Báo cáo khoa học: Epitope analysis of the rat dipeptidyl peptidase IV monoclonal antibody 6A3 that blocks pericellular fibronectin-mediated cancer cell adhesion pot

Epitope analysis of the rat dipeptidyl peptidase IV monoclonal antibody 6A3 that blocks pericellular fibronectin-mediated cancer cell adhesion Ting-Ting Hung1,*, Jun-Yi Wu1,*, Ju-Fang Liu1 and Hung-Chi Cheng1,2 Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan National Cheng Kung University Hospital Cancer Center, National Cheng Kung University, Tainan, Taiwan Keywords dipeptidyl peptidase IV; epitope mapping; monoclonal antibody; polymeric fibronectin; steric hindrance Correspondence H.-C Cheng, Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan Fax: +886 274 1694 Tel: +886 235353 E-mail: hungchi@mail.ncku.edu.tw *These authors contributed equally to this work (Received June 2009, revised 27 July 2009, accepted September 2009) doi:10.1111/j.1742-4658.2009.07352.x We previously showed that the rat dipeptidyl peptidase IV (rDPP IV) monoclonal antibody (mAb) 6A3 greatly inhibits the pericellular polymeric fibronectin-mediated metastatic cancer cell adhesion to rDPP IV L311QWLRRI in rDPP IV has been proposed as the putative fibronectinbinding site However, the inhibitory mechanism of 6A3 has been elusive Epitope mapping of 6A3 may help to understand the interaction between fibronectin and rDPP IV In the present study, we showed that 6A3 species-specifically recognized rDPP IV but inhibited fibronectin ⁄ rDPP IVmediated cell adhesions of various cancer types and species, which was independent of rDPP IV enzymatic activity The 6A3 epitope was stably exposed in both native and denatured rDPP IV On the basis of the resolved structures and the species variations in DPP IV sequences, we finely mapped the 6A3 epitope to a surface-exposed Thr331-dependent motif D329KTTLVWN, only 11 amino acids away from L311QWLRRI on the same plane as the fifth b-propeller blade The functionality of 6A3 epitope in rDPP IV was ultimately demonstrated by the ability of 6A3-recognizable fragments to interfere with the inhibitory effect of 6A3 on fulllength rDPP IV binding to pericellular polymeric fibronectin On the basis of structural analysis, and the fact that the preformed fibronectin fragment ⁄ rDPP IV complex was co-immunoprecipitated by 6A3 and fixing the rDPP IV structure with paraformaldehyde did not avert the inhibitory effect, the mechanism of 6A3 inhibition may not be the result of complete competition or conformational change Structured digital abstract l MINT-7261577: DppIV (uniprotkb:P14740) binds (MI:0407) to FNIII14 (uniprotkb:P04937) by anti bait coimmunoprecipitation (MI:0006) Introduction Specific endothelial ⁄ cancer cell–cell adhesions dictate organ-preference cancer metastases [1] We previously demonstrated that the blood-borne cancer cells become arrested in the lung vasculature via adhesion between the lung endothelial adhesion receptor dipeptidyl peptidase IV (DPP IV) and pericellular polymeric Abbreviations ADA, adenosine deaminase; DPP IV, dipeptidyl peptidase IV; FACS, fluorescence-activated cell sorting; FN, fibronectin; hDPP, human dipeptidyl peptidase; Hm, human mutant; mAb, monoclonal antibody; MBP, maltose-binding protein; mDPP, mouse dipeptidyl peptidase; Mm, mouse mutant; pAb, polyclonal antibody; PFD, paraformaldehyde; polyFN, polymeric fibronectin; rDPP, rat dipeptidyl peptidase 6548 FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS T.-T Hung et al fibronectin (polyFN) [1–5] This cancer cell adhesion to DPP IV can be greatly blocked by monoclonal antibody (mAb) 6A3 directed against rat DPP IV (rDPP IV) [2,3] DPP IV is a homodimeric type II transmembrane serine protease with multiple functions [6] For example, it plays roles in proteolytic cleavage and inactivation of glucagon-like peptide to maintain glucose homeostasis [7] and in inactivating other bioactive peptides and various cytokines for sustaining normal physiological conditions [8] Much of the drug development carried out with respect to type II diabetes has aimed to effectively block the enzymatic activity of DPP IV [9] However, the binding between DPP IV and pericellular polyFN appears to be independent of this enzymatic activity [3,4] Indeed, the putative FN-binding site in DPP IV has been proposed as a seven amino acid sequence, L311QWLRRI, located in the fifth blade of the b-propeller domain, which is structurally distinct from the a ⁄ b hydrolase domain that harbors the catalytic triad [6,10] FN, a large, multifunctional glycoprotein, is secreted as the soluble, dimeric plasma FN or as the insoluble polyFN PolyFN is either deposited in the extracellular matrix or assembled on the surfaces of metastatic cancer cells in the lung to which the endothelial adhesion receptor DPP IV binds [11,12] A consensus DPPIV-binding motif in FN has been located in the 13th, 14th and 15th FN type III repeats [1] Recently, we demonstrated that the assembly of hematogenous cancer pericellular polyFN is regulated by protein kinase Ce [5] Several mAbs against DPP IV have been found to interrupt extracellular matrix attachment and to arrest the cell cycle of DPP IV-expressing cancer cells [13,14] One of these mAbs was engineered into humanized mAb and exerted an inhibitory effect on tumor growth in a xenograft model [15] Before considering 6A3 as a base for designing anti-adhesion drugs [16,17], we need to better understand how 6A3 exerts its inhibitory effect on the interaction between DPP IV and polyFN For example, although metastatic cancer cells of various species adhere to DPP IV [1], it is unclear whether the inhibitory effect of 6A3 is also a cross-species phenomenon in preventing cancer cell adhesion to DPP IV Although 6A3 strongly inhibits DPP IV ⁄ FN binding, the inhibitory mechanism of 6A3 still remains elusive Furthermore, although DPP IV enzymatic activity plays such important roles in many physiological functions [18], it is not known whether 6A3-binding affects this catalytic activity Moreover, because the structural stabilities of eptiopes recognized by mAbs are indispensable to the drug efficacy [19,20], it is important to examine the stability of the 6A3 epitope Epitope of monoclonal DPP IV antibody Epitope mapping of 6A3 may be the most direct approach for answering the above questions [21] In the present study, we first show that the inhibitory effect of 6A3 on rDPP IV ⁄ FN is a general phenomenon 6A3 species specifically recognized a structurally stable epitope in both native and denatured rDPP IV, independent of DPP IV enzymatic activity, which is suggestive of a noncompetitive inhibition mechanism The 6A3 epitope in full-length rDPP IV was finely mapped to the surface-exposed Thr331dependent eight amino acid sequence D329KTTLVWN near the putative FN-binding site, L311QWLRRI, and only 11 amino acids apart According to structural analysis and co-immunoprecipitation of the preformed FN fragment ⁄ rDPP IV complex with 6A3, we suggest that the competitive mechanism is not responsible for 6A3 inhibition Preventing conformational change of the rDPP IV structure did not avert the inhibitory effect of 6A3 These observations suggest that the inhibitory effect of 6A3 is a result of steric hindrance rather than conformational change Results The inhibitory effect of 6A3 on rDPP IV ⁄ cancer pericellular polyFN-mediated cell adhesion is a general phenomenon To determine whether the 6A3 inhibition is a general phenomenon, we selected another two metastatic cancer cell lines, human breast cancer cells (MDA-MB231) and mouse melanoma cells (B16F10), for rDPP IV adhesion assays Similar to MTF7, MDA-MB-231 and B16F10 exhibited high adhesion activities to rDPP IV, which was specifically abolished by 6A3 in a dosedependent manner (Fig 1A) The adhesion activities exerted by these cells of various species and cancer types were also greatly blocked by a polyclonal antibody (pAb) that recognizes FN of multiple species [3] (and data not shown) We next reconfirmed the inhibitory effect of 6A3 on binding between rDPP IV and pericellular polyFN After incubation with 6A3, soluble rDPP IV lost the ability to bind to immobilized MTF7 cell surfaces to the same degree that polyclonal anti-FN serum inhibited rDPP IV-binding (Fig 1B) These data suggest that the decreased adhesion activity by 6A3 was indeed a result of the blockage of binding between rDPP IV and pericellular polyFN Biochemically, we also demonstrated that the same FN co-immunoprecipitated with polyclonal anti-FN serum was affinity-precipitated by DPP IV-conjugated beads, which was abrogated by preincubating the beads with 6A3 (Fig 1C) This rDPP IV-precipitated FN was FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS 6549 Epitope of monoclonal DPP IV antibody T.-T Hung et al demonstrated to be pericellular polyFN because it was removed from the cell surfaces by pretreating the [35S]methionine metabolically labeled cells with a-chymotrypsin (Fig 1C) These results suggest that 6A3 directly inhibits binding of rDPP IV to pericellular polyFN of cancer cells % adhesion activity A Cells alone Control 6A3 50 µg·mL–1 6A3 100 µg·mL–1 6A3 300 µg·mL–1 F7 23 B- MT M AMD 10 6A3 species-specifically recognizes both native and denatured rDPP IV without interfering with its enzymatic activity 6F B1 DPP IV binding (O.D 492 nm) B IV ne l 50 300 50 300 PP alo ntro –1 ) –1 ) o DP IV Co L N P + mL g·m (µg· -D P IV o (µ b Bi -DP A3 pA o +6 N Bi IV αF + PP IV o-D PP Bi -D o Bi C DPP IV-conjugated Affi-Gel l tro on – + N αF IP: A3 +C +6 – + – + α-chymotrypsin FN monomer 180 kDa Fig 6A3 inhibits pericellular FN-mediated lung-metastatic cancer cell adhesion (A) rDPP IV adhesion activities of MDA-MB231, MTF7 and B16F10 (5 · 104 cells per well) in the absence or presence of various concentrations of 6A3 or 0.3 mgỈmL)1 nonimmune isotype IgG1 (control) at 37 °C for 30 were measured as described in the Experimental procedures (B) Binding of soluble biotinylated rDPP IV (2 lg) to MTF7 (5 · 104 cells per well), grown at 37 °C overnight on 96-well plates (after PFD fixation) in the absence or presence of 6A3 (50 or 300 lgỈmL)1), the same isotype mouse IgG1 (control; 300 lgỈmL)1) or polyclonal FN antibody (50 or 300 lgỈmL)1), was detected with horseradish peroxidase-conjugated streptavidin (C) Whole cell lysates from · 106 2-h recovered suspended S35-labeled MTF7 cells, pretreated without ()) or with (+) 10 lgỈmL)1 a-chymotrypsin (1 h at 37 °C), were immunoprecipitated with lg polyclonal FN antibody or pulled-down with DPP IV-conjugated Affi-Gel 10 beads in the presence of 0.3 mgỈmL)1 6A3 or control before being subjected to SDS–PAGE and radiography Note that Affi-Gel conjugated with control protein phosphorylase b did not pull down any pericellular polyFN [1] (and data not shown) and the FN monomeric bands shown in the radiography were reduced from high molecular weight pericellular polyFN by b-mecaptoethanol [3,37] 6550 We next examined the species-specificity of 6A3-recognition of DPP IV in immunoprecipitation and in immunoblotting assays We found that 6A3 strongly recognized rDPP IV but only negligibly bound to native human (h)DPP IV and mouse (m)DPP IV in immunoprecipitation assays (Fig 2A–C) In immunoblotting assays where DPP IVs were subjected to the denatured condition in SDS–PAGE, 6A3 exclusively recognized rDPP IV (Fig 2D) The ability of 6A3 to recognize both native and denatured rDPP IV suggests that the 6A3 epitope is stably exposed in full-length rDPP IV By contrast to the species-conserved FNbinding sequence, the rat-specific recognition of DPP IV by 6A3 implies that the 6A3 epitope does not totally overlap with the putative FN-binding site This possibility was further supported by the fact that, although the FNIII14 (a DPP IV-binding competent FN fragment) [1] did not to bind to the 6A3-preincubated rDPP IV (data not shown), the pre-bound FNIII14 ⁄ rDPP IV complex was co-immunoprecipitated by 6A3, even in the presence of high salt (200 mm NaCl) solution (Fig 2E) To determine whether the inhibitory effect of 6A3 interferes with the peptidase function, we set out to measure DPP IV enzymatic activity in the presence of 6A3 In line with the previous results showing that FN ⁄ DPP IV binding is independent of DPP IV catalytic activity [3], 6A3-binding of rDPP IV also had no effect on its enzymatic activity, indicating that this process may occur outside the enzymatic active site (Fig 2F) The eight-bladed b-propeller domain harbors the 6A3-binding site To identify the 6A3-binding site, we first generated recombinant maltose-binding protein (MBP)-fusion fragments of extracellular rDPP IV based on the reported structural and functional domains of the predicted version [22] and the resolved version [23] (Fig 3A) These two versions differ mainly in the numbers of blades in the b-propeller domain Although version was predicted to be seven-bladed FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS T.-T Hung et al Epitope of monoclonal DPP IV antibody A IP: B l tro on 6A3 C U3 C PP l tro IgG on Rat C 6A IP: 110 kDa IB: 6A3 Mouse Kidney Extract Rat Kidney Extract C IP: IV PP hD mα 6A D IV l PP tro αhD n Co m 6A Mo 110 kDa IB: 6A3 hDPP IV-Jurkat 1.00 F WT 200 mM MT O.D 405 nm 150 mM MT n e ma t Ra Hu us E NaCl MBPFNIII14 110 kDa IB: rαmDPP IV DPP IV 110 kDa IB: IF7 Mock-Jurkat IV mD rα WT 55 kDa IP: 6A3 IB: MBP 110 kDa IP: 6A3 IB: 6A3 0.75 0.50 0.25 0.00 e lon P DP a IV P DP + IV 6A Fig 6A3 specifically recognizes both native and denatured rat DPP IV independently of its enzymatic activity (A) Three hundred microliters of Sprague-Dawley (SD) rat kidney extracts (one kidney per milliliter being homogenized, as previously described [3]) was subjected to immunoprecipitation at °C overnight with lg of purified control mouse IgG1, 6A3 mAb or CU31 pAb and then to immunoblotting with 6A3 (B) Three hundred microliters of C57BL6 mouse kidney extracts (two kidneys per milliliter [3]) was subjected to immunoprecipitation at °C for overnight with lg of purified 6A3, control, mouse DPPIV (mDPP IV) mAb from rat or rat IgG, and then to immunoblotting with mDPP IV mAb from rat (C) Three hundred microliters of human Mock-Jurkat or DPP IV-Jurkat cell lysates (1 mgỈmL)1 from · 107 cells) was subjected to immunoprecipitation at °C for overnight with lg of 6A3, control, or human DPP IV (hDPP IV) mAb from mouse and then to immunoblotting with hDPP IV mAb IF7 from mouse (D) Mouse, rat kidney extracts or DPP IV-Jurkat cell lysates (made as described in A, B, and C) were subjected to immunoprecipitation at °C for overnight with lg of 6A3, mDPP IV mAb from rat or hDPP IV mAb from mouse, respectively, and then to immunoblotting with 6A3 (E) rDPP IV preincubated with DPP IV-binding competent FN fragment MBP-FNIII14 (WT) or with DPP IV-binding sequence-mutated MBP-FNIII14 (MT) [1] were co-immunoprecipitated by 6A3 in the presence of 150 or 200 mM NaCl and then subjected to immunoblotting with a-MBP pAb (upper panel) or with 6A3 (lower panel) (F) DPP IV enzymatic activities were measured in the absence or presence of 6A3 as described in the Experimental procedures (amino acids 131–502) [22], version has been shown to be eight-bladed (amino acids 49–502) [24] (Fig 3A) The reason that we took the predicted version into consideration was that other members of the propyl oligopeptidase family to which DPP IV belongs contain a seven-bladed b-propeller domain and that deleting portions of the predicted N-terminal a ⁄ b-hydrolase domain (amino acids 29–130; the first propeller blade of version 2) resulted in a loss of enzymatic activity and protein integrity, implying that this region, although structurally belonging to the eight-bladed b-propeller domain, may be functionally involved in a ⁄ b-hydrolase catalytic activity [22] Because the a ⁄ b-hydrolase domains of the two versions both include certain portions of the N- and C-terminal regions, we constructed two chimeric MBP-fusion fragments, A+D and G+D, to cover these domains (Fig 3A) After purification, we found that all but fragment A were endogenously degraded (Fig 3B), which is not uncommon in bacterial systems expressing mammalian proteins [25] The high structural stability of fragment A explains why it is required for stabilizing the a ⁄ b-hydrolase structure and enzymatic activity even within the b-propeller domain [22,24] From the results of both immunoblotting and immunoprecipitation, we found that fragments B and C, and extracellular full-length, were recognized by 6A3 (Fig 3B), suggesting that the 6A3 epitope resides within the last seven propeller blades in the eight-bladed b-propeller domain The fragments containing the a ⁄ b-hydrolase FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS 6551 Epitope of monoclonal DPP IV antibody T.-T Hung et al A 29 131 503 A B 767 D Ver 17 3749 503 G C 767 D Ver B kDa A 100 75 50 100 75 50 100 75 50 D A C B * + D G + DexFL * C kDa IB:6A3 * * * * * * Hm IB:6A3 50 * IB:MBP B-166 E B-166 kDa WT 75 IP:6A3 IB:MBP * * D 29 27 B (3 15 16 BB- B- ) 58 ~3 * I 31 Mm kDa 75 T3 T 34 V3 75 IB:6A3 IB:6A3 Fig 6A3 recognizes the linear Thr331-dependent eight-amino acid epitope in the eight-bladed b-propeller domain (A) Scheme of rDPP IV molecular dissecting for constructing MBP-fusion proteins based on the predicted version [22] and resolved version [24] of the reported structural and functional domains In both versions, black rectangles represent intracellular domains and white rectangles transmembrane domains Although fragments A and G belong to N-terminal portions of a ⁄ b-hydrolase domains in both versions of DPP IV, fragment D is the invariant C-terminal a ⁄ b-hydrolase domain Fragment B represents the seven-bladed b-propeller domain of version and fragment C is the eight-bladed b-propeller domain of version Chimera fragments A+D and G+D represent the complete a ⁄ b-hydrolase domains exFL represents the extracellular full-length DPP IV fragment (B) 6A3 immunoblotting (upper panel), 6A3 immunoprecipitation followed by anti-MBP immunoblotting (middle panel) and anti-MBP immunoblotting (lower panel) for 50 ng of amylose agarose bead-purified MBP-fusion fragments as described in the Experimental procedures As a result of general protein degradations, all fragments were loaded so that the amounts of full-length proteins were approximately equal Note that full-length fragments are indicated by an asterisk to the right of the individual protein bands (C) 6A3 immunoblotting of B-159, B-166, B-272 and B(329–358) (for detailed positioning, see Fig S2B) Note that binding of B-166 and B(329–358) to 6A3 indicates that the eight-amino acid sequence (Asp329 to Asn336) within the 106-amino acid region between Val231 and Asn336 of the eight-bladed b-propeller domain harbors the 6A3 epitope (D) 6A3 immunoblotting of T331I and V334T mutants (for detailed positioning, see Fig S2D) (E) 6A3 immunoblotting of wild-type (WT), Hm and Mm (for detailed positioning, see Fig S2E) domain were not recognized by 6A3, which is consistent with the results showing that 6A3-binding did not interfere with DPP IV enzymatic activity (Fig 2E) 6A3-binding epitope is located within a Thr331-dependent linear eight-amino acid region near the proposed FN-binding site Because 6A3 specifically recognized rDPP IV (Fig 2A–D), we next compared the aligned secondary structures and sequences of the three species and selected three nonconserved rDPP IV regions to test 6A3-binding (Fig S1A) Unfortunately, amino acid 6552 swapping in these three regions in rDPP IV with those of hDPP IV did not affect 6A3 recognition (Fig S1B) Therefore, we began with a serial deletion scheme and generated two truncated B fragments: B-144 and B-272 6A3 only bound to B-144 but not to B-272 (Fig S2A), suggesting that the 128 amino acid region between Val231 and Ala358 contains the 6A3 epitope To further narrow down the 6A3 epitope, we then identified two nonconserved clusters within this 128 amino acid region (Fig S2B) Because the first cluster harboring B ⁄ c was invalid for 6A3-binding (Fig S1), we focused on the second cluster, according to which we generated B-159 and B-166, and a 28 amino acid FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS T.-T Hung et al Epitope of monoclonal DPP IV antibody A B T331 T331 T331 R315 316 I317 R D C T331 T331 E Cells alone Control % adhesion activity Fig 6A3 epitope is in close proximity with the proposed FN-binding site L311QWLRRI but is far from the enzymatic active site (A) Ribbon representation for the eight-bladed b-propeller domain (dark yellow) of the resolved rDPP IV (Protein Data Bank code: 2GBC) The view is from the top of the propeller domain with individual propeller blades being alphabetically numbered The Thr331-dependent eight-amino acid 6A3 epitope located at upper portion of the fifth propeller blade is labeled in green with Thr331 highlighted in red The proposed FN-binding sequence L311QWLRRI in cyan is located at lower portion of the fifth propeller blade The rest of the fifth propeller blade is labeled in blue (B) Side view of the eight-bladed b-propeller domain as presented in ribbon mode (upper panel) or in surface contour mode (lower panel) (C) Top view and (D) side view of the surface contour image for the entire rDPP IV structure The catalytic triad (S631, D709 and H741) labeled in marine blue can be visualized through the narrower channel that is formed by the eight b-propeller blades (the white arrow) where the catalytic dipeptide products leave (C), or through the open active site cleft into which DPP IV substrates may enter (the yellow arrow) (D) All the views were rendered with PYMOL 0.99 and color representations are the same as those shown in (A) (E) rDPP IV-coated wells were first pre-treated with 2% PFD at room temperature for 30 Percent specific adhesion activities of MTF7 were then measured similar to Fig 1A 6A3 50 µg·mL–1 6A3 100 µg·mL–1 6A3 300 µg·mL–1 MTF7 cell adhesion to PFD-fixed DPP IV fragment B (amino acids 329–358) (Fig S2B) We found that 6A3 recognized all of them (Figs 4C and S2C), indicating that the 6A3 epitope is located within the linear eight-amino acid region between Asp329 and Asn 336 This conclusion was well supported by the fact that swapping the eight-amino acid sequence in B-166 (wild-type) with that of human (Hm) or mouse (Mm) (Fig S2D) lead to a loss of their 6A3-binding abilities (Fig 3D) We then constructed B-166 mutants, T331I and V334T, where the only amino acids distinct from those in Mm were individually swapped with Ile and Thr (Fig S2E) The results demonstrate that 6A3 recognizes V334T but T331I (Fig 3E), strongly suggesting that the 6A3-binding epitope lies within a Thr331-dependent eight-amino acid region In the compiled ribbon and surface representations of the resolved DPP IV structure, this region is located at the fifth propeller blade of the eight-bladed propeller domain (Fig 4A, B) Consistently, it resides at the FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS 6553 Epitope of monoclonal DPP IV antibody T.-T Hung et al outer surface of the eight-bladed propeller domain, which is totally opposite to the inner surface of the a ⁄ b-hydrolase domain (Fig 4C, D) Interestingly, this 6A3 epitope and the proposed FN-binding site, L311QWLRRI, both belong to exactly the same propeller blade, and are only 11 residues apart (Fig 4A, B) This structural proximity suggests that the inhibitory effect of 6A3 is a result of steric hindrance To rule out the remote possibility that the inhibitory effect is a result of conformational change, we used paraformaldehyde (PFD), which forms methylene bridges between any two residues with an amino group in their side chains [26], to prevent conformational change of the DPP IV structure 6A3 was still able to inhibit the cancer cell adhering to the PFD-fixed DPP IV (Fig 4E), further supporting the idea that 6A3 most likely exerts steric hindrance in the inhibition of FN ⁄ DPP IV binding The Thr331-dependent linear eight-amino acid region mediates the 6A3-binding in full-length DPP IV Although we identified the 6A3 epitope in a loop area of the rDPP IV molecule according to the in silico analysis (Fig 4A–D), we did not know whether this epitope is available for 6A3-binding in the full-length rDPP IV structure Therefore, we first ectopically expressed full-length rDPP IV on HEK293 cell surfaces After preincubation with 6A3, B-166 wild-type but not Hm, Mm or T331I, greatly inhibited 6A3 immunofluorescent staining of rDPP IV-expressing HEK293 cells (Fig 5A) Next, purified full-length rDPP IV together with reference mouse IgG were immunoblotted with 6A3 that was preincubated with B-166 wild-type, Hm or Mm Normalized with the 55 kDa IgG heavy chain, the wild-type demonstrated a greater blocking effect compared to the other two mutants (Fig 5B) Consistently, 6A3 ⁄ rDPP IV immunoprecipitation was inhibited in the presence of the wild-type fragment (Fig 5C) The essential role of Thr331 in 6A3-recognition was reconfirmed by the results showing that T331I failed to inhibit 6A3 ⁄ rDPP IV binding in immunofluorescent staining, immunocytochemistry, immunoblotting and immunoprecipitation assays (Fig 5A–C) To further corroborate the specific binding between 6A3 and its epitope in rDPP IV, we performed competition assays between DPP IV protein and the T331I orV334T B-166 mutant peptides for 6A3 binding T331I, but not V334T, blocked the inhibition of cancer cell adhesion to DPP IV and soluble DPP IV binding to polyFN-expressing cancer cells (Table 1) Taken together, these data indicate that the 6554 Thr331-dependent linear eight-amino acid region indeed mediates 6A3-binding in full-length DPP IV Discussion Specific adhesion between cancer cells and endothelia contributes to organ-preference cancer metastasis [1,3] We previously generated rDPP IV mAb 6A3 that blocks the DPP IV ⁄ FN-mediated adhesion of lungmetastatic cancer cells in the lungs [3] In the present study, we finely mapped the 6A3 epitope and analyzed the inhibitory mechanism of 6A3 One of the three mechanisms, namely competition, conformational change and steric hindrance, may explain the inhibitory effect of 6A3 The distinct species-specificities of the 6A3 epitope and the FN-binding and the co-immunoprecipitation of preformed FN ⁄ DPP IV complex with 6A3 (Fig 2E) make it less likely that 6A3 inhibits the FN ⁄ rDPP IV binding via competition Nevertheless, before the putative FN-binding sequence is firmly validated, we cannot totally rule out that both binding sites partially overlap On the other hand, most residues in the putative FN-binding sequence L311QWLRRI [10] are apparently buried in the propeller core except the R315RI (Fig 4B, lower panel) FN remains to bind to the PFD-fixed DPP IV, where no conformation can be changed to expose the buried residues (Fig 4E), indicating that additional residues, other than R315RI, may contribute to the FN-binding However, the DPP IV fragment B, which contains L311QWLRRI, did not exhibit significant FN-binding activity (data not shown), suggesting either that the true FN-binding site is located outside the fragment B and affected by 6A3 via conformational change or that L311QWLRRI is part of the true-binding site, the presentation of which might only be supported by the full-length DPP IV structure and affected by 6A3 via steric hindrance Although the exact inhibitory mechanism of 6A3 cannot be proclaimed for certain, the preservation of the inhibitory effect on FN binding to PFD-fixed DPP IV by 6A3 (Fig 4E) appears to disfavor the effect of conformational change By contrast, the R315RI together with several nearby sequences appear to belong to a discrete surface-exposed motif, which is located near the 6A3 epitope at a distance of approxi˚ mately 30 A (Fig S3A, C), likely representing a discontinuous FN-binding domain and favoring the mechanism of steric hindrance Interestingly, based on structural comparison and superimposition of rDPP IV (2GBC) and hDPP IV (1NU6), this putative FN-binding motif appears to be relatively conserved FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS T.-T Hung et al Epitope of monoclonal DPP IV antibody A Fig 6A3-recognizable DPP IV fragments block the bindings of 6A3 to both native and denatured full-length DPP IV (A) FACS analyses of ectopically full-length rDPP IV-expressing HEK293 cells by staining the cells with 6A3 preincubated with MBP, B-166 Hm, Mm, wild-type (WT), T331I mutants or the same isotype mouse IgG (control) as described in the Experimental procedures The dot plots were generated with PE-Cy5 fluorescent intensities (representing 6A3-binding abilities) against FSC-H The arbitrary fluorescent intensities (A.F.I.) of the DPP IV-positive HEK293 cells are calculated as average fluorescent intensity · total DPP IV-positive HEK293 cell numbers for each staining Representative images of each immunofluorescent staining results are inserted inside each corresponding dot plot (B) Immunoblotting of the purified full-length rDPP IV and the mouse IgG heavy chain as a quantitative reference protein with 6A3 preincubated with the same MBP-fusion DPP IV fragments as in (A) (C) 6A3 immunoblotting of the immunoprecipitates where 0.2 lg of purified fulllength DPP IV was immunoprecipitated with 0.5 lg of 6A3 preincubated with lg of WT, Hm, T331I or V334T mutants Note that, although the 6A3 binding-competent WT and V334T blocked rDPP IV immunoprecipitation by 6A3, Hm and T311I, which were incapable of binding to 6A3, did not exert this inhibitory effect Binding of 6A3 preincubated with B-166 mutants Control (A.F.I = 1) MBP (A.F.I = 20.26) Hm (A.F.I = 20.26) Mm (A.F.I = 20.46) WT (A.F.I = 8.75) T331I (A.F.I = 22.53) IB: 6A3 preincubated with B-166 mutants B P MB Hm Mm WT 31I T3 rDPP IV Ig heavy chain C IP: 6A3 preincubated with B-166 mutants 34T 31I Hm WT T3 V3 (Fig S3A–D), implying that this motif may be a more appropriate FN-binding domain General application of antigen-specific mAbs in cancer therapy is highly anticipated [27] For example, bevacizumab is generally used to inhibit angiogenesis in treating patients with various cancer types [28] In line with this concept, 6A3 inhibits the rDPP IV-binding of rat and human breast cancer cells, mouse melanoma cells (Fig 1A) and several other types of cancer cells (data not shown) However, the failure of 6A3 in recognizing hDPP IV makes it impossible to use for direct application in cancer therapies Nevertheless, the superimposition of rDPP IV and hDPP IV reveals that the overall conformations of the two molecules are rather similar, with subtle differences as a result of amino acid side chain variations (Fig S3D) We speculate that mAbs generated against peptides containing the 6A3 epitope-corresponding sequence D331ESSGRWN in hDPP IV may be evaluated in the future rDPP IV B-166 mutants for therapeutic purposes One important consideration is that, before clinical application, pretests of these mAbs in animal models are required Accordingly, the antigenic peptide sequences should be carefully determined so that the generated mAbs will also recognize rDPP IV and ⁄ or mDPP IV and block lung metastases of cancer cells in these animals To serve as a safe drug, a potential mAb is expected to exert its effect without causing adverse consequences of physiological functions [29] DPP IV is involved in adenosine deaminase (ADA)-mediated T cell proliferation [6] Antibodies recognizing epitopes including residues Leu294 and Val341 were found to have inhibitory effects on ADA-binding of hDPP IV [30] These epitopes not overlap with the 6A3 epitope-corresponding sequence in hDPP IV, implying that the mAb generated against this hDPP IV sequence should not interrupt ADA binding to hDPP IV Although monoclonal DPP IV antibody 4D10 arrests the cell cycle FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS 6555 Epitope of monoclonal DPP IV antibody T.-T Hung et al Table 6A3-recognizable DPP IV fragments competitively neutralize the 6A3 inhibitory effects on cancer cell adhesion to DPP IV Status of 6A3 DPP IV binding (A492)a DPP IV adhesion (%)b No 6A3 1.76 ± 0.18 6A3 + B-166 V334T 0.67 ± 0.06c 6A3 + B-166 T331I 1.60 ± 0.20 79.1 ± 6.85 11.2 ± 6.49c 69.23 ± 14.75 a Binding of biotinylated rDPP IV to MTF7 in the absence or presence of 6A3 preincubated with B-166 mutants b rDPP IV% specific adhesion activities of MTF7 in the absence or presence of 6A3 preincubated with B-166 mutants c Compared to the values of assays without 6A3, these values are significantly decreased (P < 0.05) progression of cancer cells and reduces in vivo tumor growth, it does not cause any apparent adverse effect in nude mice [15] It is likely that it causes distinct effects of cytotoxicity against DPP IV-expressing cancer cells and normal cells Altogether, we propose a hypothesis that a mAb raised against the 6A3 epitopecorresponding sequence in hDPP IV may functionally be unique and safe in cancer patients The surface-exposed mAb epitope must be stable enough in the circulation to resist those cancer-associated protein modification factors, such as the reactive oxygen species resulting from pro-inflammatory oxidative stress [31] and mechanical stress toward endothelia [32,33] Together with shear stress from the capillary circulation and the respiratory pressure of the lungs, all the above inflammatory factors are potent with respect to causing various degrees of conformation alterations of endothelial proteins [34] Because 6A3 is able to recognize either the native form of DPP IV in immunoprecipitation assays or the denatured form in immunoblotting assays (Fig 2A–D), the 6A3 epitope in DPP IV appears to be relatively stable This stability is further confirmed by the ability of 6A3 to recognize cell-surface expressed DPP IV either in live cells (Fig 5A) or formaldehyde-fixed tissues [2,4,35] In conclusion, we have successfully identified the epitope of the potent anti-metastatic mAb 6A3 that blocks metastatic cancer cell adhesions to rat lung endothelial DPP IV, most likely via steric hindrance The 6A3-epitope corresponding sequence in hDPP IV may potentially be used to generate functionally unique and safe monoclonal anti-metastatic sera Experimental procedures Cell lines, antibodies and reagents Rat lung-metastatic MTF7 cells, mouse B16F10 cells and human MDA-MB-231 cells were obtained from 6556 Dr B U Pauli (Cornell University, Ithaca, NY, USA) [1] They were grown in DMEM (Invitrogen, Carlsbad, CA, USA) containing 5% fetal bovine serum (FBS) Mock (Mock-Jurkat) or hDPP IV-expressing (hDPP IV-Jurkat) Jurkat cells were generous gifts from Dr C Morimoto (University of Tokyo, Tokyo, Japan) They were grown in RPMI 1640 medium (Invitrogen) containing 5% FBS mAb 6A3 and pAb CU31 were generated against rDPPIV [1] mDPP IV mAb from rat was from R&D Systems (Minneapolis, MN, USA); hDPPIV mAb from mouse was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); human FN pAb from rabbit was from Sigma (St Louis, MO, USA); rabbit polyclonal anti-MBP serum and amylase agarose beads were from New England Biolabs Inc (Ipswich, MA, USA); and IF7 mAb was a generous gift from Dr C Morimoto (University of Tokyo, Tokyo, Japan) All other reagents were purchased from Sigma [35S]methionine was from ICN Biochemicals (Irvine, CA, USA) and Affi-Gel 10 beads were obtained from Bio-Rad (Hercules, CA, USA) Plasmid construction pMAL-c2 vector and full-length rDPP IV cloned in pRCCMV vector were obtained from Dr B U Pauli [1] All MBP-fusion fragments of DPP IV were PCR-amplified and inserted into the EcoRI and HindIII sites of pMAL-c2 vector as described previously [1] Using wild-type B-166 as template, overlap extension PCR amplification was performed to generate B-166 Hm mutant, B-166 Mn mutant, B-166 T331I (T331) and B-166 V334T (V334T) as MBPfusion proteins, as described above [1] The amino acids included in the PCR-amplified fragments are described in Doc S1 Purification of MBP-fusion fragments and rDPP IV The fusion protein purification procedures have been described previously [1] Briefly, the cell lysates of Escherichia coli cells expressing various MBP-fusion proteins were passed through amylose columns and eluted with 10 mm maltose in column buffer [1] Immunoblotting with polyclonal anti-MBP serum was used to verify the purified proteins Rat lung DPP IV was purified from rat lung extracts by 6A3 immunoaffinity chromatography [3] [35S]methionine metabolic labeling of MTF7 for rDPP IV-conjugated Affi-Gel 10 affinity precipitation MTF7 Cells were first starved for methionine before addition of 50–100 lCi of [35S]methionine at 37 °C overnight Cells were recovered in DMEM containing 20% FBS as previously described [3] Before making cell lysates [36], FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS T.-T Hung et al some cells were first treated with 10 lgỈmL)1 a-chymotrypsin for 30 at 37 °C Cell lysates were then subjected to rDPP IV-conjugated Affi-Gel 10 affinity precipitation in the absence or presence of 0.3 mgỈmL)1 6A3 and then to SDS– PAGE and autoradiography The aliquots of cell lysates were also subjected to immunoprecipitation with polyclonal anti-FN serum [3] DPP IV enzymatic activity assays Purified rDPP IV (0.5 lg per assay) in the presence or absence of a two-fold molar ratio excess of 6A3 was incubated in 250 lL of assay buffer for 30 at 37 °C, followed by stopping the reaction with 750 lL of m acetate buffer and then measuring the absorption at 405 nm [4,35] Cancer cell adhesion assays rDPP IV adhesion activities of MDA-MB-231, MTF7 and B16F10 (5 · 104 cells per well) in the absence or presence of 0.3, 0.1 or 0.05 mgỈmL)1 6A3 or the same isotype IgG1 (control) were measured at 37 °C, for 30 in 96-well plates coated with 100 lgỈmL)1 purified rDPP IV as described previously [1,3,35] Immunoprecipitation and immunoblotting [35S]methionine metabolically labeled MTF7 cell lysates (1–3 mgỈmL)1) or purified MBP-fusion fragments (0.1 lgỈmL)1) were incubated with various antibodies (1– lg) The immunoprecipitates were subjected to immunoblotting, as described previously [36] Full-length rDPP IV (0.2 lg) and 0.05 lg of mouse IgG were subjected to 6A3 immunoblotting (dilution : 1000) in the presence of various MBP-fusion fragments (0.1 lg) ELISA A modified ELISA [1,3] was used to measure the binding of soluble biotinylated rDPP IV (2 lg) to MTF7 (5 · 104 cells per well) grown at 37 °C overnight on 96-well plates (after PFD fixation) in the absence or presence of 50 and 300 lgỈmL)1 polyclonal anti-FN serum, 6A3 or the same isotype mouse IgG1 (control) with horseradish peroxidase-conjugated streptavidin Immunofluorescent staining and fluorescenceactivated cell sorting (FACS) analysis rDPP IV-expressing HEK293 cells were stained with lgỈmL)1 control mouse IgG2a or 6A3 preincubated with 10 lgỈmL)1 MBP, B-166 wild-type, Mm, Hm, T331I or V334T mutants, followed by PE-Cy5-conjugated goat anti-mouse IgG and FACS analysis (detected using a FL3 Epitope of monoclonal DPP IV antibody photonmultiplier tube) with FACSCallibur (BD Biosciences, San Jose, CA, USA) [1,36] Software for DPP IV structural analysis The ribbon- and surface-representations of rDPP IV(Protein Data Bank code: 2GBC) X-ray structure was visualized with pymol, version 0.99 (http://www.pymol.org/) The superimposition of rDPP IV and hDPP IV was performed using friend 2.0 (http://ilyinlab.org/friend/Downloads php) Acknowledgements These studies were supported by National Science Council Grant NSC94-2314-B-006-122, National Science Council Grant NSC 95-2320-B-006-075-MY3, and The Program for Promoting Academic Excellence & Developing World Class Research Centers and Center of Excellence for Clinical Trial and Research in Oncology Specialty DOH-TD-B-111-004 The authors wish to thank Dr Iain C Bruce for his professional editing on our manuscript and Dr Ming-Derg Lai for his thoughtful discussion regarding the manuscript The authors also thank Ms Megan Cheng for her language assistance in manuscript writing and Mr Chien-Fang Kuo and Mr Chu-kuei Lin for their help in preparation and assembly of figures References Cheng HC, Abdel-Ghany M & Pauli BU (2003) A novel consensus motif in fibronectin mediates dipeptidyl peptidase IV adhesion and metastasis J Biol Chem 278, 24600–24607 Johnson RC, Zhu D, Augustin-Voss HG & Pauli BU (1993) Lung endothelial dipeptidyl peptidase IV is an adhesion molecule for lung-metastatic rat breast and prostate carcinoma cells J Cell Biol 121, 1423–1432 Cheng HC, Abdel-Ghany M, Elble RC & Pauli BU (1998) Lung endothelial dipeptidyl peptidase IV promotes adhesion and metastasis of rat breast cancer cells via tumor cell surface-associated fibronectin J Biol Chem 273, 24207–24215 Cheng HC, Abdel-Ghany M, Zhang S & Pauli BU (1999) Is the Fischer 344 ⁄ CRJ rat a protein-knock-out model for dipeptidyl peptidase IV-mediated lung metastasis of breast cancer? Clin Exp Metastasis 17, 609–615 Huang L, Cheng HC, Isom R, Chen CS, Levine RA & Pauli BU (2008) Protein kinase Cepsilon mediates polymeric fibronectin assembly on the surface of bloodborne rat breast cancer cells to promote pulmonary metastasis J Biol Chem 283, 7616–7627 FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS 6557 Epitope of monoclonal DPP IV antibody T.-T Hung et al Gorrell MD, Gysbers V & McCaughan GW (2001) CD26: a multifunctional integral membrane and secreted protein of activated lymphocytes Scand J Immunol 54, 249–264 Zhu L, Tamvakopoulos C, Xie D, Dragovic J, Shen X, Fenyk-Melody JE, Schmidt K, Bagchi A, Griffin PR, Thornberry NA et al (2003) The role of dipeptidyl peptidase IV in the cleavage of glucagon family peptides: in vivo metabolism of pituitary adenylate cyclase activating polypeptide-(1-38) J Biol Chem 278, 22418–22423 Silva AP, Cavadas C, Baisse-Agushi B, Spertini O, Brunner HR & Grouzmann E (2003) NPY, NPY receptors, and DPP IV activity are modulated by LPS, TNFalpha and IFN-gamma in HUVEC Regul Pept 116, 71–79 Pei Z (2008) From the bench to the bedside: dipeptidyl peptidase IV inhibitors, a new class of oral antihyperglycemic agents Curr Opin Drug Discov Devel 11, 512– 532 10 Gonzalez-Gronow M, Weber MR, Shearinf TV, Gawdi G, Pirie-Shepherdf SR & Pizzo SV (1998) Plasmin(ogen) carbohydrate chains mediate binding to dipeptidyl peptidase IV (CD 26) in rheumatoid arthritis human synovial fibroblasts Fibrinolysis and Proteolysis 12, 366–374 11 Mao Y & Schwarzbauer JE (2005) Fibronectin fibrillogenesis, a cell-mediated matrix assembly process Matrix Biol 24, 389–399 12 Wierzbicka-Patynowski I & Schwarzbauer JE (2003) The ins and outs of fibronectin matrix assembly J Cell Sci 116, 3269–3276 13 Inamoto T, Yamochi T, Ohnuma K, Iwata S, Kina S, Inamoto S, Tachibana M, Katsuoka Y, Dang NH & Morimoto C (2006) Anti-CD26 monoclonal antibodymediated G1-S arrest of human renal clear cell carcinoma Caki-2 is associated with retinoblastoma substrate dephosphorylation, cyclin-dependent kinase reduction, p27(kip1) enhancement, and disruption of binding to the extracellular matrix Clin Cancer Res 12, 3470– 3477 14 Ho L, Aytac U, Stephens LC, Ohnuma K, Mills GB, McKee KS, Neumann C, LaPushin R, Cabanillas F, Abbruzzese JL et al (2001) In vitro and in vivo antitumor effect of the anti-CD26 monoclonal antibody 1F7 on human CD30+ anaplastic large cell T-cell lymphoma Karpas 299 Clin Cancer Res 7, 2031–2040 15 Inamoto T, Yamada T, Ohnuma K, Kina S, Takahashi N, Yamochi T, Inamoto S, Katsuoka Y, Hosono O, Tanaka H et al (2007) Humanized anti-CD26 monoclonal antibody as a treatment for malignant mesothelioma tumors Clin Cancer Res 13, 4191–4200 16 Lafrenie RM, Buckner CA & Bewick MA (2007) Cell adhesion and cancer: is there a potential for therapeutic intervention? Expert Opin Ther Targets 11, 727–731 17 Simmons DL (2005) Anti-adhesion therapies Curr Opin Pharmacol 5, 398–404 6558 18 Flatt PR, Bailey CJ & Green BD (2008) Dipeptidyl peptidase IV (DPP IV) and related molecules in type diabetes Front Biosci 13, 3648–3660 19 Matsoukas J, Apostolopoulos V, Kalbacher H, Papini AM, Tselios T, Chatzantoni K, Biagioli T, Lolli F, Deraos S, Papathanassopoulos P et al (2005) Design and synthesis of a novel potent myelin basic protein epitope 87-99 cyclic analogue: enhanced stability and biological properties of mimics render them a potentially new class of immunomodulators J Med Chem 48, 1470–1480 20 Wilson BE, Sun S, Ozturk M & Wands JR (1991) Stability of monoclonal antibody-defined epitopes J Immunol Methods 139, 55–64 21 May C, Doody JF, Abdullah R, Balderes P, Xu X, Chen CP, Zhu Z, Shapiro L, Kussie P, Hicklin DJ et al (2005) Identification of a transiently exposed VEcadherin epitope that allows for specific targeting of an antibody to the tumor neovasculature Blood 105, 4337–4344 22 Ajami K, Abbott CA, Obradovic M, Gysbers V, Kahne T, McCaughan GW & Gorrell MD (2003) Structural requirements for catalysis, expression, and dimerization in the CD26 ⁄ DPIV gene family Biochemistry (Mosc) 42, 694–701 23 Longenecker KL, Stewart KD, Madar DJ, Jakob CG, Fry EH, Wilk S, Lin CW, Ballaron SJ, Stashko MA, Lubben TH et al (2006) Crystal structures of DPP-IV (CD26) from rat kidney exhibit flexible accommodation of peptidase-selective inhibitors Biochemistry (Mosc) 45, 7474–7482 24 Gorrell MD (2005) Dipeptidyl peptidase IV and related enzymes in cell biology and liver disorders Clin Sci (Lond) 108, 277–292 25 Sun G & Budde RJ (1995) A modified pGEX expression system that eliminates degradation products and thrombin from the recombinant protein Anal Biochem 231, 458–460 26 Dapson RW (1993) Fixation for the 1990’s: a review of needs and accomplishments Biotech Histochem 68, 75– 82 27 Reichert JM & Valge-Archer VE (2007) Development trends for monoclonal antibody cancer therapeutics Nat Rev Drug Discov 6, 349–356 28 Miller K, Wang M, Gralow J, Dickler M, Cobleigh M, Perez EA, Shenkier T, Cella D & Davidson NE (2007) Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer N Engl J Med 357, 2666–2676 29 Swann PG, Tolnay M, Muthukkumar S, Shapiro MA, Rellahan BL & Clouse KA (2008) Considerations for the development of therapeutic monoclonal antibodies Curr Opin Immunol 20, 493–499 30 Gorrell MD, Abbott CA, Kahne T, Levy MT, Church WB & McCaughan GW (2000) Relating structure to function in the beta-propeller domain of dipeptidyl pep- FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS T.-T Hung et al 31 32 33 34 35 36 tidase IV Point mutations that influence adenosine deaminase binding, antibody binding and enzyme activity Adv Exp Med Biol 477, 89–95 Dal Negro RW, Visconti M, Micheletto C & Tognella S (2008) Changes in blood ROS, e-NO, and some pro-inflammatory mediators in bronchial secretions following erdosteine or placebo: a controlled study in current smokers with mild COPD Pulm Pharmacol Ther 21, 304–308 Mook OR, van Marle J, Jonges R, Vreeling-Sindelarova H, Frederiks WM & Van Noorden CJ (2008) Interactions between colon cancer cells and hepatocytes in rats in relation to metastasis J Cell Mol Med 12, 2052–2061 Vaziri ND & Rodriguez-Iturbe B (2006) Mechanisms of disease: oxidative stress and inflammation in the pathogenesis of hypertension Nat Clin Pract Nephrol 2, 582–593 Dworakowski R, Alom-Ruiz SP & Shah AM (2008) NADPH oxidase-derived reactive oxygen species in the regulation of endothelial phenotype Pharmacol Rep 60, 21–28 Abdel-Ghany M, Cheng H, Levine RA & Pauli BU (1998) Truncated dipeptidyl peptidase IV is a potent anti-adhesion and anti-metastasis peptide for rat breast cancer cells Invasion Metastasis 18, 35–43 Abdel-Ghany M, Cheng HC, Elble RC, Lin H, DiBiasio J & Pauli BU (2003) The interacting binding domains of the beta(4) integrin and calcium-activated Epitope of monoclonal DPP IV antibody chloride channels (CLCAs) in metastasis J Biol Chem 278, 49406–49416 37 Morla A, Zhang Z & Ruoslahti E (1994) Superfibronectin is a functionally distinct form of fibronectin Nature 367, 193–196 Supporting information The following supplementary material is available: Fig S1 The putative 6A3-binding epitope is not located in the third- and the fourth-bladed b-propeller domains Fig S2 6A3 recognizes the linear Thr331-dependent eight-amino acid epitope Fig S3 Structural comparison and superimposition of rDPP IV and hDPP IV Doc S1 Amino acids included in the PCR-amplified fragments This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS 6559 ... successfully identified the epitope of the potent anti-metastatic mAb 6A3 that blocks metastatic cancer cell adhesions to rat lung endothelial DPP IV, most likely via steric hindrance The 6A3 -epitope corresponding... 6A3 that blocks the DPP IV ⁄ FN-mediated adhesion of lungmetastatic cancer cells in the lungs [3] In the present study, we finely mapped the 6A3 epitope and analyzed the inhibitory mechanism of 6A3. .. to examine the stability of the 6A3 epitope Epitope of monoclonal DPP IV antibody Epitope mapping of 6A3 may be the most direct approach for answering the above questions [21] In the present