Mass spectrometric characterization of human skin elastin peptides produced by proteolytic digestion with pepsin and thermitase

Schmelzer, Christian E.H.; Getie, Melkamu; Neubert, Reinhard H.H.

doi:10.1016/j.chroma.2005.06.034

Cited by 31 publications

(20 citation statements)

References 39 publications

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“…The data furthermore points to the absence of domain 22 as no linear peptides from this domain were identified in any of the enzymatic digests. These results are in agreement with previous studies on human skin and aortic elastin Schmelzer et al, 2005;Taddese et al, 2010;Heinz et al, 2011Heinz et al, , 2012Heinz et al, , 2014 and suggest that TE isoform 2 is present in both healthy individuals and WBS patients. Overall, comparable sequence coverages of 71% (64%) and 69% (79%), based on TE isoform 2, were obtained upon sequencing of linear peptides from enzymatic digests of skin (aortic) elastin from WBS patients and skin elastin from healthy donors, respectively (Fig.…”

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confidence: 95%

Elastins from patients with Williams–Beuren syndrome and healthy individuals differ on the molecular level

Heinz

Huertas

Schräder

et al. 2016

American J of Med Genetics Pt A

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Williams-Beuren syndrome (WBS) is a congenital disorder, which involves the heterozygous deletion of the elastin gene and other genes on chromosome 7. Clinical symptoms that are associated with hemizygosity of the essential extracellular matrix protein elastin include premature aging of the skin and supravalvular aortic stenosis. However, only little is known about the molecular basis of structural abnormalities in the connective tissue of WBS patients. Therefore, for the first time this study aimed to systematically characterize and compare the structure and amount of elastin present in skin and aortic tissue from WBS patients and healthy individuals. Elastin fibers were isolated from tissue biopsies, and it was found that skin of WBS patients contains significantly less elastin compared to skin of healthy individuals. Scanning electron microscopy and mass spectrometric measurements combined with bioinformatics data analysis were used to investigate the molecular-level structure of elastin. Scanning electron microscopy revealed clear differences between WBS and healthy elastin. With respect to the molecular-level structure, it was found that the proline hydroxylation degree differed between WBS and healthy elastin, while the tropoelastin isoform appeared to be the same. In terms of cross-linking, no differences in the content of the tetrafunctional cross-links desmosine and isodesmosine were found between WBS and healthy elastin. However, principal component analysis revealed differences between enzymatic digests of elastin from healthy probands and WBS patients, which indicates differing susceptibility toward enzymatic cleavage. Overall, the study contributes to a better understanding of the correlation between genotypic and elastin-related phenotypic features of WBS patients. © 2016 Wiley Periodicals, Inc.

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confidence: 95%

Elastins from patients with Williams–Beuren syndrome and healthy individuals differ on the molecular level

Heinz

Huertas

Schräder

et al. 2016

American J of Med Genetics Pt A

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“…Pulse-chase data suggested that after facilitating incorporation of the tropoelastin molecule into the growing elastic fiber, the C-terminal "pro" peptide is removed by a specific trypticlike protease and is not incorporated into the mature elastic structure. Other investigators, however, have identified C-terminal sequences in solubilized peptides from insoluble elastin using specific antibodies (Rosenbloom et al, 1986) or by mass spectrometry Schmelzer et al, 2005), implying that the C-terminus may not be processed off during fiber assembly.…”

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confidence: 99%

Modification and functional inactivation of the tropoelastin carboxy-terminal domain in cross-linked elastin

et al. 2008

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The carboxyl terminus of tropoelastin is a highly conserved, atypical region of the molecule with sequences that define both cell and matrix interactions. This domain also plays a critical but unknown role in the assembly and crosslinking of tropoelastin during elastic fiber maturation. Using a competitive ELISA with an antibody to an elastase-resistant epitope in the carboxy-terminus of tropoelastin (domain-36), we quantified levels of the domain-36 sequence in elastase-derived peptides from mature, insoluble elastin. We found that the amount of carboxy-terminal epitope in elastin is ∼0.2% of the expected value, assuming each tropoelastin monomer that is incorporated into the insoluble polymer has an intact carboxy-terminus. The low levels suggest that the majority of domain-36 sequence is either removed at some stage of elastin assembly or that the antigenic epitope is altered by posttranslational modification. Biochemical evidence is presented for a potential lysinederived cross-link in this region, which would alter the extractability and antigenicity of the carboxyterminal epitope. These results show that there is little or no unmodified domain-36 in mature elastin, indicating that the cell and matrix binding activities associated with this region of tropoelastin are lost or modified as elastin matures. A crosslinking function for domain-36 may serve to help register the multiple crosslinking sites in elastin and explains why mutations that alter the domain-36 sequence have detrimental effects on elastic fiber assembly.

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“…In the stomach, the stability of protein polymers depends on their particular chemical structures (19). For example, enzymatic cross-linking modification of ␤-casein results in the formation of high-molecular-mass polymers with a compact structure and enhanced resistance to in vitro pepsin digestion (20).…”

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N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Niu

Luo

Shi

et al. 2016

Appl Environ Microbiol

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N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed.A s much as 80% of the total phosphorus in cereal grains, oilseeds, and legumes exists in the form of phytate (myo-inositol hexakisphosphate), one of the most important functional ingredients in animal feed (1). However, phytate usually forms indigestible complexes with mineral cations and proteins, thereby causing reductions in nutrient utilization (2, 3). The dietary phytate undigested by monogastric animals is also released into the environment, where it causes phosphorus pollution (4, 5).The enzyme phytase catalyzes the sequential release of inorganic phosphate and lower myo-inositol phosphates from phytate (6); thus, inclusion of exogenous phytases in animal feeds as enzyme additives can enhance the nutrient uptake, lower the feedstuff production costs, and protect the environment in regions where intensive animal farming is conducted (7,8). Numerous phytases have been identified from microorganisms, animals, and plants (9-11), but only a few of them are commercially produced (12). The extensive application of phytases is limited by enzyme lability and protease sensitivity under high processing temperatures and low physiological pHs. Therefore, improving phytase stability at low pHs and high protease concentrations is desirable.Pepsin, a monomeric protein composed of two ␤-barrel-like domains, represents the main proteolytic enzyme in gastric juice (13). The acidic residues D32 and D215 are mainly responsible for proteoly...

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Mass spectrometric characterization of human skin elastin peptides produced by proteolytic digestion with pepsin and thermitase

Cited by 31 publications

References 39 publications

Elastins from patients with Williams–Beuren syndrome and healthy individuals differ on the molecular level

Elastins from patients with Williams–Beuren syndrome and healthy individuals differ on the molecular level

Modification and functional inactivation of the tropoelastin carboxy-terminal domain in cross-linked elastin

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

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