Cell surface adenosine deaminase binds and stimulates plasminogen activation on 1-LN human prostate cancer cells

Adenosine deaminase (ADA) is expressed intracellularly by all cells, but in some tissues, it is also associated with the cell surface multifunctional glycoprotein CD26/dipeptidyl peptidase IV. By modulating extracellular adenosine, this "ecto-ADA" may regulate adenosine receptor signaling implicated in various cellular functions. CD26 is expressed on the surface of human prostate cancer 1-LN cells acting as a receptor for plasminogen (Pg). Since ADA and Pg bind to CD26 at distinct but nearby sites, we investigated a possible interaction between these two proteins on the surface of 1-LN cells. Human ADA binds to CD26 on the surface 1-LN cells and immobilized CD26 isolated from the same cells with similar affinity. In both cases, ADA binding is diminished by mutation of ADA residues known to interact with CD26. ADA was also found to bind Pg 2 in the absence of CD26 via the Pg kringle 4 (K4) domain. In the presence of 1-LN cells or immobilized CD26, exogenous ADA enhances conversion of Pg 2 to plasmin by 1-LN endogenous urinary plasminogen activator (u-PA), as well as by added tissue Pg Activator (t-PA), suggesting that ADA and Pg bind simultaneously to CD26 in a ternary complex that stimulates the Pg activation by its physiologic activators. Consistent with this, in melanoma A375 cells that bind Pg, but do not express CD26, the rate of Pg activation was not affected by ADA. Thus, ADA may be a factor regulating events in prostate cancer cells that occur when Pg binds to the cell surface and is activated.     

The HIV-1 gp120 inhibits the binding of adenosine deaminase to CD26 by a mechanism modulated by CD4 and CXCR4 expression

HIV-1 external envelope glycoprotein gp120 inhibits adenosine deaminase (ADA) binding to its cell surface receptor in lymphocytes, CD26, by a mechanism that does not require the gp120-CD4 interaction. To further characterize this mechanism, we studied ADA binding to murine clones stably expressing human CD26 and/or human CD4, and transiently expressing human CXCR4. In this heterologous model, we show that both recombinant gp120 and viral particles from the X4 HIV-1 isolate IIIB inhibited the binding of ADA to wild-type or catalytically inactive forms of CD26. In cells lacking human CXCR4 expression, this gp120-mediated inhibition of ADA binding to human CD26 was completely dependent on the expression of human CD4. In contrast, when cells were transfected with human CXCR4 the inhibitory effect of gp120 was significantly enhanced and was not blocked by anti-CD4 antibodies. These data suggest that the interaction of gp120 with CD4 or CXCR4 is required for efficient inhibition of ADA binding to CD26, although in the presence of CXCR4 the interaction of gp120 with CD4 may be dispensable.     

Crystal structure of CD26/dipeptidyl-peptidase IV in complex with adenosine deaminase reveals a highly amphiphilic interface

Dipeptidyl-peptidase IV (DPPIV or CD26) is a homodimeric type II membrane glycoprotein in which the two monomers are subdivided into a beta-propeller domain and an alpha/beta-hydrolase domain. As dipeptidase, DPPIV modulates the activity of various biologically important peptides and, in addition, DPPIV acts as a receptor for adenosine deaminase (ADA), thereby mediating co-stimulatory signals in T-lymphocytes. The 3.0-A resolution crystal structure of the complex formed between human DPPIV and bovine ADA presented here shows that each beta-propeller domain of the DPPIV dimer binds one ADA. At the binding interface, two hydrophobic loops protruding from the beta-propeller domain of DPPIV interact with two hydrophilic and heavily charged alpha-helices of ADA, giving rise to the highest percentage of charged residues involved in a protein-protein contact reported thus far. Additionally, four glycosides linked to Asn229 of DPPIV bind to ADA. In the crystal structure of porcine DPPIV, the observed tetramer formation was suggested to mediate epithelial and lymphocyte cell-cell adhesion. ADA binding to DPPIV could regulate this adhesion, as it would abolish tetramerization.     

Binding to human dipeptidyl peptidase IV by adenosine deaminase and antibodies that inhibit ligand binding involves overlapping, discontinuous sites on a predicted beta propeller domain.

Dipeptidyl peptidase IV (DPPIV) is an atypical serine protease that modifies the biological activities of certain chemokines and neuropeptides. In addition, human DPPIV, also known as the T-cell activation antigen CD26, binds adenosine deaminase (ADA) to the T-cell surface, thus protecting the T-cell from adenosine-mediated inhibition of proliferation. Mutations were engineered into DPPIV (five point, 16 single point and six deletion mutations) to examine the binding of ADA and 19 monoclonal antibodies. Deletions of C-terminal residues from the 738-residue extracellular portion of DPPIV showed that the 214 residues C-terminal to Ser552 were not required for ADA binding and that peptidase activity could be ablated by deletion of 20 residues from the C-terminus. Point mutations at either of two locations, Leu294 and Val341, ablated ADA binding. Binding by six anti-DPPIV antibodies that inhibited ADA binding was found to require Leu340 to Arg343 and Thr440/Lys441 but not the 214 residues C-terminal to Ser552. The 13 other antibodies studied bound to a truncated DPPIV consisting of amino acids 1-356. Therefore, the binding sites on DPPIV of ADA and antibodies that inhibit ADA binding are discontinuous and overlapping. Moreover, the 47 and 97 residue spacing of amino acids in these binding sites concords with their location on a beta propeller fold consisting of repeated beta sheets of about 50 amino acids.     

3D structure of the CD26-ADA complex obtained by cryo-EM and single particle analysis

The specific binding of adenosine deaminase to the multifunctional membrane glycoprotein dipeptidyl peptidase IV is thought to be immunologically relevant for certain regulatory and co-stimulatory processes. In this study we present the 3D structure of the complete CD26-ADA complex obtained by single particle cryo-EM at 22A resolution. ADA binding occurs at the outer edges of the beta-propeller of CD26. Docking calculations of available CD26 and ADA crystal data into the obtained EM density map revealed that the ADA-binding site is stretched across CD26 beta-propeller blades 4 and 5 involving the outermost distal hydrophobic amino acids L294 and V341 but not T440 and K441 as suggested by antibody binding. Though the docking of the ADA orientation appears less significant due to the lack of distinct surface features, non-ambiguous conclusions can be drawn in the combination with earlier indirect non-imaging methods affirming the crucial role of the ADA alpha2-helix for binding.     

Influence of dipeptidyl peptidase IV on enzymatic properties of adenosine deaminase

The importance of ADA (adenosine deaminase) in the immune system and the role of its interaction with an ADA-binding cell membrane protein dipeptidyl peptidase IV (DPPIV), identical to the activated immune cell antigen, CD26, has attracted the interest of researchers for many years. To investigate the specific properties in the structure-function relationship of the ADA/DPPIV-CD26 complex, its soluble form, identical to large ADA (LADA), was isolated from human blood serum, human pleural fluid and bovine kidney cortex. The kinetic constants (Km and Vmax) of LADA and of small ADA (SADA), purified from bovine lung and spleen, were compared using adenosine (Ado) and 2'-deoxyadenosine (2'-dAdo) as substrates. The Michaelis constant, Km, evidences a higher affinity of both substrates (in particular of more toxic 2'-dAdo) for LADA and proves the modulation of toxic nucleoside neutralization in the extracellular medium due to complex formation between ADA and DPPIV-CD26. The values of Vmax are significantly higher for SADA, but the efficiency, Vmax/Km, in LADA-catalyzed 2'-dAdo deamination is higher than that in Ado deamination. The interaction of all enzyme preparations with derivatives of adenosine and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was studied. 1-DeazaEHNA and 3-deazaEHNA demonstrate stronger inhibiting activity towards LADA, the DPPIV-CD26-bound form of ADA. The observed differences between the properties of the two ADA isoforms may be considered as a consequence of SADA binding with DPPIV-CD26. Both SADA and LADA indicated a similar pH-profile of adenosine deamination reaction with the optimum at pHs 6.5-7.5, while the pH-profile of dipeptidyl peptidase activity of the ADA/DPPIV-CD26 complex appeared in a more alkaline region.     

Distribution and expression of A1 adenosine receptors,adenosine deaminase and adenosine deaminase-binding protein (CD26) in goldfish brain

The expression patterns of adenosine A(1) receptors (A(1)Rs), adenosine deaminase (ADA) and ADA binding protein (CD26) were studied in goldfish brain using mammalian monoclonal antibody against A(1)R and polyclonal antibodies against ADA and CD26. Western blot analysis revealed the presence of a band of 35 kDa for A(1)R in membrane preparations and a band of 43 kDa for ADA in both cytosol and membranes. Immunohistochemistry on goldfish brain slices showed that A(1) receptors were present in several neuronal cell bodies diffused in the telencephalon, cerebellum, optic tectum. In the rhombencephalon, large and medium sized neurons of the raphe nucleus showed a strong immunopositivity. A(1)R immunoreactivity was also present in the glial cells of the rhombencephalon and optic tectum. An analogous distribution was observed for ADA immunoreactivity. Tests for the presence of CD26 gave positive labelling in several populations of neurons in the rhombencephalon as well as in the radial glia of optic tectum, where immunostaining for ADA and A(1)R was observed. In goldfish astrocyte cultures the immunohistochemical staining of A(1)R, ADA and CD26, performed on the same cell population, displayed a complete overlapping distribution of the three antibodies. The parallel immunopositivity, at least in some discrete neuronal areas, for A(1)Rs, ADA and CD26 led us to hypothesize that a co-localization among A(1)R, ecto-ADA and CD26 also exists in the neurons of goldfish since it has been established to exist in the neurons of mammals. Moreover, we have demonstrated for the first time, that A(1)R, ecto-ADA and CD26 co-localization is present on the astroglial component of the goldfish brain. This raises the possibility that a similar situation is also shown in the glia of the mammalian brain.     

Charged residues at the intracellular boundary of transmembrane helices 2 and 3 independently affect constitutive activity of Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor

Because charged residues at the intracellular ends of transmembrane helix (TMH) 2 and TMH3 of G protein-coupled receptors (GPCRs) affect signaling, we performed mutational analysis of these residues in the constitutively signaling Kaposi's sarcoma-associated herpesvirus GPCR (KSHV-GPCR). KSHV-GPCR contains the amino acid sequence Val-Arg-Tyr rather than the Asp/Glu-Arg-Tyr ((D/E)RY) motif at the intracellular end of TMH3. Mutation of Arg-143 to Ala (R143A) or Gln (R143Q) abolished constitutive signaling whereas R143K exhibited 50% of the basal activity of KSHV-GPCR. R143A was not stimulated by agonist, whereas R143Q was stimulated by growth-related oncogene-alpha, and R143K, similar to KSHV-GPCR, was stimulated further. These findings show that Arg-143 is critical for signal generation in KSHV-GPCR. In other GPCRs, Arg in this position may act as a signaling switch by movement of its sidechain from a hydrophilic pocket in the TMH bundle to a position outside the bundle. In rhodopsin, the Arg of Glu-Arg-Tyr interacts with the adjacent Asp to constrain Arg outside the TMH bundle. V142D was 70% more active than KSHV-GPCR, suggesting that an Arg residue, which is constrained outside the bundle by interacting with Asp-142, leads to a receptor that signals more actively. Because the usually conserved Asp in the middle of TMH2 is not present in KSHV-GPCR, we tested whether Asp-83 at the intracellular end of TMH2 was involved in signaling. D83N and D83A were 110 and 190% more active than KSHV-GPCR, respectively. The double mutant D83A/V142D was 510% more active than KSHV-GPCR. That is, cosubstitutions of Asp-83 by Ala and Val-142 by Asp act synergistically to increase basal signaling. A model of KSHV-GPCR predicts that Arg-143 interacts with residues in the TMH bundle and that the sidechain of Asp-83 does not interact with Arg-143. These data are consistent with the hypothesis that Arg-143 and Asp-83 independently affect the signaling activity of KSHV-GPCR.     

Interaction of adenosine deaminase with inhibitors. Chemical modification by diethyl pyrocarbonate

The interaction of adenosine deaminase (adenosine aminohydrolase, ADA) from bovine spleen with inhibitors— erythro-9-(2-hydroxy-3-nonyl)adenine, erythro-9-(2-hydroxy-3-nonyl)-3-deazaadenine, and 1-deazaadenosine—was investigated. Using selective chemical modification by diethyl pyrocarbonate (DEP), the possible involvement of His residues in this interaction was studied. The graphical method of Tsou indicates that of six His residues modified in the presence of DEP, only one is essential for ADA activity. Inactivation of the enzyme, though with low rate, in complex with any of the inhibitors suggests that the adenine moiety of the inhibitors (and consequently, of the substrate) does not bind with the essential His to prevent its modification. The absence of noticeable changes in the dissociation constants of any of the enzyme–inhibitor complexes for the DEP-modified and control enzyme indicates that at least the most available His residues modified in our experiments do not participate in binding the inhibitors—derivatives of adenosine or erythro-9-(2-hydroxy-3-nonyl)adenine.     

Adenosine deaminase affects ligand-induced signalling by interacting with cell surface adenosine receptors

Adenosine deaminase (ADA) is not only a cytosolic enzyme but can be found as an ecto-enzyme. At the plasma membrane, an adenosine deaminase binding protein (CD26, also known as dipeptidylpeptidase IV) has been identified but the functional role of this ADA/CD26 complex is unclear. Here by confocal microscopy, affinity chromatography and coprecipitation experiments we show that A₁ adenosine receptor (A₁R) is a second ecto-ADA binding protein. Binding of ADA to A₁R increased its affinity for the ligand thus suggesting that ADA was needed for an effective coupling between A₁R and heterotrimeric G proteins. This was confirmed by the fact that ASA, independently of its catalytic behaviour, enhanced the ligand-induced second messenger production via A₁R. These findings demonstrate that, apart from the cleavage of adenosine, a further role of ecto-adenosine deaminase on the cell surface is to facilitate the signal transduction via A₁R.     

Expression vectors for human adenosine deaminase gene therapy

Somatic gene transfer offers a possible new approach for treatment of human genetic disease. Defects affecting blood-forming tissues are candidates for therapies involving transfer of genetic information into hematopoietic stem cells. Adenosine deaminase (ADA) deficiency is being used as a model disease for which gene transfer techniques can be developed and evaluated. We describe here the construction and testing of 20 retroviral vectors for their ability to transfer and express human ADA in vitro and in vivo via a mouse bone marrow transplantation model. After infection of primary bone marrow with one fo these vectors (p delta NN2ADA), human ADA was detected in 60-85% of spleen colonies at day 14 and maintained long term in the blood of fully reconstituted mice. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.     

The adenosine deaminase-binding region is distinct from major anti-CD26 mAb epitopes on the human dipeptidyl peptidase IV(CD26) molecule

CD26 or dipeptidyl peptidase IV (DPP-IV) is a cell surface protease involved in T cell activation. Monoclonal antibodies (mAbs) directed against the CD26 molecule are able to stimulate CD26-expressing T cells. Although many different CD26-specific mAbs exist which are able to provide a triggering signal in T cells, little is known about their specific epitopes on the CD26 molecule. Whereas some mAbs were shown to compete with each other and to inhibit the association of adenosine deaminase (ADA) and human immunodeficiency virus 1 (HIV-1)-derived Tat protein with CD26, other CD26-specific mAbs obviously bind to distinct regions on DPP-IV. In the present study we have generated truncated versions of the human CD26 molecule and expressed them in COS-1 cells to study the binding pattern of a panel of 14 CD26-specific mAbs in confocal microscopy and, thus, correlated the CD26-specific mAbs epitopes with the binding region of ADA. We show that the majority of anti-CD26 mAbs is directed against the glycosylation-rich region of the molecule whereas the ADA-binding site could be located in the cysteine-rich region of DPP-IV. In contrast to binding experiments with purified ADA, which revealed a specific association with CD26 on CD26-positive Jurkat cells, HIV-derived Tat protein did not interact specifically with CD26 on transfected Jurkat cells, nor could Tat binding be competed by anti-CD26-specific mAbs.     

Residues in the alpha subunit of human choriotropin that are important for interaction with the lutropin receptor

Synthetic peptides were used to probe the structure-function relationships between human choriotropin (hCG) and the lutropin (LH) receptor. Previously, a peptide region of the alpha subunit of hCG, residues 26-46, had been shown to inhibit binding of 125I-hCG to the LH receptor in rat ovarian membranes (Charlesworth, M.C., McCormick, D.J., Madden, B., and Ryan, R.J. (1987) J. Biol. Chem. 262, 13409-13416). To determine which residues are important for this inhibitory activity, peptides were truncated from either the amino or carboxyl terminus, or individual residues were substituted with alanine. The amino-terminal boundary was determined to be Gly-30 and the carboxyl-terminal boundary, Lys-44. This core peptide contained all the residues needed for full activity of the parent peptide 26-46. Arg-35 and Phe-33 were particularly important residues; when they were substituted with alanine, the peptide inhibitory potencies were decreased. Ser-43, Arg-42, Cys-32, and Cys-31 were also important but to a lesser degree. These results are consistent with predictions based on chemical and enzymatic modification studies and provide insight into which residues are important for interaction between hCG and the LH receptor.     

Comodulation of CXCR4 and CD26 in human lymphocytes

We provide convergent and multiple evidence for a CD26/CXCR4 interaction. Thus, CD26 codistributes with CXCR4, and both coimmunoprecipitate from membranes of T (CD4(+)) and B (CD4(-)) cell lines. Upon induction with stromal cell-derived factor 1alpha (SDF-1alpha), CD26 is cointernalized with CXCR4. CXCR4-mediated down-regulation of CD26 is not induced by antagonists or human immunodeficiency virus (HIV)-1 gp120. SDF-1alpha-mediated down-regulation of CD26 is not blocked by pertussis toxin but does not occur in cells expressing mutant CXCR4 receptors unable to internalize. Codistribution and cointernalization also occurs in peripheral blood lymphocytes. Since CD26 is a cell surface endopeptidase that has the capacity to cleave SDF-1alpha, the CXCR4.CD26 complex is likely a functional unit in which CD26 may directly modulate SDF-1alpha-induced chemotaxis and antiviral capacity. CD26 anchors adenosine deaminase (ADA) to the lymphocyte cell surface, and this interaction is blocked by HIV-1 gp120. Here we demonstrate that gp120 interacts with CD26 and that gp120-mediated disruption of ADA/CD26 interaction is a consequence of a first interaction of gp120 with a domain different from the ADA binding site. SDF-1alpha and gp120 induce the appearance of pseudopodia in which CD26 and CXCR4 colocalize and in which ADA is not present. The physical association of CXCR4 and CD26, direct or part of a supramolecular structure, suggests a role on the function of the immune system and the pathophysiology of HIV infection.     

Kinetics of inhibition of calf intestinal adenosine deaminase by (+)- and (-)-erythro-9-(2-hydroxy-3-nonyl)adenine.

The enantiomers of erythro-9-(2-hydroxy-3-nonyl)adenine [(+)- and (-)-EHNA) bound to adenosine deaminase (ADA) at pH 7 with concomitant changes in the optical properties of the enzyme. The association rate constant for (+)-EHNA was 2.9 x 10(6) M-1 s-1 and that for (-)-EHNA was 6.4 x 10(6) M-1 s-1. The dissociation of (-)-EHNA.ADA or (+)-EHNA.ADA in the presence of excess coformycin was monitored by the quenching of enzyme fluorescence as coformycin.ADA was formed. The dissociation rate constants of (+)- and (-)-EHNA.ADA were 0.0054 s-1 and 2.7 s-1, respectively. A similar value for the dissociation rate constant (0.005 s-1) for (+)-EHNA.ADA was calculated from the time course for the appearance of catalytic activity after dilution of (+)-EHNA.ADA into 100 microM adenosine. The Ki values of ADA for (+)- and (-)-EHNA were similar to the dissociation constants calculated from the ratio of the respective dissociation and association rate constants. The biphasic time-dependent inhibition of the catalytic activity of ADA by (+/- )-EHNA [Frieden, C., Kurz, L. C., & Gilbert, H. R. (1980) Biochemistry 19, 5303-5309] was confirmed. However, the catalytic activity of ADA was inhibited monophasically by (+)-EHNA. Thus, the biphasic nature of the time course for inhibition of ADA by (+/- )-EHNA was the result of the presence of both enantiomers of the inhibitor in this assay. These kinetic data were interpreted in terms of single-step mechanisms for binding of (+)- and (-)-EHNA.     

The heparin/heparan sulfate-binding site on apo-serum amyloid A. Implications for the therapeutic intervention of amyloidosis

Serum amyloid A isoforms, apoSAA1 and apoSAA2, are apolipoproteins of unknown function that become major components of high density lipoprotein (HDL) during the acute phase of an inflammatory response. ApoSAA is also the precursor of inflammation-associated amyloid, and there is strong evidence that the formation of inflammation-associated and other types of amyloid is promoted by heparan sulfate (HS). Data presented herein demonstrate that both mouse and human apoSAA contain binding sites that are specific for heparin and HS, with no binding for the other major glycosaminoglycans detected. Cyanogen bromide-generated peptides of mouse apoSAA1 and apoSAA2 were screened for heparin binding activity. Two peptides, an apoSAA1-derived 80-mer (residues 24-103) and a smaller carboxyl-terminal 27-mer peptide of apoSAA2 (residues 77-103), were retained by a heparin column. A synthetic peptide corresponding to the CNBr-generated 27-mer also bound heparin, and by substituting or deleting one or more of its six basic residues (Arg-83, His-84, Arg-86, Lys-89, Arg-95, and Lys-102), their relative importance for heparin and HS binding was determined. The Lys-102 residue appeared to be required only for HS binding. The residues Arg-86, Lys-89, Arg-95, and Lys-102 are phylogenetically conserved suggesting that the heparin/HS binding activity may be an important aspect of the function of apoSAA. HS linked by its carboxyl groups to an Affi-Gel column or treated with carbodiimide to block its carboxyl groups lost the ability to bind apoSAA. HDL-apoSAA did not bind to heparin; however, it did bind to HS, an interaction to which apoA-I contributed. Results from binding experiments with Congo Red-Sepharose 4B columns support the conclusions of a recent structural study which found that heparin binding domains have a common spatial distance of about 20 A between their two outer basic residues. Our present work provides direct evidence that apoSAA can associate with HS (and heparin) and that the occupation of its binding site by HS, and HS analogs, likely caused the previously reported increase in amyloidogenic conformation (beta-sheet) of apoSAA2 (McCubbin, W. D., Kay, C. M., Narindrasorasak, S., and Kisilevsky, R. (1988) Biochem. J. 256, 775-783) and their amyloid-suppressing effects in vivo (Kisilevsky, R., Lemieux, L. J., Fraser, P. E., Kong, X., Hultin, P. G., and Szarek, W. A. (1995) Nat. Med. 1, 143-147), respectively.     

Characterization and purification of adenosine deaminase 1 from human and chicken liver

Adenosine deaminase 1 (ADA1) was purified from human and chicken liver. The purified enzyme had a molecular weight of approximately 42,000 Da on SDS-PAGE. In humans, ADA1 was mainly purified concomitant with ADA-binding protein, dipeptidyl peptidase IV (DPP IV)/CD26; however, in chickens, only ADA1 without DPP IV was purified. Both human and chicken ADA1s showed similar properties on substrate specificities, sensitivities on inhibitors, and pH profile. However, they had different affinities with adenosine-Sepharose and IgG anti-ADA1-Sepharose. Human ADA1 was not adsorbed in adenosine-Sepharose column, but chicken ADA1 was adsorbed. As for IgG anti-ADA1-Sepharose column, the results were converse. Furthermore, human ADA1 could bind to DPP IV whereas chicken ADA1 could not.     

On the regulatory role of dipeptidyl peptidase IV (=CD=adenosine deaminase complexing protein) on adenosine deaminase activity

The molecular mechanism controlling the variable activity of the malignancy marker adenosine deaminase (ADA) is enigmatic. ADA activity was found to be modulated by the membrane-bound adenosine deaminase complexing protein (CP=DPPIV=CD26). The role of lipid-protein interactions in this modulation was sought. While direct solubilization of ADA in vesicles resulted in loss of ADA activity, the binding of ADA to CP reconstituted in vesicles restored the specific activity. The activity of ADA, free or bound to CP in solution, resulted in continuous linear Arrhenius plots. However, ADA bound to reconstituted CP exhibited two breaks associated with approximately 30% increased activity, at 25 and 13 degrees C, yielding three lines with similar apparent activation energies (E(a)). Continuum solvent model calculations of the free energy of transfer of the transmembrane helix of CP from the aqueous phase into membranes of various widths show that the most favorable orientations of the helix above and below the main phase transition may be different. We suggest that the 20% change in the thickness of the bilayer below and above the main phase transition may modify the orientation of CP in the membrane, thereby affecting substrate accessibility of ADA. This could account for ADA's reduced activity associated with increased membrane fluidity in transformed vs. normal fibroblasts.     

N-linked glycosylation of dipeptidyl peptidase IV (CD26): effects on enzyme activity, homodimer formation, and adenosine deaminase binding

The type II transmembrane serine protease dipeptidyl peptidase IV (DPPIV), also known as CD26 or adenosine deaminase binding protein, is a major regulator of various physiological processes, including immune, inflammatory, nervous, and endocrine functions. It has been generally accepted that glycosylation of DPPIV and of other transmembrane dipeptidyl peptidases is a prerequisite for enzyme activity and correct protein folding. Crystallographic studies on DPPIV reveal clear N-linked glycosylation of nine Asn residues in DPPIV. However, the importance of each glycosylation site on physiologically relevant reactions such as dipeptide cleavage, dimer formation, and adenosine deaminase (ADA) binding remains obscure. Individual Asn-->Ala point mutants were introduced at the nine glycosylation sites in the extracellular domain of DPPIV (residues 39-766). Crystallographic and biochemical data demonstrate that N-linked glycosylation of DPPIV does not contribute significantly to its peptidase activity. The kinetic parameters of dipeptidyl peptidase cleavage of wild-type DPPIV and the N-glycosylation site mutants were determined by using Ala-Pro-AFC and Gly-Pro-pNA as substrates and varied by <50%. DPPIV is active as a homodimer. Size-exclusion chromatographic analysis showed that the glycosylation site mutants do not affect dimerization. ADA binds to the highly glycosylated beta-propeller domain of DPPIV, but the impact of glycosylation on binding had not previously been determined. Our studies indicate that glycosylation of DPPIV is not required for ADA binding. Taken together, these data indicate that in contrast to the generally accepted view, glycosylation of DPPIV is not a prerequisite for catalysis, dimerization, or ADA binding.     

New insights into the heparan sulfate proteoglycan-binding activity of apolipoprotein E

Defective binding of apolipoprotein E (apoE) to heparan sulfate proteoglycans (HSPGs) is associated with increased risk of atherosclerosis due to inefficient clearance of lipoprotein remnants by the liver. The interaction of apoE with HSPGs has also been implicated in the pathogenesis of Alzheimer's disease and may play a role in neuronal repair. To identify which residues in the heparin-binding site of apoE and which structural elements of heparan sulfate interact, we used a variety of approaches, including glycosaminoglycan specificity assays, (13)C nuclear magnetic resonance, and heparin affinity chromatography. The formation of the high affinity complex required Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147 from apoE and N- and 6-O-sulfo groups of the glucosamine units from the heparin fragment. As shown by molecular modeling, using a high affinity binding octasaccharide fragment of heparin, these findings are consistent with a binding mode in which five saccharide residues of fully sulfated heparan sulfate lie in a shallow groove of the alpha-helix that contains the HSPG-binding site (helix 4 of the four-helix bundle of the 22-kDa fragment). This groove is lined with residues Arg-136, Ser-139, His-140, Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147. In the model, all of these residues make direct contact with either the 2-O-sulfo groups of the iduronic acid monosaccharides or the N- and 6-O-sulfo groups of the glucosamine sulfate monosaccharides. This model indicates that apoE has an HSPG-binding site highly complementary to heparan sulfate rich in N- and O-sulfo groups such as that found in the liver and the brain.