Protein Isoaspartyl Methyltransferase Research Articles

Structural and biochemical basis of the formation of isoaspartate in the complementarity-determining region of antibody 64M-5 Fab

The formation of the isoaspartate (isoAsp) is one of spontaneous degradation processes of proteins, affecting their stability and activity. Here, we report for the first time the crystal structures of an antibody Fab that contains isoAsp in the complementarity-determining region (CDR), along with biochemical studies to detect isoAsp. By comparing the elution profiles of cation-exchange chromatography, it was clarified that the antibody 64M-5 Fab is converted from the normal form to isoAsp form spontaneously and time-dependently under physiological conditions. The isoAsp residue was identified with tryptic peptide mapping, N-terminal sequencing, and the protein isoaspartyl methyltransferase assay. Based on the fluorescence quenching method, the isoAsp form of 64M-5 Fab shows a one order of magnitude lower binding constant for its dinucleotide ligand dT(6–4)T than the normal form. According to the structure of the isoAsp form, the conformation of CDR L1 is changed from the normal form to isoAsp form; the loss of hydrogen bonds involving the Asn28L side-chain, and structural conversion of the β-turn from type I to type II’. The formation of isoAsp leads to a large displacement of the side chain of His27dL, and decreased electrostatic interactions with the phosphate group of dT(6–4)T. Such structural changes should be responsible for the lower affinity of the isoAsp form for dT(6–4)T than the normal form. These findings may provide insight into neurodegenerative diseases (NDDs) and related diseases caused by misfolded proteins.

Differences in α-Crystallin isomerization reveal the activity of protein isoaspartyl methyltransferase (PIMT) in the nucleus and cortex of human lenses

Although it is well-known that protein turnover essentially stops in mature lens fiber cells, mapping out the ensuing protein degradation and its effects on lens function over time remains challenging. In particular, isomerization is a common, spontaneous post-translational modification that occurs over long timescales and generates products invisible to most analytical methods. Nevertheless, isomerization can significantly impact protein structure, function, and solubility, which are all necessary to maintain clarity and proper refractive index within the lens. Herein, we examine the degree of isomerization occurring in crystallin proteins in the human eye lens as a function of both age and location within the lens. A novel mass spectrometric technique leveraging radical chemistry enables detailed characterization of proteins extracted from the cortex and nucleus of the lens. It is observed that the degree of isomerization increases significantly between the cortex and nucleus and between water-soluble and water-insoluble fractions. Interestingly, the abundance of L-isoAsp is low in the water-soluble cortex despite being the dominant product generated by isomerization of Asp in vitro, suggesting that Protein L-isoaspartyl methyltransferase (PIMT) is active in the cortex and suppresses the accumulation of L-isoAsp. The abundance of L-isoAsp increases dramatically in the nucleus, revealing that PIMT activity decreases over time in the center of the lens. In addition, the growth of L-isoAsp in the nuclear fraction suggests protein isomerization continues within the nucleus, despite the fact that most of the protein within the nucleus has become insoluble. Additionally, it is demonstrated that sequential Asp residues lead to isomerization hotspots in human crystallin proteins and that the isomerization profiles for αA and αB crystallin are notably different. Although αA is more prone to isomerization, αB loses solubility more rapidly upon modification. These differences are likely related to the distribution of Asp residues within αA and αB, which are in turn connected to refractive index. The high Asp content of αA is a hazard in terms of isomerization and aging, but it serves to enhance the refractive index of αA relative to αB, and may explain why αA is only found in the eye.

Contribution of protein isoaspartate methyl transferase (PIMT) in the survival of Salmonella Typhimurium under oxidative stress and virulence

The enteric pathogen Salmonella Typhimurium (ST) survives inside the oxidative environment of phagocytic cells. Phagocyte generated oxidants primarily target proteins and modify amino acids in them. These modifications render the targeted proteins functionally inactive. Conversion of Asp to iso-Asp is one of the several known oxidant mediated amino acids modifications. By repairing iso-Asp to Asp, protein-isoaspartyl methyltransferase (PIMT) maintains the activities of proteins and thus helps in cellular survival under oxidative stress. To elucidate the role of PIMT in ST survival under oxidative stress, we have constructed a pimt gene deletion strain (Δpimt strain) of ST. The Δpimt strain grows normally in various culture media in vitro. However, in comparison to wild type ST, the Δpimt strain is found significantly (p<0.001) more susceptible to H2O2 and hypochlorite (HOCl). Further, the Δpimt mutant strain shows hypersusceptibility (p<0.001) to INF-γ stimulated macrophages. This susceptibility is reversed by pharmacological inhibition of reactive oxygen species (ROS) but not reactive nitrogen species (RNS) production. Further, plasmid based complementation enhances the survival of Δpimt mutant strain against oxidants in vitro and also inside the macrophages. In mice model, the LD50 for wild type ST and mutant Δpimt has been 1.73×104 and 1.38×105, respectively. Further, the mutant strain shows reduced dissemination to spleen and liver in mice. Following infection with a mixture of wild type ST and the Δpimt mutant (co-infection experiment), we recover significantly (p<0.001) less numbers of mutant bacteria from the spleen and liver of mice.

SY11-2ALCOHOL-INDUCED LIVER INJURY FROM ISOASPARTATE PROTEIN DAMAGE

The formation of isoaspartate peptide bonds is a major form of protein post-translational damage in vivo . The accumulation of isoaspartate damage is normally restricted by the activity of protein isoaspartyl methyltransferase (PIMT); a methylation enzyme that triggers restoration of a normal peptide bond. Alcohol consumption of 4 …

Regulation of mRNA translation controls seed germination and is critical for seedling vigor.

Regulation of mRNA translation controls seed germination and is critical for seedling vigor.

Isoaspartate, carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury

We had previously shown that alcohol consumption can induce cellular isoaspartate protein damage via an impairment of the activity of protein isoaspartyl methyltransferase (PIMT), an enzyme that triggers repair of isoaspartate protein damage. To further investigate the mechanism of isoaspartate accumulation, hepatocytes cultured from control or 4-week ethanol-fed rats were incubated in vitro with tubercidin or adenosine. Both these agents, known to elevate intracellular S-adenosylhomocysteine levels, increased cellular isoaspartate damage over that recorded following ethanol consumption in vivo. Increased isoaspartate damage was attenuated by treatment with betaine. To characterize isoaspartate-damaged proteins that accumulate after ethanol administration, rat liver cytosolic proteins were methylated using exogenous PIMT and 3H-S-adenosylmethionine and proteins resolved by gel electrophoresis. Three major protein bands of ∼75–80 kDa, ∼95–100 kDa, and ∼155–160 kDa were identified by autoradiography. Column chromatography used to enrich isoaspartate-damaged proteins indicated that damaged proteins from ethanol-fed rats were similar to those that accrued in the livers of PIMT knockout (KO) mice. Carbamoyl phosphate synthase-1 (CPS-1) was partially purified and identified as the ∼160 kDa protein target of PIMT in ethanol-fed rats and in PIMT KO mice. Analysis of the liver proteome of 4-week ethanol-fed rats and PIMT KO mice demonstrated elevated cytosolic CPS-1 and betaine homocysteine S-methyltransferase-1 when compared to their respective controls, and a significant reduction of carbonic anhydrase-III (CA-III) evident only in ethanol-fed rats. Ethanol feeding of rats for 8 weeks resulted in a larger (∼2.3-fold) increase in CPS-1 levels compared to 4-week ethanol feeding indicating that CPS-1 accumulation correlated with the duration of ethanol consumption. Collectively, our results suggest that elevated isoaspartate and CPS-1, and reduced CA-III levels could serve as biomarkers of hepatocellular injury.

Alcohol-Related Brain Damage in Humans

Chronic excessive alcohol intoxications evoke cumulative damage to tissues and organs. We examined prefrontal cortex (Brodmann’s area (BA) 9) from 20 human alcoholics and 20 age, gender, and postmortem delay matched control subjects. H & E staining and light microscopy of prefrontal cortex tissue revealed a reduction in the levels of cytoskeleton surrounding the nuclei of cortical and subcortical neurons, and a disruption of subcortical neuron patterning in alcoholic subjects. BA 9 tissue homogenisation and one dimensional polyacrylamide gel electrophoresis (PAGE) proteomics of cytosolic proteins identified dramatic reductions in the protein levels of spectrin β II, and α- and β-tubulins in alcoholics, and these were validated and quantitated by Western blotting. We detected a significant increase in α-tubulin acetylation in alcoholics, a non-significant increase in isoaspartate protein damage, but a significant increase in protein isoaspartyl methyltransferase protein levels, the enzyme that triggers isoaspartate damage repair in vivo. There was also a significant reduction in proteasome activity in alcoholics. One dimensional PAGE of membrane-enriched fractions detected a reduction in β-spectrin protein levels, and a significant increase in transmembranous α3 (catalytic) subunit of the Na+,K+-ATPase in alcoholic subjects. However, control subjects retained stable oligomeric forms of α-subunit that were diminished in alcoholics. In alcoholics, significant loss of cytosolic α- and β-tubulins were also seen in caudate nucleus, hippocampus and cerebellum, but to different levels, indicative of brain regional susceptibility to alcohol-related damage. Collectively, these protein changes provide a molecular basis for some of the neuronal and behavioural abnormalities attributed to alcoholics.

An Arabidopsis ATP-Dependent, DEAD-Box RNA Helicase Loses Activity upon IsoAsp Formation but Is Restored by PROTEIN ISOASPARTYL METHYLTRANSFERASE

Orthodox seeds are capable of withstanding severe dehydration. However, in the dehydrated state, Asn and Asp residues in proteins can convert to succinimide residues that can further react to predominantly form isomerized isoAsp residues upon rehydration (imbibition). IsoAsp residues can impair protein function and can render seeds nonviable, but PROTEIN ISOASPARTYL METHYLTRANSFERASE (PIMT) can initiate isoAsp conversion to Asp residues. The proteins necessary for translation upon imbibition in orthodox seeds may be particularly important to maintain in an active state. One such protein is the large, multidomain protein, Arabidopsis thaliana PLANT RNA HELICASE75 (PRH75), a DEAD-box helicase known to be susceptible to isoAsp residue accumulation. However, the consequences of such isomerization on PRH75 catalysis and for the plant are unknown. Here, it is demonstrated that PRH75 is necessary for successful seed development. It acquires isoAsp rapidly during heat stress, which eliminates RNA unwinding (but not rewinding) competence. The repair by PIMT is able to restore PRH75's complex biochemical activity provided isoAsp formation has not led to subsequent, destabilizing conformational alterations. For PRH75, an important enzymatic activity associated with translation would be eliminated unless rapidly repaired by PIMT prior to additional, deleterious conformational changes that would compromise seed vitality and germination.

Defying the proteasome, autophagy and convention: S. cerevisiae dodges the isoaspartyl aging bullet

The spontaneous degradation of aspartyl and asparaginyl protein amino acids to isoaspartyl residues is one of the most common types of protein damage in aging organisms. Although the protein isoaspartyl methyltransferase (PIMT) repairs this detrimental modification and is essential to many organisms, no gene homolog or equivalent enzymatic activity is present in Saccharomyces cerevisiae. We therefore used biochemical approaches to quantify isoaspartyl damage and elucidate how isoaspartyls are repaired and/or degraded in a system absent of PIMT. Surprisingly, cytosolic isoaspartyl damage quantified by base labile volatility assays was strikingly low (~70 pmol/mg of total protein) compared to cells with PIMT and decreased as the cells aged. Proteasome and autophagy inhibition by MG‐132 and 3‐methyladenine, respectively, did not significantly alter isoaspartyl protein damage levels. Yeast lacking the catalytic and regulatory proteasome proteins Rpn4p, Ubr2p, Pre3p and Pup1p, or the autophagy Atg8p protein, also did not exhibit elevated damage. However, depleting cells of ATP did increase isoaspartyl damage 5‐fold. Perturbing the intracellular pH homeostasis, an ATP‐dependent process, by incubating exponentially growing yeast in water buffered to pH 3 or 9 resulted in significant isoaspartyl accumulation (&gt;200 pmol/mg of total protein). Further analysis under these conditions using pH‐sensitive plasma membrane and vacuolar H+‐ATPase knockout cells revealed additional increases in protein isoaspartyls, suggesting that pH is a vital factor controlling damage, particularly in the vacuole. Finally, enzymological approaches with synthetic peptides have potentially identified an isoaspartyl‐targeted protease. Future investigations will identify and characterize this isoaspartyl‐directed proteolytic activity, in addition to elucidating the role of cytosolic and vacuolar pH on isoaspartyl protein damage. Ruth L. Kirschstein National Research Service Award GM007185.

Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning

The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by on-blot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An over-representation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance.

Proteomic Identification of Novel Substrates of a Protein Isoaspartyl Methyltransferase Repair Enzyme

We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein l-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1(-/-)) accumulate more isoaspartyl residues than their wild-type littermates, with the most "damaged" residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1(-/-) mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-(3)H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and two-dimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1(-/-) animals compared with Pcmt1(+/+) littermates. Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxyl-terminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, beta-synuclein, and alpha-synuclein, are all substrates for the l-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1(-/-) brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice.

Asparagine Deamidation Perturbs Antigen Presentation on Class II Major Histocompatibility Complex Molecules

Post-translational protein modifications can be recognized by B and T lymphocytes and can potentially make "self"-proteins appear foreign to the immune system. Such modifications may directly affect major histocompatibility complex-restricted T cell recognition of processed peptides or may perturb the processing events that generate such peptides. Using the tetanus toxin C fragment protein as a test case, we show that spontaneous deamidation of asparagine residues interferes with processing by the enzyme asparagine endopeptidase (AEP) and contributes to diminished antigen presentation. Deamidation inhibits AEP action either directly, when asparagine residues targeted by AEP are modified, or indirectly, when adjacent Asn residues are deamidated. Thus, deamidation of long-lived self-proteins may qualitatively or quantitatively affect the spectrum of self-peptides displayed to T cells and may thereby contribute to the onset or exacerbation of autoimmune disease.

Analysis of isoaspartate in a recombinant monoclonal antibody and its charge isoforms

Monoclonal antibody (mAb) therapy applications have been growing rapidly in recent years. Like other proteins, therapeutic mAbs can under go various enzymatic and non-enzymatic reactions that can affect their structural integrity and stability. Among the degradation reactions, isoaspartate (isoAsp) formation is one of the major sources of charge heterogeneity of mAbs. This paper reports the detection and quantification of isoAsp in a recombinant mAb and its charge isoforms resolved by cation exchange high performance liquid chromatography. The assay utilizes the enzyme protein isoaspartyl methyltransferase in conjunction with strong cation exchange separation and UV detection (at 260 nm) of S-adenosyl- l-homocysteine, which is produced stoichiometrically in the enzymatic reaction. The mAb is found to contain an average 0.2 mol of isoAsp per mol of protein, however, various charge isoforms were found to contain different levels of isoAsp. The most acidic isoforms contain approximately 0.7 mol of isoAsp per mol of protein, and no isoAsp is detected in the most basic isoform. It appears that the majority of isoAsp in the mAb is formed as a result of asparagine deamidation.

Crystal structure of human L-isoaspartyl-O-methyl-transferase with S-adenosyl homocysteine at 1.6-A resolution and modeling of an isoaspartyl-containing peptide at the active site.

Spontaneous formation of isoaspartyl residues (isoAsp) disrupts the structure and function of many normal proteins. Protein isoaspartyl methyltransferase (PIMT) reverts many isoAsp residues to aspartate as a protein repair process. We have determined the crystal structure of human protein isoaspartyl methyltransferase (HPIMT) complexed with adenosyl homocysteine (AdoHcy) to 1.6-A resolution. The core structure has a nucleotide binding domain motif, which is structurally homologous with the N-terminal domain of the bacterial Thermotoga maritima PIMT. Highly conserved residues in PIMTs among different phyla are placed at positions critical to AdoHcy binding and orienting the isoAsp residue substrate for methylation. The AdoHcy is completely enclosed within the HPIMT and a conformational change must occur to allow exchange with adenosyl methionine (AdoMet). An ordered sequential enzyme mechanism is supported because C-terminal residues involved with AdoHcy binding also form the isoAsp peptide binding site, and a change of conformation to allow AdoHcy to escape would preclude peptide binding. Modeling experiments indicated isoAsp groups observed in some known protein crystal structures could bind to the HPIMT active site.

Crystal Structure of Protein Isoaspartyl Methyltransferase: A Catalyst for Protein Repair

Background: Formation of isoaspartyl residues is one of several processes that damage proteins as they age. Protein L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) is a conserved and nearly ubiquitous enzyme that catalyzes the repair of proteins damaged by isoaspartyl formation. Results: We have determined the first structure of a PIMT from crystals of the T. maritima enzyme complexed to S-adenosyl-L-homocysteine (AdoHcy) and refined it to 1.8 Å resolution. Although PIMT forms one structural unit, the protein can be divided functionally into three subdomains. The central subdomain closely resembles other S-adenosyl-L-methionine-dependent methyltransferases but bears a striking alteration of topological connectivity, which is not shared by any other member of this family. Rather than arranged as a mixed β sheet with topology 6↑7↓5↑4↑1↑2↑3↑, the central sheet of PIMT is reorganized to 7↑6↓5↑4↑1↑2↑3↑. AdoHcy is largely buried between the N-terminal and central subdomains by a conserved and largely hydrophobic loop on one rim of the binding cleft, and a conserved Ser/Thr-rich β strand on the other. The Ser/Thr-rich strand may provide hydrogen bonds for specific interactions with isoaspartyl substrates. The side chain of Ile-206, a conserved residue, crosses the cleft, restricting access to the donor methyl group to a deep well, the putative isoaspartyl methyl acceptor site. Conclusions: The structure of PIMT reveals a unique modification of the methyltransferase fold along with a site for specific recognition of isoaspartyl substrates. The sequence conservation among PIMTs suggests that the current structure should prove a reliable model for understanding the repair of isoaspartyl damage in all organisms.

Translation of a unique transcript for protein isoaspartyl methyltransferase in haploid spermatids: implications for protein storage and repair.

The mammalian testis contains high levels of a protein, L-isoaspartyl (D-aspartyl) O-methyltransferase (PIMT), postulated to play a role in the repair of age-damaged proteins. To examine the regulation of PIMT concentrations during the development of spermatozoa, poly(A)+ RNA was isolated from purified populations of pachytene spermatocytes and round spermatids. Northern blot analysis revealed that a unique 1.1-1.3 kb PIMT transcript is present in preparations of round spermatid and pachytene spermatocyte poly (A)+ RNA. The concentration of this small PIMT transcript is at least four times higher in mRNA isolated from round spermatids than in mRNA isolated from pachytene spermatocytes, indicating that the PIMT gene is actively transcribed during the haploid phase of spermatogenesis. The germ cell-specific PIMT transcripts are distributed between the polysomal fraction and the nonpolysomal fractions of testis RNA, suggesting that translational controls also contribute to the high concentrations of PIMT in mammalian sperm. PIMT function is not essential for spermatogenesis because the testes from transgenic mice lacking PIMT activity have normal levels of protamine transcripts, and because functional sperm can be recovered from the cauda epididymis. The protein repair function of the PIMT may be more important in maintaining the fertilization competence of translationally-inactive mature sperm during the prolonged period of epididymal transit and storage in the male reproductive tract.

Development of Improved High-Performance Liquid Chromatography Conditions for Nonisotopic Detection of Isoaspartic Acid to Determine the Extent of Protein Deamidation

A rapid and accurate ion-pairing reversed-phase high-performance liquid chromatography (IP-RP-HPLC) procedure has been developed for nonisotopic detection of isoaspartic acid residues in protein or peptides resulting from deamidation of asparagine residues. The IP-RP-HPLC procedure specifically detects and quantifies S-adenosylhomocysteine (SAH). SAH is a by-product of the reaction between protein isoaspartyl methyltransferase (PIMT), S-adenosylmethionine (SAM), and isoaspartic acid residues. The HPLC conditions described in this paper have been demonstrated to offer significantly better reproducibility compared to earlier studies. The HPLC method allows determination of the extent of protein deamidation without the use of radioisotopes and therefore offers significant advantages for biopharmaceutical development laboratories.

Protein isoaspartyl methyltransferase protects from Bax-induced apoptosis.

Protein L-isoaspartyl methyltransferase (Pimt) is a highly conserved enzyme utilising S-adenosylmethionine (AdoMet) to methylate aspartate residues of proteins damaged by age-related isomerisation and deamidation. We have been particularly interested in this enzyme since addition of the compound CGP3466 to primary rat astroglia cell cultures resulted in an upregulation of Pimt at the mRNA level, as shown here by semi-quantitative RT-PCR. CGP3466 is a compound related to the anti-Parkinson's drug R-(-)-deprenyl, which has been shown to protect from neural apoptosis induced by trophic factor withdrawal [Tatton et al., 1994. J. Neurochem. 63, 1572]. The pro-apoptotic gene Bax is required in the cascade of events following withdrawal [Deckwerth et al., 1996. Neuron 17, 401]. We therefore investigated whether Pimt overexpression was able to affect Bax-induced apoptosis in primary mouse cortical neurons. Our results show that Pimt is indeed able to protect from Bax-induced apoptosis. Furthermore, this activity is not restricted to brain-specific cell types, since the same effect is also demonstrated in COS1 cells. In addition, mutational analysis suggests that the protective effect is dependent on the adenosine methionine-binding motif, which is well conserved in protein methyltransferases, and that a mutation destroying this motif crucially affects cytoskeletal structures of the cell.

Purification and characterization of recombinant rabbit cytosolic serine hydroxymethyltransferase.

A rabbit liver cDNA library in phage lambdagt10 was screened using the coding cDNA for human cytosolic serine hydroxymethyltransferase. A clone of 1754 bp was isolated and the nucleotide sequence showed an open reading frame of 1455 bp, which coded for rabbit cytosolic serine hydroxymethyltransferase and was flanked by 12 bp at the 5' end and 287 bp at the 3' end. The full-length cDNA was then cloned into a pET22b vector as a NdeI-EcoRI insert. HMS174(DE3) cells were transformed with this plasmid and, after induction with isopropyl beta-D-thiogalactopyranoside, expressed a catalytically active serine hydroxymethyltransferase. The enzyme was purified and shown to be the expressed rabbit enzyme lacking the first methionine residue. Spectral characteristics of the bound pyridoxal phosphate and kinetic constants for the natural substrates L-serine and tetrahydrofolate were essentially identical to the values obtained previously for the rabbit cytosolic enzyme. The pattern of bands shown by the pure recombinant enzyme on an isoelectric focusing gel containing 6 M urea showed a major band and a minor band representing about 15-20% of the protein. Upon incubation of the recombinant enzyme at pH 7.3 and 37 degreesC, three new bands were observed on isoelectric focusing with the concomitant formation of isoaspartyl residues, as determined by reactivity with protein isoaspartyl methyltransferase. These results are consistent with deamidation of Asn residues to isoaspartyl during the in vitro incubation. The enzyme purified from rabbit liver has previously been shown to contain isoaspartyl residues.

Complex Interactions of the Protein l-Isoaspartyl Methyltransferase and Calmodulin Revealed with the Yeast Two-hybrid System

The widely distributed protein-L-isoaspartyl, D-aspartyl carboxylmethyltransferase (EC 2.1.1.77) is hypothesized to play a role in the repair or metabolism of deamidated and isomerized proteins that are spontaneously generated during the aging of proteins in cells. The yeast two-hybrid system was used to identify proteins that potentially interact with the methyltransferase in a cellular processing pathway. Two cDNAs, both encoding calmodulin, were isolated from a human fetal brain cDNA library using the human methyltransferase as the bait. Enzymatic assays with purified components revealed a complex set of interactions between the methyltransferase and calmodulin. Calmodulin weakly stimulated protein carboxylmethyltransferase activity in vitro at concentrations of the two proteins reflecting their representation in mammalian brain. Calmodulin stimulation of methyltransferase was observed in both the presence and absence of calcium, although the effect was greater in the presence of calcium. Native calmodulin was not a substrate for the carboxylmethyltransferase, but deamidated variants of calmodulin act as substrates for the methyltransferase, with calculated Km values of 3.6 and 8.6 microM for calcium-liganded and unliganded calmodulin, respectively. Both the effector and substrate interactions of calmodulin with the protein isoaspartyl methyltransferase likely contributed to the positive results obtained with the two-hybrid system.