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.     

Identification of isoaspartyl-containing sequences in peptides and proteins that are usually poor substrates for the class II protein carboxyl methyltransferase

We have found that a chicken egg lysozyme derivative (beta-101-lysozyme) containing an L-isoaspartyl residue at position 101 has a Km for methylation by the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase (EC 2.1.1.77) of 183 microM, about 30 times higher than that expected from previous studies with isoaspartyl-containing peptides. In the course of investigating the reasons for this poor enzyme recognition, we found that charged residues on the carboxyl side of isoaspartyl residues had a large effect on the affinity of the enzyme for synthetic peptides. This is best illustrated by the lysozyme-related peptide YVSisoDGDG, which has a Km for methylation of 469 microM. When the penultimate aspartyl residue is replaced by a cysteinyl residue, the Km drops to 4.6 microM, comparable to other peptides of similar size. Furthermore, replacing it with a cysteic acid residue results in a Km of 104 microM, suggesting that a negative charge at this position may lead to a weaker affinity of the peptide substrate for the methyltransferase. Assays with additional synthetic peptides indicate that moving the negative charge to the first or third residue on the carboxyl side of the isoaspartyl residue has a similar but less severe effect in reducing its affinity for the methyltransferase. Enzymatic methylation has recently been proposed to be the first step in the conversion of abnormal isoaspartyl residues to aspartyl residues. The results reported here, however, along with previous evidence that protein tertiary structure can inhibit isoaspartyl methylation, suggest that only a subclass of damaged sites are capable of efficiently entering a putative repair pathway; the sites not recognized by the methyltransferase may accumulate in vivo.     

Regulation and subcellular distribution of a protein methyltransferase and its damaged aspartyl substrate sites in developing Xenopus oocytes

A protein methyltransferase which recognizes racemized and isomerized aspartyl residues in proteins has been identified in both the cytoplasm and nucleus of Xenopus laevis oocytes by enzymatic and immunochemical assays. The methyltransferase activity is maintained at a constant concentration of approximately 0.2 microM throughout vitellogenesis. Two forms of the enzyme can be identified on immunoblots by their cross-reactivity with an antibody prepared against the purified enzyme from bovine brain. Although both forms, with molecular weights of 27,000 and 34,000, are present in the cytoplasm, only the smaller form is found in the oocyte nucleus. A heterogeneous group of endogenous methyl-accepting proteins has been identified following the addition of S-adenosyl-L-[methyl-3H]methionine to oocyte extracts. The subcellular distribution of these methyl-accepting proteins, i.e. those proteins with unmodified or unmetabolized D- and L-isoaspartyl residues, is complementary to that of the methyltransferase. Very low levels of methyl-accepting activity are associated with nuclear proteins, which are actively methylated by the methyltransferase in vivo (O'Connor, C. M., and Germain, B. J. (1987) J. Biol. Chem. 262, 10404-10411). Yolk platelet proteins, which are inaccessible to the methyltransferase in vivo, are readily methylated by the enzyme in vitro. The specific methyl-accepting activity of the yolk proteins increases severalfold during the months required for the development of an early-to-late vitellogenic oocyte, suggesting that derivatized aspartyl residues accumulate with time in proteins which are inaccessible to the methyltransferase. The results support the hypothesis that the methyltransferase initiates either the repair or metabolism of cellular proteins which have been damaged by spontaneous racemization and deamidation processes (Clarke, S. (1985) Annu. Rev. Biochem. 54, 479-506).     

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.     

Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes

The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-[methyl-3H]methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-[methyl-3H]methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly. The results suggest the quantitative significance of carboxyl methylation in the metabolism of oocyte calmodulin.     

Modification of synthetic peptides related to lactate dehydrogenase (231-242) by protein carboxyl methyltransferase and tyrosine protein kinase: effects of introducing an isopeptide bond between aspartic acid-235 and serine-236

The possibility that isoaspartyl residues contribute to the substrate specificity of eucaryotic protein carboxyl methyltransferases and/or tyrosine protein kinases has been investigated with two synthetic oligopeptides, Lys-Gln-Val-Val-Asp/isoAsp-Ser-Ala-Tyr-Glu-Val-Ile-Lys, which correspond to amino acids 231-242 of lactate dehydrogenase. One version of the peptide contains the normal amino acid sequence of the chicken muscle M4 isozyme. The other version contains an isoaspartyl residue in position 235 in place of the normal aspartyl residue; i.e., Asp-235 is linked to Ser-236 via its side-chain beta-carboxyl group, rather than via the usual alpha-carboxyl linkage. The normal peptide corresponds to the sequence around Tyr-238 that is phosphorylated in Rous sarcoma virus infected chick embryo fibroblasts [Cooper, J. A., Esch, F. S., Taylor, S. S., & Hunter, T. (1984) J. Biol Chem. 259, 7835]. Using protein carboxyl methyltransferase purified from bovine brain, we found that the normal peptide did not serve as a methyl-accepting substrate but that the isopeptide served as an excellent substrate, exhibiting a stoichiometry of one methyl group per peptide and Km of 0.54 microM. With tyrosine protein kinase partially purified from normal rat spleen both peptides were found to serve as phosphate acceptors at Tyr-238, exhibiting Km values of 4.7 and 8.9 mM for the normal and isopeptide versions, respectively. These results support the idea that protein carboxyl methyltransferase selectively methylates the alpha-carboxyl group of atypical isoaspartyl residues. In contrast, the presence of isoaspartate had a modest negative effect on substrate activity for a tyrosine protein kinase from rat spleen.     

Methylation of erythrocyte membrane proteins at extracellular and intracellular D-aspartyl sites in vitro. Saturation of intracellular sites in vivo

A cytosolic protein carboxyl methyltransferase (S-adenosyl-L-methionine:protein O-methyltransferase, E.C. 2.1.1.24) purified from human erythrocytes catalyzes the methylation of erythrocyte membrane proteins in vitro using S-adenosyl-L-[methyl-3H]methionine as the methyl group donor. The principal methyl-accepting proteins have been identified by sodium dodecyl sulfate-gel electrophoresis at pH 2.4 and fluorography as the anion transport protein (band 3), ankyrin (band 2.1), and integral membrane proteins with molecular weights of 45,000, 28,000, and 21,000. Many of the methylation sites associated with intrinsic membrane proteins may reside in their extracellular portions, since these same proteins are methylated when intact cells are used as the substrate. The maximal number of methyl groups transferred in these experiments is approximately 30 pmol/mg of membrane protein, a value which represents less than one methyl group/50 polypeptide chains of any methyl-accepting species. The number of methylation sites associated with the membranes is increased, but not to stoichiometric levels, by prior demethylation of the membranes. The additional sites are associated primarily with bands 2.1 and 4.1, the principal methyl acceptors in vivo, suggesting that most methylation sites are fully modified in vivo. Extracellular methylation sites are not increased by demethylation of membranes. The aspartic acid beta-methyl ester which can be isolated from carboxypeptidase Y digests of [3H]methylated membranes is in the unusual D-stereoconfiguration. Similar results have been obtained with [3H]methylated membranes isolated from intact cells (McFadden, P.N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2460-2464). It is proposed that the methyltransferase recognizes D-aspartyl residues in proteins and is involved with the metabolism of damaged proteins in vivo.     

Specific recognition of altered polypeptides by widely distributed methyltransferases

Protein carboxyl methyltransferase activity has been detected in extracts prepared from bacterial cells (Salmonella typhimurium), amphibian (Xenopus laevis) oocytes, and transformed mammalian cell lines. This activity appears to specifically recognize altered aspartyl residues based on the observation that the synthetic peptide L-Val-L-Tyr-L-Pro-L-isoAsp-Gly-L-Ala is a good methyl-accepting substrate for the methyltransferase activity, but that the corresponding peptide containing a normal L-aspartyl residue is not. These activities are similar to those of the previously described human erythrocyte and bovine brain enzymes which catalyze the formation of polypeptide D-aspartyl beta-methyl esters and L-isoaspartyl alpha-methyl esters. The wide distribution of these enzymatic activites suggest that the methylation of atypical proteins is an essential function in cells.     

Kinetic and electrophoretic analysis of transmethylation reactions in intact Xenopus laevis oocytes

Transmethylation reactions in fully grown Xenopus oocytes were analyzed following the microinjection of S-adenosyl-L-[methyl-3H]methionine (AdoMet). The size of the endogenous AdoMet pool, measured by cation exchange high pressure liquid chromatography is 5.91 pmol/oocyte. The AdoMet pool turns over with a half-time of 2 h, at a rate of 2.07 pmol/h/oocyte. Fractionation experiments indicate that approximately one-third of the AdoMet in oocytes is utilized for protein carboxylmethylation reactions and another third is metabolized into small molecules which are secreted. The remainder of the intracellular AdoMet is used primarily for protein N-methylation reactions, although some methylation of phospholipids and nucleic acids also occurs. Polyacrylamide gel electrophoresis of 3H-methylated proteins at pH 2.4 in the presence of sodium dodecyl sulfate demonstrated that methyl esters are associated with a heterogeneous group of proteins in both the nucleus and cytoplasm of oocytes, coincident with the subcellular distribution of the protein D-aspartyl, L-isoaspartyl methyl transferase (O'Connor, C. M. (1987) J. Biol. Chem. 262, 10398-10403). The protein methyl esters associated with oocyte proteins turn over rapidly, as evidenced from the presence of [3H]methanol in the medium. The calculated rate of protein carboxyl methylation, 0.7 pmol/h/oocyte, is similar to that of protein synthesis in oocytes, suggesting that the modification of derivatized aspartyl residues represents a major pathway in oocyte protein metabolism. Since the formation of protein methyl esters is unaffected by cycloheximide, it is unlikely that methyl-accepting sites on oocyte proteins arise primarily from errors in protein synthesis. Unlike protein carboxyl methylation reactions, protein N-methylation reactions are closely linked to protein synthesis, and the methyl group linkages are stable over a period of at least 4 h. Numerous protein acceptors for N-methylation reactions were identified by polyacrylamide gel electrophoresis.     

Enzymatic protein carboxyl methylation at physiological pH: cyclic imide formation explains rapid methyl turnover

At pH 7.4, 37 degrees C, bovine brain protein carboxyl methyltransferase transiently methylates deamidated adrenocorticotropin. The methylation occurs at the alpha-carboxyl group of an atypical beta-carboxyl-linked isoaspartyl residue (position 25). Several lines of evidence indicate that the immediate product of demethylation is an aspartyl cyclic imide involving positions 25 and 26. The evidence includes (1) the rapid rate of methyl ester hydrolysis, which is consistent with intramolecular catalysis, (2) the inability of the demethylated product to be remethylated, (3) the charge of this product, and (4) its rate of breakdown. The eventual hydrolysis of the cyclic imide produces a 30/70 mixture of peptides containing either alpha- or beta-carboxyl-linked aspartyl residues, respectively. Cyclic imide formation is nonenzymatic and can explain the unusual lability of mammalian protein methyl esters in general. These findings suggest that protein carboxyl methylation in mammalian tissues is not a simple on/off reversible modification as it apparently is in chemotactic bacteria. Carboxyl methylation may serve to activate selected protein carboxyl groups for subsequent longer lasting modifications, possibly subserving a role in protein repair, degradation, cross-linking, or some other as yet undiscovered alteration of protein structure.     

Evidence for the in vivo deamidation and isomerization of an asparaginyl residue in cytosolic serine hydroxymethyltransferase

Rabbit liver cytosolic serine hydroxymethyltransferase exists in several subforms which have different isoelectric points. Incubation of the purified enzyme with chymotrypsin cleaves the enzyme at Trp14. The released amino-terminal 14-mer peptide was shown to exist in three forms of equal concentration. The peptides differ in structure only at the asparaginyl residue at position 5. In addition to asparagine at this position we found both aspartyl and isoaspartyl residues. The deamidation of Asn5 does not appear to occur during the purification of the enzyme. The in vitro rate of deamidation of Asn5 in the enzyme is more than 5-fold slower than the rate of deamidation of this residue in the free 14-mer peptide. The isoaspartyl residue at position 5 serves as a substrate for protein carboxyl methyltransferase both in the free 14-mer peptide and the native enzyme. The enzyme which has had the amino-terminal 14 residues removed by digestion with chymotrypsin still exists in several forms with different isoelectric points. Reaction of peptides from this enzyme with carboxyl methyltransferase suggests that there is at least one more asparaginyl residue in this enzyme other than Asn5 which has undergone deamidation with the formation of isoaspartyl bonds.     

Enzymatic methylation of L-isoaspartyl residues derived from aspartyl residues in affinity-purified calmodulin. The role of conformational flexibility in spontaneous isoaspartyl formation

We have investigated the formation of D-aspartyl and L-isoaspartyl (beta-aspartyl) residues and their subsequent methylation in bovine brain calmodulin by the type II protein carboxyl methyltransferase. Based on the results of studies with unstructured peptides and denatured proteins, it has been proposed that the major sites of carboxyl methylation in calmodulin are at L-isoaspartyl residues that originate from two Asn-Gly sequences. To test this hypothesis, we directly identified the sites of methylation in affinity-purified preparations of calmodulin by peptide mapping using the proteases trypsin, endoproteinase Lys-C, clostripain, chymotrypsin, and Staphylococcus aureus V8 protease. We found, however, that the major high-affinity sites of methylation originate from aspartyl residues at position 2 and at positions 78 and/or 80. The methylatable residue in the first case was shown to be L-isoaspartate by comparison of the properties of a synthetic peptide corresponding to the N-terminal 13 residues substituted with an L-iso-Asp residue at position 2. The second methylatable residue, probably derived from Asp78, also appears to be an L-isoaspartyl residue. These sites appear to be readily accessible to the methyltransferase and are present in relatively flexible regions of calmodulin that may allow the spontaneous degradation reactions to occur that generate L-isoaspartyl residues via succinimide intermediates. Interestingly, the four calcium binding regions, each containing 3-4 aspartyl and asparaginyl residues (including the two Asn-Gly sequences), do not appear to contribute to the high-affinity methyl acceptor sites, even when calcium is removed prior to the methylation reaction. We propose that methylatable residues do not form at these sites because of the inflexibility of these regions when calcium is bound.     

The carboxyl methylation of aspartyl residues in eledoisin and tetragastrin by rabbit erythrocyte aspartyl β-carboxyl methyltransferase

Rabbit erythrocytes contain a soluble aspartyl β-carboxyl methyltransferase capable of specifically carboxyl methylating the β-carboxyl group of an internal aspartyl residue in the synthetic polypeptide eledoisin, a hypotensively active peptide from the cephalopodsEledone moschata andE. aldrovandi, and tetragastrin, the biologically active C-terminal tetrapeptide of human gastrin. However, the aspartyl residue in delta sleep-inducing peptide (DSIP) could not be carboxyl methylated, nor could glutamyl residues in any polypeptide tested.     

Stoichiometric methylation of porcine adrenocorticotropin by protein carboxyl methyltransferase requires deamidation of asparagine 25. Evidence for methylation at the alpha-carboxyl group of atypical L-isoaspartyl residues

The enzymatic methylation of porcine adrenocorticotropin (ACTH) in both its native form and a form which is deamidated at asparagine 25 has been compared using purified protein carboxyl methyltransferase from bovine brain. Incubation of deamidated ACTH with high concentrations of methyltransferase resulted in near stoichiometric levels of methyl incorporation (78 mol %), while the methylation of native ACTH was highly substoichiometric (3-12 mol %). The Km and Vmax for deamidated ACTH were 1.9 microM and 11,200 pmol/min/mg, respectively, making this peptide the most specific substrate known for the mammalian methyltransferase. Deamidation of asparagine 25 leads to the formation of an atypical isopeptide bond in which the resulting aspartyl residue is linked to the adjacent glycine 26 via its side-chain beta-carboxyl group rather than the usual alpha-carboxyl linkage (Gráf, L., Bajusz, S., Patthy A., Barát, E., and Cseh, G. (1971) Acta Biochim. Biophys. Acad. Sci. Hung. 6, 415-418; Bornstein, P., and Balian, G. (1977) Methods Enzymol. 47, 132-145). A synthetic isopeptide (beta-linked) analog of deamidated ACTH serves as a highly effective substrate for the methyltransferase, but the corresponding normal (alpha-linked) peptide does not, indicating that this enzyme selectively recognizes the alpha-carboxyl group of atypical beta-linked L-aspartyl residues (see also accompanying paper (Murray, E.D., Jr., and Clarke, S. (1984) J. Biol. Chem. 259, 10722-10732]. Methylation of atypical beta-linked L-aspartyl residues resulting from deamidation can account for previous observations that in vitro protein carboxyl methylation in mammalian systems almost always occurs with a low stoichiometry and that these protein methyl esters are considerably less stable than most chemically formed protein methyl esters.     

Conversion of a bacterial warm sensor to a cold sensor by methylation of a single residue in the presence of an attractant

The aspartate chemoreceptor (Tar) of Escherichia coli also serves as a thermosensor, and it is very amenable to genetic and biochemical analysis of the thermosensing mechanism. Its thermosensing properties are controlled by reversible methylation of the cytoplasmic signalling/adaptation domain of the protein. The unmethylated and the fully methylated (aspartate-bound) receptors sense, as attractant stimuli, increases (warm sensor) and decreases (cold sensor) in temperature respectively. To learn more about the mechanism of thermosensing, we replaced the four methyl-accepting glutamyl residues with non-methylatable aspartyl residues in all possible combinations. In a strain defective in both methyltransferase (CheR) and methylesterase (CheB) activities, all of the mutant Tar proteins functioned as warm sensors. To create a situation in which all of the remaining glutamyl residues were methylated, we expressed the mutant proteins in a CheB-defective, CheR-overproducing strain. The fully glutamyl-methylated proteins were designed to mimic the full range of methylation states possible for wild-type Tar. Almost all of the methylated mutant receptors, including those with single glutamyl residues, were cold sensors in the presence of aspartate. Thus, binding of aspartate to Tar and methylation of its single glutamyl residue can invert its temperature-dependent signalling properties.     

Protein carboxyl methylation in kidney brush-border membranes.

Protein carboxyl methylation activity was detected in the cytosol and in purified brush-border membranes (BBM) from the kidney cortex. The protein carboxyl methyltransferase (PCMT) activity associated with the BBM was specific for endogenous membrane-bound protein substrates, while the cytosolic PCMT methylated exogenous substrates (ovalbumin and gelatin) as well as endogenous proteins. The apparent Km for S-adenosyl-L-methionine with endogenous proteins as substrates were 30 microM and 4 microM for the cytosolic and BBM enzymes, respectively. These activities were sensitive to S-adenosyl-L-homocysteine, a well known competitor of methyltransferase-catalyzed reactions, but were not affected by the presence of chymostatin and E-64, two protein methylesterase inhibitors. The activity of both cytosolic and BBM PCMT was maximal at pH 7.5, while BBM-phospholipid methylation was predominant at pH 10.0. Separation of the = methylated proteins by acidic gel electrophoresis in the presence of the cationic detergent benzyldimethyl-n-hexadecylammonium chloride revealed distinct methyl accepting proteins in the cytosol (14, 17, 21, 27, 31, 48, 61 and 168 kDa) and in the BBM (14, 60, 66, 82, and 105 kDa). Most of the labelling was lost following electrophoresis under moderately alkaline conditions, except for a 21 kDa protein in the cytosol and a 23 kDa protein in the BBM fraction. These results suggest the existence of two distinct PCMT in the kidney cortex: a cytosolic enzyme with low selectivity and affinity, methylating endogenous and exogenous protein substrates, and a high-affinity BBM-associated methylating activity.     

In vivo and in vitro chemotactic methylation in Bacillus subtilis

Two doublets of Bacillus subtilis membrane proteins with molecular weights of 69,000 and 71,000 and of 30,000 and 30,800, were labeled by C3H3 transfer in the absence of protein synthesis. In addition, there was intense methylation of several low-molecular-weight substances. Both doublets were missing in a chemotaxis mutant. The equivalent proteins in Escherichia coli and Salmonella typhimurium are believed to be the methyl-accepting chemotaxis proteins. The higher-molecular-weight doublet bands were increased in degree of methylation upon addition of attractant to the bacteria. A methyltransferase from B. subtilis that methylates the wild-type membrane significantly better than the mutant membrane, using S-adenosylmethionine, has been partly purified. The methylated product was alkali labile and is probably a gamma-glutamyl methyl ester, as in E. coli and S. typhimurium. Ca2+ ion inhibited the methyltransferase, with a Ki of about 80 nM. Analysis of the in vitro methylation product showed labeling of the 69,000-dalton methyl-accepting chemotaxis protein and a low-molecular-weight protein, using wild-type membrane. Labeling of the low-molecular-weight protein but not of the 69,000 dalton protein was observed when the mutant membrane was used. The chemotaxis mutant tumbled much longer than the wild type when diluted away from attractant.     

Effects of adenosine dialdehyde treatment on in vitro and in vivo stable protein methylation in HeLa cells

Adenosine dialdehyde (AdOx) is an indirect methyltransferase inhibitor broadly used in cell culture to accumulate methyl-accepting proteins in hypomethylated states for in vitro protein methylation analyses. In this study we included a translation inhibitor, cycloheximide, in the AdOx treatment of HeLa cells. The methyl-accepting proteins disappeared in the double treatment, indicating that they were most likely newly synthesized in the AdOx incubation period. AdOx treatment could also be used in combination with in vivo methylation, another technique frequently used to study protein methylation. AdOx treatment prior to in vivo methylation accumulated methyl-accepting proteins for the labeling reaction. The continued presence of AdOx in the in vivo labeling period decreased the methylation of the majority of in vivo methyl-accepting polypeptides. The level and pattern of the in vivo methylated polypeptides did not change after a 12-h chase, supporting the notion that the methylated polypeptide as well as the methyl groups on the modified polypeptides are stable. On the other hand, methylarginine-specific antibodies detected limited but consistent reduction of the methylarginine-containing proteins in AdOx-treated samples compared to the untreated ones. Thus, AdOx treatment probably only blocked a small fraction of stable protein methylation. Overall, it is likely that base-stable methylation are formed soon after the synthesis of the polypeptide and remain stable after the modification.     

Identification of a C-terminal protein carboxyl methyltransferase in rat liver membranes utilizing a synthetic farnesyl cysteine-containing peptide substrate

Polypeptides synthesized in eucaryotic cells with a C-terminal -Cys-Xaa-Xaa-Xaa (-CXXX) sequence are candidates for post-translational modifications that include the removal of the last 3 amino acids and the lipidation and methyl esterification of the cysteinyl residue. To characterize the methylation reaction in vitro, the peptide Leu-Ala-Arg-Tyr-Lys-Cys (LARYKC) and its S-isoprenylated and S-alkylated derivatives were synthesized and assayed as methyl-accepting substrates with subcellular fractions of rat tissues including liver microsomal membranes. While little or no peptide-specific methyltransferase activity was detected in the latter preparation using the unmodified hexapeptide, the C10, C15, and C20 isoprenylated derivatives were substrates with Km values of 389 microM for S-geranyl-LARYKC, 2.2 microM for S-farnesyl-LARYKC, and approximately 10.9 microM for S-geranylgeranyl-LARYKC. The methyl-acceptor activities of a variety of n-alkyl S-derivatives of LARYKC (C8, C10, C13, C15) were also tested; all of these compounds were poorer substrates than the S-geranyl derivative. This enzyme activity uses S-adenosyl-L-methionine as the methyl donor (Km = 2.1 microM) and can be inhibited by S-adenosylhomocysteine (Ki = 9.2 microM), a product of the methylation reaction. The S-farnesyl-LARYKC peptide can inhibit the carboxyl methylation of bovine retinal rod outer segment membrane proteins that was previously shown to occur at the alpha-carboxyl group of C-terminal cysteine residues, demonstrating that the same enzyme can methylate both peptides and proteins. These results suggest that the methyl esterification of proteins containing a C-terminal -CXXX sequence requires not only the removal of the 3 terminal amino acids, but the isoprenylation of the sulfhydryl group as well.     

COOH-terminal methylation of lamin B and inhibition of methylation by farnesylated peptides corresponding to lamin B and other CAAX motif proteins.

Previous reports from this laboratory have demonstrated that lamin B is reversibly methylesterified in a cell cycle-dependent manner. The site of this methylation, however, was not identified. In this report, we describe a single major methylated product obtained following reversed-phase high-performance liquid chromatographic analysis of peptides generated by proteolytic digestion of lamin B from rat liver nuclear envelopes. This peptide was retained on a lamin B COOH-terminal-specific antibody-affinity column, and COOH-terminal localization was confirmed by amino acid sequencing. Two other COOH-terminal peptides were found but were not methylated and differed in sequence by at least a single residue from the methylated peptide, indicating the existence of two lamin B gene products. Tetrapeptides, representing the putative mature COOH termini of lamin B, K-ras-2A, and unprocessed lamin A, were synthesized with or without farnesyl modification of the COOH-terminal cysteines. All three farnesylated peptides served as substrates for the partially purified lamin B methyltransferase with apparent Km values of 4.5, 0.69, and 21 microM, respectively. Nonfarnesylated peptides were not substrates for the enzyme. The three farnesylated peptides were also effective to varying degrees at inhibiting the methylation of lamin B and other cellular proteins in cell lysates.