Autocatalytic maturation of the prohormone convertase PC2.

Matthews, Glenn; Shennan, K. I. J.; Seal, AJ; Taylor, Nick; Colman, Alan; Docherty, Kevin

doi:10.1016/s0021-9258(17)42389-1

Cited by 65 publications

(11 citation statements)

References 46 publications

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“…We have confirmed our previous results (Matthews et al, 1994) that pro-PC2 maturation occurs by an autocatalytic reaction. Our present results show that mature PC2 can activate catalytically inactive pro-PC2 mutants by an intermolecular reaction.…”

Section: Discussionsupporting

confidence: 92%

“…A similar pattern of processing was shown to occur in GH4 cells (Benjannet et al, 1992) and in islets of Langerhans (Guest et al, 1992). Maturation of pro-PC2 has been shown to occur by way of an autocatalytic reaction (Matthews et al, 1994).…”

mentioning

confidence: 53%

“…When expressed in Xenopus oocytes, pro-PC2 undergoes cleavage at the sequence KRRR 5 6 to generate a 71-kDa intermediate that is further cleaved at the sequence RKKR 84 to generate the 68-kDa mature enzyme. Processing of the 75-kDa propolypeptide can also occur directly by way of cleavage at RKKR 8 4 (Shennan et al, 1991b;Matthews et al, 1994). A similar pattern of processing was shown to occur in GH4 cells (Benjannet et al, 1992) and in islets of Langerhans (Guest et al, 1992).…”

mentioning

confidence: 66%

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Differences in pH Optima and Calcium Requirements for Maturation of the Prohormone Convertases PC2 and PC3 Indicates Different Intracellular Locations for These Events

Shennan

Taylor

Jermany

et al. 1995

Journal of Biological Chemistry

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PC2 and PC3, which is also known as PC1, are subtilisin-like proteases that are involved in the intracellular processing of prohormones and proneuropeptides. Both enzymes are synthesized as propolypeptides that undergo proteolytic maturation within the secretory pathway. An in vitro translation/translocation system from Xenopus egg extracts was used to investigate mechanisms in the maturation of pro-PC3 and pro-PC2. Pro-PC3 underwent rapid (t1/2 < 10 min) processing of the 88-kDa propolypeptide at the sequence RSKR83 to generate the 80-kDa active form of the enzyme. This processing was blocked when the active site aspartate was changed to asparagine, suggesting that an autocatalytic mechanism was involved. In this system, processing of pro-PC3 was optimal between pH 7.0 and 8.0 and was not dependent on additional calcium. These results are consistent with pro-PC3 maturation occurring at an early stage in the secretory pathway, possibly within the endoplasmic reticulum, where the pH would be close to neutral and the calcium concentration less than that observed in later compartments. Processing of pro-PC2 in the Xenopus egg extract was much slower than that of pro-PC3 (t1/2 = 8 h). It exhibited a pH optimum of 5.5-6.0 and was dependent on calcium (K0.5 = 2-4 mM). The enzymatic properties of pro-PC2 processing were similar to that of the mature enzyme. Further studies using mutant pro-PC2 constructs suggested that cleavage of pro-PC2 was catalyzed by the mature 68-kDa PC2 molecule. The results were consistent with pro-PC2 maturation occurring within a late compartment of the secretory pathway that contains a high calcium concentration and low pH.

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Section: Discussionsupporting

confidence: 92%

mentioning

confidence: 53%

mentioning

confidence: 66%

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Differences in pH Optima and Calcium Requirements for Maturation of the Prohormone Convertases PC2 and PC3 Indicates Different Intracellular Locations for These Events

Shennan

Taylor

Jermany

et al. 1995

Journal of Biological Chemistry

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“…We found that proPC2 required 2-3 h for half-maximal conversion to mature PC2 in AtT-20/PC2 cells; these kinetics are in good agreement with those obtained by Zhou and Mains (1994) for the same cell line. Similarly slow processing of proPC2 was observed in other neuroendocrine cell lines and in rat pancreatic islets (Guest et al, 1992;Shen et al, 1993;Benjannet et al, 1993;Shennan et al, (Lindberg et al, 1995). Cells were plated at 500,000 cells/well in a six-well plate; 2 d later, serum-free medium was placed on the cells and assayed in duplicate the following day for the expression of enzymatic activity.…”

Section: Discussionmentioning

confidence: 66%

“…The proteolytic activation of PC2 has been the subject of several previous investigations. Studies using oocyte extracts and site-directed mutants of proPC2 have shown that the conversion of proPC2 (75 kD) to mature PC2 (64 kD) is autocatalytic, and that it is apparently mediated through an intermolecular reaction which is extremely slow in this system (Matthews et al, 1994;Shennan et al, 1995). In previous experiments using a CHO cell overexpression system, we were unable to demonstrate any autocatalysis of proPC2, and proPC2 was largely secreted as an intact, inactive form (Shen et al, 1993).…”

Section: Discussionmentioning

confidence: 99%

7B2 facilitates the maturation of proPC2 in neuroendocrine cells and is required for the expression of enzymatic activity.

Zhu

Lindberg

1995

The Journal of Cell Biology

156

217

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Abstract. The prohormone convertase PC2, which is thought to mediate the proteolytic conversion of many peptide hormones, has recently been shown to interact with the neuroendocrine-specific polypeptide 7B2 in Xenopus intermediate lobe (Braks, J. A. M., and G. J. M. Martens. Cell 78:263. 1994). In the present work we have stably transfected neuroendocrine cell lines with rat 7B2 constructs and found that overexpression of 27 kD 7B2 greatly facilitates the kinetics of maturation of proPC2, both in AtT-20/PC2 cells and in Rin5f cells. The half-life of conversion of proPC2 was reduced from 2.7 to 1.7 h in AtT-20/PC2 cells stably transfected with 27 kD 7B2 cDNA. The previously proposed "chaperone" domain was not sufficient for this facilitation event; however, a construct corresponding to the 21-kD 7B2 protein (which represents the naturally occurring maturation product) functioned well. A 7B2 construct in which maturation of 27 kD 7B2 to its 21-kD form was blocked was unable to facilitate maturation of proPC2.To correlate effects on PC2 maturation with the actual generation of PC2 enzymatic activity, a similar transfection of 21 kD 7B2 was performed using CHO cells previously amplified for the expression of proPC2. Enzymatic activity cleaving the fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC was highly correlated with the expression of immunoreactive 21 kD 7B2 in the conditioned medium; medium obtained from the parent cell line was completely inactive. Enzymatic activity was identified as PC2 on the basis of inhibition by the carboxy-terminal peptide of 7B2, which has previously been shown to represent a potent and specific PC2 inhibitor. Taken together, our in vivo results indicate that the interesting secretory protein 7B2 is a bifunctional molecule with an amino-terminal domain involved in proPC2 transport as well as activation.

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Secretion and pH-dependent self-processing of the pro-form of the Yarrowia lipolytica acid extracellular protease

McEwen

Young

1998

Yeast

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Autocatalytic maturation of the prohormone convertase PC2.

Cited by 65 publications

References 46 publications

Differences in pH Optima and Calcium Requirements for Maturation of the Prohormone Convertases PC2 and PC3 Indicates Different Intracellular Locations for These Events

Differences in pH Optima and Calcium Requirements for Maturation of the Prohormone Convertases PC2 and PC3 Indicates Different Intracellular Locations for These Events

7B2 facilitates the maturation of proPC2 in neuroendocrine cells and is required for the expression of enzymatic activity.

Secretion and pH-dependent self-processing of the pro-form of the Yarrowia lipolytica acid extracellular protease

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