Calmodulin carboxylmethyl ester formation in intact human red cells and modulation of this reaction by divalent cations in vitro

Runte, L.; Jürgensmeier, H.-L.; Unger, Clemens; Söling, Hans‐Dieter

doi:10.1016/0014-5793(82)81025-9

Cited by 23 publications

(16 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…CaM is carboxyl-methylated in situ in frog oocytes (Desrosiers et al, 1990), in cultured pituitary cells (Vincent & Siegel, 1987), and in human erythrocytes (Runte et al, 1982;Brunauer & Clarke, 1986), albeit to a very small extent. Asn-Gly sequences are notorious for their propensity toward deamidation, both in peptides and in proteins (reviewed in Wright, 1991).…”

mentioning

confidence: 99%

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

Potter

Henzel²,

Aswad

1993

Protein Science

View full text Add to dashboard Cite

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 methyL3H from S-adenosyl-~-[methyl-~H]methionine to the isoaspartyl (a-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 "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 111, IV, and I1 accumulated 0.72, 0.60, and 0.13 mol of isoaspartate per mol of calmodulin, respectively. The Asn-97-Gly-98 sequence (domain 111) 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 11. 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 11, and to Asp-93 and/or Asp-95 in domain 111. 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.

show abstract

mentioning

confidence: 99%

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

Potter

Henzel²,

Aswad

1993

Protein Science

View full text Add to dashboard Cite

show abstract

“…4) CaM, it is probable that the stimulatory interactions of native CaM with PCMT are principally responsible for our positive results with the two-hybrid system. At the high expression levels achieved in yeast cells, however, substrate interactions of deamidated CaM and PCMT could also contribute to the results, confirming CaM as a significant physiological methyl-acceptor (45)(46)(47)(48) and validating the use of CaM as a model substrate for PCMT (45).…”

Section: Discussionmentioning

confidence: 91%

“…In the absence of calcium, CaM also binds to a number of cellular proteins, including the abundant neuroprotein neuromodulin (42), which is associated with neuronal growth and neurite extension, and the 110-kDa myosin from intestinal microvilli (43,44). There are no previous reports of PCMT activation by CaM, although the distribution of the two proteins in mammalian tissues is similar (18,36) and CaM serves as a physiological substrate for PCMT (45)(46)(47)(48)(49). Calmodulin methylation in intact cells is markedly substoichiometric, however, presumably reflecting the fact that only a subpopulation of cellular CaM molecules would contain isoaspartyl residues that can serve as methyl acceptors.…”

Section: Resultsmentioning

confidence: 99%

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

O'Connor

O’Connor

1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

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 K m values of 3.6 and 8.6 M 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. Protein-L-isoaspartyl, D-aspartyl carboxyl methyltransferase (PCMT)1 (E.C. 2.1.1.77), a well conserved enzyme detected in a broad spectrum of organisms from Escherichia coli to humans (1-6), specifically modifies L-isoaspartyl and D-aspartyl residues that arise in proteins due to spontaneous deamidation and racemization reactions involving asparaginyl and aspartyl residues (7,8). Because the formation of L-isoaspartate can adversely affect the stability and biological potency of many proteins (9 -13), it has been proposed that methylation of the damaged protein initiates either the repair or degradation of the substrate. Experimental support for a structural repair function for PCMT has been obtained in purified systems using isoaspartyl peptides as substrates for the PCMT (14 -16). The esterified isoaspartyl peptides formed by PCMT hydrolyze into both aspartyl peptides and isoaspartyl peptides, the latter of which can be remethylated by the PCMT. The nearly quantitative conversion of isoaspartyl to aspartyl forms of the peptides has been interpreted as evidence for a repair function, although the process lacks the efficiency characteristic of enzymatic processes, and no isoaspartyl residues are converted into asparaginyl residues, the likely origin of most isoaspartyl sites in cellular proteins (2).Because of the structural complexity of protein substrates for the PCMT, very little information is available regarding the fate of isoaspartyl residues in protein substrates. Deamidation of some proteins, including calmodulin (11) and the bacterial HPr phosphocarrier protein (17), generate...

show abstract

“…It has been suggested that carboxyl methylation can influence neurotransmitter synthesis and release (5-7), leukocyte chemotaxis (8,9), and repair/recognition of Daspartate residues in protein (10)(11)(12)(13)(14)(15). More recently, effects of carboxyl methylation on the function of calmodulin (16)(17)(18) and calmodulin-binding proteins have been investigated (19,20). Carboxyl methylation of calmodulin was observed both in vivo (16,18) and in vitro (16)(17)(18)(19)(20).…”

mentioning

confidence: 99%

“…More recently, effects of carboxyl methylation on the function of calmodulin (16)(17)(18) and calmodulin-binding proteins have been investigated (19,20). Carboxyl methylation of calmodulin was observed both in vivo (16,18) and in vitro (16)(17)(18)(19)(20). However, carboxyl methylation of calmodulin was consistently substoichiometric (18,20,21), as is the case for other substrates (10).…”

mentioning

confidence: 99%

Stoichiometric methylation of calcineurin by protein carboxyl O-methyltransferase and its effects on calmodulin-stimulated phosphatase activity.

Billingsley¹,

Kincaid²,

Lovenberg³

1985

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Calcineurin, a calmodulin-stimulated protein phosphatase, was a substrate for purified bovine brain protein carboxyl O-methyltransferase (protein O-methyltransferase; EC 2.1.1.24) and incorporated up to 2 mol of CH3 per mol of calcineurin. Carboxyl methylation was dependent on the concentrations of S-adenosyl-L-[methyl-3H~methionine and was prevented by addition of the carboxyl methylation inhibitor S-adenosylhomocysteine. The stoichiometry of methyl group incorporation was related to the ratio of methyltransferase/calcineurin. The rate of spontaneous hydrolysis of carboxyl methylester groups on calcineurin increased rapidly above pH 6.5 with those on native carboxyl-methylated calcineurin substantially more labile than for tricholoracetic acid-precipitated calcineurin. Polyacrylamide gel electrophoresis in the presence of NaDodSO4 (pH 2.4) confirmed that the A subunit of calcineurin (Mr = 61,000) was the primary site of carboxyl methylation with little, ifany, modification of the B subunit (Mr = 18,000). When carboxyl-methylated calcineurin (r1-2 mol of CH3 per mol of protein) was assayed for p-nitrophenyl phosphatase activity at pH 6.5, a marked inhibition of calmodulin-stimulated activity was observed while there was little effect on Mn2+-stimulated phosphatase activity. Thus, calcineurin appears to be an excellent substrate for protein carboxyl O-methylation and this modification, which impairs calmodulin stimulation of phosphatase activity, may be of functional significance.Carboxyl methylation of glutamic and/or aspartic acid residues of proteins by the enzyme protein carboxyl 0-methyltransferase (protein O-methyltransferase; EC 2.1.1.24) is well documented (1-3), although the functional significance of this reversible modification of proteins remains an enigma (4). It has been suggested that carboxyl methylation can influence neurotransmitter synthesis and release (5-7), leukocyte chemotaxis (8, 9), and repair/recognition of Daspartate residues in protein (10)(11)(12)(13)(14)(15) METHODS Materials. S-Adenosyl-L-[methyl-3H]methionine (Ado[3H]-Met) (62 Ci/mmol and 15 Ci/mmol; Ci = 37 GBq) was purchased from Amersham; 2-(N-morpholino)ethanesulfonic acid (Mes) was from Calbiochem; AdoMet and Sadenosylhomocysteine (AdoHcy) were from Sigma; AdoHcy-agarose was from Bethesda Research Laboratories; electrophoresis supplies were from Bio-Rad; and frozen bovine brains were from Hazelton Labs (Denver, PA). Allreagents were of the highest available purity. CalmodulinSepharose was prepared according to the method of Kincaid and Vaughan (25).Enzyme Preparations. Bovine brain protein carboxyl 0-methyltransferase was purified to apparent homogeneity by using minor modifications of previously published techniques (26,27). Bovine brain calcineurin was purified to homogeneity (28) and contained subunits of Mr 61,000 and Mr 18,000 in approximately equal proportions, which accounted for -95% of Coomassie-stained protein after NaDodSO4 gel electrophoresis.Carboxyl Methylation Assays. Reactions were carried out in a fi...

show abstract

Calmodulin carboxylmethyl ester formation in intact human red cells and modulation of this reaction by divalent cations in vitro

Cited by 23 publications

References 12 publications

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

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

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

Stoichiometric methylation of calcineurin by protein carboxyl O-methyltransferase and its effects on calmodulin-stimulated phosphatase activity.

Contact Info

Product

Resources

About