author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19009035 doi: medRxiv preprint S y n o p s i s 26 The oxidative stress and the post-translational modification of deamidation in lens crystallins 27 seem to play a significant role in the formation of age-related cataract in human. 28 29 30 All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. S  i  g  n  i  f  i  c  a  n  c  e  o  f  t  h  e  s  t  u  d  y   57   58 The crystallins undergo extensive post-translational modifications in order to interact and 59 maintain the soluble basis for lenticular transparency. Severe perturbations of this balance of 60 modifications and interactions lead to inappropriate protein folding, association and 61 aggregation. Those protein aggregates, if a certain threshold is surpassed and perpetuated, 62 diffract incident light leading to lens opacification and cataractogenesis. In addition, the 63 crystallins in high concentration are more than physical structural components of the 64 crystalline lens, and their protein species have additional multiple catalytical or regulatory 65 functions affecting the transparency of the lens and thus, cataractogenesis. Therefore, it is 66 necessary to understand the dynamics of this protein superfamily. Proteomics research is an 67 ideal way to characterize the healthy lenticular protein constituents and elucidate the complex 68 factors involved in cataractogenesis, given that the lens consists mainly of proteins and water. 69
Although considerable advances have been made in the field, and significant proteomic 70 studies have been published to date, this is a systematic review of all proteomic studies on 71 cataractous human lens compared to healthy human lens. The present systematic review aims 72 to shed light on the complex pathophysiology of cataractogenesis and set the basis for future 73 research on possible prevention or even new non-invasive treatment of age-related cataract. 74 75 All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19009035 doi: medRxiv preprint  I  n  t  r  o  d  u  c  t  i  o  n   76   77 Cataract is an opacification of normally clear crystalline lens that may cause a decrease in 78 vision or even blindness if left untreated (1). Cataract is responsible for the 33% of blindness 79 worldwide (2-4). Age is the most important aetiological factor of cataract, due to the 80 cumulative damage of environmental insults on proteins and cells of the crystalline lens 81 (5,6,7). 82
The lens fibers have a high protein content (~34%) and relatively low water content (~65%). In particular, many studies were focused on lens proteomics of various species, such as mice. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19009035 doi: medRxiv preprint Although considerable advances have been made to clarify the pathophysiology of age-related 110 cataract formation, additional high-quality data focused on the human lens need to be 111 exported using proteomics. Thereby, it is necessary to collect and summarise all proteomic 112 researches to date that provide data on cataractous human lens compared to a healthy human 113 lens in order to elucidate the complex factors involved in cataractogenesis and suggest 114 possible prevention or even new non-invasive treatment of age-related cataract. 115
The present systematic review aims to identify all the available data on human lens 119 proteomics with a critical role to age-related cataract formation in order to elucidate the 120 different physiopathological states that lead to the aging of the crystalline lens and thus, 121 cataractogenesis. 122 123 All rights reserved. No reuse allowed without permission.
author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the 
In this systematic review were included English full-text peer-reviewed articles that used 130 proteomics methodologies to study in vivo or in vitro human lens proteins and define possible 131 pathways for age-related cataract formation. 132
In particular, studies were selected according to the criteria outlined below. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19009035 doi: medRxiv preprint Type of outcome: Aging of the crystalline lens and cataract formation was the outcome of 148 interest in the proteomic analysis. Articles that studied other types of cataract except for the 149 age-related one will be excluded. 150
The eligibility criteria are summarized in Table 1 Two reviewers independently screened the titles and the abstracts against the eligibility 168 criteria. Our search strategy was based on proteomics and the biological context of interest 169 (human crystalline lens). Our literature search strategies were developed using medical 170 subject headings (MeSH) and free-text terms related to proteomics and crystalline lens. The 171 "English" filter was applied via the Filter section. 172
We used the following search terms in all databases: proteomics; proteome; "proteom*"; 173 "proteomic analysis"; "proteomic study"; "lens, crystalline"; "Anterior Capsule of the Lens"; 174 "Posterior Capsule of the Lens"; "Lens Nucleus, Crystalline"; "Lens Cortex, Crystalline"; 175 "Lens Capsule, Crystalline"; "Crystallins"; "Cataract" and "Cataract, Age-Related Nuclear". 176
The detailed research strategy can be found in Appendix 1. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19009035 doi: medRxiv preprint eligible studies or in case of any uncertainty. Then, the two reviewers assessed the full-text 188 forms and chose those that met the predefined inclusion criteria. In order to avoid data 189 duplication, multiple publications from the same research group were not extracted (if 190 reporting the same methodology and the same results); instead, the paper that provided the 191 most robust evidence was chosen as the primary evidence source. 192
Any potential disagreements were resolved through discussion with a senior reviewer. • Study Characteristics (the type of study: proteomic analysis) 208
• Type of methods used for separation and isolation of proteins (Electrophoresis or other 209 methods) 210
• Type of methods used for the obtainment of protein identification, structural 211 characterization and relative quantitation (Mass spectrometry) 212 All rights reserved. No reuse allowed without permission.
author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19009035 doi: medRxiv preprint author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19009035 doi: medRxiv preprint author/funder, who has granted medRxiv a license to display the preprint in perpetuity. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19009035 doi: medRxiv preprint Intervention: They performed proteomic analysis to compare normal human lenses to 268 cataractous human lenses. 269
Outcomes: The included studies obtained information on proteins with a key role in the aging 270 of the crystalline lens. In all studies, the primary outcome assessed was this information, 271 which was either identified proteins, or protein structural characteristics, or post-translational 272 modifications, or protein expression, or protein function, or protein localization, or protein-273 protein interactions. 274
The conditions in basic science studies could be completely controlled, in contrast to the 277 clinical studies. As a result, the risk of bias is minimum in basic science studies. However, we 278 created a risk of bias tool to assess the included studies. 279
The results can be found in table 4. 280 281 All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19009035 doi: medRxiv preprint previously unreported sites. Based on their results, large numbers of asparagine and glutamine 292 sites were found to be significantly more deamidated in the water-insoluble 293 proteins of aged lenses and deamidation was found to be the major age-related PTM. They 294 also confirmed the far greater extent of S-methylation known in lens, while there was no 295 methylation in the 3-day-old lens. They identified specific Carboxymethyl lysine (CML) and 296 carboxyethyl lysine (CEL) sites in lens crystallins, which are advanced glycation end products 297 and have been suggested as general markers of advanced age and oxidation, although these 298 modifications were far less abundant than deamidation or methylation. Firstly, they stated that the number of deamidated residues varies considerably between the 339 various crystallins. In β B2-and γ C-crystallin, only one residue was found to be 340 deamidated in the aged normal lenses. By contrast, nine sites of deamidation were found in 341 All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the They also observed that the sites of deamidation were often localized to discrete regions. In 349 α A-crystallin, deamidation was found only in the putative C-terminal domain. In β A1/A3-350 crystallin, the most substantial deamidation was found at three sites contained within a region 351 spanning residues 103 to 133. In author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19009035 doi: medRxiv preprint degree of conversion of Asn 76 to isoAsp in cataract lenses was approximately double that of 367 normal lenses at every age. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19009035 doi: medRxiv preprint oxidized in vitro with H2O2 as a model of crystallin aggregation and opacification. They used 392 as control 3 young normal human lens nuclei (at 15, 7, and 393 3 years of age), and 3 old normal human lens nuclei (at 74, 72, and 68 394 years of age). They used as well 9 cataractous lenses, 3 in each category of grade II-V of Lens 395
Opacities Classification System III. The age of grade II group was 64, 67, and 60 years old, 396 the grade III was 60, 59, and 69 years old, the grade IV was 56, 63, and 77 years old, and the 397 grade V was 75, 80, and 78 years old. Four types of cysteine residues were identified based on their susceptibility to oxidation with 469 age and as a function of presence and severity of cataract graded from II to V. The first 470 includes cysteine residues that are mildly oxidized with age and not further oxidized in 471 cataracts; the second includes cysteine residues with mild oxidation in old normal lenses but 472 massive increase in cataracts regardless of cataract severity; the third includes cysteine 473 residues with oxidation in old normal lenses and further increase in cataract lenses, and the 474 fourth includes cysteine residues that are barely oxidized with age but oxidized in cataracts, 475 and also associated with cataract severity. However, all studies used proteomic analysis that has a tremendous favorable potential for 491 detecting candidate proteins and, so, facilitating the development of precision medicine for 492 All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. suggesting that it could as well be associated with the development of age-related cataract in 505 human. Such a dominant age-related modification may contribute to the denaturation of a 506 crystallin, and its ability to act as a chaperone. 507
Based on the results from their proteome analysis of human foetal, aged and advanced nuclear 508 cataract lenses, Hains and Truscott suggested there are no major differences between aged 509 normal and advanced cataract lenses in terms of proteins present. 510
The results of their other study showed that the level of Trp oxidation appeared to increase in 511 cataract lenses when these were compared to age-matched normal lenses and this may 512 indicate that oxidation of Trp may be associated with nuclear cataract formation in man. 513
Deamidation appears to be important for protein denaturation in the aging lens and it is 514 possible that the few sites they observed to be differentially deamidated in cataractous lenses 515 may be sufficient to cause opacification. 516
The results of Wilmarth et al. show that large numbers of asparagine and glutamine sites were 517 All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19009035 doi: medRxiv preprint significantly more deamidated in the water-insoluble proteins of aged lenses, and since 518 cataract can be thought of as a crystallin insolubility disease, modifications exclusively 519 associated with water-insoluble proteins may have implications in cataract formation. 520
Finally, the results of Wang et al. and fan et al. support the important role of oxidant stress in 521 age-related cataract formation, and in particular, the importance of GSH and that 522 pharmacological GSH mimetics are probably needed to protect the aging lens from oxidation. 523 524 All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the The authors report no conflicts of interest. The authors alone are responsible for the content 536 and writing of this article. 537 538 539 All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/19009035 doi: medRxiv preprint