenting activity that involves calveolin-1
`binding [93]. DPIV activity probably does not contribute
`as the DPIV inhibitor concentrations needed to diminish
`T-cell proliferation in vitro are well above the Ki. More-
`over, we found that srhCD26 rendered catalytically inert
`by point mutation produced the same effects on T-cell
`proliferation as wild-type srhCD26 [9].
`
`FUNCTIONS OF DPIV
`
`CD26/DPIV in activated and
`memory T-cells
`Both the percentage of cells expressing CD26 and the
`number of molecules/cell are increased following activ-
`ation of T-cells [94]. The strongest lymphocytic CD26
`expression is found on cells co-expressing high densities
`of other activation markers such as CD25, CD71,
`+
`CD45RO and CD29 [95,96]. The CD26brightCD4
`popu-
`+
`+
`CD29
`memory/helper subset.
`lation is the CD45RO
`The immunological synapse contains cholesterol-rich
`rafts and at least some CD26 lies in these rafts [97].
`The signal transduced by CD26 overlaps with the T-cell
`receptor/CD3 pathway, increasing tyrosine phosphoryl-
`ation of p56lck, p59fyn, ZAP-70, phospholipase C-γ ,
`MAPK (mitogen-activated protein kinase) and c-Cbl in
`that pathway (for review, see [14]).
`The co