Cleavage C-terminal to Asp leads to covalent crosslinking of long-lived human proteins

doi:10.1016/j.bbapap.2019.06.009

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

Volume 1867, Issue 9, September 2019, Pages 831-839

https://doi.org/10.1016/j.bbapap.2019.06.009 Get rights and content

Highlights

•
Aging is associated with the formation of covalently crosslinked proteins.
•
A novel protein-protein crosslinking mechanism was identified.
•
Crosslinking increased with lens age and cataract.
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The mechanism involves peptide bond cleavage at Asp and formation of an anhydride.
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The anhydride reacts with free amine groups to form a protein-protein crosslinks.

Abstract

With age, long-lived proteins in the human body deteriorate, which can have consequences both for aging and disease. The aging process is often associated with the formation of covalently crosslinked proteins. Currently our knowledge of the mechanism of formation of these crosslinks is limited. In this study, proteomics was used to characterize sites of covalent protein-protein crosslinking and identify a novel mechanism of protein-protein crosslinking in the adult human lens. In this mechanism, Lys residues are crosslinked to C-terminal Asp residues that are formed by non-enzymatic protein truncation. Ten different crosslinks were identified in major lens proteins such as αA-crystallin, αB-crystallin and AQP0. Crosslinking in AQP0 increased significantly with age and also increased significantly in cataract lenses compared with normal lenses. Using model peptides, a mechanism of formation of the Lys-Asp crosslink was elucidated. The mechanism involves spontaneous peptide cleavage on the C-terminal side of Asp residues which can take place in the pH range 5–7.4. Cleavage appears to involve attack by the side chain carboxyl group on the adjacent peptide bond, resulting in the formation of a C-terminal Asp anhydride. This anhydride intermediate can then either react with water to form Asp, or with a nucleophile, such as a free amine group to form a crosslink. If an ε-amino group of Lys or an N-terminal amine group attacks the anhydride, a covalent protein-protein crosslink will be formed. This bi-phasic mechanism represents the first report to link two spontaneous events: protein cleavage and crosslinking that are characteristic of long-lived proteins.

Introduction

The human body contains numerous long-lived proteins (LLPs) [[2], [3], [4]]. With age, certain amino acids within these proteins; in particular Asn, Asp, Gln, Cys, Thr and Ser, undergo deterioration [[5], [6], [7]] and these spontaneous modifications can result in racemization, deamidation and protein cleavage [2]. Another common modification associated with aged LLPs is non-disulphide covalent crosslinking. Protein-protein crosslinking is increasingly observed with age in tissues such as the lens [8], brain [9], heart [10], arteries [11], cartilage [12] and has been associated with decreased function and disease [8,13,14]. Several crosslinking mechanisms have been proposed such as via advanced glycation end products [15,16], transglutaminase activity [17], and through dehydroalanine (DHA) intermediates [8,18]; however, identification of novel age-related protein-protein crosslinking processes is challenging due to the large number of potential crosslink combinations that could form coupled with technical difficulties in sequencing crosslinked peptides.

With advances in proteomics technology and data analysis methodology, identification of novel crosslinking sites formed in aged tissue become increasingly feasible [8]. Recently, one mechanism for crosslinking of aspartic acid and lysine residues in the human eye lens, involving the formation of a succinimide intermediate, was described [19]. This discovery provided the first link between the age-related processes of protein-protein crosslinking and protein racemisation/isomerisation. Racemisation and isomerisation of Asp and Asn are two of the most common modifications present in LLPs [[20], [21], [22]] and the reaction pathway is thought to involve the peptide bond NH attacking the Asn or Asp side chains to form a cyclic succinimide intermediate [23,24]. Hydrolysis of this succinimide intermediate can result in the formation of four Asp isomers: L- Asp, D-Asp, L-iso Asp and D-isoAsp [25]. Whilst, formation of a succinimide is a major pathway of Asp degradation, a competing mechanism can take place where the side chain carboxylic acid attacks the adjacent peptide bond carbonyl resulting in peptide cleavage and the formation of a C-terminal succinic anhydride. This anhydride can then hydrolyse to yield a C-terminal Asp. Such cleavages, are typically observed under low pH conditions, for example, those employed for storage of protein pharmaceuticals such as monoclonal antibodies [[26], [27], [28]]. The mechanism of this peptide bond cleavage has been examined in detail, and it appears to involve attack of the ionized carboxyl side chain on the protonated carbonyl group of the peptide bond [29]. A separate computational study outlined a similar mechanism of peptide bond cleavage that involved the protonated side chain of Asp which also resulted in the formation of a C-terminal anhydride [30].

As part of a project to characterize the covalent crosslinking of LLPs and their mechanisms of formation, we used proteomic methods to examine proteins from adult lenses with the aim of detecting sites of novel crosslinks. When aged human lenses were examined for the presence of non- disulphide covalently crosslinked proteins, several sites were found that involved Lys residues crosslinked to C-terminal Asp residues. This paper describes the mechanism of formation of these crosslinks.

Section snippets

Materials and methods

Frozen human lenses were obtained from NDRI (Philadelphia, PA) or from the Kansas Eye Bank (Wichita, KS). All lenses were isolated from the donor no later than 8 h post mortem and shipped on dry ice. Human lens work was conducted in compliance with the Declaration of Helsinki. The lenses were classified as normal or cataract lenses by the eye bank. All lenses received were stored at −80 °C until use. All chemicals were purchased from Sigma (St. Louis, MO). All HPLC grade solvents were purchased

Identification of protein-protein crosslinking in human lenses

Previously, DHA and DHB-mediated protein-protein crosslinking was identified as one of the mechanisms that contributes to irreversible protein-protein crosslinking in human lenses [8]. During our searching protocol a strong signal was detected in tryptic digest of the urea-insoluble fraction from the nucleus region of a 68 year old cataract lens corresponding to crosslinking between peptides AQP0 227–233 (LK*SISER) and AQP0 239–243 (GAKPD*). The crosslink appeared to involve an amide bond

Discussion

Since LLPs undergo a range of modifications with time, degradation of LLPs is of major importance for human aging and age-related diseases. Some modifications, such as glycation [41] and oxidation [42] are the result of external reactive molecules. Many other modifications are spontaneous and occur due to the structures of the amino acid side chains and their local environment e.g. unstructured regions permit more allowable conformations. As we age, non-disulphide crosslinks involving proteins

Conclusions

Overall this investigation has elucidated one mechanism responsible for spontaneous cleavage and crosslinking of proteins in the lens. Cleavage of the peptide bond adjacent to Asp via the formation of a C-terminal anhydride was a prerequisite. This is the first reported case where two of the main reactions of LLPs, crosslinking and cleavage, are linked directly. Asp residues in other long-lived proteins, should be considered as potential sites of cleavage and crosslinking.

Declaration of Competing Interest

The authors declare no conflict of interest

Acknowledgements

The authors acknowledge use of the UOW Mass Spectrometry User Resource and Research Facility (MSURRF), University of Wollongong and the Proteomics Core Facility of the Vanderbilt University Mass Spectrometry Research Center.

Author contribution statement

ZW, MGF, RJWT & KLS contributed to writing and preparation of the manuscript. MGF & ZW undertook all experiments. MGF, RJWT, KLS & ZW were involved in planning of experiments.

Funding sources

Funding for this study was provided by National Institutes of Health by grants R01 EY024258 and P30 EY008126.

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¹: Indicates joint first authorship.

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Cleavage C-terminal to Asp leads to covalent crosslinking of long-lived human proteins

Highlights

Abstract

Introduction

Section snippets

Materials and methods

Identification of protein-protein crosslinking in human lenses

Discussion

Conclusions

Declaration of Competing Interest

Acknowledgements

Author contribution statement

Funding sources

Trends Biochem. Sci.

Cell

Biochem. Biophys. Res. Commun.

Matrix Biol.

J. Biol. Chem.

Exp. Eye Res.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Chromatogr. B

J. Pharm. Sci.

Exp. Eye Res.

Trends Biochem. Sci.

Vis. Res.

Matrix Biol.

Exp. Eye Res.

Biochim. Biophys. Acta

Exp. Eye Res.

Biochem. Biophys. Rep.

Biochim. Biophys. Acta

J. Pharm. Biomed. Anal.

The Proteomics Identifications (PRIDE) database and associated tools: status in 2013

Nucleic Acids Res

Identification of long-lived synaptic proteins by proteomic analysis of synaptosome protein turnover

Proc. Natl. Acad. Sci.

Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins

Int. J. Pept. Protein Res.

Racemisation and human cataract. d-Ser, d-Asp/Asn and d-Thr are higher in the lifelong proteins of cataract lenses than in age-matched normal lenses

Age

Spontaneous cleavage of proteins at serine and threonine is facilitated by zinc

Aging Cell

Human protein aging: modification and crosslinking through dehydroalanine and dehydrobutyrine intermediates

Aging Cell

Molecular aging of tau: disulfide-independent aggregation and non-enzymatic degradation in vitro and in vivo

J. Neurochem.

Elastin and collagen fibre microstructure of the human aorta in ageing and disease: a review

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Collagen, cross-linking, and advanced glycation end products in aging human skeletal muscle

J. Appl. Physiol.