Mechanism of protein cleavage at asparagine leading to protein–protein cross-links

Long-lived proteins (LLPs) are present in numerous tissues within the human body. With age, they deteriorate, often leading to the formation of irreversible modifications such as peptide bond cleavage and covalent cross-linking. Currently understanding of the mechanism of formation of these cross-links is limited. As part of an ongoing study, proteomics was used to characterise sites of novel covalent cross-linking in the human lens. In this process, Lys residues were found cross-linked to C-terminal aspartates that had been present in the original protein as Asn residues. Cross-links were identified in major lens proteins such as αA-crystallin, αB-crystallin and aquaporin 0. Quantification of the level of an AQP0/AQP0 cross-linked peptide showed increased cross-linking with age and in cataract lenses. Using model peptides, a mechanism of cross-link formation was elucidated that involves spontaneous peptide bond cleavage on the C-terminal side of Asn residues resulting in the formation of a C-terminal succinimide. This succinimide does not form cross-links, but can hydrolyse to a mixture of C-terminal Asn and C-terminal Asp amide peptides. The C-terminal Asp amide is unstable at neutral pH and decomposes to a succinic anhydride. If the side chain of Lys attacks the anhydride, a covalent cross-link will be formed. This multi-step mechanism represents a link between two spontaneous events: peptide bond cleavage at Asn and covalent cross-linking. Since Asn deamidation and cleavage are abundant age-related modifications in LLPs, this finding suggests that such susceptible Asn residues should also be considered as potential sites for spontaneous covalent cross-linking.

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Age-related changes in the physical properties, cross-linking, and glycation of collagen from mouse tail tendon

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011031 ◽

2020 ◽

Vol 295 (31) ◽

pp. 10562-10571 ◽

Cited By ~ 4

Author(s):

Melanie Stammers ◽

Irina M. Ivanova ◽

Izabella S. Niewczas ◽

Anne Segonds-Pichon ◽

Matthew Streeter ◽

...

Keyword(s):

The Other ◽

Cross Linking ◽

Structural Protein ◽

Cross Link ◽

Age Related ◽

Cross Links ◽

Tail Tendon ◽

Collagen Cross Linking ◽

Age Related Changes ◽

Tendon Collagen

Collagen is a structural protein whose internal cross-linking critically determines the properties and functions of connective tissue. Knowing how the cross-linking of collagen changes with age is key to understanding why the mechanical properties of tissues change over a lifetime. The current scientific consensus is that collagen cross-linking increases with age and that this increase leads to tendon stiffening. Here, we show that this view should be reconsidered. Using MS-based analyses, we demonstrated that during aging of healthy C57BL/6 mice, the overall levels of collagen cross-linking in tail tendon decreased with age. However, the levels of lysine glycation in collagen, which is not considered a cross-link, increased dramatically with age. We found that in 16-week-old diabetic db/db mice, glycation reaches levels similar to those observed in 98-week-old C57BL/6 mice, while the other cross-links typical of tendon collagen either decreased or remained the same as those observed in 20-week-old WT mice. These results, combined with findings from mechanical testing of tendons from these mice, indicate that overall collagen cross-linking in mouse tendon decreases with age. Our findings also reveal that lysine glycation appears to be an important factor that contributes to tendon stiffening with age and in diabetes.

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Collagen cross-linking in human bone and articular cartilage. Age-related changes in the content of mature hydroxypyridinium residues

Biochemical Journal ◽

10.1042/bj2520495 ◽

1988 ◽

Vol 252 (2) ◽

pp. 495-500 ◽

Cited By ~ 288

Author(s):

D R Eyre ◽

I R Dickson ◽

K Van Ness

Keyword(s):

Cortical Bone ◽

Cancellous Bone ◽

Human Subjects ◽

Adult Life ◽

Bone Collagen ◽

Cross Linking ◽

Cross Link ◽

Age Related ◽

Cross Links ◽

And Cartilage

The concentration in collagen of hydroxypyridinium cross-linking amino acids was measured in samples of bone and cartilage from human subjects aged from 1 month to 80 years. Cortical and cancellous bone samples were dissected and analysed separately. In both bone and cartilage, the content of this mature form of cross-link reached a maximum by 10-15 years of age (the amount in cartilage being 5-10 times that in bone), then stayed essentially in the same range throughout adult life. In bone the ratio of the two chemical variants of the mature cross-link, hydroxylysylpyridinoline to lysylpyridinoline, was constant throughout adult life at 3.5:1, whereas in cartilage it was always greater than 10:1. The ratio of hydroxypyridinium cross-links to borohydride-reducible keto-amine cross-links also changed with age. The reducible cross-links in bone collagen decreased steeply in content between birth and 25 years, but persisted in significant amounts throughout adult life. Reducible cross-links had virtually disappeared from cartilage by 10-15 years of age, being replaced by hydroxypyridinium residues, their maturation products. Cancellous and cortical bone collagens showed similar trends with age in their content of mature cross-links, though for each subject the concentration in cancellous bone was always lower than in cortical bone, presumably reflecting the higher turnover rate and hence the more immature state of cancellous bone.

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Serine protease dynamics revealed by NMR analysis of the thrombin-thrombomodulin complex

Scientific Reports ◽

10.1038/s41598-021-88432-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Riley B. Peacock ◽

Taylor McGrann ◽

Marco Tonelli ◽

Elizabeth A. Komives

Keyword(s):

Active Site ◽

Serine Proteases ◽

Protein C ◽

Catalytic Mechanism ◽

Bond Cleavage ◽

Peptide Bond ◽

Peptide Bond Cleavage ◽

Exchange Mass Spectrometry ◽

Catalytically Active ◽

Hydrogen Deuterium

AbstractSerine proteases catalyze a multi-step covalent catalytic mechanism of peptide bond cleavage. It has long been assumed that serine proteases including thrombin carry-out catalysis without significant conformational rearrangement of their stable two-β-barrel structure. We present nuclear magnetic resonance (NMR) and hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments on the thrombin-thrombomodulin (TM) complex. Thrombin promotes procoagulative fibrinogen cleavage when fibrinogen engages both the anion binding exosite 1 (ABE1) and the active site. It is thought that TM promotes cleavage of protein C by engaging ABE1 in a similar manner as fibrinogen. Thus, the thrombin-TM complex may represent the catalytically active, ABE1-engaged thrombin. Compared to apo- and active site inhibited-thrombin, we show that thrombin-TM has reduced μs-ms dynamics in the substrate binding (S1) pocket consistent with its known acceleration of protein C binding. Thrombin-TM has increased μs-ms dynamics in a β-strand connecting the TM binding site to the catalytic aspartate. Finally, thrombin-TM had doublet peaks indicative of dynamics that are slow on the NMR timescale in residues along the interface between the two β-barrels. Such dynamics may be responsible for facilitating the N-terminal product release and water molecule entry that are required for hydrolysis of the acyl-enzyme intermediate.

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Activation of Bovine Factor V by Thrombin and A Protease From Russell's Viper Venom (RVV)

10.1055/s-0039-1687642 ◽

1979 ◽

Author(s):

M.J. Lindhout ◽

C. M. Jackson

Keyword(s):

Bond Cleavage ◽

Peptide Bond ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Factor V ◽

Peptide Bond Cleavage ◽

Prothrombinase Complex ◽

Factor Va ◽

Activation Products ◽

Ca Oxalate

In order to understand the function of activated factor V in the prothrombinase complex, we isolated the activation products obtained by action of thrombin and RVV-V on factor V and studied their functional properties. Factor V isolated from plasma by means of ion-exchange chromatography, a Ca-oxalate adsorption step and gelfiltration was homogenous in SDS-gelelectrophoresis (apparent MW 360,000, with and without reduction). Increase in factor V activity upon action by RVV-V is correlated with a single peptide bond cleavage, resulting in a 270,000 dalton and a 80,000 dalton component. Additional proteolysis of factor Va(RVV/V)’ by thrombin results in a further cleavage of the high MW component into peptides with MW's of 72,000, 94,000 and about 150,000 without a furth~r increase in factor V activity. Whereas none of the isolated peptides reveal factor Va activity, activity would be generated by a recombination in the presence of Ca2+ of the 94,000 MW or 270,000 MW component with the 80,000 component. Action of thrombin alone on factor V results in peptides of MW 72,000, 80,000, 94,000 and a peptide very rich in carbohydrate with an apparent MW of 150,000.

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EXPERIMENTAL VALIDATION OF THE CHARLESBY AND HORIKX MODELS APPLIED TO DE-VULCANIZATION OF SULFUR AND PEROXIDE VULCANIZATES OF NR AND EPDM

Rubber Chemistry and Technology ◽

10.5254/rct.16.83776 ◽

2016 ◽

Vol 89 (4) ◽

pp. 671-688 ◽

Cited By ~ 8

Author(s):

M. A. L. Verbruggen ◽

L. van der Does ◽

W. K. Dierkes ◽

J. W. M. Noordermeer

Keyword(s):

Experimental Validation ◽

Main Chain ◽

Sol Gel ◽

Chain Scission ◽

Cross Linking ◽

Cross Link ◽

Practical Reality ◽

Cross Links ◽

The Cross ◽

Theoretical Considerations

ABSTRACT The theoretical model developed by Charlesby to quantify the balance between cross-links creation of polymers and chain scission during radiation cross-linking and further modifications by Horikx to describe network breakdown from aging were merged to characterize the balance of both types of scission on the development of the sol content during de-vulcanization of rubber networks. There are, however, disturbing factors in these theoretical considerations vis-à-vis practical reality. Sulfur- and peroxide-cured NR and EPDM vulcanizates were de-vulcanized under conditions of selective cross-link and random main-chain scissions. Cross-link scission was obtained using thiol-amine reagents for selective cleavage of sulfur cross-links. Random main-chain scission was achieved by heating peroxide vulcanizates of NR with diphenyldisulfide, a method commonly employed for NR reclaiming. An important factor in the analyses of these experiments is the cross-linking index. Its value must be calculated using the sol fraction of the cross-linked network before de-vulcanization to obtain reliable results. The values for the cross-linking index calculated with sol-gel data before de-vulcanization appear to fit the experimentally determined modes of network scission during de-vulcanization very well. This study confirms that the treatment of de-vulcanization data with the merged Charlesby and Horikx models can be used satisfactorily to characterize the de-vulcanization of NR and EPDM vulcanizates.

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The Protein Core of the Largest Proteoglycan Monomer of Articular Cartilage. Gel Electrophoretic Pattern and Tryptophanyl Peptide Bond Cleavage

Connective Tissue Research ◽

10.3109/03008208709005622 ◽

1987 ◽

Vol 16 (4) ◽

pp. 377-384 ◽

Cited By ~ 1

Author(s):

Victor Stanescu ◽

Françoise Chaminade ◽

Marie-Paule Muriel

Keyword(s):

Articular Cartilage ◽

Bond Cleavage ◽

Electrophoretic Pattern ◽

Peptide Bond ◽

Protein Core ◽

Peptide Bond Cleavage

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The Nature of Non-Specific Peptide Bond Cleavage During the Isothiocyanate Degradation of Proteins

Methods in Protein Sequence Analysis ◽

10.1007/978-1-4612-5832-2_6 ◽

1982 ◽

pp. 101-110 ◽

Cited By ~ 4

Author(s):

Wolf F. Brandt ◽

Agnes Henschen ◽

Claus Von Holt

Keyword(s):

Bond Cleavage ◽

Peptide Bond ◽

Peptide Bond Cleavage ◽

Specific Peptide

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Nitrogen-15 nuclear magnetic resonance spectroscopy. The state of histidine in the catalytic triad of .alpha.-lytic protease. Implications for the charge-relay mechanism of peptide-bond cleavage by serine proteases

Journal of the American Chemical Society ◽

10.1021/ja00494a001 ◽

1978 ◽

Vol 100 (26) ◽

pp. 8041-8047 ◽

Cited By ~ 196

Author(s):

William W. Bachovchin ◽

John D. Roberts

Keyword(s):

Nuclear Magnetic Resonance ◽

Magnetic Resonance ◽

Magnetic Resonance Spectroscopy ◽

Nuclear Magnetic Resonance Spectroscopy ◽

Serine Proteases ◽

Bond Cleavage ◽

Peptide Bond ◽

Catalytic Triad ◽

Resonance Spectroscopy ◽

Peptide Bond Cleavage

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Two DD-Carboxypeptidases fromMycobacterium smegmatisAffect Cell Surface Properties through Regulation of Peptidoglycan Cross-Linking and Glycopeptidolipids

Journal of Bacteriology ◽

10.1128/jb.00760-17 ◽

2018 ◽

Vol 200 (14) ◽

Cited By ~ 7

Author(s):

Satya Deo Pandey ◽

Shilpa Pal ◽

Ganesh Kumar N ◽

Ankita Bansal ◽

Sathi Mallick ◽

...

Keyword(s):

Cell Surface ◽

Mycobacterium Smegmatis ◽

Biofilm Formation ◽

Cross Linking ◽

Surface Lipid ◽

Cross Link ◽

Content Type ◽

Murine Macrophages ◽

Cross Links ◽

Link Formation

ABSTRACTDuring the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between twomeso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (betweend-Ala andmeso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. Thedd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used byld-transpeptidases as substrates to form 3-3 cross-links. Therefore,dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology ofdd-CPases in mycobacteria is relatively unexplored. In this study, we deleted twodd-CPase genes,msmeg_2433andmsmeg_2432, both individually and in combination, fromMycobacterium smegmatismc2155. Though the singledd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant,viz., a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to β-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of thedd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCEThe glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. Thedd-CPases generate tetrapeptides by acting on the pentapeptides, andld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of twodd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, includingMycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology ofdd-CPases might help us design antimycobacterial therapeutics in the future.

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