Lobe-specific heterogeneity in asymmetric dimethylarginine and matrix metalloproteinase levels in a rat model of obstructive cholestasis.

We investigated the effects of obstructive cholestasis in different hepatic lobes by evaluating asymmetric dimethylarginine (ADMA) (a nitric oxide synthase inhibitor), protein methyltransferase (PRMT) and dimethylarginine dimethylaminohydrolase (DDAH) (enzymes involved, resp., in its synthesis and degradation), the cationic transporter (CAT), and metalloproteinase (MMP) activity. Sixteen male Wistar rats underwent a 3-day cholestasis by common bile duct ligation (BDL) or sham operation. Blood samples and hepatic biopsies from left lobe (LL), median lobe (ML), and right lobe (RL) were collected. Serum hepatic enzymes, tissue ADMA, DDAH activity, CAT-2 protein, mRNA expression of DDAH and PRMT, and MMP-2 and MMP-9 activity were monitored. Cholestasis was confirmed by altered serum hepatic enzymes. Higher levels of tissue ADMA were detected in RL and ML as compared with LL. PRMT mRNA expression and DDAH activity did not differ among the lobes after BDL. CAT-2 levels are higher in the RL and ML in the sham-operated group. Higher activity in MMP-2 and MMP-9 was found in RL. In conclusion, after cholestasis an increase in hepatic ADMA in RL and ML was detected as well as tissue MMP-2 and MMP-9 activation in RL, supporting the evidence of functional heterogeneity among the liver lobes also occurring in an obstructive cholestasis model.

A high-performance liquid chromatography method for the quantification of cysmethynil, an inhibitor of isoprenylcysteine carboxylmethyl transferase, in mouse plasma.

Cysmethynil, a newly identified small molecule inhibitor of isoprenylcysteine carboxylmethyl transferase (Icmt) is involved in the post-translational modification of CaaX proteins. Cysmethynil causes cell death in many human cancer cells in vitro, and inhibits tumor growth in the xenograft mouse model in vivo. A HPLC method for the quantification of cysmethynil in mouse plasma was developed and validated. The lower limit of quantification of this method was 0.01microg/ml. Inter- and intra-day variability ranged from 0.38-8.5% and accuracy was between 86% and 98%. This sensitive method was used to quantify cysmethynil in plasma of mice after intraperitoneal dosing for preliminary pharmacokinetic studies.

Influence of osmotic stress on protein methylation in resealed erythrocytes.

Resealed erythrocytes are a useful means of targeting exogenous proteins or extending their activity. We tested human resealed erythrocytes as a model system for studying protein methyl esterification, a reaction involved in the processing of spontaneously deamidated/isomerized polypeptides. Our results show that resealed erythrocytes are still active in the metabolic processes that lead to the formation of methyl-esterified proteins. The methylation pattern of endogenous membrane proteins appeared to be similar to that of normal erythrocytes, with bands 2.1, 3, 4.1 and 4.2 as the major methyl acceptors. We detected methyl esterification of ovalbumin, as an exogenous substrate trapped within resealed erythrocytes. Methyl incorporation was almost completely inhibited by simultaneously loading red cells with adenosine and homocysteine thiolactone, in vivo precursors of the transmethylation inhibitor S-adenosylhomocysteine. We investigated the effects of repeated resealing procedures on methyl acceptability of endogenous membrane proteins. We found that methyl-incorporation levels increased, despite an apparent conserved protein composition of the membrane. This result suggests that osmotic stress to the membrane may be responsible for increased protein methylation due to the appearance of new sites or an increased accessibility of existing sites.

Characterization of a plasma membrane-associated prenylcysteine-directed alpha carboxyl methyltransferase in human neutrophils.

Signal transduction in human neutrophils requires prenylcysteine-directed carboxyl methylation of ras-related low molecular weight GTP-binding proteins. We now report the subcellular localization and characterization of a neutrophil prenylcysteine alpha carboxyl methyltransferase. The highest carboxyl methyltransferase activity copurified with biotinylated neutrophil surface membranes, supporting a plasma membrane localization of the enzyme. Neutrophil nuclear fractions contained little or no methyltransferase activity. Methyltransferase activity was detergent-sensitive but could be reconstituted by removal of detergent in the presence of phosphatidyl choline and an anionic phospholipid. N-Acetyl-S-trans,trans-farnesyl-L-cysteine (AFC) and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine (AGGC) were effective substrates for neutrophil prenylcysteine-directed methyltransferase; Vmax values for AFC and AGGC (16.4 and 22.1 pmol of methylated/mg protein/min, respectively) are among the highest yet reported. Although both GTP gamma S and the chemoattractant fMet-Leu-Phe stimulated methylation of ras-related proteins, neither affected methylation of AFC. These data suggest that neutrophil plasma membranes contain a phospholipid-dependent, prenylcysteine-directed carboxyl methyltransferase of relatively high specific activity that modifies ras-related protein substrates in the GTP-bound, activated state.

Automethylation of protein (D-aspartyl/L-isoaspartyl) carboxyl methyltransferase, a response to enzyme aging.

A question that is central to understanding the mechanisms of aging and cellular deterioration is whether enzymes involved in recognition and metabolism of spontaneously damaged proteins are themselves damaged, either becoming substrates for their own activity; or being unable to act upon themselves, initiating cascades of cellular damage. We show here by in vitro experiments that protein (D-aspartyl/L-isoaspartyl) carboxyl methyltransferase (PCM) from bovine erythrocytes does methylate age-dependent amino acid damage in its own sequence. The subpopulation that is methylated, termed the alpha PCM fraction, appears to be formed through age-dependent deamidation of an asparaginly site to either an L-isoaspartyl or D-aspartyl site because (a) the stoichiometry of automethylation of purified PCM is less than 1%, a value typical of the substoichiometric methylation of many other aged protein substrates, (b) alpha PCM is slightly more acidic than the bulk of PCM, and (c) the methyl esterified site in alpha PCM has the characteristic base-lability of this type of methyl ester. Also, the methyl group is not incorporated into the enzyme as an active site intermediate because the incorporated methyl group is not chased onto substrate protein. The effect of enzyme dilution on the rate of the automethylation reaction is consistent with methylation occurring between protein molecules, showing that the pool of PCM is autocatalytic even though individual molecules may not be. The automethylation and possible self-repair of the PCM pool has implications for maintaining the in vivo efficiency of methylation-dependent protein repair.

Recognition of D-aspartyl residues in polypeptides by the erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase. Implications for the repair hypothesis.

We provide here the first direct evidence that D-aspartyl residues in peptides are substrates for the L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77). We do this by showing that D-aspartic acid beta-methyl ester can be isolated from carboxypeptidase Y digests of enzymatically methylated D-aspartyl-containing synthetic peptides. The specificity of this reaction is supported by the lack of methylation of L-aspartyl-containing peptides under similar conditions. Methylation of D-aspartyl residues in synthetic peptides was not observed previously because with Km values ranging from 2.5 to 4.8 mM, these peptides are recognized by the methyltransferase with 700-10,000-fold lower affinity than are their L-isoaspartyl-containing counterparts. The physiological significance of D-aspartyl methylation was investigated in two ways. First, analysis of in situ methylated human erythrocyte proteins showed that at least 22% of the methyl groups associated with the proteins ankyrin and band 4.1 are on D-aspartyl residues, suggesting that D-aspartyl methylation is an important function of the methyltransferase in vivo. Second, mathematical modeling of the protein aging and methylation reactions occurring in intact erythrocytes indicated that the accumulation of D-aspartyl residues can be reduced as much as 2-5-fold by the methyltransferase activity. Although this reduction is much less than that predicted for L-isoaspartyl residues, it may be significant in maintaining functional proteins throughout the 120-day life span of these cells.

Distinct C-terminal sequences of isozymes I and II of the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase.

We have purified the more acidic major isozyme (II) of the human erythrocyte L-isoaspartyl/D-aspartyl methyltransferase and compared its structure to that of the previously sequenced isozyme I. These isozymes are both monomers of 25,000 molecular weight polypeptides and have similar enzymatic properties, but have isoelectric points that differ by one pH unit. Analysis of 16 tryptic peptides of isozyme II accounting for 89% of the sequence of isozyme I revealed no differences between these enzyme forms. However, analysis of a Staphylococcal V8 protease C-terminal fragment revealed that the last two residues of these proteins differed. The Trp-Lys-COOH terminus of isozyme I is replaced by a Asp-Asp-COOH terminus in isozyme II. Southern blot analysis of genomic DNA suggests that the human genome [corrected] may contain only a single gene encoding the enzyme. We propose that the distinct C-termini of isozymes I and II can arise from the generation of multiple mRNA's by alternative splicing.

Sequence of the D-aspartyl/L-isoaspartyl protein methyltransferase from human erythrocytes. Common sequence motifs for protein, DNA, RNA, and small molecule S-adenosylmethionine-dependent methyltransferases.

A widely distributed protein methyltransferase catalyzes the transfer of a methyl group from S-adenosyl-methionine to the free carboxyl groups of D-aspartyl and/or L-isoaspartyl derivatives of L-aspartyl and L-asparaginyl residues. This enzyme has been postulated to function in the repair or the catabolism of age-damaged proteins. We present here the complete amino acid sequence of the more basic isozyme I of this enzyme from human erythrocytes. The sequence was determined by Edman degradation and mass spectral analysis of overlapping trypsin, Staphylococcus aureus V8 protease, Pseudomonas fragi endoproteinase Asp-N, cyanogen bromide, and hydroxylamine-generated fragments. The NH2-terminus is modified by acetylation and the protein contains 226 amino acids for a calculated molecular weight of 24,575. This value is in good agreement with the molecular weight determined for the purified protein by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and by gel filtration chromatography under nondenaturing conditions. The identification of 2 different amino acid residues at both positions 22 and 119 may indicate the presence of allelic variants or of two or more closely related structural genes. Finally, comparison of this sequence with those of methyltransferases for RNA, DNA, and small molecules, as well as other S-adenosylmethionine-utilizing enzymes, shows that many of these proteins share elements of three regions of sequence similarity and may be structurally or evolutionarily related.

Two major isozymes of the protein D-aspartyl/L-isoaspartyl methyltransferase from human erythrocytes.

We have been able to separate protein carboxyl methyltransferase activity from human erythrocyte cytosol into two major fractions by DEAE-cellulose chromatography. These isozymes, designated I and II, are characterized by their isoelectric points of approximately 6.6 and 5.5 as determined by isoelectric focusing in polyacrylamide gels. The ratio of the isozymes (II/I) was found to range from 0.52 to 1.2 in blood samples from 14 individuals. We did not detect differences in this ratio between males and females. We also found no differences between freshly drawn and outdated blood samples. Both isozymes catalyzed the methylation of proteins such as ovalbumin as well as synthetic L-isoaspartyl-containing peptides.

Protein-O-carboxylmethyltransferase from cytosol and membranes of chicken erythrocytes.

Cytosolic protein-O-carboxylmethyltransferase was purified more than 4,000-fold in specific activity and membrane-associated protein-O-carboxylmethyltransferase carboxymethylase about 900-fold from chicken erythrocytes by use of a combination of affinity chromatography on immobilized S-adenosyl-L-homocysteine and gel filtration on Sephacryl S-200 (Pharmacia), together with 3-((3-cholamidopropyl)-dimethylammonio)-1-propane-sulfonate as a detergent to solubilize the membrane-associated enzyme. The two enzymes were characterized by examining the dependence of their activity on pH and on concentration of S-adenosyl-L-methionine using fetuin as an exogenous methyl-acceptor substrate, and were found to differ somewhat. The cytosolic enzyme had a pH optimum of 6.0 with an apparent Km value of 2.1 microM for S-adenosyl-L-methionine, whereas corresponding values for the membrane-associated enzyme were 6.5 and 0.71 microM. This report deals with the biochemical differences between purified cytosolic and membrane-associated protein carboxymethylase from the same cell source.

Conversion of isoaspartyl peptides to normal peptides: implications for the cellular repair of damaged proteins.

The hypothesis that cellular protein carboxyl-methylation reactions recognize altered aspartyl residues as part of a protein repair pathway has been tested in an in vitro system using tetragastrin (Trp-Met-Asp-Phe-NH2) as a model sequence. The L-isoaspartyl form of tetragastrin, where the phenylalanine residue is linked to the side-chain carboxyl group of the aspartate residue ([iso-Asp3]tetragastrin), is a substrate for the erythrocyte protein carboxyl methyltransferases, while the normal form is not. The enzymatically produced alpha-methyl ester of [iso-Asp3]tetragastrin, [iso-Asp(OMe)3]tetragastrin, is unstable at pH 7.4 and 37 degrees C and spontaneously demethylates with a half-time of 41 min to an intermediate L-succinimide form ([Asu3]tetragastrin) that, in turn, spontaneously hydrolyzes with a half time of 116 min to give a mixture of normal tetragastrin (20%) and [iso-Asp3]tetragastrin (80%). This sequence of enzymatic and nonenzymatic reactions can be coupled in a single reaction mixture; the [iso-Asp3]tetragastrin that is produced upon succinimide hydrolysis can reenter the reaction sequence by enzymatic methylation, and the net result of the process is the conversion of the isomerized peptide to the normal peptide. The efficiency of this "repair" reaction is limited by a side reaction of racemization at the alpha-carbon of the succinimide (half-time = 580 min). In a 24-hr time period, normal L-aspartyl-containing tetragastrin is obtained in about 50% yield from the coupled reaction mixture; other products include [D-iso-Asp3]tetragastrin and [D-Asp3]tetragastrin. The versatile chemistry of succinimide peptides suggests that methylated L-isoaspartyl sites (and possibly methylated D-aspartyl sites) in cellular polypeptides can eventually yield "repaired" normal L-aspartyl sites through succinimide intermediates.

Enzymatic methylation of band 3 anion transporter in intact human erythrocytes.

Band 3, the anion transport protein of erythrocyte membranes, is a major methyl-accepting substrate of the intracellular erythrocyte protein carboxyl methyltransferase (S-adenosyl-L-methionine: protein-D-aspartate O-methyltransferase; EC 2.1.1.77) [Freitag, C., & Clarke, S. (1981) J. Biol. Chem. 256, 6102-6108]. The localization of methylation sites in intact cells by analysis of proteolytic fragments indicated that sites were present in the cytoplasmic N-terminal domain as well as the membranous C-terminal portion of the polypeptide. The amino acid residues that serve as carboxyl methylation sites of the erythrocyte anion transporter were also investigated. 3H-Methylated band 3 was purified from intact erythrocytes incubated with L-[methyl-3H]methionine and from trypsinized and lysed erythrocytes incubated with S-adenosyl-L-[methyl-3H]methionine. After proteolytic digestion with carboxypeptidase Y, D-aspartic acid beta-[3H]methyl ester was isolated in low yields (9% and 1%, respectively) from each preparation. The bulk of the radioactivity was recovered as [3H]methanol, and the amino acid residue(s) originally associated with these methyl groups could not be determined. No L-aspartic acid beta-[3H]methyl ester or glutamyl gamma-[3H]methyl ester was detected. The formation of D-aspartic acid beta-[3H]methyl esters in this protein in intact cells resulted from protein carboxyl methyltransferase activity since it was inhibited by adenosine and homocysteine thiolactone, which increases the intracellular concentration of the potent product inhibitor S-adenosylhomocysteine, and cycloleucine, which prevents the formation of the substrate S-adenosyl-L-[methyl-3H]methionine.

Chemical conversion of aspartyl peptides to isoaspartyl peptides. A method for generating new methyl-accepting substrates for the erythrocyte D-aspartyl/L-isoaspartyl protein methyltransferase.

Mammalian protein carboxyl methyltransferases have recently been proposed to recognize atypical configurations of aspartic acid and may possibly function in the metabolism of covalently altered cellular proteins. Consistent with this proposal, the tetrapeptide tetragastrin, containing a single "normal" L-aspartyl residue (L-Trp-L-Met-L-Asp-L-Phe-NH2) was found here not to be an in vitro substrate for erythrocyte carboxyl methyltransferase activity. However, chemical treatment of tetragastrin by methyl esterification and then de-esterification of the aspartic acid residue yielded a mixture of peptide products, the major one of which could now be enzymatically methylated. We show here that this new peptide species is the isomeric beta-aspartyl form of tetragastrin (L-iso-tetragastrin; L-Trp-L-Met-L-Asp-L-Phe-NH2), and it appears that isomerization proceeds via an intramolecular succinimide intermediate during the de-esterification procedure. L-iso-Tetragastrin is stoichiometrically methylated (up to 90% in these experiments) with a Km for the enzyme of 5.0 microM. Similar chemical treatment of several other L-aspartyl peptides also resulted in the formation of new methyltransferase substrates. This general method for converting normal aspartyl peptides to isoaspartyl peptides may have application in the reverse process as well.

Enzymatic basis for the calcium-induced decrease of membrane protein methyl esterification in intact erythrocytes. Evidence for an impairment of S-adenosylmethionine synthesis.

The effect of Ca2+ loading, induced by the ionophore A23187, on methyl esterification of membrane proteins (i.e. bands 2.1, 3, 4.1 and 4.5) has been investigated in intact human erythrocytes. When the cells were incubated with L-[methyl-3H]methionine, 40 microM CaCl2 and 10 microM A23187 induce a 50% inhibition of membrane protein methyl esterification. This effect is selectively due to the increased intracellular Ca2+ concentration, as it is antagonized by 10 mM EGTA, and other divalent cations such as Mn2+ do not exert any inhibition. In order to clarify the mechanism(s) of the reported inhibition, the various events involved in the methyl esterification process in vivo were analyzed. L-Methionine uptake as well as protein methylase II activity are not directly affected by altered intracellular Ca2+ concentrations. Conversely in the Ca2+-loaded erythrocytes the conversion of [3H]methionine into [3H]AdoMet, catalyzed by AdoMet synthetase, decreases up to 25%. When the undialyzed erythrocyte cytosolic fraction is assayed in vitro for AdoMet synthetase the activity of the enzyme from the CaCl2/A23187-treated erythrocytes is significantly lower than the control, up to 5 mM ATP. This result suggests that in the Ca2+-loaded erythrocytes the ATP intracellular concentration is significantly lowered. The direct evaluation of ATP intracellular concentration, by HPLC, confirms a significant drop of ATP level, as a consequence of the Ca2+ loading. The removal of Ca2+ from the cells quantitatively restores both the AdoMet synthesis and the methyl esterification levels. The possible role of altered ATP intracellular concentrations as a regulatory factor in the AdoMet-dependent reactions as well as in post-translational protein methylation related to the ageing process is also discussed.

Synthetic peptide substrates for the erythrocyte protein carboxyl methyltransferase. Detection of a new site of methylation at isomerized L-aspartyl residues.

Four hexapeptides of sequence L-Val-L-Tyr-L-Pro-(Asp)-Gly-L-Ala containing D- or L-aspartyl residues in normal or isopeptide linkages have been synthesized by the Merrifield solid-phase method as potential substrates of the erythrocyte protein carboxyl methyltransferase. This enzyme has been shown to catalyze the methylation of D-aspartyl residues in proteins in red blood cell membranes and cytosol. Using a new vapor-phase methanol diffusion assay, we have found that the normal hexapeptides containing either D- or L-aspartyl residues were not substrates for the human erythrocyte methyltransferase. On the other hand, the L-aspartyl isopeptide, in which the glycyl residue was linked in a peptide bond to the beta-carboxyl group of the aspartyl residue, was a substrate for the enzyme with a Km of 6.3 microM and was methylated with a maximal velocity equal to that observed when ovalbumin was used as a methyl acceptor. The enzyme catalyzed the transfer of up to 0.8 mol of methyl groups/mol of this peptide. Of the four synthetic peptides, only the L-isohexapeptide competitively inhibits the methylation of ovalbumin by the erythrocyte enzyme. This peptide also acts as a substrate for both of the purified protein carboxyl methyltransferases I and II which have been previously isolated from bovine brain (Aswad, D. W., and Deight, E. A. (1983) J. Neurochem. 40, 1718-1726). The L-isoaspartyl hexapeptide represents the first defined synthetic substrate for a eucaryotic protein carboxyl methyltransferase. These results demonstrate that these enzymes can not only catalyze the formation of methyl esters at the beta-carboxyl groups of D-aspartyl residues but can also form esters at the alpha-carboxyl groups of isomerized L-aspartyl residues. The implications of these findings for the metabolism of modified proteins are discussed.

Inhibition of protein carboxyl methylation by S-adenosyl-L-homocysteine in intact erythrocytes. Physiological consequences.

S-Adenosyl-L-homocysteine was used to inhibit the methylation of carboxylic acid residues of membrane proteins in intact human erythrocytes. Incubation of erythrocytes for 24 h with 5 mM each of adenosine and L-homocysteine resulted in the intracellular accumulation of S-adenosyl-L-homocysteine and substantially inhibited membrane protein carboxyl methylation. From the degree of inhibition and from the observed turnover of methylated proteins, we estimate that the number of protein methyl esters in cells incubated with adenosine and L-homocysteine for 20 h is less than 20% that of cells incubated without these inhibitors. No significant differences in the physical deformability properties of the membrane of these hypomethylated cells were detected. However, there was a small but significant (p less than 0.001) increase in the amount of membrane protein D-aspartyl residues in these cells compared to control cells. These observations are consistent with the hypothesis that methylation of membrane proteins at D-aspartyl residues may result in the selective removal or repair of these uncommon residues.