Spontaneous Cleavage at Glu and Gln Residues in Long-Lived Proteins

Abstract

Long-lived proteins (LLPs) are prone to deterioration with time, and one prominent breakdown process is the scission of peptide bonds. These cleavages can either be enzymatic or spontaneous. In this study, human lens proteins were examined and many were found to have been cleaved on the C-terminal side of Glu and Gln residues. Such cleavages could be reproduced experimentally by in vitro incubation of Glu- or Gln-containing peptides at physiological pHs. Spontaneous cleavage was dependent on pH and amino acid sequence. These model peptide studies suggested that the mechanism involves a cyclic intermediate and is therefore analogous to that characterized for cleavage of peptide bonds adjacent to Asp and Asn residues. An increased amount of some Glu/Gln cleaved peptides in the insoluble fraction of human lenses suggests that cleavage may act to destabilize proteins. Spontaneous cleavage at Glu and Gln, as well as recently described cross-linking at these residues, can therefore be added to the similar processes affecting long-lived proteins that have already been documented for Asn and Asp residues.

Figure 1:. Relative quantification of truncation at Gln and Glu in middle-aged normal human lenses.

Relative truncation of αA E165 (A), αA E156 (B), βA3 Q208 (C), βA4 Q189 (D), βB1 E249 (E) and βB2 Q197 (F) in different regions of middle-aged normal human lenses. * indicates a statistically significant difference in truncation compared with WSF of the same regions of the lens (p < 0.05). $ indicates a statistically significant difference in truncation compared with cortex region of the lens. The error bars indicate standard deviation of four biological replicates (WSF = Water soluble fraction, USF = Urea soluble fraction and UIF=Urea insoluble fraction.)

Figure 2.. Cleavage on the C-terminal side of Glu in Ac-FAEPA.

a) HPLC profile of Ac-FAEPA at time zero (bottom) and after incubation for 25 days at 60°C, pH 4.0 (Top). The identification of Ac-FAE was confirmed by mass spectrometry. b) Time course of Ac-FAE formation at pH 6.7, 7.0 and 7.4. Ac-FAE was calculated as a percentage of total HPLC peak area. All samples were run in triplicate. Error bars +/− SD.

Figure 3.. MS/MS spectrum of Ac-FAE and Ac-FAQ isolated from the Ac-FAEPA and Ac-FAQPA incubation.

a) Ac-FAE b) Ac-FAQ following incubation at pH 6.7.

Figure 4.. Cleavage on the C-terminal side of Glu in Ac-FAEDA, Ac-FAEPA and Ac-FAEAA.

a) Time course of Ac-FAE from the three peptides at pH 4.0. Ac-FAE formation was calculated as a percentage of total HPLC peak area b) Apparent rate of Glu cleavage in Ac-FAEDA Ac-FAEPA and Ac-FAEAA incubated at pH 6.7, 7.0 and 7.4. Cleavage rate was determined by the slope of the curve over a 25 day incubation period. All samples were run in triplicate at 60°C. Error bars +/− SD.

Figure 5.. Cleavage on the C-terminal side of Gln in Ac-FAQDA, Ac-FAQPA and Ac-FAQAA

a) Time course of formation of Ac-FAQ from Ac-FAQDA and Ac-FAQPA following incubation at pH 4.0, 60°C. No cleavage of Ac-FAQAA was observed at pH 4.0. b) Apparent rate of formation of Ac-FAQ from Ac-FAQDA and Ac-FAQAA incubated at pH 6.7, 7.0 and 7.4. No cleavage of Ac-FAQPA was observed at these pHs. Cleavage rate was determined by the slope of the line over 25 days at 60°C. All samples were run in triplicate. Error bars +/− SD.

Figure 6.. Proposed mechanism of spontaneous cleavage at Gln and Glu in polypeptides.

a) The side chain nitrogen atom of Gln attacks the peptide carbonyl on the C-terminal side of Gln. A glutarimide is formed which is accompanied by cleavage of the peptide bond leading ultimately, after hydrolysis, to the formation of a C-terminal Gln. b) The side chain oxygen atom of Glu attacks the peptide carbonyl on the C-terminal side of Gln. A glutaric anhydride is formed accompanied by cleavage of the peptide bond ultimately leading to the formation of a C-terminal Glu residue.