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Autocatalytic activation of the furin zymogen requires removal of the emerging enzyme's N-terminus from the active site.

Gawlik K, Shiryaev SA, Zhu W, Motamedchaboki K, Desjardins R, Day R, Remacle AG, Stec B, Strongin AY - PLoS ONE (2009)

Bottom Line: Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg(107) downward arrowAsp(108), but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg(75) downward arrowSer(76)) cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108.Collectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms.In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases.

View Article: PubMed Central - PubMed

Affiliation: Burnham Institute for Medical Research, La Jolla, California, United States of America.

ABSTRACT

Background: Before furin can act on protein substrates, it must go through an ordered process of activation. Similar to many other proteinases, furin is synthesized as a zymogen (profurin) which becomes active only after the autocatalytic removal of its auto-inhibitory prodomain. We hypothesized that to activate profurin its prodomain had to be removed and, in addition, the emerging enzyme's N-terminus had to be ejected from the catalytic cleft.

Methodology/principal findings: We constructed and analyzed the profurin mutants in which the egress of the emerging enzyme's N-terminus from the catalytic cleft was restricted. Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg(107) downward arrowAsp(108), but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg(75) downward arrowSer(76)) cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108. These correctly processed furin mutants, however, remained self-inhibited by the constrained N-terminal sequence which continuously occupied the S' sub-sites of the catalytic cleft and interfered with the functional activity. Further, using the in vitro cleavage of the purified prodomain and the analyses of colon carcinoma LoVo cells with the reconstituted expression of the wild-type and mutant furins, we demonstrated that a three-step autocatalytic processing including the cleavage of the prodomain at the previously unidentified Arg-Leu-Gln-Arg(89) downward arrowGlu(90) site, is required for the efficient activation of furin.

Conclusions/significance: Collectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms. In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases.

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Related in: MedlinePlus

Furin proteolysis in trans of its inhibitory prodomain.(A) The recombinant furin prodomain was cloned into the pTrcHis A vector and expressed in E. coli. The sequence of the furin prodomain Gln27-Arg107 is in bold. The cleavage sites are indicated by the arrows. The N-terminal peptide sequence of the prodomain starts from Gly2 because of the cleavage of the initiating Met1 in E. coli. The sequence of the cleavage products determined by N-terminal sequencing is underlined. The sequence of the Xpress tag and His6 tag is italicized. The numbering used is for intact translated human furin. (B) The recombinant furin prodomain (1.8 µg) was incubated at 37°C for 1 h with furin at a molar ratio of 1:1 or 2:1. The digest samples were analyzed by Tricine gel electrophoresis followed by Coomassie staining. Where indicated, dec-RVKR-cmk (50 µM) was added. The sequence numbering of prodomain and cleavage products identified by MS analysis is shown on the left side. (C) The molecular mass of the intact recombinant furin prodomain and the cleavage products was determined by MALDI-TOF MS. The calculated and measured molecular mass of the peptides is shown in Da. (D) A Western blotting analysis of colon carcinoma LoVo cells transiently transfected with the WT furin (WT), the catalytically inert D153N mutant (D153N) and the furin mutants in which the primary (R107G), the secondary (K74G/R75G) and the tertiary (R89G) cleavage sites were inactivated in the prodomain sequence. The R89G/R107G; K74G/R75G/R89G and K74G/R75G/R107G, and K74G/R75G/R89G/R107G are the mutants in which two and three cleavage sites, respectively, were inactivated by mutations. Mock cells are LoVo cells transfected with the original plasmid. The antibodies to the catalytic domain and to the cytoplasmic tail of furin (MON-148 and MON-139, respectively) were used for Western blotting. To show equal loading, the samples were also analyzed by Western blotting with an α-tubulin antibody (bottom panel).
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pone-0005031-g007: Furin proteolysis in trans of its inhibitory prodomain.(A) The recombinant furin prodomain was cloned into the pTrcHis A vector and expressed in E. coli. The sequence of the furin prodomain Gln27-Arg107 is in bold. The cleavage sites are indicated by the arrows. The N-terminal peptide sequence of the prodomain starts from Gly2 because of the cleavage of the initiating Met1 in E. coli. The sequence of the cleavage products determined by N-terminal sequencing is underlined. The sequence of the Xpress tag and His6 tag is italicized. The numbering used is for intact translated human furin. (B) The recombinant furin prodomain (1.8 µg) was incubated at 37°C for 1 h with furin at a molar ratio of 1:1 or 2:1. The digest samples were analyzed by Tricine gel electrophoresis followed by Coomassie staining. Where indicated, dec-RVKR-cmk (50 µM) was added. The sequence numbering of prodomain and cleavage products identified by MS analysis is shown on the left side. (C) The molecular mass of the intact recombinant furin prodomain and the cleavage products was determined by MALDI-TOF MS. The calculated and measured molecular mass of the peptides is shown in Da. (D) A Western blotting analysis of colon carcinoma LoVo cells transiently transfected with the WT furin (WT), the catalytically inert D153N mutant (D153N) and the furin mutants in which the primary (R107G), the secondary (K74G/R75G) and the tertiary (R89G) cleavage sites were inactivated in the prodomain sequence. The R89G/R107G; K74G/R75G/R89G and K74G/R75G/R107G, and K74G/R75G/R89G/R107G are the mutants in which two and three cleavage sites, respectively, were inactivated by mutations. Mock cells are LoVo cells transfected with the original plasmid. The antibodies to the catalytic domain and to the cytoplasmic tail of furin (MON-148 and MON-139, respectively) were used for Western blotting. To show equal loading, the samples were also analyzed by Western blotting with an α-tubulin antibody (bottom panel).

Mentions: To gain additional insight into the proteolytic pathway that prevents re-inhibition of furin by the residual amount of its inhibitory prodomain (Figure 7A), we re-examined proteolysis of the prodomain sequence by furin. For this purpose, we subjected the purified individual prodomain sequence to furin proteolysis in trans (Figure 7B). Because the prodomain construct was expressed in frame with a His6 tag sequence and a short Xpress tag, the sequence of the actual prodomain commenced from Gln27 (preprofurin numbering). In contrast to the expected single cleavage of the 13 kDa tagged prodomain at the Arg-Gly-Val-Thr-Lys-Arg75↓Ser76 that should result in two peptide products [18], [19], [21], furin proteolysis of the prodomain followed by SDS-PAGE demonstrated the existence of two cleavage sites and the presence of three distinct products with apparent molecular weights of ∼10 kDa, ∼5 kDa and ∼3 kDa in the cleavage reactions. Dec-RVKR-cmk reversed the effect of furin and rescued the prodomain from furin proteolysis. N-terminal sequencing determined that the 5 kDa fragment commenced from Ser76 and therefore represented the 76–107 region of the prodomain. The determined N-terminal sequence (EPQVQ) suggested that the 3 kDa fragment represented the 90–107 C-terminal sequence of the prodomain (Figure 7C).


Autocatalytic activation of the furin zymogen requires removal of the emerging enzyme's N-terminus from the active site.

Gawlik K, Shiryaev SA, Zhu W, Motamedchaboki K, Desjardins R, Day R, Remacle AG, Stec B, Strongin AY - PLoS ONE (2009)

Furin proteolysis in trans of its inhibitory prodomain.(A) The recombinant furin prodomain was cloned into the pTrcHis A vector and expressed in E. coli. The sequence of the furin prodomain Gln27-Arg107 is in bold. The cleavage sites are indicated by the arrows. The N-terminal peptide sequence of the prodomain starts from Gly2 because of the cleavage of the initiating Met1 in E. coli. The sequence of the cleavage products determined by N-terminal sequencing is underlined. The sequence of the Xpress tag and His6 tag is italicized. The numbering used is for intact translated human furin. (B) The recombinant furin prodomain (1.8 µg) was incubated at 37°C for 1 h with furin at a molar ratio of 1:1 or 2:1. The digest samples were analyzed by Tricine gel electrophoresis followed by Coomassie staining. Where indicated, dec-RVKR-cmk (50 µM) was added. The sequence numbering of prodomain and cleavage products identified by MS analysis is shown on the left side. (C) The molecular mass of the intact recombinant furin prodomain and the cleavage products was determined by MALDI-TOF MS. The calculated and measured molecular mass of the peptides is shown in Da. (D) A Western blotting analysis of colon carcinoma LoVo cells transiently transfected with the WT furin (WT), the catalytically inert D153N mutant (D153N) and the furin mutants in which the primary (R107G), the secondary (K74G/R75G) and the tertiary (R89G) cleavage sites were inactivated in the prodomain sequence. The R89G/R107G; K74G/R75G/R89G and K74G/R75G/R107G, and K74G/R75G/R89G/R107G are the mutants in which two and three cleavage sites, respectively, were inactivated by mutations. Mock cells are LoVo cells transfected with the original plasmid. The antibodies to the catalytic domain and to the cytoplasmic tail of furin (MON-148 and MON-139, respectively) were used for Western blotting. To show equal loading, the samples were also analyzed by Western blotting with an α-tubulin antibody (bottom panel).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2662429&req=5

pone-0005031-g007: Furin proteolysis in trans of its inhibitory prodomain.(A) The recombinant furin prodomain was cloned into the pTrcHis A vector and expressed in E. coli. The sequence of the furin prodomain Gln27-Arg107 is in bold. The cleavage sites are indicated by the arrows. The N-terminal peptide sequence of the prodomain starts from Gly2 because of the cleavage of the initiating Met1 in E. coli. The sequence of the cleavage products determined by N-terminal sequencing is underlined. The sequence of the Xpress tag and His6 tag is italicized. The numbering used is for intact translated human furin. (B) The recombinant furin prodomain (1.8 µg) was incubated at 37°C for 1 h with furin at a molar ratio of 1:1 or 2:1. The digest samples were analyzed by Tricine gel electrophoresis followed by Coomassie staining. Where indicated, dec-RVKR-cmk (50 µM) was added. The sequence numbering of prodomain and cleavage products identified by MS analysis is shown on the left side. (C) The molecular mass of the intact recombinant furin prodomain and the cleavage products was determined by MALDI-TOF MS. The calculated and measured molecular mass of the peptides is shown in Da. (D) A Western blotting analysis of colon carcinoma LoVo cells transiently transfected with the WT furin (WT), the catalytically inert D153N mutant (D153N) and the furin mutants in which the primary (R107G), the secondary (K74G/R75G) and the tertiary (R89G) cleavage sites were inactivated in the prodomain sequence. The R89G/R107G; K74G/R75G/R89G and K74G/R75G/R107G, and K74G/R75G/R89G/R107G are the mutants in which two and three cleavage sites, respectively, were inactivated by mutations. Mock cells are LoVo cells transfected with the original plasmid. The antibodies to the catalytic domain and to the cytoplasmic tail of furin (MON-148 and MON-139, respectively) were used for Western blotting. To show equal loading, the samples were also analyzed by Western blotting with an α-tubulin antibody (bottom panel).
Mentions: To gain additional insight into the proteolytic pathway that prevents re-inhibition of furin by the residual amount of its inhibitory prodomain (Figure 7A), we re-examined proteolysis of the prodomain sequence by furin. For this purpose, we subjected the purified individual prodomain sequence to furin proteolysis in trans (Figure 7B). Because the prodomain construct was expressed in frame with a His6 tag sequence and a short Xpress tag, the sequence of the actual prodomain commenced from Gln27 (preprofurin numbering). In contrast to the expected single cleavage of the 13 kDa tagged prodomain at the Arg-Gly-Val-Thr-Lys-Arg75↓Ser76 that should result in two peptide products [18], [19], [21], furin proteolysis of the prodomain followed by SDS-PAGE demonstrated the existence of two cleavage sites and the presence of three distinct products with apparent molecular weights of ∼10 kDa, ∼5 kDa and ∼3 kDa in the cleavage reactions. Dec-RVKR-cmk reversed the effect of furin and rescued the prodomain from furin proteolysis. N-terminal sequencing determined that the 5 kDa fragment commenced from Ser76 and therefore represented the 76–107 region of the prodomain. The determined N-terminal sequence (EPQVQ) suggested that the 3 kDa fragment represented the 90–107 C-terminal sequence of the prodomain (Figure 7C).

Bottom Line: Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg(107) downward arrowAsp(108), but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg(75) downward arrowSer(76)) cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108.Collectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms.In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases.

View Article: PubMed Central - PubMed

Affiliation: Burnham Institute for Medical Research, La Jolla, California, United States of America.

ABSTRACT

Background: Before furin can act on protein substrates, it must go through an ordered process of activation. Similar to many other proteinases, furin is synthesized as a zymogen (profurin) which becomes active only after the autocatalytic removal of its auto-inhibitory prodomain. We hypothesized that to activate profurin its prodomain had to be removed and, in addition, the emerging enzyme's N-terminus had to be ejected from the catalytic cleft.

Methodology/principal findings: We constructed and analyzed the profurin mutants in which the egress of the emerging enzyme's N-terminus from the catalytic cleft was restricted. Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg(107) downward arrowAsp(108), but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg(75) downward arrowSer(76)) cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108. These correctly processed furin mutants, however, remained self-inhibited by the constrained N-terminal sequence which continuously occupied the S' sub-sites of the catalytic cleft and interfered with the functional activity. Further, using the in vitro cleavage of the purified prodomain and the analyses of colon carcinoma LoVo cells with the reconstituted expression of the wild-type and mutant furins, we demonstrated that a three-step autocatalytic processing including the cleavage of the prodomain at the previously unidentified Arg-Leu-Gln-Arg(89) downward arrowGlu(90) site, is required for the efficient activation of furin.

Conclusions/significance: Collectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms. In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases.

Show MeSH
Related in: MedlinePlus