Roles of cysteines in rat dipeptidyl peptidase IV/CD26 in processing and proteolytic activity.

The multifunctional type II transmembrane glycoprotein, dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5), is expressed by almost all mammalian cells and is identical to the adenosine deaminase binding protein CD26 on lymphocytes. The extracellular part of rat DPPIV can be divided into three domains the middle part of which harbors 10 of the 12 highly conserved cysteine residues. The cysteine-rich domain is responsible for DPPIV-binding to collagen I and to extracellular ADA. The participation of distinct cysteines in disulfide bridges is not yet known. Titration experiments have shown the presence of six free cysteines and three disulfide bridges in native rat DPPIV. To investigate the role of distinct cysteines in the structure-function relationships of rat DPPIV we constructed 12 different cysteine point mutations (C299, C326, C383, C455, C650 mutated to G; C337, C395, C445, C448, C473, C552, C763 mutated to S). Intracellular translocation to the cell surface of stable transfected Chinese hamster ovary cells was examined with antibodies against different epitopes of DPPIV. Surface expression of mutants C326G, C445S and C448S is inhibited totally; mutants C337S, C455G, C473S and C552S show weak expression only. In parallel, the half-life of these mutants is reduced to < 10% compared with wild-type enzyme. We were able to show that the specific peptidase activity of the mutant protein depends on cell-surface expression, dimerization and the existence of a 150-kDa form demonstrable by nondenaturing SDS/PAGE. We conclude that cysteine residues 326, 337, 445, 448, 455, 473 and 552 in rat DPPIV are essential for the correct folding and intracellular trafficking of this glycoprotein, and therefore for its normal biological properties.     

Apical cell surface expression of rat dipeptidyl peptidase IV in transfected Madin-Darby canine kidney cells.

Dipeptidyl peptidase IV (DPPIV) is a type II membrane glycoprotein that is predominantly localized to the apical plasma membrane in various epithelial cells. In order to understand in more detail the biogenesis and sorting of DPPIV, the cDNA for rat DPPIV was inserted into a mammalian plasmid expression vector so that DPPIV expression was driven by a control region composed of the SV40 early promoter region fused to the enhancer of the Rous sarcoma virus. Madin-Darby canine kidney cells transfected with this construct were found to express the DPPIV protein. In these transfected cells, the majority of DPPIV was present on the apial cell surface. This observation suggests that the information for apical surface localization is inherent in the DPPIV molecule itself and that this sorting information is decipherable in the epithelial cells of a different species. DPPIV is transported efficiently from the endoplasmic reticulum to the Golgi apparatus as assessed by pulse-chase experiments. Furthermore, evidence is presented which suggests that the majority of DPPIV is sorted intracellularly to the apical cell surface. The same protein has, however, been reported to be sorted by an indirect pathway through transcytosis from the basolateral to the apical cell surface in hepatocytes (Bartles, J.R., Feracci, H., M., Stinger, B., and Hubbard, A.L. (1987) J. Cell Biol. 105, 1241-1251). This study suggests that the same protein can take two different pathways in different cell types for its correct apical cell surface localization.     

CD26/DPPIV signal transduction function, but not proteolytic activity, is directly related to its expression level on human Th1 and Th2 cell lines as detected with living cell cytochemistry.

CD26/DPPIV is a cell surface glycoprotein that functions both in signal transduction and as a proteolytic enzyme, dipeptidyl peptidase IV (DPPIV). To investigate how two separate functions of one molecule are regulated, we analyzed CD26 protein expression and DPPIV enzyme activity on living human T-helper 1 (Th1) and Th2 cells that express different levels of CD26/DPPIV. DPPIV activity was specifically determined with the synthetic fluorogenic substrate ala-pro-cresyl violet and CD26 protein expression was demonstrated with an FITC-conjugated CD26-specific antibody. Fluorescence of liberated cresyl violet (red) and FITC (green) was detected simultaneously on living T-cells using flow cytometry and spectrofluorometry. Th1 cells expressed three- to sixfold more CD26 protein than Th2 cells. The signal transduction function of the CD26/DPPIV complex, tested by measuring its co-stimulatory potential for proliferation, was directly related to the amount of CD26 protein at the cell surface. However, DPPIV activity was similar in both cell populations at physiological substrate concentrations because of differences in K(m) and V(max) values of DPPIV on Th1 and Th2 cells. Western blotting and zymography of Th1 and Th2 whole-cell lysates demonstrated similar patterns. This study shows that two functions of one molecule can be controlled differentially.     

An active-site mutation (Gly633-->Arg) of dipeptidyl peptidase IV causes its retention and rapid degradation in the endoplasmic reticulum.

Dipeptidyl peptidase IV (DPPIV), a serine protease expressed on the cell surface, is deficient in a Fischer rat substrain. Northern blot analysis showed no difference in the size and amount of DPPIV mRNA between normal (344/NC) and deficient (344/CRJ) rats. Cloning and sequencing of DPPIV cDNAs revealed a G to A transition at nucleotide 1897 in the cDNA sequence of 344/CRJ, which leads to substitution of Gly633-->Arg in the active-site sequence Gly629-Trp-Ser-Tyr-Gly633 determined for the wild-type DPPIV [Ogata, S., Misumi, Y., Takami, N., Oda, K., & Ikehara, Y. (1992) Biochemistry 31, 2582-2587]. Pulse-chase experiments with hepatocytes showed that the wild-type DPPIV was initially synthesized as a 103-kDa form with high-mannose-type oligosaccharides, which was processed to a mature form of 109 kDa with the complex type during intracellular transport. In contrast, the mutant DPPIV, although being synthesized as the 103-kDa form, was rapidly degraded without being processed to the mature form. Site-directed mutagenesis of the wild-type and mutant cDNAs and their transfection/expression in COS-1 cells confirmed that the single substitution of Gly633-->Arg is sufficient to cause the rapid intracellular degradation of DPPIV. Immunoelectron-microscopic observations showed that the mutant DPPIV was detectable only in the endoplasmic reticulum (ER), in contrast to the distribution of the wild-type DPPIV in the Golgi complex and on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective cell-surface expression of dipeptidyl peptidase IV with mutations at the active site sequence.

The cell surface expression of dipeptidyl peptidase IV (DPPIV) was examined in COS-1 cells transfected with its cDNA with or without mutations at the active site sequence Gly-Trp-Ser-Tyr-Gly (positions 629-633). Mutants with substitution of Trp630----Glu or Ser631----Ala were expressed on the cell surface as normally as the wild-type DPPIV, although the mutant with Ala631 had no enzyme activity. In contrast, any single substitutions of Gly at positions 629 and 633 resulted in no surface expression of the mutants, which were, instead, detected within the cells. When Tyr632 was substituted, one mutant (Tyr----Phe) was expressed on the surface, whereas the others (Tyr----Gly or Leu) were intracellularly retained. These results indicate that the surface expression of DPPIV is critically influenced by mutations at the active site sequence.     

Involvement of both vectorial and transcytotic pathways in the preferential apical cell surface localization of rat dipeptidyl peptidase IV in transfected LLC-PK1 cells.

Dipeptidyl peptidase IV (DPPIV) is a membrane glycoprotein with type II orientation. It is predominantly localized to the apical surface in epithelial cells. Previous studies (Bantles, J. P., Feracci, H. M., Shinger, B., and Hubbard, A. L. (1987) J. Cell Biol. 105, 1241-1251) using cellular fractionation and immunoprecipitation in rat liver suggest that DPPIV is targeted to the apical surface by an indirect pathway through transient appearance in the basolateral surface followed by specific transcytosis to the apical domain. In transfected Madin-Darby canine kidney (MDCK) cells using domain-selective biotinylation and streptavidin absorption, it was, however, shown that DPPIV is directly sorted to the apical surface (Low, S. H., Wong, S. H., Tang, B. L. Subramaniam, V. N., and Hong, W. (1991) J. Biol. Chem, 266, 13391-13396). These studies suggest that the sorting pathway for DPPIV may be cell type-specific, but it cannot be ruled out that the observed difference in the DPPIV sorting pathway may be due to different methods employed for dissecting the sorting pathway. In this study, we have expressed rat DPPIV, using an expression system driven by the Rous sarcoma virus enhancer and the SV40 early promoter region, in another epithelial cell line, LLC-PK1. As in MDCK cells, DPPIV is preferentially (about 90%) localized to the apical surface. Employing identical methods used previously in MDCK cells, it was found that both direct and transcytotic pathways are involved in the apical surface localization of DPPIV in this epithelial cell type. These observations clearly illustrate that the sorting pathway of rat DPPIV is cell type-specific.     

Biosynthesis and characterization of the brain-specific membrane protein DPPX, a dipeptidyl peptidase IV-related protein

Dipeptidyl peptidase IV-related protein (DPPX) was found to be preferentially expressed in the brain tissue. We isolated two rat cDNA clones encoding DPPX-S and DPPX-L from a brain cDNA library, of which DPPX-L had a longer sequence at the NH2 terminus. The biosynthesis of DPPXs was examined in both in vitro and in vivo systems. In the cell-free translation system, DPPX-S and DPPX-L were synthesized as 93-kDa and 97-kDa forms, respectively, which are in good agreement with the molecular masses estimated from their primary structure. In COS-1 cells transfected with the cDNAs, DPPX-S and DPPX-L were initially synthesized as 113-kDa and 117-kDa forms, respectively, with high-mannose type oligosaccharides, which were then converted to 115-kDa and 120-kDa forms, mostly with the complex-type sugar chains. Immunofluorescence-microscopic observations confirmed that both DPPXs were expressed on the cell surface. DPPXs were found to have no enzyme activity of DPPIV, even when they were mutated to have the consensus active-site sequence Gly-X-Ser-X-Gly for serine proteases. Immunoblot analysis of samples prepared from various rat tissues demonstrated that DPPX-S, but not DPPX-L, was detectable only in the brain tissue. These results indicate that, of the two isoforms, DPPX-S is preferentially expressed in the brain tissue as the surface glycoprotein without protease activity, although its function remains unknown at present.     

Fluorogenic substrate [Ala-Pro]2-cresyl violet but not Ala-Pro-rhodamine 110 is cleaved specifically by DPPIV activity: a study in living Jurkat cells and CD26/DPPIV-transfected Jurkat cells.

Fluorogenic substrates [Ala-Pro](2)-cresyl violet and Ala-Pro-rhodamine 110 have been tested for microscopic detection of protease activity of dipeptidyl peptidase IV (DPPIV) in living cells. DPPIV activity is one of the many functions of the multifunctional or moonlighting protein CD26/DPPIV. As a model we used Jurkat cells, which are T-cells that lack CD26/DPPIV expression, and CD26/DPPIV-transfected Jurkat cells. Ala-Pro-rhodamine 110 is not fluorescent, but after proteolytic cleavage rhodamine 110 fluoresces. [Ala-Pro](2)-cresyl violet is fluorescent by itself but proteolytic cleavage into cresyl violet induces a shift to longer wavelengths. This phenomenon enables the simultaneous determination of local (intracellular) substrate and product concentrations, which is important for analysis of kinetics of the cleavage reaction. [Ala-Pro](2)-cresyl violet, but not Ala-Pro-rhodamine 110, appeared to be specific for DPPIV. When microscopic analysis is performed on living cells during the first minutes of the enzyme reaction, DPPIV activity can be precisely localized in cells with the use of [Ala-Pro](2)-cresyl violet. Fluorescent product is rapidly internalized into submembrane granules in transfected Jurkat cells and is redistributed intracellularly via internalization pathways that have been described for CD26/DPPIV. We conclude that [Ala-Pro](2)-cresyl violet is a good fluorogenic substrate to localize DPPIV activity in living cells when the correct wavelengths are used for excitation and emission and images are captured in the early stages of the enzyme reaction.     

Dipeptidyl peptidase IV in tumor progression

Dipeptidyl peptidase IV (DPPIV) is a 110-kDa glycoprotein with ubiquitous expression. Several recent studies have shown that DPPIV affects tumor progression in several human malignancies. We found that ovarian carcinoma cell lines with higher DPPIV expression showed less invasive potential. Furthermore, introduction of DPPIV cDNA into SKOV3 cells (SKDPIV), derived from serous cystadenocarcinoma showing little DPPIV expression, caused a significant decrease in both migration and invasive potential. In addition, nude mice inoculated with SKDPIV cells showed significantly less peritoneal dissemination and longer survival time than those inoculated with parental or vector-transfected cells. We further examined the mechanisms of anti-invasive ability of DPPIV. The expression of E-cadherin was positively correlated with DPPIV expression among five independent ovarian carcinoma cell lines. The SKDPIV cells showed enhanced expression of E-cadherin with a cellular morphological change from a fibroblastic and motile phenotype to an epithelial phenotype compared to parental and MOCK cells. In addition, matrix metalloproteinase 2 (MMP-2) and membrane type 1 matrix metalloprotease (MT1-MMP), which are important markers associated with invasive and metastatic potential, were remarkably reduced in SKDPIV cells. In contrast, tissue inhibitors of matrix metalloproteinases (TIMPs) were enhanced by DPPIV transfection. These findings imply that DPPIV may functionally suppress peritoneal dissemination and progression of ovarian carcinoma by regulating the expression levels of several molecules associated with carcinoma cell invasion and progression.     

Cloning, expression and chromosomal localization of a novel human dipeptidyl peptidase (DPP) IV homolog, DPP8.

Dipeptidyl peptidase (DPP) IV has roles in T-cell costimulation, chemokine biology, type-II diabetes and tumor biology. Fibroblast activation protein (FAP) has been implicated in tumor growth and cirrhosis. Here we describe DPP8, a novel human postproline dipeptidyl aminopeptidase that is homologous to DPPIV and FAP. Northern-blot hybridization showed that the tissue expression of DPP8 mRNA is ubiquitous, similar to that of DPPIV. The DPP8 gene was localized to chromosome 15q22, distinct from a closely related gene at 19p13.3 which we named DPP9. The full-length DPP8 cDNA codes for an 882-amino-acid protein that has about 27% identity and 51% similarity to DPPIV and FAP, but no transmembrane domain and no N-linked or O-linked glycosylation. Western blots and confocal microscopy of transfected COS-7 cells showed DPP8 to be a 100-kDa monomeric protein expressed in the cytoplasm. Purified recombinant DPP8 hydrolyzed the DPPIV substrates Ala-Pro, Arg-Pro and Gly-Pro. Thus recombinant DPP8 shares a postproline dipeptidyl aminopeptidase activity with DPPIV and FAP. DPP8 enzyme activity had a neutral pH optimum consistent with it being nonlysosomal. The similarities between DPP8 and DPPIV in tissue expression pattern and substrates suggests a potential role for DPP8 in T-cell activation and immune function.     

Seprase: an overview of an important matrix serine protease

Seprase or Fibroblast Activation Protein (FAP) is an integral membrane serine peptidase, which has been shown to have gelatinase activity. Seprase has a dual function in tumour progression. The proteolytic activity of Seprase has been shown to promote cell invasiveness towards the ECM and also to support tumour growth and proliferation. Seprase appears to act as a proteolytically active 170-kDa dimer, consisting of two 97-kDa subunits. It is a member of the group type II integral serine proteases, which includes dipeptidyl peptidase IV (DPPIV/CD26) and related type II transmembrane prolyl serine peptidases, which exert their mechanisms of action on the cell surface. DPPIV and Seprase exhibit multiple functions due to their abilities to form complexes with each other and to interact with other membrane-associated molecules. Localisation of these protease complexes at cell surface protrusions, called invadopodia, may have a prominent role in processing soluble factors and in the degradation of extracellular matrix components that are essential to the cellular migration and matrix invasion that occur during tumour invasion, metastasis and angiogenesis.     

Association of Na(+)-H(+) exchanger isoform NHE3 and dipeptidyl peptidase IV in the renal proximal tubule

In an attempt to identify proteins that assemble with the apical membrane Na(+)-H(+) exchanger isoform NHE3, we generated monoclonal antibodies (mAbs) against affinity-purified NHE3 protein complexes isolated from solubilized renal microvillus membrane vesicles. Hybridomas were selected based on their ability to immunoprecipitate NHE3. We have characterized in detail one of the mAbs (1D11) that specifically co-precipitated NHE3 but not villin or NaPi-2. Western blot analyses of microvillus membranes and immunoelectron microscopy of kidney sections showed that mAb 1D11 recognizes a 110-kDa protein highly expressed on the apical membrane of proximal tubule cells. Immunoaffinity chromatography was used to isolate the antigen against which mAb 1D11 is directed. N-terminal sequencing of the purified protein identified it as dipeptidyl peptidase IV (DPPIV) (EC ), which was confirmed by assays of DPPIV enzyme activity. We also evaluated the distribution of the NHE3-DPPIV complex in microdomains of rabbit renal brush border. In contrast to the previously described NHE3-megalin complex, which principally resides in a dense membrane population (coated pits) in which NHE3 is inactive, the NHE3-DPPIV complex was predominantly in the microvillar fraction in which NHE3 is active. Serial precipitation experiments confirmed that anti-megalin and anti-DPPIV antibodies co-precipitate different pools of NHE3. Taken together, these studies revealed an unexpected association of the brush border Na(+)-H(+) exchanger NHE3 with dipeptidyl peptidase IV in the proximal tubule. These findings raise the possibility that association with DPPIV may affect NHE3 surface expression and/or activity.     

Regulation of human extravillous trophoblast function by membrane-bound peptidases

During human placentation, the invasion of extravillous trophoblasts (EVTs) into maternal decidual tissues, especially toward maternal spiral arteries, is considered an essential process for subsequent normal fetal development. However, the precise regulatory mechanisms to induce EVT invasion toward arteries and/or to protect EVTs from further invasion have not been well understood. Recently, we found that two cell surface peptidases, dipeptidyl peptidase IV (DPPIV) and carboxypeptidase-M (CP-M,) are differentially expressed on EVTs. DPPIV expression was mainly observed on EVTs that had already ceased invasion. CP-M was detected on migrating EVTs including endovascular trophoblasts in the maternal arteries. The enzymatic inhibition of these peptidases affected the invasive property of choriocarcinoma-derived cell lines, BeWo and JEG3 cells. In addition, a chemokine, RANTES, that is one of the substrates for DPPIV, enhanced invasion of EVTs isolated from primary villous explant culture and its receptor, CCR1, was specifically expressed on migrating EVTs toward maternal arteries. Furthermore, a novel membrane-bound cell surface peptidase, named laeverin, was found to be specifically expressed on EVTs that had almost ceased invasion. These findings suggest that membrane-bound peptidases are important factors regulating EVT invasion during early placentation in humans.     

Expression of dipeptidyl peptidase IV in rat tissues is mainly regulated at the mRNA levels

Dipeptidyl peptidase IV (DPPIV) is a serine peptidase that cleaves N-terminal dipeptides from polypeptides when the second residue is a proline or an alanine. We have recently cloned cDNAs for rat gp110, a membrane glycoprotein with Mr of 110,000 isolated initially from rat liver. Studies reported here establish that the gp110 for which we have cloned cDNAs is DPPIV. Using the antibodies against and cDNA for DPPIV, we have assessed the tissue distribution of DPPIV by molecular approaches. Immunoblot analysis demonstrated that DPPIV is present in the kidney, lung, and small intestine at high levels, in the liver and spleen at moderate levels, and in the heart at low levels. The highest levels of mRNA for DPPIV were detected in the kidney and small intestine as compared to moderate levels found in the lung, liver, and spleen. The lowest levels of DPPIV mRNA were found in the stomach, testis, and heart. No detectable DPPIV protein and mRNA were found in brain or muscle. LDPPIV protein and mRNA are present at much lower levels in fetal livers as compared to the adult liver. Indirect immunofluorescence microscopy demonstrated that DPPIV is localized in the bile canaliculus of hematocytes and in the apical membrane domains of kidney tubule and small intestine. Further studies by Southern blot analysis indicate that DPPIV is encoded by a single gene.     

Regulation of fibroblast migration on collagenous matrix by a cell surface peptidase complex

The invasion of migratory cells through connective tissues involves metallo- and serine types of cell surface proteases. We show that formation of a novel protease complex, consisting of the membrane-bound prolyl peptidases seprase and dipeptidyl peptidase IV (DPPIV), at invadopodia of migratory fibroblasts is a prerequisite for cell invasion and migration on a collagenous matrix. Seprase and DPPIV form a complex on the cell surface that elicits both gelatin binding and gelatinase activities localized at invadopodia of cells migrating on collagenous fibers. The protease complex participates in the binding to gelatin and localized gelatin degradation, cellular migration, and monolayer wound closure. Serine protease inhibitors can block the gelatinase activity and the localized gelatin degradation by cells. Antibodies to the gelatin-binding domain of DPPIV reduce the cellular abilities of the proteases to degrade gelatin but do not affect cellular adhesion or spreading on type I collagen. Furthermore, expression of the seprase-DPPIV complex is restricted to migratory cells involved in wound closure in vitro and in connective tissue cells during closure of gingival wounds but not in differentiated tissue cells. Thus, we have identified cell surface proteolytic activities, which are non-metalloproteases, seprase and DPPIV, that are responsible for the tissue-invasive phenotype.     

Dipeptidyl peptidase IV (DPPIV) enzyme activity on immature T-cell line R1.1 is down-regulated by dynorphin-A(1-17) as a non-substrate inhibitor

In this study we examined surface expression of CD26 and the corresponding enzyme activity of dipeptidyl peptidase IV (DPPIV) on the cells of immature murine T-cell line, R1.1. The data obtained have shown that R1.1 cells express high density of surface CD26 as compared to normal thymus cells. This was associated with strong enzyme activity, which, based on substrates and inhibitor specificity, corresponded to DPPIV. The DPPIV enzyme activity of R1.1 cells was 10 times stronger than that found on normal murine thymus cells (V(max) = 39 micromol/min/10(6) cells, vs 3.7 micromol/min/10(6) cells, respectively). Upon activation with anti-CD3, up-regulation of both membrane CD26, as well as of DPPIV enzyme activity on R1.1 cells were observed. The finding of strong DPPIV on R1.1 cells makes them suitable model for testing putative substrates/inhibitors of the enzyme in its natural microenvironment. Since in addition to strong DPPIV, R1.1 cells also express kappa opioid receptors (KOR) [European Journal of Pharmacology 227 (1992) 257], we tested the effect of dynorphin-A(1-17), an endogenous opioid peptide with KOR selectivity, on DPPIV of R1.1 cells. Dynorphin-A(1-17) down-regulated DPPIV in a dose-dependent manner, with the potency similar to that of substance P, a known natural DPPIV substrate [Journal of Pharmacology and Experimental Therapeutics 260 (1992) 1257]. DPPIV down-regulation was resistant to bestatin and thiorphan, the inhibitors of two cell surface peptidases (APN and NEP, respectively) with potential of dynorphin-A(1-17) degradation, suggesting that the mechanism underlying the observed effect does not involve degradative products of dynorphin-A(1-17). DPPIV down-regulation was also resistent to KOR antagonist, NBI, suggesting that the mechanism underlying the observed phenomenon involves neither cointernalization of KOR and DPPIV. Collectively, cells of immature T cell line, R1.1 exert strong DPPIV enzyme activity, which could be down-regulated in the presence of dynorphin-A(1-17) by mechanism that presumably includes non-substrate inhibition. By down-regulating DPPIV, dynorphin-A(1-17) may indirectly affect activity and/or specificity of natural substrates of DPPIV, such as substance P, RANTES, and endomorphins.     

Cloning and functional expression of the T cell activation antigen CD26.

A cDNA encoding the T cell activation Ag CD26 was isolated from human PHA-activated T cells by using an expression cloning method. The nucleotide sequence obtained predicts a protein of 766 amino acids of type II membrane topology, with six amino acids in the cytoplasmic region. The predicted amino acid sequence of the Ag was 85% homologous to that of the dipeptidyl peptidase IV enzyme isolated from rat liver. Derivatives of the human leukemic T cell line Jurkat transfected with a CD26 expression plasmid were established. Characterization of the CD26 Ag expressed by the transfected Jurkat cells revealed that the Ag could be immunoprecipitated as a 110-kDa molecule similar to that found on peripheral blood T cells and that the Ag had dipeptidyl peptidase IV activity. Functional analysis of these Jurkat transfectants showed that cross-linking of the CD26 and CD3 Ag with their respective antibodies resulted in enhanced intracellular calcium mobilization and IL-2 production. These results provide direct evidence that the CD26 Ag plays a role in T cell activation.     

Soluble CD26/dipeptidyl peptidase IV enhances transendothelial migration via its interaction with mannose 6-phosphate/insulin-like growth factor II receptor

CD26 is a T cell surface molecule with dipeptidyl peptidase IV (DPPIV) enzyme activity in its extracellular region. In addition to its membrane form, CD26 exists in plasma as a soluble form (sCD26), which is the extracellular domain of the molecule thought to be cleaved from the cell surface. In this paper, we demonstrate that sCD26 mediates enhanced transendothelial T cell migration, an effect that requires its intrinsic DPPIV enzyme activity. We also show that sCD26 directly targets endothelial cells and that mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR) on the endothelial cell surface acts as a receptor for sCD26. Our findings therefore suggest that sCD26 influences T cell migration through its interaction with M6P/IGFIIR.     

Molecular characterization of dipeptidyl peptidase activity in serum: soluble CD26/dipeptidyl peptidase IV is responsible for the release of X-Pro dipeptides.

Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) is a serine type protease with an important modulatory activity on a number of chemokines, neuropeptides and peptide hormones. It is also known as CD26 or adenosine deaminase (ADA; EC 3.5.4.4) binding protein. DPPIV has been demonstrated on the plasmamembranes of T cells and activated natural killer or B cells as well as on a number of endothelial and differentiated epithelial cells. A soluble form of CD26/DPPIV has been described in serum. Over the past few years, several related enzymes with similar dipeptidyl peptidase activity have been discovered, raising questions on the molecular origin(s) of serum dipeptidyl peptidase activity. Among them attractin, the human orthologue of the mouse mahogany protein, was postulated to be responsible for the majority of the DPPIV-like activity in serum. Using ADA-affinity chromatography, it is shown here that 95% of the serum dipeptidyl peptidase activity is associated with a protein with ADA-binding properties. The natural protein was purified in milligram quantities, allowing molecular characterization (N-terminal sequence, glycosylation type, CD-spectrum, pH and thermal stability) and comparison with CD26/DPPIV from other sources. The purified serum enzyme was confirmed as CD26.