Protein carboxyl methylation and methyl ester turnover in density-fractionated human erythrocytes

Abstract

A widely distributed methyltransferase modifies protein d-aspartyl and l-isoaspartyl residues which arise spontaneously as proteins age. Protein carboxyl methylation reactions were analyzed in human erythrocytes which had been separated on density gradients, a procedure which provides fractions enriched in older cells in the denser areas of the gradient. The total flux of methyl groups through the carboxyl methylation pathway was monitored by incubating cells from each fraction with l-[methyl- 3 H]methionine and measuring the formation of both protein [ 3H]methyl esters and [ 3H]methanol, derived from the hydrolysis of protein [ 3H]methyl esters in vivo. Cells isolated from denser areas of the gradient showed progressively higher rates of both protein carboxyl methylation and methanol production. In all cases, only 10–20% of the total methyl groups transferred were still present as intact protein [ 3H]methyl esters, consistent with the rapid hydrolysis of protein methyl esters in erythrocytes of all ages. The total flux of methyl groups through the carboxyl methylation pathway was approximately 3-fold higher in cell isolated from densest areas of the gradient compared to cells isolated from least dense areas of the gradient. Increases of a similar magnitude were observed in the numbers of both membrane protein carboxyl methyl esters and cytosolic protein carboxyl methyl esters. The only protein whose methylation was unchanged in denser cell was a 35 000 Da cytosolic protein. It has been proposed that protein carboxyl methyl esters are intermediates in either the repair or metabolism of structurally damaged proteins. The increased methylation observed in older erythrocytes may reflect either an elevated rate of protein damage in older cells or the inefficient metabolism of methyl-accepting substrates subsequent to the removal of the methyl ester.