©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differences in pH Optima and Calcium Requirements for Maturation of the Prohormone Convertases PC2 and PC3 Indicates Different Intracellular Locations for These Events (*)

(Received for publication, July 27, 1994; and in revised form, October 31, 1994)

Kathleen I. J. Shennan (1) Neil A. Taylor (1) Joanne L. Jermany (1) Glenn Matthews (2) Kevin Docherty (1)(§)

From the  (1)Department of Medicine, University of Birmingham, Queen Elizabeth Hospital, Birmingham, B15 2TH and the (2)School of Biochemistry, University of Birmingham, P. O. Box 363, Birmingham, B15 2TT, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 (t < 10 min) processing of the 88-kDa propolypeptide at the sequence RSKR 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 (t = 8 h). It exhibited a pH optimum of 5.5-6.0 and was dependent on calcium (K(0.5) = 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.


INTRODUCTION

PC2 and PC3 are members of the eukaryotic family of subtilisin-like proteases that are involved in the intracellular processing of propolypeptides within the secretory pathway (Smeekens and Steiner, 1990; Seidah et al., 1990; Smeekens et al., 1991). Expression of PC2 and PC3 is restricted to neuroendocrine cells, which is compatible with their role in the processing of proneuropeptides and prohormones. Both enzymes require pairs of basic amino acids at their cleavage site. Both enzymes are synthesized as larger precursor molecules that undergo proteolytic maturation within the secretory pathway. PC2 is synthesized as a 69-kDa prepropolypeptide. The NH(2)-terminal signal sequence is removed during segregation within the endoplasmic reticulum, where glycosylation generates a 75-kDa propolypeptide. When expressed in Xenopus oocytes, pro-PC2 undergoes cleavage at the sequence KRRR to generate a 71-kDa intermediate that is further cleaved at the sequence RKKR to generate the 68-kDa mature enzyme. Processing of the 75-kDa propolypeptide can also occur directly by way of cleavage at RKKR (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). Maturation of pro-PC2 has been shown to occur by way of an autocatalytic reaction (Matthews et al., 1994).

PC3 is synthesized as an 88-kDa propolypeptide that undergoes cleavage at the sequence RSKR to generate an 80-kDa active form of the enzyme (Benjannet et al., 1992; Zhou and Lindberg, 1993). Further cleavage occurs at the COOH terminus to generate 75- and 66-kDa forms (Zhou and Lindberg, 1993; Rufaut et al., 1993; Benjannet et al., 1993). Processing of pro-PC3 occurs very rapidly in its biosynthesis (Zhou and Lindberg, 1993; Benjannet et al., 1993), while pro-PC2 processing is very slow (Shennan et al., 1991b; Guest et al., 1992).

Here we describe in detail the calcium and pH dependence for pro-PC2 and pro-PC3 maturation and show that optimal conditions for processing are very different for the two enzymes. The results suggest that pro-PC3 may undergo autocatalytic processing within the endoplasmic reticulum or early secretory compartment where the pH is near neutral and the calcium concentration in the micromolar range, while pro-PC2 processing occurs in a late secretory compartment where the pH is low (5.5) and the calcium concentration within the millimolar range.


MATERIALS AND METHODS

Chemicals and Reagents

[S]Methionine (1000 Ci/mmol) and Rainbow ^14C-methylated protein markers (molecular weight range, 14,300-200,000) were obtained from Amersham International, Little Chalfont, United Kingdom. Rabbit reticulocyte lysate was purchased from Promega, Southampton, U. K. SMURFT DNA linker was purchased from Pharmacia, St. Albans, U. K. Full-length cDNAs encoding human PC2 and mouse PC3 were provided by Dr. D. F. Steiner, Howard Hughes Medical Institute, University of Chicago. A tripeptide to inhibit glycosylation (Asn-Tyr-Thr) was synthesized by Alta Bioscience, University of Birmingham.

Site-directed Mutagenesis

The PC2 mutants PC2M3 and PC2M4 have been described previously (Shennan et al., 1991b). In PC2M3 the tetrabasic sequence at position 81-84 (where position 1 is the first amino acid in the propolypeptide) was deleted, and in PC2M4 the active-site aspartate was changed to an asparagine. PC2M5, PC3M2, and PC3M3 were made by oligonucleotide site-directed mutagenesis using the T(7)-GEN mutagenesis kit (U. S. Biochemical Corp.) according to the manufacturer's protocols. Oligonucleotides for use in mutagenesis were synthesized on a laboratory PCR MATE and purified using OPC cartridges. The following oligonucleotides were used: PC2M5, 5`TGCCGCCGTTCCCGGAGGC3`, which changed the Asp at position 285 to an Asn; PC3M2, 5`GCTTCTTCCGGAGCTCCGAGG3`, which changed the sequence Arg-Ser-Arg-Arg at position 51-54 to Ser-Ser-Gly-Arg; and PC3M3, 5`AACTGAACTTTTACTTC3`, which changed the tetrabasic sequence Arg-Ser-Lys-Arg at position 80-83 to Arg-Ser-Lys-Ser. PC3M4 was constructed by polymerase chain reaction mutagenesis of pPC3-B64T, which has a BglII site inserted at position 107-108, using two complementary oligonucleotides encoding the Asp to Asn mutation (5` CGTTACTGAATGATGGC3` and 5`GCCATCATTCAGTACAG3`) and two primers that anneal to the SP64T vector on either side of the PC3-B insert. Mutant cDNA was amplified and digested with BglII and ApaI, and the BglII/ApaI fragment was ligated into BglII/ApaI cut-pPC3-B64T. The construction of PC2M7 has previously been described (Shennan et al., 1994).

In Vitro Transcription

mRNA was synthesized from BamHI-linearized vector in a transcription reaction containing 40 mM Tris (pH 8.0), 15 mM MgCl(2), 4 mM spermidine, 5 mM dithiothreitol, 1 mM each of ATP, CTP, and UTP, 0.1 mM GTP, 0.5 mM m^7G(5`)ppp(5`)G, 0.25 mg/ml bovine serum albumin (RNase and DNase-free), 10 units of RNase guard, 60 units of SP6 RNA polymerase, and 1 µg of plasmid DNA in a final volume of 50 µl. The mixture was incubated at 37 °C for 60 min and then extracted once with phenol:chloroform:isoamyl alcohol (25:24:1) and once with chloroform:isoamyl alcohol (24:1), ethanol-precipitated with 0.1 volume of 7 M ammonium acetate and 2.5 volumes of absolute ethanol, and resuspended in H(2)O at a concentration of 0.5 mg/ml.

Xenopus Egg Extract

The preparation of the Xenopus egg extract was as described previously (Matthews and Colman, 1992). Translations were performed by addition of mRNA to an aliquot of extract containing 10% (v/v) nuclease-treated rabbit reticulocyte lysate, 10 µM creatine phosphate, 0.2 µM spermidine, and 1 mCi/ml [S]methionine. To determine the effect of calcium and pH on the processing of PC2, translations were performed at 21 °C for 2 h, designated the pulse period, and then the calcium concentration or pH was adjusted and the reactions continued for a given time at 21 °C, designated the chase period. In the case of PC3 the initial translation reaction was reduced to 30 min, and then RNase was added to a concentration of 40 µg/ml to inhibit further translation. After the chase period, the membranes of the extract were pelleted by centrifugation at 12,000 times g for 15 min at 4 °C, washed in phosphate-buffered saline containing 10% (w/v) sucrose, and resuspended in phosphate-buffered saline containing 10% (w/v) sucrose, 1 mM phenylmethylsulfonyl fluoride, and 1% (v/v) Triton X-100. An aliquot of this membrane preparation was then analyzed by SDS-PAGE (^1)and fluorography. In some experiments the protein bands were quantified using an LKB scanning densitometer.


RESULTS

The wild-type and mutant PC2 and PC3 constructs used in the present study are shown in Fig. 1. PC2 and PC3 contain a highly conserved subtilisin domain with a catalytic triad of Asp, His, and Ser residues. In addition, PC2 has an Asp at residue 285 while PC3 has an Asn residue at 282 that are thought to stabilize the transition complex. PC3M2 contains a mutation of the sequence RSRR to SSGR thereby removing a potential cleavage site. PC2M3 and PC3M3 contain mutations that remove the propolypeptide processing site, i.e. deletion of RKKR in PC2M3 and R83S in PC3M3. PC2M4 contains a change of Asp to Asn, which renders the enzyme catalytically inactive, while PC3M4 contains the equivalent mutation of Asp to Asn. PC2M5 contains a mutation of Asn to Asp, while PC2M7 has a major deletion of the entire P-domain and C-terminal sequences. All cDNAs were subcloned into the expression plasmid SP64T, and mRNA was generated by in vitro transcription. mRNAs were then translated in a cell-free system prepared from Xenopus eggs. This system has previously been shown to cleave signal peptides, segregate proteins within membranes, perform some glycosylation and phosphorylation events, and to assemble polypeptide chains of multisubunit proteins (Matthews and Colman, 1991).


Figure 1: Schematic representation of wild-type PC3 (A) and PC2 (B) and the mutants used in this study. The figure indicates the propeptide, the subtilisin domain (dark area), and the P-domain (hatched area) as well as the propeptide cleavage sites, the Asp-His-Ser of the catalytic site, and the Asp/Asn of the oxyanion hole.



To investigate PC3 propolypeptide processing, mRNA encoding PC3 was translated in the egg extract in the presence of [S]methionine for 30 min. RNase was then added to inhibit further translation and the reactions chased for periods of time (Fig. 2). An 88-kDa polypeptide was observed that underwent partial processing within the 30-min pulse labeling period. Complete processing of the 88-kDa polypeptide was observed within a 30-40-min chase period. Mutagenesis of the putative processing site RSRR to SSGR had no effect on processing to the 80-kDa form (Fig. 3), while mutation of the alternative potential processing site RSKR to RSKS prevented processing of the 88-kDa precursor at the 3-h time point, suggesting that processing was occurring at this site and was compatible with proteolytic cleavage of an NH(2)-terminal propeptide. Some processing to an intermediate 84-kDa form did occur after prolonged chase (Fig. 3, track 6), which may be the result of cleavage at the site RSRR by an endogenous extract protease. Maximal processing of the 88-kDa pro-PC3 polypeptide occurred at pH 7.0 (Fig. 4). Over the pH range 6.5-8.0, a broad peak of activity was observed between pH 7.0 and 8.0 with little activity above pH 8.0 (data not shown). Processing was not dependent on added calcium over the range 0-10 mM (Fig. 5). The mutant PC3M4, in which the Asp of the catalytic triad had been altered to an Asn, remained unprocessed over the 24-h time period (Fig. 6), suggesting that pro-PC3 maturation was autocatalytic.


Figure 2: Time course of pro-PC3 processing. PC3 mRNA was translated in the egg extract for 30 min, and translation was stopped by the addition of RNase (40 µg/ml final concentration). Aliquots were taken at various times, membranes were prepared as described under ``Materials and Methods,'' and samples were analyzed by SDS-PAGE and fluorography. Track M denotes ^14C-labeled protein markers, with the molecular mass in kDa. These bands were quantified by laser scanning densitometry and plotted to show the percentage processing (ratio of 88- to 80-kDa bands) against time.




Figure 3: Identification of the processing site of pro-PC3. PC3M2 and PC3M3 mRNAs were translated in the egg extract for 1 h, RNase was added, and the reactions were chased for 0, 3, and 24 h. Membranes were prepared as described under ``Materials and Methods,'' and samples were analyzed by SDS-PAGE and fluorography. Track M denotes ^14C-labeled protein markers, with the molecular mass in kDa.




Figure 4: pH dependence of pro-PC3 processing. PC3 mRNA was translated in the egg extract for 30 min, and RNase was added to inhibit further translation. Aliquots were then made 0.1 M with respect to buffer at different pH using MES for pH 5.0-6.5 and TES for pH 7.0. Reactions were then chased for 15 min, membranes prepared as described under ``Materials and Methods,'' and samples were analyzed by SDS-PAGE and fluorography. Track M denotes ^14C-labeled protein markers, with the molecular mass in kDa. Bands were quantified by laser scanning densitometry and plotted to show the percentage processing (ratio of 88- to 80-kDa bands) against pH.




Figure 5: Effect of calcium on pro-PC3 processing. PC3 mRNA was translated in the egg extract for 30 min, and RNase was added to inhibit further translation. Aliquots were then adjusted to the calcium concentrations shown by addition of a 10-fold stock of calcium chloride. Reactions were then chased for 15 min, membranes prepared as described under ``Materials and Methods,'' and samples analyzed by SDS-PAGE and fluorography. Arrows indicate positions of ^14C-labeled protein markers, with the molecular mass in kDa. Bands were quantified by laser scanning densitometry and plotted to show the percentage processing (ratio of 88- to 80-kDa bands) against calcium concentration.




Figure 6: Processing of pro-PC3 is autocatalytic. PC3 and PC3M4 RNA were translated in the egg extract for 1 h, RNase was added, and the reactions were chased for 0, 3, and 24 h. Membranes were prepared and analyzed by SDS-PAGE and fluorography. Track M denotes ^14C-labeled protein markers, with molecular mass in kDa.



We have previously shown that the maturation of pro-PC2 involves cleavage of a 75-kDa glycosylated propolypeptide at the sequence RKKR to generate the 68-kDa mature form of the enzyme (Matthews et al., 1994). To investigate the pH optimum for pro-PC2 processing, PC2 mRNA was translated in the egg extract for 2 h followed by a chase period of 6 h in different pH buffers (Fig. 7). Processing occurred within a narrow pH range of 5.5-6.0. The effect of calcium on pro-PC2 processing was determined by translating PC2 mRNA for a pulse period of 2 h followed by a chase period of 20 h at different calcium concentrations (Fig. 8). Pro-PC2 processing was stimulated by calcium in the millimolar range with processing being nearly complete within 20 h in 10 mM calcium. Using these optimum conditions of pH and calcium, the time course of pro-PC2 processing was investigated (Fig. 9). Processing of the 75-kDa pro-PC2 polypeptide to the mature 68-kDa polypeptide was slow (t = 8 h).


Figure 7: pH dependence of pro-PC2 processing. PC2 mRNA was translated in the egg extract for 2 h. Aliquots were then taken and adjusted to the pH shown by addition of 0.1 vol of 1 M sodium acetate (pH 4.0 and 4.5), 1 M MES (pH 5.5, 6.0, and 6.5) or 1 M TES (pH 7.0). The reactions were then incubated at 21 °C for 6 h. Membranes were prepared as described under ``Materials and Methods,'' and samples were analyzed by SDS-PAGE and fluorography. Track M denotes ^14C-labeled protein markers, with the molecular mass in kDa. Arrowheads on the right point to the 75- and 68-kDa forms of PC2. These bands were quantified by laser scanning densitometry and plotted to show the percentage processing (ratio of 68- to 75-kDa bands) against pH of the chase buffer.




Figure 8: Effect of calcium on pro-PC2 processing. PC2 mRNA was translated in the egg extract for 2 h. Aliquots were then adjusted to the calcium concentrations shown by addition of a 10-fold stock of CaCl(2). The reactions were then chased for 20 h before membranes were prepared as described under ``Materials and Methods,'' and samples were analyzed by SDS-PAGE and fluorography. Arrowheads on the left indicate the positions of ^14C-labeled protein markers, with the molecular mass in kDa. The arrowheads on the right indicate the 75- and 68-kDa forms of PC2. These bands were quantified by scanning densitometry and plotted as percentage processing (ratio of 68- to 75-kDa bands) against concentration of added calcium.




Figure 9: Time course of pro-PC2 processing. PC2 mRNA was translated in the egg extract for 2 h. The translation reaction mix was then adjusted to pH 5.5 and 10 mM CaCl(2), and the reaction mix was incubated at 21 °C. Aliquots were removed at the indicated times, and membranes were prepared as described under ``Materials and Methods.'' Samples were analyzed by SDS-PAGE and fluorography. Track M denotes ^14C-labeled protein markers, with the molecular mass in kDa. The arrowheads on the right indicate the 75- and 68-kDa forms of PC2. The bands were quantified by scanning laser densitometry and plotted to show the percentage processing (ratio of 68- to 75-kDa bands) with time.



We have previously shown that the maturation of PC2 involves an autocatalytic reaction (Matthews et al., 1994). We now wished to determine whether the same mechanism occurred under optimal conditions of pH and Ca. This was achieved by using two mutant PC2 molecules: PC2M4, which contained a change of the catalytically important Asp to Asn, and PC2M7, which contains a deletion of 214 amino acids from the COOH-terminal end of the molecule. This part of the protein has been shown to be important for furin activity (Creemers et al., 1993). When translated in the Xenopus egg extract, PC2M7 remained unprocessed after an 18-h chase period (Fig. 10, tracks 1 and 2). Wild-type pro-PC2 was completely processed to the 68-kDa polypeptide (Fig. 10, tracks 3 and 4). PC2M4 underwent a small degree of processing, principally to a 71-kDa intermediate (Fig. 10, track 6). This is likely to be due to the activity of an endogenous egg extract protease during the chase conditions. When PC2M7 and PC2M4 were translated in the same reaction, there was no change in the pattern of processing (Fig. 10, tracks 7 and 8), confirming that both PC2M7 and PC2M4 are catalytically inactive. However, PC2M7 was completely processed in trans by cotranslating PC2M7 mRNA with wild-type PC2 mRNA (Fig. 10, tracks 9 and 10). As shown previously (Matthews et al., 1994), the mutant PC2M4 pro-PC2 polypeptide migrates slightly faster than the wild-type pro-PC2 polypeptide. Co-incubation of pro-PC2M4 with wild type pro-PC2 resulted in the appearance of two processed molecules indicating that pro-PC2M4 had also undergone processing in trans by the wild-type pro-PC2 or PC2 molecule(s) (Fig. 10, tracks 11 and 12).


Figure 10: Processing of PC2 is autocatalytic and intermolecular. PC2 (W/T), PC2M7, and PC2M4 were translated separately (tracks 1-6) and in the combinations shown (tracks 7-12) for 2 h. The pH was adjusted to pH 5.5 by the addition of 1 M MES, pH 5.5, the calcium concentration was adjusted to 10 mM by the addition of CaCl(2), and the reactions were continued for 18 h. Membranes were prepared after the 2-h pulse and 18-h chase periods as described under ``Materials and Methods,'' and were analyzed by SDS-PAGE and fluorography.



These results confirm that cleavage of pro-PC2 can occur via an intermolecular reaction. The question remained as to whether the pro-PC2 itself could cleave other pro-PC2 molecules or whether following an initial activation step further cleavage involved the newly formed mature enzyme. To investigate whether the pro-PC2 could cleave in an intermolecular reaction we used the mutant PC2M3 (Fig. 1), which contains a deletion of the tetrabasic processing site RKKR. This mutation blocks processing of the 75-kDa pro-PC2 polypeptide to the mature 68-kDa form. Translation of PC2M3 mRNA in the Xenopus egg extract resulted in accumulation of the unprocessed 75-kDa propolypeptide and the 71-kDa intermediate (Fig. 11, tracks 1 and 2). The 71-kDa intermediate is generated through cleavage of the 75-kDa PC2M3 polypeptide at the sequence KRRR. When PC2M3 mRNA was cotranslated in the egg extract with PC2M7 mRNA, the 75- and 71-kDa pro-PC2M3 polypeptides had no effect on the processing of pro-PC2M7 (Fig. 11, tracks 5 and 6). Pro-PC2M7 was, however, completely processed when cotranslated with wild-type PC2 mRNA (Fig. 11, tracks 7 and 8). These results indicated that the pro-PC2 molecules were not involved in the processing of other pro-PC2 molecules in trans.


Figure 11: The 75- and 71-kDa molecular forms of pro-PC2 are not catalytically active. PC2M3 (tracks 1 and 2), PC2M7 (tracks 3 and 4), PC2M3 plus PC2M7 (tracks 5 and 6), and PC2WT plus PC2M7 (tracks 7 and 8) were translated for 2 h. The pH was adjusted to pH 5.5 by the addition of 1 M MES, pH 5.5, the calcium concentration was adjusted to 10 mM by the addition of CaCl(2), and the reactions were chased for 18 h. Membranes were prepared after the pulse and the chase period as described under ``Materials and Methods,'' and were analyzed by SDS-PAGE and fluorography. Track M denotes ^14C-labeled protein markers, with the molecular mass in kDa.



To determine the class of protease responsible for the maturation of pro-PC2 and pro-PC3, chase reactions were performed in the presence of various protease inhibitors. Maturation of pro-PC2 and pro-PC3 was inhibited by high (1 mM) but not low (1 µM) concentrations of leupeptin, unaffected by pepstatin A or phenylmethylsulfonyl fluoride, and inhibited by high (10 mM) but not low (2 mM) concentrations of EDTA (data not shown). In these respects the inhibitor profile of maturation was similar to that of PC2 and PC3 activity against peptide substrates (Shennan et al., 1991a; Bailyes et al., 1992).

We next looked for structural differences between pro-PC2 and pro-PC3 that might explain the different rate of maturation of the two molecules. Most members of the subtilisin family of proteases have a conserved asparagine that stabilizes the oxyanion transition state. PC2 differs from other members of the subtilisin-like family in that it contains an aspartate in place of asparagine in this position. We therefore changed Asp to Asn in PC2M5. PC2M5 was translated in the Xenopus egg extract for 2 h and then chased for up to 18 h in buffer at pH 7.0 in the absence of added Ca, i.e. the conditions that favored rapid processing of pro-PC3. Under these conditions pro-PC2M5 was processed slightly more efficiently than pro-PC2, but the rate of processing was nonetheless more like PC2 than PC3 (Fig. 12).


Figure 12: Effect of D285N mutation on time course of processing of pro-PC2 at neutral pH. PC2 and PC2M5 mRNA were translated in the egg extract for 1 h, and then RNase was added. Chase reactions were performed at pH 7.0, and aliquots were removed at various times. Membranes were prepared as described under ``Materials and Methods,'' and were analyzed by SDS-PAGE and fluorography. Track M denotes ^14C-labeled protein markers, with the molecular mass in kDa.



Pro-PC2 and pro-PC3 are glycosylated at three and two sites, respectively. To determine whether the glycosylation state would affect maturation of either protease, translations were carried out in the presence of a tripeptide (Asn-Tyr-Thr) that acts as a substrate for glycosylation, competing for glycosylation of newly synthesized proteins (Lau et al., 1983). While the tripeptide inhibited glycosylation of both pro-PC2 (Fig. 13A, tracks 1 and 3) and pro-PC3 (Fig. 13B), the nonglycosylated pro-PC2 and pro-PC3 were processed to their mature forms with similar efficiencies as glycosylated pro-PC2 and pro-PC3.


Figure 13: Effect of glycosylation on pro-PC2 and pro-PC3 maturation. A, PC2 mRNA was translated in the egg extract for 2 h in the presence or absence of 1.67 mM Asn-Tyr-Thr. The extract was then adjusted to pH 5.5 and 10 mM calcium and the reaction chased for 18 h at 21 °C. Membranes were then prepared and analyzed by SDS-PAGE and fluorography. Track M denotes ^14C-labeled protein markers, with the molecular mass in kDa. B, PC3 mRNA was translated in the egg extract for 2 h in the presence or absence of 1.67 mM Asn-Tyr-Thr. Membranes were then prepared and analyzed by SDS-PAGE and fluorography. Track M denotes ^14C-labeled protein markers, with the molecular mass in kDa.




DISCUSSION

We show here that the processing of pro-PC3 and pro-PC2 exhibits different pH optima and calcium requirements. Pro-PC3 (88-kDa form) was rapidly processed (t < 10 min) to the active 80-kDa form of the enzyme in an autocatalytic reaction that occurred at pH 7.0-8.0, and which was not dependent on additional Ca. The exact Ca concentration in the Xenopus egg extract was not measured, but it is likely to be within the micromolar range. The enzymic properties of autocatalytic activation are, therefore, very different from those of the mature enzyme, which has been shown to be optimally active in the pH range 5.5-6.5 and to exhibit a requirement for Ca in the millimolar range (K(0.5) = 2 mM) (Jean et al., 1993; Rufaut et al., 1993; Zhou and Lindberg, 1993; Vindrola and Lindberg, 1993; Bailyes et al., 1992). We have not determined whether cleavage involves an intra- or intermolecular reaction. If it is intramolecular, then our results would suggest that conformational changes occur on removal of the propeptide, which may result in different pH and calcium requirements of the mature protease. Differences have previously been noted between autoprocessing of furin and processing of substrates by furin (Creemers et al., 1993) and similarly between autoprocessing of Kex2 and processing of substrates by Kex2 (Brenner et al., 1993).

The time course of pro-PC3 processing in the Xenopus egg extract is similar to that observed when PC3 was expressed in GH4 cells (Benjannet et al., 1992; Zhou and Lindberg, 1993) and in Cos-7 cells. (^2)Endogenous pro-PC3 in AtT20 cells is also rapidly processed (Lindberg, 1994), suggesting that these events occur in an early secretory compartment, most likely the endoplasmic reticulum. This may be important for PC3 involvement in early processing events of prohormone maturation. For example, PC3 is implicated in some of the earliest events in pro-opiomelanocortin processing (Bloomquist et al., 1991), and in proinsulin processing, PC3 is thought to perform the initial processing step at the B-chain/C-peptide junction before PC2 can act at the C-peptide/A-chain junction (Rhodes et al., 1992). The observation that pro-PC3 processing occurs efficiently at pH 7.0 and is not dependent on the addition of Ca is therefore compatible with current views on the internal ionic environment of the endoplasmic reticulum (Anderson and Orci, 1988). However, simple removal of the propeptide may not be sufficient for enzyme activation. A furin mutant containing an endoplasmic reticulum retention signal was efficiently processed to the mature form in the endoplasmic reticulum, but this was enzymatically inactive (Molloy et al., 1994) suggesting that movement through the secretory pathway may confer further modifications leading to activation. In the case of PC3, although it may be processed in the endoplasmic reticulum, because of the low pH and high calcium requirements of the mature protease, it is unlikely to be active until later in the secretory pathway.

In GH4, AtT20, or mouse L cells, the 80-kDa form of PC3 undergoes further cleavage to generate 75- and 66-kDa polypeptides that are secreted. These molecules are enzymatically active and are generated by COOH-terminal cleavage of the 80-kDa polypeptide, possibly by an autocatalytic mechanism that takes place in the secretory vesicles of the regulated pathway (Zhou and Lindberg, 1994). These smaller PC3-related peptides were not generated in the Xenopus egg extract under various conditions of pH and Ca concentration. In COS-7 cells^2 and microinjected Xenopus oocytes (Bailyes et al., 1992) the major secreted form of PC3 exhibits a molecular size of 80 kDa with no further cleavage observed.

Pro-PC2 (75 kDa) was processed only slowly (t = 8 h) to the active (68 kDa) form in a reaction that required acidic pH and millimolar concentrations of calcium. The enzymic properties of pro-PC2 maturation were therefore identical to those of the mature enzyme when assayed both against small peptide fluorogenic substrates (Shennan et al., 1991a) and potential in vivo substrates (Rhodes et al., 1993). The conditions required for pro-PC2 maturation (acidic pH and high calcium) would suggest that pro-PC2 is not activated until late in the secretory pathway, i.e. in the trans-Golgi network or possibly not until the secretory granules. This interpretation, however, is at variance with the results of Guest et al.(1992) who showed that mature PC2 was present in subcellular fractions enriched in Golgi and endoplasmic reticulum elements.

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. This is in contrast to Kex2 (Germain et al., 1992) and furin (Leduc et al., 1992; Creemers et al., 1993) where activation of the precursor molecules has been shown to be exclusively intramolecular. Our data would favor a two-step (intermolecular) reaction rather than a simple one-step (intramolecular) reaction for the maturation of pro-PC2. However, to define a process as intramolecular, the reaction must be shown to be independent of the enzyme concentration as, for example, in the maturation of pro-cathepsin B (Mach et al., 1994). A definitive answer to this question must await full kinetic analysis of pro-PC2 processing following overexpression and purification of pro-PC2 on a larger scale.

The catalytic importance of the middle/P-domain of pro-PC2 has been shown by the inability of the deletion mutant PC2M7 either to process itself or another catalytically inactive mutant in trans. It is unlikely that this was simply due to inappropriate folding as wild-type PC2 was able to process PC2M7 in trans. The P-domain of both Kex2 (Gluschankof and Fuller, 1994) and furin (Creemers et al., 1993) has previously been shown to be necessary for catalytic activity; in furin the essential region has been mapped to a 21-amino acid stretch from Glu to Glu.

Pro-PC2 differs from the other subtilisin-like serine proteases in having an aspartate residue, instead of an asparagine residue, in a position which is thought to stabilize the oxyanion transition state formed during substrate processing. It has been suggested that the aspartate in PC2 might act as an oxyanion hole when protonated, restricting its activity to acidic pH (Steiner, 1991); however, the fact that PC3 activity is also restricted to acidic pH despite having an asparagine as the oxyanion hole might discount this view. The major differences we have found in pH requirements for activation of pro-PC2 and pro-PC3 suggested that the aspartate of the oxyanion hole of PC2 might be important in restricting the pH at which maturation occurs. The mutant PC2M5, in which the oxyanion aspartate was altered to an asparagine, was more efficiently processed to its mature form at neutral pH, but the time course of processing was still slow compared with that of pro-PC3. The reverse substitution in pro-Kex2, i.e. Asn to Asp, had no effect either on the rate of maturation of pro-Kex2 or its pH requirements, but it did affect the activity of the mature protease against substrates (Brenner et al., 1993). It is possible that pro-PC2 has an additional oxyanion binding site and, indeed, there are in pro-PC2 further residues around the Asp site that are different in pro-PC3. For example PC2 has an Asp residue at position 291 that is an Asn in PC3 and, conversely, PC2 has an Asn at position 293 whereas PC3 has an Asp at the equivalent position. The importance of these substitutions in the maturation or substrate specificity of PC2 and PC3 is currently being investigated.


FOOTNOTES

*
This work was supported by grants from the Medical Research Council and the British Diabetic Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

()
Present address: Dept. of Molecular and Cell Biology, University of Aberdeen, Marischal College, Aberdeen, AB9 1AS, United Kingdom.

§
To whom correspondence should be addressed. Fax: 44-224-273144.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; MES, 4-morpholineethanesulfonic acid.

(^2)
N. A. Taylor and K. Docherty, unpublished observations.


ACKNOWLEDGEMENTS

We thank Don Steiner for providing the hPC2 and mPC3 cDNAs.


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