High-performance liquid chromatographic method to measure protein L-isoaspartyl/D-aspartyl o-methyltransferase activity in cell lysates

Protein l-isoaspartyl/d-aspartyl o-methyltransferase (PIMT) is a widely expressed protein repair enzyme that restores isomerized aspartyl residues to their normal configuration. Current methods for measuring PIMT activity have limited sensitivity or require radioactivity. We have developed a highly sensitive new assay method to measure PIMT activity in cell lysates. As a substrate, we used a fluorescently labeled delta sleep-inducing peptide (DSIP) that contains an isoaspartyl residue: 7-nitro-2,1,3-benzoxadiazole (NBD)-DSIP(isoAsp). The PIMT-catalyzed transfer of a methyl group onto this substrate can be detected with a simple high-performance liquid chromatography (HPLC) procedure. After the enzyme reaction, the methylated form of the peptide is stable and can be reproducibly separated from the unmethylated form in an acidic solvent and fluorometrically detected by HPLC. The limit of detection was estimated to be approximately 1 pmol of NBD-DSIP(isoAsp) (signal/noise ratio [S/N] = 3), and the quantitation limit of the activity was approximately 18 μg of total cell lysate from HEK293 cells (10.7 pmol/min/mg protein). This assay method is sensitive enough to detect PIMT activity in biological samples without the use of radioisotopes, offering significant advantages over previously reported methods.     

Reactive oxygen species generated by thiol-modifying phenylarsine oxide stimulate the expression of protein L-isoaspartyl methyltransferase

Expression of the repair enzyme protein l-isoaspartyl methyltransferase (PIMT) has been reported to play important roles in brain. However, little is known about the regulation of PIMT expression following protein damage by oxidation in brain. Phenylarsine oxide (PAO) is an arsenical compound that alters proteins by forming disulfide bond with vicinal cysteinyl residues. Here we report that PIMT was rapidly up-regulated by PAO in U-87 human astroglioma cells. We also confirmed that PIMT up-regulation by PAO was mediated by the reaction with vicinal cysteines. Furthermore, we showed that PIMT induction by PAO was dependent on formation of reactive oxygen species (ROS). Crucially, both ROS formation and PIMT induction by PAO were inhibited by antioxidant N-acetyl-l-cysteine and NADPH oxidase inhibitor diphenyleneiodonium chloride. Importantly, down-regulation of PIMT by siRNA strikingly enhanced PAO-induced ROS. Together, these results highlight that PIMT expression is regulated by ROS and could primarily act as an antioxidant enzyme.     

Suppression of protein l-isoaspartyl (d-aspartyl) methyltransferase results in hyperactivation of EGF-stimulated MEK-ERK signaling in cultured mammalian cells

l-Aspartyl (l-Asp) and l-asparaginyl residues in proteins isomerize or racemize to d,l-isoaspartyl (d,l-isoAsp) or d-aspartyl (d-Asp) residues during protein aging. These atypical aspartyl residues can interfere with the biological function of the protein and lead to cellular dysfunction. Protein l-isoaspartyl (d-aspartyl) methyltransferase (PIMT) is a repair enzyme that facilitates conversion of l-isoAsp and d-Asp to l-Asp. PIMT deficient mice exhibit accumulation of l-isoAsp in several tissues and die, on average, 12 days after birth from progressive epileptic seizures with grand mal and myoclonus features. However, little is known about the molecular mechanisms by which accumulation of the aberrant residues leads to cellular abnormalities. In this study, we established PIMT-knockdown cells using a short interfering RNA expression system and characterized the resultant molecular abnormalities in intracellular signaling pathways. PIMT-knockdown cells showed significant accumulation of proteins with isomerized residues, compared to control cells. In the PIMT-knockdown cells, Raf-1, MEK, and ERK, members of the MAPK cascade, were hyperphosphorylated after EGF stimulation compared to control cells. These results suggest that PIMT repair of abnormal proteins is necessary to maintain normal MAPK signaling.     

Down-regulation of protein L-isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin

Protein L-isoaspartyl methyltransferase (PIMT) repairs the damaged proteins which have accumulated abnormal aspartyl residues during cell aging. Gene targeting has elucidated a physiological role for PIMT by showing that mice lacking PIMT died prematurely from fatal epileptic seizures. Here we investigated the role of PIMT in human mesial temporal lobe epilepsy. Using surgical specimens of hippocampus and neocortex from controls and epileptic patients, we showed that PIMT activity and expression were 50% lower in epileptic hippocampus than in controls but were unchanged in neocortex. Although the protein was down-regulated, PIMT mRNA expression was unchanged in epileptic hippocampus, suggesting post-translational regulation of the PIMT level. Moreover, several proteins with abnormal aspartyl residues accumulate in epileptic hippocampus. Microtubules component beta-tubulin, one of the major PIMT substrates, had an increased amount (two-fold) of L-isoaspartyl residues in the epileptic hippocampus. These results demonstrate that the down-regulation of PIMT in epileptic hippocampus leads to a significant accumulation of damaged tubulin that could contribute to neuron dysfunction in human mesial temporal lobe epilepsy.     

Regulation of protein L-isoaspartyl methyltransferase by cell-matrix interactions: involvement of integrin alphavbeta3, PI 3-kinase, and the proteasome.

The enzyme L-isoaspartyl methyltransferase (PIMT) is known to repair damaged proteins that have accumulated abnormal aspartyl residues during cell aging. However, little is known about the mechanisms involved in the regulation of PIMT expression. Here we report that PIMT expression in bovine aortic endothelial cells is regulated by cell detachment and readhesion to a substratum. During cell detachment, the PIMT level was rapidly and strongly increased and correlated with a stimulation of protein synthesis. Aside from endothelial cells, PIMT levels were also regulated by cell adhesion in various cancer cell lines. The upregulation of PIMT expression could be prevented by an anti-alphavbeta3 antibody (LM609) or by a cyclic RGD peptide (XJ735) specific to integrin alphavbeta3, indicating that this integrin was likely involved in PIMT regulation. Moreover, we found that PIMT expression returned to the basal level when cells were replated on a substratum after detachment, though downregulation of PIMT expression could be partly prevented by the PI3K inhibitors LY294002 and wortmannin, as well as by the proteasome inhibitors MG-132, lactacystin, and beta-lactone. These findings support the assumption that the PIMT level was downregulated by proteasomal degradation, involving the PI3K pathway, during cell attachment. This study reports new insights on the molecular mechanisms responsible for the regulation of PIMT expression in cells. The regulation of PIMT level upon cell-substratum contact suggests a potential role for PIMT in biological processes such as wound healing, cell migration, and tumor metastasis dissemination.     

Injury-Induced Enzymatic Methylation of Aging Collagen in the Extracellular Matrix of Blood Vessels

As a result of blood vessel injury, protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT), a normally intracellular enzyme, becomes trapped within the meshwork of the vascular extracellular matrix where it can methylate substrate proteins. In this investigation we examined the distribution of such altered aspartyl-containing substrate proteins in the vascular wall. Nearly 90% of all the altered aspartyl residues were inaccessible to intracellular PIMT. Proteins of the extracellular matrix were found to be the major repository of altered aspartyl-containing polypeptides in the blood vessel wall, accounting for 70% of the total amount. Proteolytic cleavage of extracellular matrix proteins with cyanogen bromide (CNBr) revealed that collagens account for most of the altered aspartyl-containing proteins of the ECM. As a consequence of blood vessel injury, both type I and type III collagen along with other proteins were found to become methylated by injury-released PIMT. It is estimated that 1 cm of vein contains on the order of 5×10¹⁴ altered aspartyl residues involving between 1% and 5% of the total extracellular protein.     

The structure of the antigenic polysaccharide produced by Eubactrium saburreum T15

The antigenic polysaccharide was obtained from the cell wall of Eubacterium saburreum strain T15 by trypsin digestion followed by gel permeation and ion-exchange chromatography. Its structure was determined using acid hydrolysis, methylation analysis, and 1D and 2D NMR spectroscopy. It contained L-threo-pent-2-ulose (Xul), D-fucose (Fuc), and D-glycero-D-galacto-heptose (Hep) in 2:3:3 ratio. Methylation analysis indicated an octasaccharide repeating-unit containing five branches. The 1H and 13C signals in NMR spectra of the sugar residues were assigned by COSY, HOHAHA, and HMQC 2D experiments, and the sequence of sugar residues in the repeating unit was determined by NOESY and HMBC experiments. The polysaccharide also contains two O-acetyl groups in the repeating unit, located on the Hep residue. The repeating structure can be written as: [see text for equation]. This is a novel structure in bacterial cell-wall polysaccharides from Gram-positive bacteria.     

Altered aspartate in Alzheimer neurofibrillary tangles

Normal protein-boundl-aspartyl/l-asparaginyl residues may undergo post-translational modification by racemization tod-aspartate, or by isomerization to thel-isoaspartyl form in which the peptide chain links through the beta carboxyl group of the residue. Based on preliminary results reported here, proteins associated with Alzheimer neurofibrillary tangle preparations contain a significantly greater number of these modified aspartyl residues than the unaffected proteins from the surrounding gray matter or in comparable preparations from normal brains.     

L-Glutamate in the extracellular space regulates endogenous D-aspartate homeostasis in rat pheochromocytoma MPT1 cells

In previous studies [FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92], we demonstrated for the first time that D-aspartate (D-Asp) is synthesized in cultured mammalian cell lines, such as pheochromocytoma 12 (PC12) and its subclone, MPT1. Our current focus is analysis of the dynamics of D-Asp homeostasis in these cells. In this communication, we show that L-glutamate (Glu) and L-Glu transporter substrates in the extracellular space regulate the homeostasis of endogenous D-Asp in MPT1 cells. D-Asp is apparently in dynamic homeostasis, whereby endogenous D-Asp is constantly released into the extracellular space by an undefined mechanism, and continuously and intensively taken up into cells by an L-Glu transporter. Under these conditions, L-Glu and its transporter substrates in the medium may competitively inhibit the uptake of D-Asp via the transporter, resulting in accumulation of the amino acid in the extracellular space. We additionally demonstrate that DL-TBOA, a well-established L-Glu transporter inhibitor, is taken up by the transporter during long time intervals, but not on a short time-scale.     

Production of L-xylose from L-xylulose using Escherichia coli L-fucose isomerase

l-Xylulose was used as a raw material for the production of l-xylose with a recombinantly produced Escherichia colil-fucose isomerase as the catalyst. The enzyme had a very alkaline pH optimum (over 10.5) and displayed Michaelis-Menten kinetics for l-xylulose with a K_m of 41 mM and a V_max of 0.23 μmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6 h 50 min and 1 h 31 min, respectively. The reaction equilibrium between l-xylulose and l-xylose was 15:85 at 35 °C and thus favored the formation of l-xylose. Contrary to the l-rhamnose isomerase catalyzed reaction described previously [14]l-lyxose was not detected in the reaction mixture with l-fucose isomerase. Although xylitol acted as an inhibitor of the reaction, even at a high ratio of xylitol to l-xylulose the inhibition did not reach 50%.     

Detection and Characterization of a Protein Isoaspartyl Methyltransferase Which Becomes Trapped in the Extracellular Space During Blood Vessel Injury

Injury to rat blood vessels in vivo was found to release intracellular pools of protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT) into the extracellular milieu, where it becomes trapped. This trapped cohort of PIMT is able to utilize radiolabeled S-adenosyl-L-methionine (AdoMet) introduced into the circulation to methylate blood vessel proteins containing altered aspartyl residues. As further shown in this study, methylated substrates are detected only at the specific site of injury. In vitro studies more fully characterized this endogenous PIMT activity in thoracic aorta and inferior vena cava. Methylation kinetics, immunoblotting, and the lability of methylated substrates at mild alkaline pH were used to demonstrate that both types of blood vessel contain an endogeneous protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT). At least 50% of the PIMT activity is resistant to nonionic detergent extraction, suggesting that the enzyme activity becomes trapped within or behind the extracellular matrix (ECM). Quantities of lactate dehydrogenase (LDH), another soluble enzyme of presumed intracellular origin, were found to be similarly trapped in the extracellular space of blood vessels.     

A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

Dictyostelium phenylalanine hydroxylase is activated by its substrate phenylalanine

We have studied the regulatory function of Dictyostelium discoideum Ax2 phenylalanine hydroxylase (dicPAH) via characterization of domain structures. Including the full-length protein, partial proteins truncated in regulatory, tetramerization, or both, were prepared from Escherichia coli as his-tag proteins and examined for oligomeric status and catalytic parameters for phenylalanine. The proteins were also expressed extrachromosomally in the dicPAH knockout strain to examine their in vivo compatibility. The results suggest that phenylalanine activates dicPAH, which is functional in vivo as a tetramer, although cooperativity was not observed. In addition, the results of kinetic study suggest that the regulatory domain of dicPAH may play a role different from that of the domain in mammalian PAH.

Structured summary of protein interactions

dicPAH and dicPAHbind by molecular sieving (View Interaction: 1, 2, 3, 4)     

A novel glycoside hydrolase family 105: the structure of family 105 unsaturated rhamnogalacturonyl hydrolase complexed with a disaccharide in comparison with family 88 enzyme complexed with the disaccharide

YteR, a hypothetical protein with unknown functions, is derived from Bacillus subtilis strain 168 and has an overall structure similar to that of bacterial unsaturated glucuronyl hydrolase (UGL), although it exhibits little amino acid sequence identity with UGL. UGL releases unsaturated glucuronic acid from glycosaminoglycan treated with glycosaminoglycan lyases. The amino acid sequence of YteR shows a significant homology (26% identity) with the hypothetical protein YesR also from B. subtilis strain 168. To clarify the intrinsic functions of YteR and YesR, both proteins were overexpressed in Escherichia coli, purified, and characterized. Based on their gene arrangements in genome and enzyme properties, YteR and YesR were found to constitute a novel enzyme activity, "unsaturated rhamnogalacturonyl hydrolase," classified as new glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) obtained from RG type-I treated with RG lyases and releases an unsaturated galacturonic acid. The crystal structure of YteR complexed with unsaturated chondroitin disaccharide (UGL substrate) was obtained and compared to the structure of UGL complexed with the same disaccharide. The UGL substrate is sterically hindered with the active pocket of YteR. The protruding loop of YteR prevents the UGL substrate from being bound effectively. The most likely candidate catalytic residues for general acid/base are Asp143 in YteR and Asp135 in YesR. This is supported by three-dimensional structural and site-directed mutagenesis studies. These findings provide molecular insights into novel enzyme catalysis and sequential reaction mechanisms involved in RG-I depolymerization by bacteria.     

Crystal structure of a novel beta-lactam-insensitive peptidoglycan transpeptidase

During the final stages of cell-wall synthesis in bacteria, penicillin-binding proteins (PBPs) catalyse the cross-linking of peptide chains from adjacent glycan strands of nascent peptidoglycan. We have recently shown that this step can be bypassed by an L,D-transpeptidase, which confers high-level beta-lactam-resistance in Enterococcus faecium. The resistance bypass leads to replacement of D-Ala4-->D-Asx-L-Lys3 cross-links generated by the PBPs by L-Lys3-->D-Asx-L-Lys3 cross-links generated by the L,D-transpeptidase. As the first structure of a member of this new transpeptidase family, we have determined the crystal structure of a fragment of the L,D-transpeptidase from E.faecium (Ldt(fm217)) at 2.4A resolution. Ldt(fm217) consists of two domains, the N-terminal domain, a new mixed alpha-beta fold, and the ErfK_YbiS_YhnG C-terminal domain, a representative of the mainly beta class of protein structures. Residue Cys442 of the C-terminal domain has been proposed to be the catalytic residue implicated in the cleavage of the L-Lys-D-Ala peptide bond. Surface analysis of Ldt(fm217) reveals that residue Cys442 is localized in a buried pocket and is accessible by two paths on different sides of the protein. We propose that the two paths to the catalytic residue Cys442 are the binding sites for the acceptor and donor substrates of the L,D-transpeptidase.     

The influence of selected O-alkyl derivatives of cyclodextrins on the enzymatic decomposition of l-tryptophan by l-tryptophan indole-lyase

A series of O-alkyl derivatives of cyclodextrin: heksakis[2,3,6-tri-O-(2′-methoxyethyl)]-α-cyclodextrin; heksakis(2,3-di-O-methyl)-α-cyclodextrin; heptakis(2,3-di-O-methyl)-β-cyclodextrin; heksakis[2,3-di-O-methyl-6-O-(2′-methoxyethyl)]-α-cyclodextrin; heptakis[2,3-di-O-methyl-6-O-(2′-methoxyethyl)]-β-cyclodextrin; heksakis[2,3-di-O-(2′-methoxyethyl)]-α-cyclodextrin and heptakis[2,3-di-O-(2′-methoxyethyl)]-β-cyclodextrin have been synthesized. Purity and composition of the obtained substances were examined. The cyclodextrin derivatives listed above as well as (2-hydroxypropyl)-α-cyclodextrin and (2-hydroxypropyl)-β-cyclodextrin, the two commercially available ones, have been investigated as the additives in the course of enzymatic decomposition of l-tryptophan by l-tryptophan indole-lyase. It has been found that each of cyclodextrin derivatives causes the inhibition of enzymatic process, both competitive and non-competitive. The competitive inhibition is connected with the formation of inclusion complexes between cyclodextrins and l-tryptophan, related to the geometry of these complexes. The mechanism of the non-competitive inhibition is not so evident; it could be related to the formation of the cyclodextrin complexes on the surface of the enzyme, leading to the change in the flexibility of the enzyme molecule.     

Binding of different monosaccharides by lectin PA-IIL from Pseudomonas aeruginosa: thermodynamics data correlated with X-ray structures

The lectin from Pseudomonas aeruginosa (PA-IIL) is involved in host recognition and biofilm formation. Lectin not only displays an unusually high affinity for fucose but also binds to L-fucose, L-galactose and D-arabinose that differ only by the group at position 5 of the sugar ring. Isothermal calorimetry experiments provided precise determination of affinity for the three methyl-glycosides and revealed a large enthalpy contribution. The crystal structures of the complexes of PA-IIL with L-galactose and Met-beta-D-arabinoside have been determined and compared with the PA-IIL/fucose complex described previously. A combination of the structures and thermodynamics provided clues for the role of the hydrophobic group in affinity.     

Oxidation of L-serine and L-threonine by bis(hydrogen periodato)argentate(III) complex anion: a mechanistic study

Oxidation of l-serine and l-threonine by a silver(III) complex anion, [Ag(HIO(6))(2)](5-), has been studied in aqueous alkaline medium. The oxidation products of the amino acids have been identified as ammonia, glyoxylic acid and aldehyde (formaldehyde for serine and acetaldehyde for threonine). Kinetics of the oxidation reactions has been followed by the conventional spectrophotometry in the temperature range of 20.0-35.0 degrees C and the reactions display an overall second-order behavior: first-order with respect to both Ag(III) and the amino acids. Analysis of influences of [OH(-)] and [periodate] on the second-order rate constants k' reveals an empirical rate expression: k(')=(k(a)+k(b)[OH(-)])K(1)/([H(2)IO(6)(3-)](e)+K(1)), where [H(2)IO(6)(3-)](e) is equilibrium concentration of periodate, and where k(a)=6.1+/-0.5M(-1)s(-1), k(b)=264+/-6M(-2)s(-1), and K(1)=(6.5+/-1.3)x10(-4)M for serine and k(a)=12.6+/-1.7M(-1)s(-1), k(b)=(5.5+/-0.2)x10(2)M(-2)s(-1), and K(1)=(6.2+/-1.5)x10(-4)M for threonine at 25.0 degrees C and ionic strength of 0.30M. Activation parameters associated with k(a) and k(b) have also been derived. A reaction mechanism is proposed to involve two pre-equilibria, leading to formation of an Ag(III)-periodato-amino acid ternary complex. The ternary complex undergoes a two-electron transfer from the coordinated amino acid to the metal center via two parallel pathways: one pathway is spontaneous and the other is assisted by a hydroxide ion. Potential applications of the Ag(III) complex as a reagent for modifications of peptides and proteins are implicated.     

Analytical validation of a microplate reader-based method for the therapeutic drug monitoring of L-asparaginase in human serum

The enzyme L-asparaginase (ASNASE), which hydrolyzes L-asparagine (L-Asn) to ammonia and L-aspartic acid (L-Asp), is commonly used for remission induction in acute lymphoblastic leukemia. To correlate ASNASE activity with L-Asn reduction in human serum, sensitive methods for the determination of ASNASE activity are required. Using L-aspartic beta-hydroxamate (AHA) as substrate we developed a sensitive plate reader-based method for the quantification of ASNASE derived from Escherichia coli and Erwinia chrysanthemi and of pegylated E. coli ASNASE in human serum. ASNASE hydrolyzed AHA to L-Asp and hydroxylamine, which was determined at 710 nm after condensation with 8-hydroxyquinoline and oxidation to indooxine. Measuring the indooxine formation allowed the detection of 2 x 10(-5)U ASNASE in 20 microl serum. Linearity was observed within 2.5-75 and 75-1,250 U/L with coefficients of correlation of r(2)>0.99. The coefficients of variation for intra- and interday variability for the three different ASNASE enzymes were 1.98 to 8.77 and 1.73 to 11.0%. The overall recovery was 101+/-9.92%. The coefficient of correlation for dilution linearity was determined as r(2)=0.986 for dilutions up to 1:20. This method combined with sensitive methods for the quantification of L-Asn will allow bioequivalence studies and individualized therapeutic drug monitoring of different ASNASE preparations.     

Structural basis of substrate specificity in human glyoxylate reductase/hydroxypyruvate reductase

Human glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is a D-2-hydroxy-acid dehydrogenase that plays a critical role in the removal of the metabolic by-product glyoxylate from within the liver. Deficiency of this enzyme is the underlying cause of primary hyperoxaluria type 2 (PH2) and leads to increased urinary oxalate levels, formation of kidney stones and renal failure. Here we describe the crystal structure of human GRHPR at 2.2 A resolution. There are four copies of GRHPR in the crystallographic asymmetric unit: in each homodimer, one subunit forms a ternary (enzyme+NADPH+reduced substrate) complex, and the other a binary (enzyme+NADPH) form. The spatial arrangement of the two enzyme domains is the same in binary and ternary forms. This first crystal structure of a true ternary complex of an enzyme from this family demonstrates the relationship of substrate and catalytic residues within the active site, confirming earlier proposals of the mode of substrate binding, stereospecificity and likely catalytic mechanism for these enzymes. GRHPR has an unusual substrate specificity, preferring glyoxylate and hydroxypyruvate, but not pyruvate. A tryptophan residue (Trp141) from the neighbouring subunit of the dimer is projected into the active site region and appears to contribute to the selectivity for hydroxypyruvate. This first crystal structure of a human GRHPR enzyme also explains the deleterious effects of naturally occurring missense mutations of this enzyme that lead to PH2.