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Mechanistic insight into the elastin degradation process by the metalloprotease myroilysin from the deep-sea bacterium Myroides profundi D25.

Yang J, Zhao HL, Tang BL, Chen XL, Su HN, Zhang XY, Song XY, Zhou BC, Xie BB, Weiss AS, Zhang YZ - Mar Drugs (2015)

Bottom Line: In this study, we examined the elastin degradation mechanism of myroilysin.Consistent with this, analysis of the cleavage pattern of myroilysin on bovine elastin and recombinant tropoelastin revealed that myroilysin preferentially cleaves peptide bonds with hydrophobic residues at the P1 and/or P1' positions.Our results are helpful for developing biotechnological applications for myroilysin.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Jinan 250100, China. yangjie199102@163.com.

ABSTRACT
Elastases have been widely studied because of their important uses as medicine and meat tenderizers. However, there are relatively few studies on marine elastases. Myroilysin, secreted by Myroides profundi D25 from deep-sea sediment, is a novel elastase. In this study, we examined the elastin degradation mechanism of myroilysin. When mixed with insoluble bovine elastin, myroilysin bound hydrophobically, suggesting that this elastase may interact with the hydrophobic domains of elastin. Consistent with this, analysis of the cleavage pattern of myroilysin on bovine elastin and recombinant tropoelastin revealed that myroilysin preferentially cleaves peptide bonds with hydrophobic residues at the P1 and/or P1' positions. Scanning electron microscopy (SEM) of cross-linked recombinant tropoelastin degraded by myroilysin showed preferential damages of spherules over cross-links, as expected for a hydrophobic preference. The degradation process of myroilysin on bovine elastin fibres was followed by light microscopy and SEM, revealing that degradation begins with the formation of crevices and cavities at the fibre surface, with these openings increasing in number and size until the fibre breaks into small pieces, which are subsequently fragmented. Our results are helpful for developing biotechnological applications for myroilysin.

No MeSH data available.


Related in: MedlinePlus

Binding of myroilysin to insoluble elastin fibres through hydrophobic interaction. (A) SDS-PAGE analysis of the ability of myroilysin to bind to insoluble elastin-orcein. Bovine serum albumin in place of myroilysin was used as a negative control. The bound and unbound fractions were analysed by 12.5% SDS-PAGE. The numbers at the bottom of the gel are the densitometric ratios of each band compared with that of the control band. (B) The elastin-degrading activity of myroilysin bound to insoluble elastin-orcein. The total activity of 0.25 mL myroilysin solution was taken as 100%. The activity of the mixture of 5 mg elastin-orcein with buffer served as a control. The data are from three experimental repeats (mean ± S.D.). (C) Effects of NaCl and nonionic detergents on the binding of myroilysin to insoluble elastin-orcein.
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marinedrugs-13-01481-f001: Binding of myroilysin to insoluble elastin fibres through hydrophobic interaction. (A) SDS-PAGE analysis of the ability of myroilysin to bind to insoluble elastin-orcein. Bovine serum albumin in place of myroilysin was used as a negative control. The bound and unbound fractions were analysed by 12.5% SDS-PAGE. The numbers at the bottom of the gel are the densitometric ratios of each band compared with that of the control band. (B) The elastin-degrading activity of myroilysin bound to insoluble elastin-orcein. The total activity of 0.25 mL myroilysin solution was taken as 100%. The activity of the mixture of 5 mg elastin-orcein with buffer served as a control. The data are from three experimental repeats (mean ± S.D.). (C) Effects of NaCl and nonionic detergents on the binding of myroilysin to insoluble elastin-orcein.

Mentions: Proteases that degrade insoluble proteins initially bind to their insoluble substrates [16,17,18]. Our previous work showed that myroilysin has a high activity towards insoluble elastin [3]. To investigate whether myroilysin can bind to insoluble elastin, the activity of myroilysin was first inhibited with exogenous Zn2+, and the elastin-binding ability of myroilysin was then assessed by SDS-PAGE. As shown in Figure 1A, the amount of bound myroilysin increased with increasing elastin-orcein concentration, consistent with myroilysin binding to insoluble elastin. Moreover, when the exogenous Zn2+ was removed, significant degradation of the treated elastin-orcein was detected after incubation (Figure 1B), indicating that the myroilysin molecules that bound to elastin during the mixing step degraded the substrate after the removal of exogenous Zn2+. Taken together, these results demonstrated that myroilysin has the ability to bind to insoluble elastin.


Mechanistic insight into the elastin degradation process by the metalloprotease myroilysin from the deep-sea bacterium Myroides profundi D25.

Yang J, Zhao HL, Tang BL, Chen XL, Su HN, Zhang XY, Song XY, Zhou BC, Xie BB, Weiss AS, Zhang YZ - Mar Drugs (2015)

Binding of myroilysin to insoluble elastin fibres through hydrophobic interaction. (A) SDS-PAGE analysis of the ability of myroilysin to bind to insoluble elastin-orcein. Bovine serum albumin in place of myroilysin was used as a negative control. The bound and unbound fractions were analysed by 12.5% SDS-PAGE. The numbers at the bottom of the gel are the densitometric ratios of each band compared with that of the control band. (B) The elastin-degrading activity of myroilysin bound to insoluble elastin-orcein. The total activity of 0.25 mL myroilysin solution was taken as 100%. The activity of the mixture of 5 mg elastin-orcein with buffer served as a control. The data are from three experimental repeats (mean ± S.D.). (C) Effects of NaCl and nonionic detergents on the binding of myroilysin to insoluble elastin-orcein.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4377995&req=5

marinedrugs-13-01481-f001: Binding of myroilysin to insoluble elastin fibres through hydrophobic interaction. (A) SDS-PAGE analysis of the ability of myroilysin to bind to insoluble elastin-orcein. Bovine serum albumin in place of myroilysin was used as a negative control. The bound and unbound fractions were analysed by 12.5% SDS-PAGE. The numbers at the bottom of the gel are the densitometric ratios of each band compared with that of the control band. (B) The elastin-degrading activity of myroilysin bound to insoluble elastin-orcein. The total activity of 0.25 mL myroilysin solution was taken as 100%. The activity of the mixture of 5 mg elastin-orcein with buffer served as a control. The data are from three experimental repeats (mean ± S.D.). (C) Effects of NaCl and nonionic detergents on the binding of myroilysin to insoluble elastin-orcein.
Mentions: Proteases that degrade insoluble proteins initially bind to their insoluble substrates [16,17,18]. Our previous work showed that myroilysin has a high activity towards insoluble elastin [3]. To investigate whether myroilysin can bind to insoluble elastin, the activity of myroilysin was first inhibited with exogenous Zn2+, and the elastin-binding ability of myroilysin was then assessed by SDS-PAGE. As shown in Figure 1A, the amount of bound myroilysin increased with increasing elastin-orcein concentration, consistent with myroilysin binding to insoluble elastin. Moreover, when the exogenous Zn2+ was removed, significant degradation of the treated elastin-orcein was detected after incubation (Figure 1B), indicating that the myroilysin molecules that bound to elastin during the mixing step degraded the substrate after the removal of exogenous Zn2+. Taken together, these results demonstrated that myroilysin has the ability to bind to insoluble elastin.

Bottom Line: In this study, we examined the elastin degradation mechanism of myroilysin.Consistent with this, analysis of the cleavage pattern of myroilysin on bovine elastin and recombinant tropoelastin revealed that myroilysin preferentially cleaves peptide bonds with hydrophobic residues at the P1 and/or P1' positions.Our results are helpful for developing biotechnological applications for myroilysin.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Jinan 250100, China. yangjie199102@163.com.

ABSTRACT
Elastases have been widely studied because of their important uses as medicine and meat tenderizers. However, there are relatively few studies on marine elastases. Myroilysin, secreted by Myroides profundi D25 from deep-sea sediment, is a novel elastase. In this study, we examined the elastin degradation mechanism of myroilysin. When mixed with insoluble bovine elastin, myroilysin bound hydrophobically, suggesting that this elastase may interact with the hydrophobic domains of elastin. Consistent with this, analysis of the cleavage pattern of myroilysin on bovine elastin and recombinant tropoelastin revealed that myroilysin preferentially cleaves peptide bonds with hydrophobic residues at the P1 and/or P1' positions. Scanning electron microscopy (SEM) of cross-linked recombinant tropoelastin degraded by myroilysin showed preferential damages of spherules over cross-links, as expected for a hydrophobic preference. The degradation process of myroilysin on bovine elastin fibres was followed by light microscopy and SEM, revealing that degradation begins with the formation of crevices and cavities at the fibre surface, with these openings increasing in number and size until the fibre breaks into small pieces, which are subsequently fragmented. Our results are helpful for developing biotechnological applications for myroilysin.

No MeSH data available.


Related in: MedlinePlus