Partial repair of deamidation-damaged calmodulin by protein carboxyl methyltransferase

Modification of calmodulin by protein carboxyl methyltransferase requires deamidation of one or more labile asparagine residues (Johnson, B.A., Freitag, N. E., and Aswad, D. W. (1985) J. Biol. Chem. 260, 10913-10916). We now show that deamidation results in the generation of two altered forms of calmodulin, designated A and B, which can be separated by electrophoresis under nondenaturing conditions. The A form is characterized by a larger apparent molecular radius, has only 10% the activity of native calmodulin when assayed for its ability to activate a Ca2+/calmodulin-dependent protein kinase from rat brain, and serves as an excellent substrate for the methyltransferase. The B form more closely resembles native calmodulin: it has an apparent molecular radius more like the native, exhibits about 40% the activity of native calmodulin, and is a relatively poor methyl acceptor. Evidence suggests that the A and B forms probably contain isoaspartate (A) and aspartate (B) in place of Asn-60 and/or Asn-97. Incubation of the A form with methyltransferase and S-adenosyl-L-methionine converts about half of the A form to an electrophoretic band indistinguishable from the B form. The activity of this partly converted calmodulin rises to 30-50% that of native calmodulin. These observations imply that the methyltransferase may have a biological role in restoring activity to proteins which contain abnormal isoaspartyl peptide bonds resulting from asparagine deamidation.     

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.     

Protein carboxyl methyltransferase activity specific for age-modified aspartyl residues in mouse testes and ovaries: Evidence for translation during spermiogenesis

An antiserum prepared against the purified protein carboxyl methltransferase (PCMT) from bovine brain has been used to compare testicular and ovarian levels of the enzyme and to study the regulation of PCMT concentrations during spermatogenesis. The PCMT, which specifically modifies age-damaged aspartyl residues, is present at a significantly higher concentration in mature mouse testis than in ovary. However, the PCMT is present at nearly equal concentrations in extracts of germ cell-deficient ovaries and testes obtained from mutant atrichosislatrichosis mice. In normal testis, the concentration of the PCMT increases severalfold during the first 4–5 weeks after birth, paralleling the appearance and maturation of testicular germ cells. Both immunochemical and enzymatic measurements of PCMT specific activities in purified spermatogenic cell preparations indicate that PCMT levels are twofold and 3.5-fold higher in round spermatids and residual bodies, respectively, than in pachytene spermatocytes. The results are consistent with the enhanced synthesis and/or stability of the PCMT in spermatogenic cells and with the continued translation of the PCMT during the haploid portion of spermatogenesis. The relatively high levels of PCMT in spermatogenic cells may be important for the extensive metabolism of proteins accompanying spermatid condensation or for the repair of damaged proteins in translationally inactive spermatozoa.     

Formation of isoaspartate at two distinct sites during in vitro aging of human growth hormone

In vitro aging at pH 7.4, 37 degrees C causes natural sequence recombinant human growth hormone (rhGH), methionyl rhGH, and human pituitary growth hormone to become substrates for bovine brain protein carboxyl methyltransferase, an enzyme that modifies the "side chain" alpha-carboxyl group present at atypical isoaspartyl linkages. The substrate capacity of rhGH increased at a rate of 1.8 methyl-accepting sites/day/100 molecules of hormone. Reversed-phase high performance liquid chromatography (HPLC) of trypsin digests of aged rhGH revealed two altered peptides not present in digests of control rhGH. These two fragments, which had the amino acid compositions of residues 128-134 (Leu-Glu-Asp-Gly-Ser-Pro-Arg) and 146-158 (Phe-Asp-Thr-Asn-Ser-His-Asn-Asp-Asp-Ala-Leu-Leu-Lys), contained the majority of the induced methylation sites, 22 and 58%, respectively. Isoaspartate can result from deamidation of asparagine or isomerization of aspartate. Isomerization of Asp-130, the only candidate site in 128-134, was corroborated by coelution of the altered fragment with the synthetic isoaspartyl peptide upon reversed-phase HPLC. Evidence is presented that the altered 146-158 fragment is a mixture of two peptides resulting from deamidation of Asn-149 to form 70-80% isoaspartate and 20-30% aspartate at this position. The position of isoaspartate in the altered 146-158 fragment was deduced from mass spectrometry, which indicated a single deamidated asparagine; from methylation stoichiometry, which indicated only one methylation site; and from automated Edman degradation, which showed an absence of asparagine and a low yield of aspartate at position 149. These results show that isoaspartate formation from both aspartate and asparagine is a significant, and possibly the major, source of spontaneous covalent alteration of rhGH and that enzymatic carboxyl methylation provides a powerful tool for assessing this type of modification.     

The primary structure of a protein carboxyl methyltransferase from bovine brain that selectively methylates L-isoaspartyl sites

Protein L-isoaspartyl methyltransferase (PIMT) transfers the methyl group of S-adenosyl-L-methionine to free alpha-carboxyl groups of atypical L-isoaspartyl residues in proteins. The complete primary structure of the type I isoform of bovine brain PIMT was determined by sequence analysis of peptides generated by endoprotease Lys-C, trypsin, cyanogen bromide, and endoprotease Asp-N digests. The correct composition of every peptide was verified by fast atom bombardment mass spectrometry. The efficiency of sequencing by tandem mass spectrometry was examined for several peptides by comparing its speed and accuracy with automated Edman degradation. Tandem mass spectrometry was used to determine the structure of the NH2-terminal blocked peptide derived from a hydroxylamine cleavage. PIMT is 226 residues with Mr = 24,500 and contains acetyl alanine as the amino-terminal residue. The partial sequence (141 residues from 8 tryptic peptides) of a homologous human red cell PIMT (Gilbert, J. M., Fowler, A., Bleibaum, J., and Clarke, S. (1988) Biochemistry 27, 5227-5233) shows a 97% identity with the corresponding peptides of the bovine brain enzyme. The complete brain enzyme sequence reported here bears no significant homology to any other known class of methyltransferase including those which methylate the side chain gamma-carboxyl group of receptor proteins involved in bacterial chemotaxis.     

In vitro aging of calmodulin generates isoaspartate at multiple Asn-Gly and Asp-Gly sites in calcium-binding domains II, III, and IV.

We have determined the major sites responsible for isoaspartate formation during in vitro aging of bovine brain calmodulin under mild conditions. Protein L-isoaspartyl methyltransferase (EC 2.1.1.77) was used to quantify isoaspartate by the transfer of methyl-3H from S-adenosyl-L-[methyl-3H]methionine to the isoaspartyl (alpha-carboxyl) side chain. More than 1.2 mol of methyl-acceptor sites per mol of calmodulin accumulated during a 2-week incubation without calcium at pH 7.4, 37 degrees C. Analysis of proteolytic peptides of aged calmodulin revealed that > 95% of the methylation capacity is restricted to residues in the four calcium-binding domains, which are predicted to be highly flexible in the absence of calcium. We estimate that domains III, IV, and II accumulated 0.72, 0.60, and 0.13 mol of isoaspartate per mol of calmodulin, respectively. The Asn-97-Gly-98 sequence (domain III) is the greatest contributor to isoaspartate formation. Other major sites of isoaspartate formation are Asp-131-Gly-132 and Asp-133-Gly-134 in domain IV, and Asn-60-Gly-61 in domain II. Significant isoaspartate formation was also localized to Asp-20, Asp-22, and/or Asp-24 in domain I, to Asp-56 and/or Asp-58 in domain II, and to Asp-93 and/or Asp-95 in domain III. All of these residues are calcium ligands in the highly conserved EF-hand calcium-binding motif. Thus, other EF-hand proteins may also be subject to isoaspartate formation at these ligands. The results support the idea that isoaspartate formation in structured proteins is strongly influenced by both the C-flanking residue and by local flexibility.     

Fragmentation of isoaspartyl peptides and proteins by carboxypeptidase Y: release of isoaspartyl dipeptides as a result of internal and external cleavage

Isoaspartate-containing versions of sea urchin sperm-activating peptide, delta sleep-inducing peptide, and lactate dehydrogenase (231-242) were cleaved at internal sites by carboxypeptidase Y. Cleavage occurred between the isoaspartate and the preceding amino acid, and it was accompanied by sequential digestion of amino acids from the two resulting carboxyl termini. Because the isoaspartyl bonds were not cleaved, isoaspartyl dipeptides were among the final products. The rate of release of isoaspartyl dipeptides was different for the three peptides, a 24-h digestion yielding 0.32 mol of isoaspartylglycine/mol of isoaspartyl sperm-activating peptide, 0.50 mol of isoaspartylalanine/mol of isoaspartyl delta sleep-inducing peptide, and 1.15 mol of isoaspartylserine/mol of isoaspartyl lactate dehydrogenase (231-242). The different rates could be explained by the slow cleavage of amino acids preceded by glycine. Isoaspartyl dipeptides were not detected in digests of the corresponding aspartate- or asparagine-containing forms of the peptides. Release of isoaspartyl dipeptides by carboxypeptidase Y was used to demonstrate the presence of isoaspartylglycine sequences in deamidated adrenocorticotropin (0.54 mol/mol), in a mixture of trypic fragments of base-treated calmodulin (0.20 mol/mol), and in a mixture of tryptic fragments of base-treated triosephosphate isomerase (0.08 mol/mol). These results confirm earlier work suggesting that isoaspartylglycine formation is prevalent in proteins exposed to alkaline conditions. They also provide a methodology that should prove useful in the characterization of natural substrates for protein L-isoaspartyl methyltransferase.     

Effect of intralipid infusion on lecithin:cholesterol acyltransferase and lipoprotein lipase in young rats

We compared the effects of Intralipid and dextrose infusion on plasma lecithin:cholesterol acyltransferase (LCAT), plasma lipid profiles and lipolytic activity. We used 5-week-old male Sprague-Dawley rats which were given total parenteral nutrition (TPN) with either Intralipid (3 g/kg body weight) or an equicaloric amount of 25% dextrose in the presence or absence of heparin (1 or 10 IU/ml of TPN). 40 min after the end of 4 h of infusion, plasma LCAT activity was significantly decreased (P less than 0.001), while total cholesterol and free fatty acid levels were significantly (P less than 0.05) increased in rats given Intralipid as compared to those given dextrose. We found associations (P less than 0.005) between LCAT activity and total cholesterol and between LCAT and free fatty acid levels; the coefficients of negative correlation were 0.543 and 0.607, respectively. Concomitantly to the increment in plasma total cholesterol levels, there was a decrease in the high-density lipoprotein (HDL) cholesterol fraction; the latter, which was 40% of the total plasma cholesterol in control and dextrose-infused rats, declined to 9% in rats given Intralipid. Administration of heparin during Intralipid infusion, even up to 10 IU/ml of TPN, did not affect any of these changes. After dextrose infusion, the values of all three parameters were similar to those of the control group. Plasma lipolytic activity was not significantly different between rats given infusion (Intralipid or dextrose) and controls. However, in the presence of heparin, plasma lipolytic activity increased similarly in both infused groups. These data indicate that in young rats, Intralipid infusion leads to an increase in plasma total cholesterol and free fatty acid levels, which correlates with a decrease in LCAT activity; the concurrent decrease in HDL cholesterol levels might account, in part, for the loss of LCAT activity. The administration of heparin results in an elevation of plasma lipolytic activity; however, it does not prevent the hypercholesterolemia, nor the decline in LCAT activity associated with Intralipid infusion.