Spontaneous Cleavage of Proteins at Serine Residues

Brian Lyons • Joanne Jamie • Roger J.W. Truscott

Accepted: 19 April 2011 / Published online: 3 May 2011

� Springer Science+Business Media, LLC 2011

Abstract Long-lived proteins are found at several sites in

the body and they undergo numerous changes as a result of

prolonged exposure to physiological conditions. Trunca-

tion is a common modification and many cleavages appear

to be non-enzymatic, however little is known about the

processes involved. In this study we demonstrate, using

synthetic peptides that incorporate the sequence of a pro-

tein that is known to cleave in older lenses, that truncation

on the N-terminal side of serine residues can occur at

neutral pH. A mechanism that incorporates an N,O-acyl

shift, which is analogous to intein cleavage, is proposed.

Such cleavages may explain the origin of abundant pep-

tides derived from crystallins in aged human lenses.

Keywords Old proteins � Age � Hydrolysis �Posttranslational modification � Human lens

Introduction

Long-lived proteins are widespread in the human body

and their age-related modifications may contribute to the

deterioration in human health and fitness (Truscott 2010).

Major modifications to such long-lived proteins include

racemisation (Hooi and Truscott 2010; Masters et al.

1977; Fujii et al. 2000); deamidation (Hains and Truscott

2010; Robinson and Robinson 2001; Miesbauer et al.

1994; Groenen et al. 1993; Wilmarth et al. 2006) and

truncation (Takemoto 1995; Srivastava and Srivastava

2003; Harrington et al. 2004). Although some, such as the

formation of succinimides from Asn and Asp residues,

(Clarke 1987) have been well characterised, our under-

standing of the processes that govern these posttransla-

tional modifications, and which of them are of most

importance, is incomplete.

The human lens is an ideal tissue for studying age-

related protein modifications because the lens crystallins

are lifelong (Lynnerup et al. 2008) and it is possible to

examine lenses across the age range. Recent publications

(Srivastava and Srivastava 2003; Santhoshkumar et al.

2008) have described the presence of shortened forms

of crystallins in aged lenses, as well as the possible

involvement of crystallin peptides in modulating the

action of the lens chaperone a crystallin (Santhoshkumar

et al. 2008). In the case of a crystallin, one notable

feature of the sequences of two of the most abundant

peptides (aA 67–80 and aB 1–18) was that sites of

cleavage were adjacent to Ser residues (Santhoshkumar

et al. 2008; Su et al. 2010). Since enzyme activity is

absent in the nuclei of adult human lenses (Scharf et al.

1987; Charlton and van Heyningen 1971; Dovrat et al.

1984; Zhu et al. 2010), we investigated whether such

cleavages may result from spontaneous reactions involv-

ing Ser. In this study a peptide that encompasses the

sequence of the cleavage site in aB crystallin (aB 16-21

Fig. 1) was incubated at neutral pH and the products

characterised.

B. Lyons � R. J.W.Truscott (&)

Save Sight Institute, University of Sydney, Sydney Eye Hospital,

8 Macquarie St., Sydney, NSW 2001, Australia

e-mail: roger.truscott@sydney.edu.au

B. Lyons

e-mail: brian.lyons@sydney.edu.au

J. Jamie

Department of Chemistry and Biomolecular Sciences,

Macquarie University, Sydney, NSW 2109, Australia

e-mail: joanne.jamie@mq.edu.au

123

Int J Pept Res Ther (2011) 17:131–135

DOI 10.1007/s10989-011-9250-3

Materials

Peptides (PFHSPSY, SPSY and SY) were synthesised by

Peptide 2.0 (Chantilly, VA, USA) to a purity of [95%.

Peptide PFHAPAY was synthesised by GLS Biochem

(Shanghai 200241, China). TFA (Sigma) was spectropho-

tometric grade and Na2HPO4 and NaH2PO4 were pur-

chased from Amresco (Solon, OH, USA). All solutions

were prepared in MilliQ water (Waters, Billerica, MA,

USA).

Methods

The peptide PFHSPSY was synthesized by peptide 2.0 with

a tyrosine residue incorporated as the C-terminal residue to

facilitate detection of C-terminal fragments by HPLC at

280 nm. Triplicate samples of this peptide, and a homol-

ogous peptide where Ser was replaced by Ala, as well as

SPSY were dissolved in 0.1 M phosphate buffer pH 7.4

(1 mg/ml) and incubated at either 37 or 60�C. In other

experiments phosphate buffer was replaced by HEPES

buffer pH 7.4. Aliquots (10 ll) were taken at various times

and analysed using HPLC as detailed below.

HPLC Analysis

An Agilent 1100 HPLC system controlled using Chem-

station software and equipped with a PDA detector was

used. Incubations were monitored at 280 nm and 216 nm.

Separation of the peptides was achieved using a Kinetex

2.6u C18 100 A´

column (100 9 4.6 mm2 I.D) at 40�C.

The gradient was 0% B (0.1% TFA) to 60% B (0.1% TFA

in acetonitrile) over 25 min followed by 2 min at 60% B at

a flow rate of 1 ml/min. The column was equilibrated at

0% B for 10 min at the end of each run.

Mass Spectrometric Analysis

Peaks collected from RP-HPLC were lyophilised, resus-

pended in 50% (v/v) acetonitrile, 0.5% (v/v) formic acid

and analysed in positive ion mode using a Micromass

Q-TOF2 equipped with a nanospray source. For MS/MS

analysis, ions were subjected to a range of collision ener-

gies (typically between 10 and 25 eV). Peptides were also

analysed by MALDI-TOF MS/MS. A Shimadzu TOF2 MS

was used in positive ion reflectron mode. Peptides were

prepared in a-cyano-4-hydroxycinnamic acid (8 mg/ml) in

50% (v/v) acetonitrile, 1.0% (v/v) TFA.

Results

Lens proteins do not turnover after their incorporation into

mature fibre cells (Lynnerup et al. 2008) and several

studies have reported the presence of peptides in older

human lenses that are derived from the breakdown of

the original crystallins (Srivastava and Srivastava 2003;

Santhoshkumar et al. 2008). A common feature of many of

these abundant peptides is that they contain an N-terminal

Ser or Thr residue (Santhoshkumar et al. 2008; Su et al.

2010). In this study we investigated whether non-enzy-

matic processes could be responsible. To examine this we

used the model peptide PFHSPSY, which incorporates a

major cleavage site that has been found in aged a B crys-

tallin (aB 16-21, PFHSPS Fig. 1). a B crystallin is one of

the most abundant lens proteins. a B 1-18 and a B 2-18 are

two of the most prominent peptides detected in extracts of

older human lenses (Fig. 1) and both result from cleavage

between His 18 and Ser19 (Su et al. 2010). A tyrosine

residue was incorporated as the C-terminal residue in the

model peptide to facilitate detection of C-terminal frag-

ments by HPLC. The peptide was incubated in phosphate

buffer at pH 7.4. Elevated temperature (60�C) was used to

promote reaction and aliquots removed at various times

and monitored by HPLC.

The peptide PFHSPSY underwent progressive cleavage

with the generation of two new HPLC peaks (Fig. 2a)

whose HPLC retention times corresponded to those of

SPSY and SY. These peaks were collected and analysed by

both electrospray and MALDI mass spectrometry and their

MS/MS spectra matched those of synthetic standards. The

time course of their appearance is shown in Fig. 3. After

3 weeks at 60�C, SPSY ? SY accounted for 24.4% of the

starting material. At all time points the major product of

incubation was found to correspond to the cis Pro form of

the starting material PFHSPSY, as determined by NMR

characterisation of the collected peak. The characterisation

of this, and its kinetics, as well as the removal of the

N-terminal Pro will be reported in a separate publication.

10 20 30 40 50 60MDIAIHHPWI RRPFFPFHSP SRLFDQFFGE HLLESDLFPT STSLSPFYLR PPSFLRAPSW

70 80 90 100 110 120FDTGLSEMRL EKDRFSVNLD VKHFSPEELK VKVLGDVIEV HGKHEERQDE HGFISREFHR

130 140 150 160 170 KYRIPADVDP LTITSSLSSD GVLTVNGPRK QVSGPERTIP ITREEKPAVT AAPKK

Fig. 1 Sequence of human

alpha B crystallin. The site of

cleavage in older human lenses

is shown with an arrow and the

peptide region examined in this

investigation is in bold

132 Int J Pept Res Ther (2011) 17:131–135

123

In order to confirm that Ser was implicated in the

cleavage, the homologous peptide, PFHAPAY, was sepa-

rately incubated under the same conditions. PFHAPAY

showed no evidence of peptide bond cleavage at the sites

corresponding to those found in the Ser-containing peptide.

As was observed for PFHSPSY, prominent peaks were

observed due to conversion to the cis Pro form of PFHA-

PAY, as well as truncation of the N-terminal Pro residue

(Fig. 2b).

Since the SPSY sequence additionally contains an

internal Ser residue that may also potentially cleave, SPSY

was incubated separately. As shown in Fig. 4, it was found

that SPSY was rapidly cleaved with the major product

being SY; its identity being confirmed by mass spectrom-

etry. Therefore, SY can be generated by direct cleavage of

PFHSPSY as well as by a secondary process involving an

initial truncation to yield SPSY.

In order to establish whether the same non-enzymatic

processes can take place under physiological conditions,

the incubations were repeated at 37�C. The same peptides

were generated under these conditions as had been

observed at the higher temperature, however the rates of

appearance of both major products was approximately 10

cis PFHAPAY

FHAPAY

PFHAPAY

cis PFHSPSYPFHSPSY

FHSPSY

SPSY

SY

(a)

(b)

* * *

Fig. 2 a HPLC trace showing the products formed following

incubation of PFHSPSY in 0.1 M phosphate buffer pH 7.4 at 60�C for

3 weeks. The identity of each product was confirmed by mass

spectrometry and NMR for the cis Pro peptide. FHSPSY may form as

result of separate processes that affect the stability of the Pro-Phe

bond. *Unidentified degradation products of SY. b HPLC trace

showing the products formed following incubation of PFHAPAY in

0.1 M phosphate buffer pH 7.4 at 60�C for 3 weeks. The identity of

each product was confirmed by mass spectrometry and NMR for the

cis Pro peptide. FHAPAY may form as a result of separate processes

that affect the stability of the Pro-Phe bond. The peak eluting at

8.5 min was found not to be APAY or AY

Fig. 3 Time course showing the appearance of SY and SPSY

following incubation of PFHSPSY (1.2 lmole) in 0.1 M phosphate

buffer pH 7.4 at 60�C. Under identical conditions, neither AY nor

APAY were observed following incubation of PFHAPAY. The slopes

of the lines for SPSY and SY were compared with those of AY/APAY

using ANCOVA and the differences in both cases were found to be

statistically significant (P \ 0.0001). Graphs in Fig. 3 and Fig. 4 are

lines of best fit determined by Sigmaplot sigmoidal 3 parameter

Fig. 4 Decrease of SPSY and appearance of SY as a function of

time. SPSY (0.22 lmole) was incubated in 0.1 M phosphate buffer

pH 7.4 at 60�C

Int J Pept Res Ther (2011) 17:131–135 133

123

times slower at 37�C. Incubation of peptides at 60�C using

0.1 M HEPES buffer pH7.4, showed almost identical

results to those seen for phosphate buffer, indicating that

any specific buffer effect on the cleavage appears

negligible.

Discussion

This work has demonstrated that cleavage of peptides at

Ser residues can be observed following incubation at

neutral pH. Peptide bond hydrolysis was promoted by

exposure to elevated temperature, however, since the same

products were observed at 37�C, it is likely that similar

processes occur under physiological conditions.

The mechanism for this process is currently under

investigation in our laboratory. The most likely one

involves an intermolecular attack involving the hydroxyl

group of Ser as depicted in Fig. 5. This mechanism is

analogous to one part of the well-known intein cleavage

(Mills and Paulus 2005; Wallace 1993; Noren et al. 2000).

Intein cleavage is an intrinsic property of certain poly-

peptides and requires no additional cofactors. One site of

the splicing involves an N,O-acyl shift adjacent to either a

Ser or Thr residue, as is depicted for Ser in Fig. 5. Other

factors such as trace metal ions (Yashiro et al. 2003) could

potentially contribute to the cleavage, however in our

experiments, incorporation of 1 mM EDTA into the buffer

did not affect the time course of the degradation of the

peptide (data not shown) indicating that metals may not

play an important role. Selective peptide bond scission near

to Ser residues has also been reported for proteins exposed

to chemical reagents (Kamo and Tsugita 1998) including

acids such as HCl (Sanger and Tuppy 1951).

It is likely that the processes outlined in this paper for

crystallins may also apply to other old proteins. Old pro-

teins are present at many sites in the human body (Truscott

2010). The results shown here demonstrate that cleavage

adjacent to Ser residues can occur in the absence of

enzymes. It is probable that such intrinsic instability is

responsible for the appearance of numerous peptides in

older lenses that contain N-terminal Ser. Although not

examined directly in this study, analogous processes may

be responsible for the formation of peptides that terminate

in Thr e.g. aA 43-56 (Santhoshkumar et al. 2008). It is

apparent that other factors such as neighbouring residues

and functional groups will affect the rate of peptide bond

hydrolysis (Powell 1994). This may well explain the

enhanced rate of cleavage of SPSY. Other processes may

take place in addition to cleavage next to Ser residues. This

was illustrated in our experiments by the appearance of a

new form of the starting material PFHSPSY that contained

cis Pro, as well as cleavage of the amino terminal amino

acid (Fig. 3).

In conclusion, this study has demonstrated that cleavage

of the peptide bond adjacent to Ser residues can occur at

physiological pH. This cleavage takes place in the absence

of enzymes and results in peptides that possess N-terminal

Ser. Such intein-like cleavages are likely to be responsible

for a number of the crystallin peptides that have been found

in aged human lenses. Such cleavages may be a general

feature of other long-lived proteins.

Acknowledgement Funding for this work was provided by a grant

from the NHMRC (#512334). RJWT is an NHMRC senior research

fellow.

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