Identification of isoaspartate-modified transthyretin as potential target for selective immunotherapy of transthyretin amyloidosis.

BACKGROUND: Numerous studies suggest a progressive accumulation of post-translationally modified peptides within amyloid fibrils, including isoaspartate (isoD) modifications. Here, we generated and characterised novel monoclonal antibodies targeting isoD-modified transthyretin (TTR). The antibodies were used to investigate the presence of isoD-modified TTR in deposits from transthyretin amyloidosis patients and to mediate antibody-dependent phagocytosis of TTR fibrils. METHODS: Monoclonal antibodies were generated by immunisation of mice using an isoD-modified peptide and subsequent hybridoma generation. The antibodies were characterised in terms of affinity and specificity to isoD-modified TTR using surface plasmon resonance, transmission electron microscopy and immunohistochemical staining of human cardiac tissue. The potential to elicit antibody-dependent phagocytosis of TTR fibrils was assessed using THP-1 cells. RESULTS: We developed two mouse monoclonal antibodies, 2F2 and 4D4, with high nanomolar affinity for isoD-modified TTR and strong selectivity over the unmodified epitope. Both antibodies show presence of isoD-modified TTR in human cardiac tissue, but not in freshly purified recombinant TTR, suggesting isoD modification only present in aged fibrillar deposits. Likewise, the antibodies only facilitated phagocytosis of TTR fibrils and not TTR monomers by THP-1 cells. CONCLUSIONS: These antibodies label aged, non-native TTR deposits, leaving native TTR unattended and thereby potentially enabling new therapeutic approaches.

Helical ultrastructure of the metalloprotease meprin α in complex with a small molecule inhibitor.

The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal tissue homeostasis meprin α performs key roles in inflammation, immunity, and extracellular matrix remodelling. Dysregulated meprin α is associated with acute kidney injury, sepsis, urinary tract infection, metastatic colorectal carcinoma, and inflammatory bowel disease. Accordingly, meprin α is the target of drug discovery programs. In contrast to meprin ß, meprin α is secreted into the extracellular space, whereupon it oligomerises to form giant assemblies and is the largest extracellular protease identified to date (~6 MDa). Here, using cryo-electron microscopy, we determine the high-resolution structure of the zymogen and mature form of meprin α, as well as the structure of the active form in complex with a prototype small molecule inhibitor and human fetuin-B. Our data reveal that meprin α forms a giant, flexible, left-handed helical assembly of roughly 22 nm in diameter. We find that oligomerisation improves proteolytic and thermal stability but does not impact substrate specificity or enzymatic activity. Furthermore, structural comparison with meprin ß reveal unique features of the active site of meprin α, and helical assembly more broadly.

Immunogenicity of the Envelope Surface Unit of Human Endogenous Retrovirus K18 in Mice.

The triggers for the development of multiple sclerosis (MS) have not been fully understood to date. One hypothesis proposes a viral etiology. Interestingly, viral proteins from human endogenous retroviruses (HERVs) may play a role in the pathogenesis of MS. Allelic variants of the HERV-K18 env gene represent a genetic risk factor for MS, and the envelope protein is considered to be an Epstein-Barr virus-trans-activated superantigen. To further specify a possible role for HERV-K18 in MS, the present study examined the immunogenicity of the purified surface unit (SU). HERV-K18(SU) induced envelope-specific plasma IgG in immunized mice and triggered proliferation of T cells isolated from these mice. It did not trigger phenotypic changes in a mouse model of experimental autoimmune encephalomyelitis. Further studies are needed to investigate the underlying mechanisms of HERV-K18 interaction with immune system regulators in more detail.

Structure and Dynamics of Meprin ß in Complex with a Hydroxamate-Based Inhibitor.

The astacin protease Meprin ß represents an emerging target for drug development due to its potential involvement in disorders such as acute and chronic kidney injury and fibrosis. Here, we elaborate on the structural basis of inhibition by a specific Meprin ß inhibitor. Our analysis of the crystal structure suggests different binding modes of the inhibitor to the active site. This flexibility is caused, at least in part, by movement of the C-terminal region of the protease domain (CTD). The CTD movement narrows the active site cleft upon inhibitor binding. Compared with other astacin proteases, among these the highly homologous isoenzyme Meprin α, differences in the subsites account for the unique selectivity of the inhibitor. Although the inhibitor shows substantial flexibility in orientation within the active site, the structural data as well as binding analyses, including molecular dynamics simulations, support a contribution of electrostatic interactions, presumably by arginine residues, to binding and specificity. Collectively, the results presented here and previously support an induced fit and substantial movement of the CTD upon ligand binding and, possibly, during catalysis. To the best of our knowledge, we here present the first structure of a Meprin ß holoenzyme containing a zinc ion and a specific inhibitor bound to the active site. The structural data will guide rational drug design and the discovery of highly potent Meprin inhibitors.

Amyloid-Beta Peptides Trigger Aggregation of Alpha-Synuclein In Vitro.

Alzheimer's disease (AD) and Parkinson's disease (PD), including dementia with Lewy bodies (DLB), account for the majority of dementia cases worldwide. Interestingly, a significant number of patients have clinical and neuropathological features of both AD and PD, i.e., the presence of amyloid deposits and Lewy bodies in the neocortex. The identification of α-synuclein peptides in amyloid plaques in DLB brain led to the hypothesis that both peptides mutually interact with each other to facilitate neurodegeneration. In this article, we report the influence of Aß(1-42) and pGlu-Aß(3-42) on the aggregation of α-synuclein in vitro. The aggregation of human recombinant α-synuclein was investigated using thioflavin-T fluorescence assay. Fibrils were investigated by means of antibody conjugated immunogold followed by transmission electron microscopy (TEM). Our data demonstrate a significantly increased aggregation propensity of α-synuclein in the presence of minor concentrations of Aß(1-42) and pGlu-Aß(3-42) for the first time, but without effect on toxicity on mouse primary neurons. The analysis of the composition of the fibrils by TEM combined with immunogold labeling of the peptides revealed an interaction of α-synuclein and Aß in vitro, leading to an accelerated fibril formation. The analysis of kinetic data suggests that significantly enhanced nucleus formation accounts for this effect. Additionally, co-occurrence of α-synuclein and Aß and pGlu-Aß, respectively, under pathological conditions was confirmed in vivo by double immunofluorescent labelings in brains of aged transgenic mice with amyloid pathology. These observations imply a cross-talk of the amyloid peptides α-synuclein and Aß species in neurodegeneration. Such effects might be responsible for the co-occurrence of Lewy bodies and plaques in many dementia cases.

Dipeptidyl-Peptidase Activity of Meprin ß Links N-truncation of Aß with Glutaminyl Cyclase-Catalyzed pGlu-Aß Formation.

The formation of amyloid-ß (Aß) peptides is causally involved in the development of Alzheimer's disease (AD). A significant proportion of deposited Aß is N-terminally truncated and modified at the N-terminus by a pGlu-residue (pGlu-Aß). These forms show enhanced neurotoxicity compared to full-length Aß. Although the truncation may occur by aminopeptidases after formation of Aß, recently discovered processing pathways of amyloid-ß protein precursor (AßPP) by proteases such as meprin ß may also be involved. Here, we assessed a role of meprin ß in forming Aß3-40/42, which is the precursor of pGlu-Aß3-40/42 generated by glutaminyl cyclase (QC). Similar to QC, meprin ß mRNA is significantly upregulated in postmortem brain from AD patients. A histochemical analysis supports the presence of meprin ß in neurons and astrocytes in the vicinity of pGlu-Aß containing deposits. Cleavage of AßPP-derived peptides by meprin ß in vitro results in peptides Aß1-x, Aß2-x, and Aß3-x. The formation of N-truncated Aß by meprin ß was also corroborated in cell culture. A subset of the generated peptides was converted into pGlu-Aß3-40 by an addition of glutaminyl cyclase, supporting the preceding formation of Aß3-40. Further analysis of the meprin ß cleavage revealed a yet unknown dipeptidyl-peptidase-like activity specific for the N-terminus of Aß1-x. Thus, our data suggest that meprin ß contributes to the formation of N-truncated Aß by endopeptidase and exopeptidase activity to generate the substrate for QC-catalyzed pGlu-Aß formation.

Continuous assays for meprin alpha and beta using prolyl tripeptidyl aminopeptidase (PtP) from Porphyromonas gingivalis.

Common assays for endoprotease activity of meprin α and ß are based on cleavage of internally quenched substrates. Although direct and convenient, for meprins these assays bear disadvantages such as, e.g., significant substrate inhibition or potential fluorescence quenching by compounds applied in inhibitor analysis. Here, we present a novel continuous assay by introducing an auxiliary enzyme, prolyl tripeptidyl aminopeptidase (PtP) and the chromogenic substrate KKGYVADAP-p-nitroanilide. We provide a quick strategy for expression and one-step-purification of the auxiliary enzyme. The enzyme kinetic data for meprin α and ß suggest hyperbolic v/S-characteristics, the kinetic parameters of substrate conversion by meprin ß were Km = 184 ±â€¯32 µM and kcat = 20 ±â€¯4 s-1. We also present conditions for the use of the fluorogenic substrate KKGYVADAP-AMC to assess meprin ß activity. The assays were applied for determination of inhibitory parameters of the natural inhibitor actinonin and two recently published hydroxamates. Hence, we present two novel methods, which can be applied to assess inhibitory mechanism and potency with the attractive current drug targets meprin α and ß. Furthermore, the assay might also provide implications for analysis of other endoproteases as well as their inhibitors.

Purification of recombinant Aß(1-42) and pGlu-Aß(3-42) using preparative SDS-PAGE.

Recombinant expression and purification of amyloid peptides represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. However, the isolation of the recombinant peptides is hampered by inefficient separation from contaminants such as the fusion protein required for efficient expression in E. coli. Here, we present a new approach for the isolation of highly purified Aß(1-42) and pGlu-Aß(3-42), which is based on a separation using preparative SDS-PAGE. The method relies on the purification of the Aß fusion protein by affinity chromatography followed by preparative SDS-PAGE under reducing conditions and subsequent removal of detergents by precipitation. The application of preparative SDS-PAGE represents the key step to isolate highly pure recombinant Aß, which has been applied for characterization of aggregation and toxicity. Thereby, the yield of the purification strategy was  >60%. To the best of our knowledge, this is the first description of an electrophoresis-based method for purification of a recombinant Aß peptide. Therefore, the method might be of interest for isolation of other amyloid peptides, which are critical for conventional purification strategies due to their aggregation propensity.

Probing secondary glutaminyl cyclase (QC) inhibitor interactions applying an in silico-modeling/site-directed mutagenesis approach: implications for drug development.

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate-modified amyloid peptides deposited in neurodegenerative disorders such as Alzheimer's disease. Inhibitors of QC are currently in development as potential therapeutics. The crystal structures of the potent inhibitor PBD150 bound to human and murine QC (hQC, mQC) have been described recently. The binding modes of a dimethoxyphenyl moiety of the inhibitor are significantly different between the structures, which contrasts with a similar K(i) value. We show the conformation of PBD150 prone to disturbance by protein-protein interactions within the crystals. Semi-empirical calculations of the enzyme-inhibitor interaction within the crystal suggest significant differences in the dissociation constants between the binding modes. To probe for interactions in solution, a site-directed mutagenesis on hQC was performed. The replacement of F325 and I303 by alanine or asparagine resulted in a 800-fold lower activity of the inhibitor, whereas the exchange of S323 by alanine or valine led to a 20-fold higher activity of PBD150. The results provide an example of deciphering the interaction mode between a target enzyme and lead substance in solution, if co-crystallization does not mirror such interactions properly. Thus, the study might provide implications for rapid screening of binding modes also for other drug targets.

Crystal structures of glutaminyl cyclases (QCs) from Drosophila melanogaster reveal active site conservation between insect and mammalian QCs.

Glutaminyl cyclases (QCs), which catalyze the formation of pyroglutamic acid (pGlu) at the N-terminus of a variety of peptides and proteins, have attracted particular attention for their potential role in Alzheimer's disease. In a transgenic Drosophila melanogaster (Dm) fruit fly model, oral application of the potent competitive QC inhibitor PBD150 was shown to reduce the burden of pGlu-modified Aß. In contrast to mammals such as humans and rodents, there are at least three DmQC species, one of which (isoDromeQC) is localized to mitochondria, whereas DromeQC and an isoDromeQC splice variant possess signal peptides for secretion. Here we present the recombinant expression, characterization, and crystal structure determination of mature DromeQC and isoDromeQC, revealing an overall fold similar to that of mammalian QCs. In the case of isoDromeQC, the putative extended substrate binding site might be affected by the proximity of the N-terminal residues. PBD150 inhibition of DromeQC is roughly 1 order of magnitude weaker than that of the human and murine QCs. The inhibitor binds to isoDromeQC in a fashion similar to that observed for human QCs, whereas it adopts alternative binding modes in a DromeQC variant lacking the conserved cysteines near the active center and shows a disordered dimethoxyphenyl moiety in wild-type DromeQC, providing an explanation for the lower affinity. Our biophysical and structural data suggest that isoDromeQC and human QC are similar with regard to functional aspects. The two Dm enzymes represent a suitable model for further in-depth analysis of the catalytic mechanism of animal QCs, and isoDromeQC might serve as a model system for the structure-based design of potential AD therapeutics.

The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions.

Acute and chronic inflammatory disorders are characterized by detrimental cytokine and chemokine expression. Frequently, the chemotactic activity of cytokines depends on a modified N-terminus of the polypeptide. Among those, the N-terminus of monocyte chemoattractant protein 1 (CCL2 and MCP-1) is modified to a pyroglutamate (pE-) residue protecting against degradation in vivo. Here, we show that the N-terminal pE-formation depends on glutaminyl cyclase activity. The pE-residue increases stability against N-terminal degradation by aminopeptidases and improves receptor activation and signal transduction in vitro. Genetic ablation of the glutaminyl cyclase iso-enzymes QC (QPCT) or isoQC (QPCTL) revealed a major role of isoQC for pE(1) -CCL2 formation and monocyte infiltration. Consistently, administration of QC-inhibitors in inflammatory models, such as thioglycollate-induced peritonitis reduced monocyte infiltration. The pharmacologic efficacy of QC/isoQC-inhibition was assessed in accelerated atherosclerosis in ApoE3*Leiden mice, showing attenuated atherosclerotic pathology following chronic oral treatment. Current strategies targeting CCL2 are mainly based on antibodies or spiegelmers. The application of small, orally available inhibitors of glutaminyl cyclases represents an alternative therapeutic strategy to treat CCL2-driven disorders such as atherosclerosis/restenosis and fibrosis.

Isolation of dipeptidyl peptidase IV (DP 4) isoforms from porcine kidney by preparative isoelectric focusing to improve crystallization.

Abstract In the present studies we resolved the post-translational microheterogeneity of purified porcine dipeptidyl peptidase IV (DP 4) from kidney cortex. Applying SDS-homogeneous DP 4 onto an analytical agarose isoelectric focusing (IEF) gel, pH 4-6, activity staining resulted in at least 17 isoforms between pH 4.8-6.0. These could be separated into fractions with only two to six isoforms by means of preparative liquid-phase IEF, using a Rotofor cell. Starting off with three parallel Rotofor runs under the same conditions at pH 5-6, the fractions were pooled according to the specific activity of DP 4, pH and analytical IEF profile, and further refractionated without any additional ampholytes. Since excessive dilution of ampholytes and proteins was kept to the minimum, a second refractionation step could be introduced, resulting in pH gradients between 0.022 and 0.028 pH increments per fraction. By performing two consecutive refractionation steps, the high resolution necessary for the separation of DP 4 isoforms could be achieved. This represents an alternative method if isolation of isoforms with similar pI's results in precipitation and denaturation in presence of a narrow pH range. Furthermore, it demonstrates that preparative IEF is a powerful tool to resolve post-translational microheterogeneity of a purified protein required for crystallization processing.

Structures of glycosylated mammalian glutaminyl cyclases reveal conformational variability near the active center.

Formation of N-terminal pyroglutamate (pGlu or pE) from glutaminyl or glutamyl precursors is catalyzed by glutaminyl cyclases (QC). As the formation of pGlu-amyloid has been linked with Alzheimer's disease, inhibitors of QCs are currently the subject of intense development. Here, we report three crystal structures of N-glycosylated mammalian QC from humans (hQC) and mice (mQC). Whereas the overall structures of the enzymes are similar to those reported previously, two surface loops in the neighborhood of the active center exhibit conformational variability. Furthermore, two conserved cysteine residues form a disulfide bond at the base of the active center that was not present in previous reports of hQC structure. Site-directed mutagenesis suggests a structure-stabilizing role of the disulfide bond. At the entrance to the active center, the conserved tryptophan residue, W(207), which displayed multiple orientations in previous structure, shows a single conformation in both glycosylated human and murine QCs. Although mutagenesis of W(207) into leucine or glutamine altered substrate conversion significantly, the binding constants of inhibitors such as the highly potent PQ50 (PBD150) were minimally affected. The crystal structure of PQ50 bound to the active center of murine QC reveals principal binding determinants provided by the catalytic zinc ion and a hydrophobic funnel. This study presents a first comparison of two mammalian QCs containing typical, conserved post-translational modifications.

Mammalian glutaminyl cyclases and their isoenzymes have identical enzymatic characteristics.

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate residues at the N-terminus of several peptides and proteins from plants and animals. Recently, isoenzymes of mammalian QCs have been identified. In order to gain further insight into the biochemical characteristics of isoQCs, the human and murine enzymes were expressed in the secretory pathway of Pichia pastoris. Replacement of the N-terminal signal anchor by an alpha-factor prepropeptide from Saccharomyces cerevisiae resulted in poor secretion of the protein. Insertion of an N-terminal glycosylation site and shortening of the N-terminus improved isoQC secretion 100-fold. A comparison of different recombinant isoQC proteins did not reveal an influence of mutagenic changes on catalytic activity. An initial characterization showed identical modes of substrate conversion of human isoQC and murine isoQC. Both proteins displayed a broad substrate specificity and preference for hydrophobic substrates, similar to the related QC. Likewise, a determination of the zinc content and reactivation of the apo-isoQC revealed equimolar zinc present in QC and isoQC. Far-UV CD spectroscopic analysis of murine QC and isoQC indicated virtually identical structural components. The present investigation provides the first enzymatic characterization of mammalian isoQCs. QC and isoQC represent very similar proteins, which are both present in the secretory pathway of cells. The functions of QCs and isoQC probably complement each other, suggesting a pivotal role of pyroglutamate modification for protein and peptide maturation.

Developmental expression and subcellular localization of glutaminyl cyclase in mouse brain.

Glutaminyl cyclase (QC) converts N-terminal glutaminyl residues into pyroglutamate (pE), thereby stabilizing these peptides/proteins. Recently, we demonstrated that QC also plays a pathogenic role in Alzheimer's disease by generating the disease-associated pE-Abeta from N-terminally truncated Abeta peptides in vivo. This newly identified function makes QC an interesting pharmacological target for Alzheimer's disease therapy. However, the expression of QC in brain and peripheral organs, its cell type-specific and subcellular localization as well as developmental profiles in brain are not known. The present study was performed to address these issues in mice. In brain, QC mRNA expression was highest in hypothalamus, followed by hippocampus and cortex. In liver, QC mRNA concentration was almost as high as in brain while lower QC mRNA levels were detected in lung and heart and very low expression levels were found in kidney and spleen. In the developmental course, stable QC mRNA levels were detected in hypothalamus from postnatal day 5 to 370. On the contrary, in cortex and hippocampus QC mRNA levels were highest after birth and declined during ontogenesis by 20-25%. These results were corroborated by immunocytochemical analysis in mouse brain demonstrating a robust QC expression in a subpopulation of lateral and paraventricular hypothalamic neurons and the labeling of a significant number of small neurons in the hippocampal molecular layer, in the hilus of the dentate gyrus and in all layers of the neocortex. Hippocampal QC-immunoreactive neurons include subsets of parvalbumin-, calbindin-, calretinin-, cholecystokinin- and somatostatin-positive GABAergic interneurons. The density of QC labeled hippocampal neurons declined during postnatal development matching the decrease in QC mRNA expression levels. Subcellular double immunofluorescent analysis localized QC within the endoplasmatic reticulum, Golgi apparatus and secretory granules, consistent with a function of QC in protein maturation and/or modification. Our results are in compliance with a role of QC in hypothalamic hormone maturation and suggest additional, yet unidentified QC functions in brain regions relevant for learning and memory which are affected in Alzheimer's disease.

Inhibition of glutaminyl cyclase prevents pGlu-Abeta formation after intracortical/hippocampal microinjection in vivo/in situ.

Modified amyloid beta (Abeta) peptides represent major constituents of the amyloid deposits in Alzheimer's disease and Down's syndrome. In particular, N-terminal pyroglutamate (pGlu) following truncation renders Abeta more stable, increases hydrophobicity and the aggregation velocity. Recent evidence based on in vitro studies suggests that the cyclization of glutamic acid, leading to pGlu-Abeta, is catalyzed by the enzyme glutaminyl cyclase (QC) following limited proteolysis of Abeta at the N-terminus. Here, we studied the pGlu-formation by rat QC in vitro as well as after microinjection of Abeta(1-40) and Abeta(3-40) into the rat cortex in vivo/in situ with and without pharmacological QC inhibition. Significant pGlu-Abeta formation was observed following injection of Abeta(3-40) after 24 h, indicating a catalyzed process. The generation of pGlu-Abeta from Abeta(3-40) was significantly inhibited by intracortical microinjection of a QC inhibitor. The study provides first evidence that generation of pGlu-Abeta is a QC-catalyzed process in vivo. The approach per se offers a strategy for a rapid evaluation of compounds targeting a reduction of pGlu formation at the N-terminus of amyloid peptides.

Isolation of an isoenzyme of human glutaminyl cyclase: retention in the Golgi complex suggests involvement in the protein maturation machinery.

Mammalian glutaminyl cyclase isoenzymes (isoQCs) were identified. The analysis of the primary structure of human isoQC (h-isoQC) revealed conservation of the zinc-binding motif of the human QC (hQC). In contrast to hQC, h-isoQC carries an N-terminal signal anchor. The cDNAs of human and murine isoQCs were isolated and h-isoQC, lacking the N-terminal signal anchor and the short cytosolic tail, was expressed as a fusion protein in Escherichia coli. h-isoQC exhibits 10fold lower activity compared to hQC. Similar to hQC, h-isoQC was competitively inhibited by imidazoles and cysteamines. Inactivation by metal chelators suggests a conserved metal-dependent catalytic mechanism of both isoenzymes. A comparison of the expression pattern of m-isoQC and murine QC revealed ubiquitous expression of both enzymes. However, murine QC transcript formation was higher in neuronal tissue, whereas the amount of m-isoQC transcripts did not vary significantly between different organs. h-isoQC was exclusively localized within the Golgi complex, obviously retained by the N-terminus. Similar resident enzymes of the Golgi complex are the glycosyltransferases. Golgi apparatus retention implies a "housekeeping" protein maturation machinery conducting glycosylation and pyroglutamyl formation. For these enzymes, apparently similar strategies evolved to retain the proteins in the Golgi complex.

Isolation and characterization of glutaminyl cyclases from Drosophila: evidence for enzyme forms with different subcellular localization.

Glutaminyl cyclases (QCs) present in plants and vertebrates catalyze the formation of pyroglutamic acid (pGlu) from N-terminal glutamine. Pyroglutamyl hormones also identified in invertebrates imply the involvement of QC activity during their posttranslational maturation. Database mining led to the identification of two genes in Drosophila, which putatively encode QCs, CG32412 (DromeQC) and CG5976 (isoDromeQC). Analysis of their primary structure suggests different subcellular localizations. While DromeQC appeared to be secreted due to an N-terminal signal peptide, isoDromeQC contains either an N-terminal mitochondrial targeting or a secretion signal due to generation of different transcripts from gene CG5976. According to the prediction, homologous expression of the corresponding cDNAs in S2 cells revealed either secreted protein in the medium or intracellular QC activity. Subcellular fractionation and immunochemistry support export of isoDromeQC into the mitochondrion. For enzymatic characterization, DromeQC and isoDromeQC were expressed heterologously in Pichia pastoris and Escherichia coli, respectively. Compared to mammalian QCs, the specificity constants were about 1 order of magnitude lower for most of the analyzed substrates. The pH dependence of the specificity constant was similar for both enzymes, indicating the necessity of an unprotonated substrate amino group and two protonated groups of the enzyme, resulting in an asymmetric bell-shaped characteristic. The determination of the metal content of DromeQC revealed equimolar protein-bound zinc. These results prove conserved enzymatic mechanisms between QCs from invertebrates and mammals. Drosophila is the first organism for which isoenzymes of glutaminyl cyclase have been isolated. The identification of a mitochondrial QC points toward yet undiscovered physiological functions of these enzymes.

Isolation and characterization of the glutaminyl cyclases from Solanum tuberosum and Arabidopsis thaliana: implications for physiological functions.

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamic acid at the N-terminus of several peptides and proteins. On the basis of the amino acid sequence of Carica papaya QC, we identified cDNAs of the putative counterparts from Solanum tuberosum and Arabidopsis thaliana. Upon expression of the corresponding cDNAs from both plants via the secretory pathway of Pichia pastoris, two active QC proteins were isolated. The specificity of the purified proteins was assessed using various substrates with different amino acid composition and length. Highest specificities were observed with substrates possessing large hydrophobic residues adjacent to the N-terminal glutamine and for fluorogenic dipeptide surrogates. However, compared to Carica papaya QC, the specificity constants were approximately one order of magnitude lower for most of the QC substrates analyzed. The QCs also catalyzed the conversion of N-terminal glutamic acid to pyroglutamic acid, but with approximately 10(5)- to 10(6)-fold lower specificity. The ubiquitous distribution of plant QCs prompted a search for potential substrates in plants. Based on database entries, numerous proteins, e.g., pathogenesis-related proteins, were found that carry a pyroglutamate residue at the N-terminus, suggesting QC involvement. The putative relevance of QCs and pyroglutamic acid for plant defense reactions is discussed.

Does human attractin have DP4 activity?

Mutations in the mouse ATRN gene, which encodes attractin, offer links between this protein and pigmentation, metabolism, immune status and neurodegeneration. However, the mechanisms of attractin action are not understood. The protein was first identified in humans in a circulating form in serum. A protease activity was postulated similar to the membrane-bound ectoenzyme DP4/CD26. In the last decade, both DP4/CD26 and attractin were controversially described to be the major source of human serum DP4 activity. We purified attractin from human plasma, and found that the DP4-like activity of the preparation shows nearly identical kinetic properties to that of recombinant human DP4. In contrast, the native electrophoretic behavior of this activity is clearly different from human and porcine DP4, but co-migrates with the protein band identified as attractin by Western blotting and N-terminal sequencing. Nevertheless, a DP4 impurity could be demonstrated in purified plasma attractin and the activity could be removed by ADA affinity chromatography, resulting in a homogenous attractin preparation without DP4 activity. These results are substantiated by expression of different attractin isoforms, in which no DP4 activity was found either. This indicates that the multidomain protein attractin acts as a receptor or adhesion protein rather than a protease.