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The centre of the human lens, which is composed of proteins that were synthesized prior to birth, is an ideal model for the evaluation of long-term protein stability and processes responsible for the degradation of macromolecules. By analysing the sequences of peptides present in human lens nuclei, characteristic features of intrinsic protein instability were determined. Prominent was the cleavage on the N-terminal side of serine residues. Despite accounting for just 9% of the amino acid composition of crystallins, peptides with N-terminal Ser represented one-quarter of all peptides. Nonenzymatic cleavage at Ser could be reproduced by incubating peptides at elevated temperatures. Serine residues may thus represent susceptible sites for autolysis in polypeptides exposed to physiological conditions over a period of years. Once these sites are cleaved, other chemical processes result in progressive removal or ‘laddering’ of amino acid residues from newly exposed N- and C-termini. As N-terminal Ser peptides originated from several crystallins with unrelated sequences, this may represent a general feature of long-lived proteins.
Long-lived polypeptides are present in heart, lung, brain, lens (Lynnerup et al., 2008), teeth, blood vessels, skin and connective tissue (Ritz-Timme & Collins, 2002). Most, like elastin and collagen, are extracellular, although some, for example, crystallins are cytosolic. Over decades at body temperature, persistent proteins undergo modifications that may affect their structure and function. These include racemization, deamidation, modification by reactive metabolites and truncation (Cloos & Fledelius, 2000; Truscott, 2011), although little is known about which processes are most significant. Enzymatic cleavage of long-lived polypeptides is a significant route for decomposition; however, the centre of the adult human lens is devoid of active proteases. In their absence, it is possible to identify amino acids that are prone to spontaneous cleavage under physiological conditions. One well-characterized reaction at Asn residues (Geiger & Clarke, 1987) involves intramolecular attack, followed by ring opening and peptide bond cleavage with the formation of a new C-terminal Asp residue. To uncover cleavage sites of long-lived proteins, we examined the terminal residues of peptides present in the centre of aged human lenses.
A total of 211 unique peptides were identified in the nuclei of four human lenses aged 16, 44, 75 and 83 years (Table S1, Supporting information), with a false discovery rate of 0.09. Peptides from αA- and αB-crystallin were most numerous (Fig. S1, Supporting information), while β- and γS-crystallin peptides were also present (Fig. S2). Interestingly, peptides originating from γC- and γD-crystallins were not detected. The diversity of peptides suggested that either extensive chemically mediated hydrolysis was taking place or proteases remain active in the mature nucleus. As no protease activity could be detected in the nuclear region of fresh (< 24 h post-mortem) lenses above age 40 (Fig. S3), a nonenzymatic mechanism seems likely.
Although it was not possible to quantify peptides using our methodology, we were able to gain some insights into their crystallin origin. Figure 1 is a graphical summary of Mascot ion scores for each peptide identified, presented according to their position on the heat gradient scale at right. For both the water-soluble (WS) (Fig. 1A) and urea-soluble (Fig. 1B) fractions, a clear correlation between age and peptide identification is evident. Among the WS peptides, there are two localized regions of high ion scores: one in αA-crystallin comprising residues 34–65 and spanning the latter third of the N-terminal domain, the adjoining interdomain linker (57–62) and the first three residues of the α-crystallin domain and the second, residues 238–252 within the C-terminal arm of βΒ1-crystallin. Both of these ‘hot spots’ are in regions of the protein that lack significant secondary structure.
Figure 1. Endogenous peptides present in (A) water-soluble and (B) urea-soluble human lens nuclear extracts. Peptides are classified by Mascot ion score according to the heat scale shown at right. The results demonstrate a correlation between the total number of peptides identified and increasing lens age.
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To determine whether particular amino acids are more susceptible to cleavage, the occurrence of each amino acid as a peptide terminal residue was expressed as a ratio of the abundance of that amino acid in the combined full-length crystallin sequences. The resulting residue frequency ratios (FR) are presented in Fig. 2 and Table S2. Asp, Ser and His were particularly abundant as N-terminal residues, with FR values of 0.32, 0.30 and 0.28, respectively, compared to a mean FR of 0.15 ± 0.08. At the C-terminus, Asp (0.28), Lys (0.25) and Arg (0.32) had FR values up to two standard deviations above the mean (0.16 ± 0.08). Thus, Asp residues were found at statistically significant levels as both N- and C-termini, whereas His, Lys, Arg and Ser were overwhelmingly localized to a single peptide terminus. In the case of Asp, C-terminal cleavage has been observed in peptides (Li et al., 2009), but the N-terminal cleavage may represent a novel phenomenon in long-lived proteins. Another prominent feature of the peptides was sequential residue loss, or ‘laddering’, which was observed from both the N- and C-termini (Figs S1 and S2).
Figure 2. Residue frequency ratio (FR) histograms for amino acids occurring at the N- and C-termini of endogenous peptides. Mean FR values are indicated by horizontal dashed lines, while shaded regions represent one standard deviation (SD) from the mean. Asterisks denote FR values greater than one (*) and two (**) SDs above the mean.
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The finding that Ser was a major N-terminal residue is consistent with Ser being involved in spontaneous peptide bond cleavage (Lyons et al., 2011). To examine this, the heptapeptide PFHSPAY, containing a known cleavage site in αB-crystallin (H18-S19) (Su et al., 2010), was synthesized and incubated at 60 °C. The result (Fig. 3) demonstrates that cleavage at Ser can occur in the absence of proteases. One mechanism postulated for this nonenzymatic lysis involves an NO acyl shift, as documented for intein splicing (Paulus, 2000). Inteins are internal self-splicing regions of proteins where one splice site is a Ser, Cys or Thr. This is significant in the context of our study of the lens, as many peptides resulted from bond cleavage on the N-terminal side of Ser. In addition, the progressive loss of terminal amino acids, or ‘laddering’, observed for the lens peptides was reproduced in vitro using the model peptide. The mechanism for sequential removal of terminal amino acids from peptides is unclear; however, involvement of the α-amino group at the N-terminal ends is likely. For example, incubation of peptides at neutral pH results in nucleophilic attack of the α-amino group on a peptide bond and removal of the two amino terminal residues as a diketopiperazine (Steinberg & Bada, 1983). The phenomenon of ‘laddering’ of peptides has been noted in other biological systems. The basis for the removal of amino acids from the C-terminus is less clear; however, incubation of recombinant R120G αB-crystallin at room temperature results in truncations from the C-terminus (Treweek et al., 2005).
Do processes outlined here occur with other long-lived proteins? To date, information is scarce; however, such polypeptides are widespread in man (Truscott, 2011). It will be of interest to discover whether the changes outlined here can be used as molecular signatures to determine whether a protein is long-lived. This will lead to a greater understanding of the breakdown of these molecules over time and their impact on human health.
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Fig. S1 Endogenous low molecular weight peptides identified in the lens derived from cleavage of, (A) αA-crystallin, and (B) αB-crystallin.
Fig. S2 Endogenous low molecular weight peptides in the lens derived from cleavage of (A) βA1/A3-crystallin, (B) γS-crystallin, (C) βB1-crystallin and (D) βB2-crystallin.
Fig. S3 Protease activity is not detectable in the centre of older lenses.
Table S1 Endogenous LMW peptides present in human lens extracts.
Table S2 Totals of each of the 20 amino acid residues are tabulated as they occur in the combined sequences of all human lens crystallins – ƒP(protein).
Appendix S1 Experimental procedures.
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