Differences in the autocatalytic cleavage of pro-PC2 and pro-PC3 can be attributed to sequences within the propeptide and Asp310 of pro-PC2

K Scougall, N A Taylor, J L Jermany, K Docherty, K I Shennan

Research output: Contribution to journalArticle

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Abstract

PC2 and PC3 are subtilisin-like proteases involved in the maturation of prohormones and proneuropeptides within neuroendocrine cells. They are synthesized as zymogens that undergo autocatalytic maturation within the secretory pathway. Maturation of pro-PC2 is slow (t12 >8 h), exhibits a pH optimum of 5.5 and is dependent on calcium (K0.5 2 mM), while pro-PC3 maturation is relatively rapid (t12 15 min), exhibits a neutral pH optimum and is not calcium dependent. These differences in the rates and optimal conditions for activation of the proteases may contribute to the diversity of products generated by these proteases in different cell types. Although highly similar, there are two major differences between pro-PC2 and pro-PC3: the presence of an aspartate at position 310 in pro-PC2 compared with asparagine at the equivalent position in pro-PC3 (and all other members of the subtilisin family), and the N-terminal propeptides, which exhibit low sequence identity (30%). With a view to establishing the structural features that might be responsible for these differences in the maturation of pro-PC2 and pro-PC3, Asp310 in pro-PC2 was mutated to Asn, and Asn309 in pro-PC3 was mutated to Asp. Chimaeric proteins were also made consisting of the pro-region of PC2 fused to the mature portion of PC3 and the pro-region of PC3 fused to the mature region of PC2. The wild-type and mutant DNA constructs were then transcribed and translated in an in vitro system capable of supporting maturation of pro-PC2 and pro-PC3. The results demonstrated that Asp310 of pro-PC2 is responsible for the acidic pH optimum for maturation. Thus changing Asp310 to Asn shifted the pH optimum for maturation to pH 7.0. However, changing Asn309 of pro-PC3 to Asp had no effect on the optimum pH for maturation of pro-PC3. A chimaeric construct containing the propeptide of pro-PC2 attached to PC3 shifted the pH optimum for maturation from pH 7.0 to 6.0 and slowed down the rate of maturation (t12 >8 h). When attached to PC2, the pro-region of pro-PC3 had no effect on the optimum pH for maturation (pH 5.5-6.0), but it did accelerate the rate of maturation (t12 2 h). These results demonstrate that Asp310 and the pro-region of pro-PC2 contribute to the acidic pH optimum and low rate of maturation of this zymogen relative to its closely related homologue PC3.
Original languageEnglish
Pages (from-to)531-7
Number of pages7
JournalBiochemical Journal
Volume334 ( Pt 3)
Publication statusPublished - 15 Sep 1998

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Subtilisin
Enzyme Precursors
Peptide Hydrolases
Calcium
Asparagine
Aspartic Acid
Chemical activation
DNA
Proteins
Neuroendocrine Cells
Secretory Pathway
Viperidae

Keywords

  • Amino Acid Sequence
  • Animals
  • Aspartic Acid Endopeptidases
  • Base Sequence
  • Catalytic Domain
  • DNA Primers
  • Enzyme Precursors
  • Female
  • Hydrogen-Ion Concentration
  • Kinetics
  • Mutagenesis, Site-Directed
  • Oocytes
  • Proprotein Convertase 2
  • Proprotein Convertases
  • Recombinant Fusion Proteins
  • Subtilisins
  • Xenopus laevis

Cite this

Differences in the autocatalytic cleavage of pro-PC2 and pro-PC3 can be attributed to sequences within the propeptide and Asp310 of pro-PC2. / Scougall, K; Taylor, N A; Jermany, J L; Docherty, K; Shennan, K I.

In: Biochemical Journal, Vol. 334 ( Pt 3), 15.09.1998, p. 531-7.

Research output: Contribution to journalArticle

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abstract = "PC2 and PC3 are subtilisin-like proteases involved in the maturation of prohormones and proneuropeptides within neuroendocrine cells. They are synthesized as zymogens that undergo autocatalytic maturation within the secretory pathway. Maturation of pro-PC2 is slow (t12 >8 h), exhibits a pH optimum of 5.5 and is dependent on calcium (K0.5 2 mM), while pro-PC3 maturation is relatively rapid (t12 15 min), exhibits a neutral pH optimum and is not calcium dependent. These differences in the rates and optimal conditions for activation of the proteases may contribute to the diversity of products generated by these proteases in different cell types. Although highly similar, there are two major differences between pro-PC2 and pro-PC3: the presence of an aspartate at position 310 in pro-PC2 compared with asparagine at the equivalent position in pro-PC3 (and all other members of the subtilisin family), and the N-terminal propeptides, which exhibit low sequence identity (30{\%}). With a view to establishing the structural features that might be responsible for these differences in the maturation of pro-PC2 and pro-PC3, Asp310 in pro-PC2 was mutated to Asn, and Asn309 in pro-PC3 was mutated to Asp. Chimaeric proteins were also made consisting of the pro-region of PC2 fused to the mature portion of PC3 and the pro-region of PC3 fused to the mature region of PC2. The wild-type and mutant DNA constructs were then transcribed and translated in an in vitro system capable of supporting maturation of pro-PC2 and pro-PC3. The results demonstrated that Asp310 of pro-PC2 is responsible for the acidic pH optimum for maturation. Thus changing Asp310 to Asn shifted the pH optimum for maturation to pH 7.0. However, changing Asn309 of pro-PC3 to Asp had no effect on the optimum pH for maturation of pro-PC3. A chimaeric construct containing the propeptide of pro-PC2 attached to PC3 shifted the pH optimum for maturation from pH 7.0 to 6.0 and slowed down the rate of maturation (t12 >8 h). When attached to PC2, the pro-region of pro-PC3 had no effect on the optimum pH for maturation (pH 5.5-6.0), but it did accelerate the rate of maturation (t12 2 h). These results demonstrate that Asp310 and the pro-region of pro-PC2 contribute to the acidic pH optimum and low rate of maturation of this zymogen relative to its closely related homologue PC3.",
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T1 - Differences in the autocatalytic cleavage of pro-PC2 and pro-PC3 can be attributed to sequences within the propeptide and Asp310 of pro-PC2

AU - Scougall, K

AU - Taylor, N A

AU - Jermany, J L

AU - Docherty, K

AU - Shennan, K I

PY - 1998/9/15

Y1 - 1998/9/15

N2 - PC2 and PC3 are subtilisin-like proteases involved in the maturation of prohormones and proneuropeptides within neuroendocrine cells. They are synthesized as zymogens that undergo autocatalytic maturation within the secretory pathway. Maturation of pro-PC2 is slow (t12 >8 h), exhibits a pH optimum of 5.5 and is dependent on calcium (K0.5 2 mM), while pro-PC3 maturation is relatively rapid (t12 15 min), exhibits a neutral pH optimum and is not calcium dependent. These differences in the rates and optimal conditions for activation of the proteases may contribute to the diversity of products generated by these proteases in different cell types. Although highly similar, there are two major differences between pro-PC2 and pro-PC3: the presence of an aspartate at position 310 in pro-PC2 compared with asparagine at the equivalent position in pro-PC3 (and all other members of the subtilisin family), and the N-terminal propeptides, which exhibit low sequence identity (30%). With a view to establishing the structural features that might be responsible for these differences in the maturation of pro-PC2 and pro-PC3, Asp310 in pro-PC2 was mutated to Asn, and Asn309 in pro-PC3 was mutated to Asp. Chimaeric proteins were also made consisting of the pro-region of PC2 fused to the mature portion of PC3 and the pro-region of PC3 fused to the mature region of PC2. The wild-type and mutant DNA constructs were then transcribed and translated in an in vitro system capable of supporting maturation of pro-PC2 and pro-PC3. The results demonstrated that Asp310 of pro-PC2 is responsible for the acidic pH optimum for maturation. Thus changing Asp310 to Asn shifted the pH optimum for maturation to pH 7.0. However, changing Asn309 of pro-PC3 to Asp had no effect on the optimum pH for maturation of pro-PC3. A chimaeric construct containing the propeptide of pro-PC2 attached to PC3 shifted the pH optimum for maturation from pH 7.0 to 6.0 and slowed down the rate of maturation (t12 >8 h). When attached to PC2, the pro-region of pro-PC3 had no effect on the optimum pH for maturation (pH 5.5-6.0), but it did accelerate the rate of maturation (t12 2 h). These results demonstrate that Asp310 and the pro-region of pro-PC2 contribute to the acidic pH optimum and low rate of maturation of this zymogen relative to its closely related homologue PC3.

AB - PC2 and PC3 are subtilisin-like proteases involved in the maturation of prohormones and proneuropeptides within neuroendocrine cells. They are synthesized as zymogens that undergo autocatalytic maturation within the secretory pathway. Maturation of pro-PC2 is slow (t12 >8 h), exhibits a pH optimum of 5.5 and is dependent on calcium (K0.5 2 mM), while pro-PC3 maturation is relatively rapid (t12 15 min), exhibits a neutral pH optimum and is not calcium dependent. These differences in the rates and optimal conditions for activation of the proteases may contribute to the diversity of products generated by these proteases in different cell types. Although highly similar, there are two major differences between pro-PC2 and pro-PC3: the presence of an aspartate at position 310 in pro-PC2 compared with asparagine at the equivalent position in pro-PC3 (and all other members of the subtilisin family), and the N-terminal propeptides, which exhibit low sequence identity (30%). With a view to establishing the structural features that might be responsible for these differences in the maturation of pro-PC2 and pro-PC3, Asp310 in pro-PC2 was mutated to Asn, and Asn309 in pro-PC3 was mutated to Asp. Chimaeric proteins were also made consisting of the pro-region of PC2 fused to the mature portion of PC3 and the pro-region of PC3 fused to the mature region of PC2. The wild-type and mutant DNA constructs were then transcribed and translated in an in vitro system capable of supporting maturation of pro-PC2 and pro-PC3. The results demonstrated that Asp310 of pro-PC2 is responsible for the acidic pH optimum for maturation. Thus changing Asp310 to Asn shifted the pH optimum for maturation to pH 7.0. However, changing Asn309 of pro-PC3 to Asp had no effect on the optimum pH for maturation of pro-PC3. A chimaeric construct containing the propeptide of pro-PC2 attached to PC3 shifted the pH optimum for maturation from pH 7.0 to 6.0 and slowed down the rate of maturation (t12 >8 h). When attached to PC2, the pro-region of pro-PC3 had no effect on the optimum pH for maturation (pH 5.5-6.0), but it did accelerate the rate of maturation (t12 2 h). These results demonstrate that Asp310 and the pro-region of pro-PC2 contribute to the acidic pH optimum and low rate of maturation of this zymogen relative to its closely related homologue PC3.

KW - Amino Acid Sequence

KW - Animals

KW - Aspartic Acid Endopeptidases

KW - Base Sequence

KW - Catalytic Domain

KW - DNA Primers

KW - Enzyme Precursors

KW - Female

KW - Hydrogen-Ion Concentration

KW - Kinetics

KW - Mutagenesis, Site-Directed

KW - Oocytes

KW - Proprotein Convertase 2

KW - Proprotein Convertases

KW - Recombinant Fusion Proteins

KW - Subtilisins

KW - Xenopus laevis

M3 - Article

VL - 334 ( Pt 3)

SP - 531

EP - 537

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

ER -