enzymatic properties of carboxyl-terminally truncated prohormone

THE JOURNAL OF BIOLCGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 28, Issue of July 15, pp. 18408-18413, 1994 Printed in U.S.A.

Enzymatic Properties of Carboxyl-terminally Truncated Prohormone Convertase 1 (PCl/SPC3) and Evidence for Autocatalytic Conversion*

(Received for publication, December 28, 1993, and in revised form, April 25, 1994)

Yi Zhou and Iris LindbergS From the Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70112

Previous studies have shown that the prohormone convertase 1 (PC1, or SPC3), a member of the new eu- karyotic subtilisin-like proteinase family, undergoes a series of proteolytic processing events during its biosynthesis. The first cleavage, of the amino-terminal proseg- ment, is probably involved in enzyme activation, while the secondary cleavages at the carboxyl terminus are of unknown significance and occur mainly in cells possessing a regulated secretory pathway. In this work, we found that 87-kDa PC1, a homogeneous recombinant protein, could spontaneously convert to 74- and 66-kDa forms in uitro. Limited digestion of 87-kDa PC1 using chymotrypsin and trypsin could also generate 74- and 66-kDa-like PCls, which were enzymatically active against the fluorogenic peptide carbobenzoxy-Arg-Tyr- Lys-Arg-aminomethylcoumarin. The 7466-kDa PC1 generated by spontaneous conversion was purified away from the 87-kDa form and enzymatically characterized. Compared to the 87-kDa form, 7466-kDa PC1 was more active but less stable. In addition, 7466-kDa PC1 exhibited a narrower pH optimum (between 5.0 and 5.5) and was activated by higher concentrations of calcium. Car- boxyl-terminally truncated PC1 also appeared to be more sensitive to certain protease inhibitors than 87- kDa PC1. Taken together, our results suggest that autocatalysis could be involved in carboxyl-terminal cleavages of PC1. These carboxyl-terminal cleavages of PC1 result in alterations in certain PC1 properties and may therefore possess potential significance with respect to prohormone processing.

Most polypeptide hormones are initially synthesized in inactive precursor forms that require a series of proteolytic processing steps for activation. These proteolytic cleavages occur specifically at basic residues, frequently at paired basic residues, and are thought to take place in the trans-Golgi network or regulated secretory granules (reviewed by Hutton (1990) and Mains et al . (1990)). Progress in understanding the mechanism of these highly specific proteolytic events has been made by recent studies documenting a mammalian subtilisin-like protease family (reviewed by Lindberg (1991) and Steiner et al.

* This work was supported in part by Grant 05084 from NIDA, Na- tional Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by a Research Career Development Award from NIDDK, National Institutes of Health. To whom correspondence should be directed at the following address: Dept. of Biochemistry and Molecular Biology, LSU Medical Center, 1901 Perdido St., New Orleans, LA 70112. Tel: 504-568-4799; Fax: 504-568-3370.

(1992)). I t has been shown that prohormone convertase 1 (PC1,’ also known as SPC3) and prohormone convertase 2 (PC2, or SPC21, two members of the family, are expressed predomi- nantly within neuroendocrine cells and tissues (Seidah et al., 1990, 1991; Smeekens et al., 1991; Day et al., 1992). Co-transfection of cDNA coding for PC1 or PC2 together with cDNA coding for opioid peptide precursors can generate properly cleaved peptide products (Benjannet et al., 1991; Thomas et al., 1991; Breslin et al., 1993). Transfection of antisense sequences of PC enzymes can block endogenous prohormone conversion (Bloomquist et al . , 1991). Collectively the available evidence strongly suggests that PC1 and PC2 are involved in the proteolytic processing of prohormones.

To understand the biosynthesis and biochemical properties of PC1, we have overexpressed recombinant mouse PC1 in Chinese hamster ovary cells (CHO/mPCl cells). In a previous study, we found that PC1 was initially synthesized in a precursor form, which quickly underwent an amino-terminal cleavage event at a very early stage of biosynthesis to generate an 87-kDa form (Zhou and Lindberg, 1993). Purified 87-kDa PC1 represents an enzymatically active form that is calcium-dependent and exhibits optimal activity between pH 5.0 and 6.5. At a later stage of biosynthesis, 87-kDa PC1 could be further converted to 74- and 66-kDa forms by carboxyl-terminal cleavages. In CHO/mPCl cells, these carboxyl-terminal cleavages are largely incomplete, and 87-kDa PC1 represents the major released form (Zhou and Lindberg, 1993). Similar results have recently been published by other groups (Benjan- net et al., 1992, 1993; Jean et al., 1993; Rufaut et al., 1993). In AtT2O and bovine chromaffin cells, which contain endogenous PC1, 66-kDa PC1 is the predominant PC1 form in the regulated secretory pathway (Christie et al., 1991; Vindrola and Lindberg, 1992; Milgram and Mains, 1994). These results suggest that carboxyl-terminal cleavage of PC1 takes place chiefly within regulated secretory granules, although a very limited amount of cleavage can occur within the constitutive secretory pathway. Regulated secretory granules have been considered as a very important location for prohormone processing; therefore, the carboxyl-terminal cleavage of PC1 may have special significance for the regulation of PC1 activity. In this work we studied possible mechanisms of PC1 carboxyl- terminal cleavages in vitro using homogeneous recombinant 87-kDa PC1. We found that carboxyl-terminal cleavage of PC1

nomethylcoumarin; CHO, Chinese hamster ovary; E-64, L-trans-epoxy- The abbreviations used are: PC, prohormone convertase; AMC, ami-

succinic acid; pCMS, p-chloromercuriphenylsulfonate; TLCK, N”-p-to- syl-L-lysine chloromethyl ketone (~-l-chloro-3-[4-tosylamidol-7-amino- 2-heptanone); TPCK, tosylphenylalanyl chloromethyl ketone (L-1- chloro-3-[4-tosylamino]-4-phenyl-2-butanone); Boc, t-butoxycarbonyl; Cbz, carbobenzoxy; Bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxy- methy1)methane.

18408

Carboxyl-terminally Duncated PC1 18409

could result from autocatalysis. Enzymatic and physical properties of the cleavage products, 74/66-kDa PC1, were somewhat different from those of the 87-kDa form, suggesting that carboxyl-terminal cleavages might play an important role in prohormone processing.

EXPERIMENTAL PROCEDURES Preparation of 87-kDa PCI-87-kDa PC1 was obtained as described

previously (Zhou and Lindberg, 1993). Briefly, 87-kDa PC1 was purified from the conditioned medium of methotrexate-amplified CHO/mPCl cells, which express high levels of mouse PC1, using fast performance liquid chromatography (Pharmacia Biotech Inc.) at 4 "C. ADEAE-Blue cartridge (Bio-Rad), as well as phenyl-Superose (HR5/5, Pharmacia) and Mono Q columns (HR5/5, Pharmacia), were sequentially employed in the purification procedure. 87-kDa PC1, which was eluted from the Mono Q column a t 700 mM sodium acetate, 20 mM Bis-Tris, 0.1% Brij 35 (polyoxyethylene 10 cetyl alcohol), pH 6.5, was pooled and stored frozen a t -20 "C. This 87-kDa PC1 was homogeneous as judged by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining (Zhou and Lindberg, 1993). For some experiments 87-kDa PC1 was further purified by gel filtration chromatography (Superdex G200; Pharmacia) using 20 mM Bis-Tris, pH 6.5, with 150 mM sodium acetate, 0.1% Brij 35 as eluant (Zhou and Lindberg, 1993). All experiments were carried out a t least twice on separate enzyme preparations.

Spontaneous Carboxyl-terminal Cleavage of 87-kDa PCI-The reactions were camed out in 80 mM Bis-Tris, pH 5.5, with 150 m~ sodium acetate and 10 mM calcium chloride. A 10-pl aliquot of pooled 87-kDa PC1 (about 0.2 pg of protein) was added to each reaction in a final volume of 50 pl. The reaction mixtures were incubated at 37 "C for different periods of time and analyzed by Western blotting using polyclonal PC1 antiserum directed against residues 84-100 (representing the amino terminus of 87-kDa mouse PCl) (Vindrola and Lindberg, 1992) or PC1 carboxyl-terminal antiserum directed toward residues 714-726 (representing the carboxyl terminus of 87-kDa PC1). To meas- ure the effects of different proteinase inhibitors, the control reaction (without inhibitors) was compared to reactions that contained either 5 mM EDTA, 1 mM pCMS (p-chloromercuriphenylsulfonate), 0.1 mM D- Tyr-Ala-Lys-kg-chloromethyl ketone (YAKR-CH,Cl) or a mixture of 2.5

E-64 (L-trans-epoxysuccinic acid), 1 p~ pepstatin A, 50 pg/ml TLCK, and 100 pg/ml TPCK. The competition of fluorogenic substrates on carboxyl-terminal conversions of PC1 was also studied by adding 0.4 mM Boc-Ala-Ala-Ala-aminomethylcoumarin (AAA-AMC), Boc-Arg-Val-Arg- Arg-aminomethylcoumarin (RVRR-AMC), or Cbz-Arg-Qr-Lys-Arg-aminomethylcoumarin (RYKR-AMC) to the reaction mixture. The mixtures were incubated for 16 h at 37 "C and analyzed by Western blotting.

Purification of 74/66-kDa PC1 Generated by Spontaneous Cleau- ages-Homogeneous 87-kDa PC1 was incubated for 16 h a t 37 "C in 10 mM Bis-Tris buffer, pH 6.5, in 350 mM sodium acetate and 1 mM calcium chloride. The reaction mixture was then diluted to 30 mM sodium acetate with deionized water and applied to a Mono Q column (HR5/5, Pharmacia). Elution was performed using a two-step gradient from 20 mM Bis-Tris, pH 6.5, with 0.1% Brij 35 to 20 mM Bis-Tris, pH 6.5, containing 0.1% Brij 35 and 1 M sodium acetate, as shown in Fig. 4A. The flow rate was 0.5 mumin, and 1-ml fractions were collected.

Characterization of 74166-kDa PCI-Enzymatic activity of 74/66- kDa PC1 was characterized by the cleavage of a fluorogenic substrate, Cbz-Arg-Tyr-Lys-kg-aminomethylcoumarin (custom-synthesized by Enzyme Systems Products, Dublin, CAI. For direct comparison with the properties of 87-kDa PC1, reactions containing 74/66-kDa PC1 were always carried out in parallel with reactions containing 87-kDa PC1. Enzyme assays were typically performed in duplicate in 80 mM Bis-Tris buffer, pH 5.5, containing 0.4 mM RYKR-AMC and 10 mM calcium chloride (final concentrations). A 10-pl aliquot of each fraction or of pooled PC1 was added to generate a 50-pl reaction mixture. Reaction mixtures were incubated at 37 "C in polypropylene 96-well plates (Costar, Cam- bridge, MA) sealed with parafilm, and released AMC (aminomethylcoumarin) was measured a t multiple time points using a 7600 Microplate Fluorometer (Cambridge Technology, Inc, Cambridge, MA). Relative fluorescence at 460 nm was converted to nanomoles of AMC by refer- ence to a standard curve of free AMC (Peninsula Laboratories, Belmont, CA). In the time-dependent experiment, 87- and 74/66-kDa PC1 were preincubated in the reaction buffer for the periods of time indicated prior to adding fluorogenic substrate. Incubation was carried out for 1 h following substrate addition and the release of free AMC quantitated. Measurements of optimal pH and effects of proteinase inhibitors were performed as described previously (Zhou and Lindberg, 1993); the final concentrations of individual inhibitors are indicated in Table I. All re-

actions were started by adding enzyme, and data points were always represented from the linear range.

Limited Digestion of 87-kDa PC1 with Chymotrypsin-Ten-pl aliquots of 87-kDa PC1 (about 0.3 pg of protein, in 20 mM Bis-'his, 700 mM sodium acetate, and 0.1% Brij 35, pH 6.5) were digested in a 25-pl final volume with various amounts of TLCK-pretreated chymotrypsin (Sigma), as indicated in Fig. 3. After a 1.5-h incubation a t 37 "C, the reactions were stopped by the addition of aprotinin (Sigma) to a final concentration of 4 pg/ml. Half of each sample was boiled with 2% SDS (sodium dodecyl sulfate) and stored at -20 "C for direct Western blotting. The other half of each sample was incubated at 37 "C in a mixture of 50 pl containing 0.4 mM RYKR-AMC, 80 m~ Bis-Tris, pH 5.5, and 10 mM calcium chloride for assay of activity. Samples were analyzed by Western blotting with polyclonal PC1 antisera directed against either the amino or carboxyl terminus. Parallel digestion experiments without PC1 were performed to control for potential residual activity of aprotinin-blocked chymotrypsin against the fluorogenic substrate.

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting- Electrophoresis was performed using 1.5-mm minigels (Bio-Rad) in a discontinuous Tris glycine buffer system (Laemmli et al., 1970). 8.8% polyacrylamide gels were used for all PC1 samples, while 15% polyacrylamide gels were used for assaying the cleavage products of proenkephalin. Western blots were camed out using affinity-purified alka- line phosphatase-coupled goat anti-rabbit second antibody as described previously (Vindrola and Lindberg, 1992).

Protein Assay-Protein contents were assayed using the Bradford method (Bradford, 1976). Bovine serum albumin was used as a standard.

Cleavage of Proenkephalin-Purified recombinant rat proenkephalin (Lindberg et al., 1991) was cleaved by PCls obtained through Mono Q chromatography. The reaction mixture contained 80 mM Bis-"is buffer, pH 5.5, 20 m~ calcium, 10 pg of proenkephalin, and 0.75 pg of 87- or 74/66-kDa PC1 in a 50-pl final volume a t 37 C. At the indicated time points, 5-pl aliquots of reaction mixtures were removed and cleavage extent determined using Western blotting with polyclonal antiserum against peptide F (Christie et al., 1984).

RESULTS

Spontaneous Conuersion-By sequentially performing three different chromatographic procedures, we obtained 87-kDa PC1 in homogeneous form as demonstrated by silver and Coo- massie Blue staining of SDS-polyacrylamide gels (Zhou and Lindberg, 1993). As shown in Fig. 1, we observed that 87-kDa PC1 could spontaneously convert to 74- and 66-kDa forms in vitro during incubation with 10 mM calcium a t pH 5.5. Anti- serum directed against residues 84-100, which represents the extreme amino terminus of 87-kDa PC1 (Zhou and Lindberg, 19931, efficiently recognized all three forms of PC1 (Fig. 11, while antiserum directed toward the carboxyl terminus (residues 714-726) could recognize only the 87-kDa form (data not shown), indicating that the observed truncations generating the 74/66-kDa forms must occur at the carboxyl terminus of 87-kDa PC1. The carboxyl-terminal cleavage of 87-kDa PC1 occurred gradually in a time-dependent fashion (Fig. 1).

Effects of Proteinase Inhibitors and Fluorogenic Sub- strates-As shown in Fig. 2, EDTAcould substantially block the conversion of 87-kDa PC1 to the 74- and 66-kDa forms, imply- ing that these cleavages represent calcium-activated events. Millimolar calcium was required for maximal conversion (data not shown). Similarly to 87-kDa PC1 activity, the carboxyl- terminal conversions were effectively blocked by pCMS and YAKR-chloromethyl ketone, but not by an inhibitor mixture containing E-64, pepstatin, TPCK, and TLCK. In addition, the conversions could also be substantially blocked (in a dose-dependent manner) by inclusion of the fluorogenic substrates RYKR-AMC and RVRR-AMC, which represent good substrates for 87-kDa PC1, but not by AAA-AMC, which is not a substrate for PC1 (Zhou and Lindberg, 1993). These results indicate that PC1 carboxyl-terminal cleavages can be competitively inhibited by substrate.

Limited Digestion of 87-kDa PC1 by Chymotrypsin-Aliquots of 87-kDa PC1 were digested with different concentrations of

18410 Carboxyl-terminally Duncated PC1

B

A

0 10 20 30 40

(h)

FIG. 1. Time dependence of spontaneous carboxyl-terminal cleavage of 87-kDa PC1. Spontaneous cleavages of PC1 were carried out as described under "Experimental Procedures." The reactions were stopped at different times as indicated above each lane. Products were analyzed by Western blotting using PC1 amino-terminal antiserum

B. 0, 87-kDa form; 0, 74-kDa form; A, 66-kDa form. (panel A). Densitometric analysis of Western blotting is shown in panel

chymotrypsin at pH 6.5 for 1.5 h and the products analyzed by Western blotting. Using antiserum directed toward the extreme amino terminus of PC1, we found that two products, 74- and 66-kDa-like PCls, were gradually generated (Fig. 3A). However, using PC1 carboxyl-terminal antiserum, 87-kDa PC1 was the only detectable form (data not shown), indicating that cleavages occurred from the carboxyl terminus. Limited digestion with trypsin also produced similar results.2 The enzymatic activity of cleaved products was assayed using Cbz-Arg-Tyr- Lys-Arg-AMC. The maximal value represented less than 15% of the substrate consumed. We found that both the 74- and 66- kDa-like forms were enzymatically active (Fig. 3). Carboxyl- terminal cleavages of PC1 appeared to increase initial enzymatic activity but decrease activity at later time points. Western blotting of the 24-h incubated samples showed that the 66-kDa PC1 generated remained intact, i.e. the decrease in activity at long times was not due to chymotryptic degradation of 66-kDa PC1 during the incubation.

Purification of 74166-kDa PC1 on Anion Exchange Chroma- tography-87-kDa PC1 was purified as described previously (Zhou and Lindberg, 1993). Spontaneous carboxyl-terminal cleavage of PC1 was carried out at pH 5.5 with 1 mM calcium. After a 16-h incubation at 37 "C, the reaction mixture was separated using anion exchange chromatography (Mono Q HR5/5, Pharmacia). Both Western blotting (using PC1 amino-

* Y. Zhou and I. Lindberg, unpublished results.

n i i . FIG. 2. Effects of proteinase inhibitors and fluorogenic sub-

strates on spontaneous cleavage of 87-kDa PC1. Spontaneous cleavage of PC1 was carried out in the presence of different proteinase inhibitors or fluorogenic substrates; samples were analyzed by Western blotting (panel A). Ctr., non-incubated control PC1; AAA, 0.4 mM Boc- Ala-Ala-Ala-AMC; RVKR, 0.4 mM Boc-Arg-Val-kg-kg-AMC; RYKR, 0.4 mM Cbz-kg-Tyr-Lys-Arg-AMC; Ctr.0, without proteinase inhibitors or fluorogenic substrates; Mix, mixture of proteinase inhibitors containing 2.5 PM E-64, 1 PM pepstatin, 50 pg/ml TLCK, and 100 pg/ml TPCK, Y M , 0.1 mM o-Tyr-Ala-Lys-kg-CH2Cl; pCMS, 1 mM pCMS; EDTA, 5

B. 0, 87-kDa form; El, 74-kDa form; W, 66-kDa form. mM EDTA. Densitometric analysis of Western blotting is shown in panel

terminal antiserum) and enzymatic activity against Cbz-Arg- Tyr-Lys-Arg-AMC were used to screen all fractions. Two peaks of enzymatic activity were detected. The first peak contained a mixture of 74/66-kDa PC1, while the second peak contained uncleaved 87-kDa PC1 as in the original sample (Fig. 4, A and B ) . All attempts to separate 74- from 66-kDa PC1 were unsuc- cessful.

Enzymatic Characterization of Carboxyl-terminally Cleaved 74166-kDa PC1-The purified 74/66-kDa PC1 was characterized in many aspects as compared with the 87-kDa form. In a time-dependent study, we found that after a lag phase, the specific activity of 74/66-kDa PC1 was about 4-fold higher than the original 87-kDa PC1; however, 74/66-kDa PC1 was much less stable than the 87-kDa form (Fig. 5) . The optimal pH for 74/66-kDa activity was between pH 5.0 and pH 5.5, somewhat narrower than that observed using 87-kDa PC1 (Fig. 6) (Zhou and Lindberg, 1993). Maximal activity of 74/66-kDa PC1 required a much higher calcium concentration than 87-kDa PC1 (Fig. 7). The for 74/66-kDa PC1 was 3.8 mM, about 15-fold higher than for the 87-kDa PC1 (0.25 mM) studied in the same experiment. (We previously reported 5 mM calcium for maximal activity of 87-kDa PC1 (Zhou and Lindberg, 19931, possibly because we used a mixture of 87- and 66-kDa PCls as a result of spontaneous conversion occurring in stored, unfrozen samples.) We also found that the potency of certain proteinase inhibitors on 74/66-kDa PC1 was different from their potency

Carboxyl-terminally Duncated PC1 18411

A

97 kDa- I m a

66 k D a “ c .- -.I)mo

Time (h) FIG. 3. Limited digestion by chymotrypsin and enzymatic ac-

tivity of cleaved products. After 1.5 h of limited digestion with chymotrypsin, the enzymatic activity of 0.3 pg of protein against RYKR- AMC was assayed as described under “Experimental Procedures.” Panel A shows the results of Western blotting of parallel samples. The concentration of chymotrypsin used is shown above each lane. Panel B shows the enzymatic activity of PCls. Open circles, control digestion without chymotrypsin; solid triangles, digested with 120 ng/ml chymotrypsin; solid diamonds, digested with 1,200 ng/ml chymotrypsin; solid circles, digested with 12,000 ng/ml chymotrypsin.

on 87-kDa PC1. As shown in Table I (see also Zhou and Lind- berg (199311, the activities of both 74166- and 87-kDa PC1 were substantially inhibited by EDTA and pCMS, but were not affected by thiol, aspartyl, and certain serine proteinase specific inhibitors. However, 74f66-kDa PC1 showed greater sensitivity to phenylmethylsulfonyl fluoride and D-m-Ma-Lys-hg-chloromethyl ketone than did 87-kDa PC1. Similar results were obtained in two independent experiments on separate preparations of 74166- and 87-kDa PCls.

Cleavage of Proenkephalin by 74 /66-kDa PC1-The ability of purified 74166-kDa PC1 to cleave recombinant proenkephalin in vitro was compared with that of 87-kDa PC1; cleavage products were detected by Western blotting using antibody against peptide F. We found that 74166- and 87-kDa PC1 produced similar cleavage products and that both enzymes acted in a time-dependent manner (Fig. 8). In agreement with results using RYKR-AMC, at short incubation times 74f66-kDa PC1 was more active against proenkephalin than the 87-kDa form; however, activity at long incubation times was comparatively diminished, indicating a relative lack of stability of truncated PC1 as compared to the 87-kDa form.

DISCUSSION It has been previously shown that the generation of mature

kexin, as well as that of mammalian kexin-like proteinases such as furin, PC1, and PC2, all require proteolytic removal of amino-terminal prosegements, a step that is likely to represent enzyme activation. These cleavages occur at a very early stage of protein biosynthesis, and it is thought that autocatalysis is involved for at least two of these proteinases (Shennan et al.,

A h ,I 1100

301 peak I peak 11,s’ I

Fractions

66 kDa

FIG. 4. Purification of 74/66-kDa PC1. After spontaneous cleavage of PC1, the incubation mixture was applied to a Mono Q column to separate 74/66-kDa PC1 from the 87-kDa form. Panel A shows the eluting gradient and enzymatic activity against RYKR-AMC. Panel B shows the results of Western analysis of PC1 immunoreactivity in the applied sample ( A P , 10 pl), peak I(30 pl), and in peak I1 (10 p1) with PC1 amino-terminally directed antiserum.

Tme of preincubation (h)

FIG. 5. Time dependence of enzymatic activity of PCls against RYKR-AMC. The enzymatic activity of repurified 74/66-kDa and 87- kDa PCls was assayed as described under “Experimental Procedures.” The enzyme was incubated in the reaction buffer for the indicated periods of time prior to addition of fluorogenic substrate. The production of free AMC at the end of the first hour of incubation with RYKR-AMC was used to calculate the specific activity of the enzyme following each preincubation period. Solid line with solid circles, 74/66-kDa PC1; dashed line with open circles, 87-kDa PC1.

1991; Brenner and Fuller, 1992; Leduc et al., 1992; Rehemtulla et al., 1992; Wilcox and Fuller, 1992; Zhou and Lindberg, 1993; Lindberg, 1994). At late biosynthetic stages, carboxyl-terminal cleavages also occur for kexin, furin, and PC1 (Fuller et al., 1989; Hatsuzawa et al., 1992; Vindrola and Lindberg, 1992). The carboxyl-terminal cleavage of kexin is dependent on an intact active site, suggesting that it may be generated by a mechanism similar to that for the amino-terminal cleavage of prokexin (Germain et al., 1992a, 1992b).

In the work presented here, we demonstrate that autocatalysis is possibly involved in the carboxyl-terminal cleavages of PC1. An alternative explanation for the spontaneous generation of the smaller forms is trace contamination of the 87-kDa PC1 preparation with other co-purifying proteinases. This hy-

18412 Carboxyl-terminally Duncated PC1

75

50 m E N

& 25

" 3 4 5 6 7 8 9 1 0 PH

RYKR-AMC. Solid line with solid circles, 74/66-kDa PC1; dashed line FIG. 6. pH dependence of enzymatic activity of PCls against

with open circles, 87-kDa PC1.

Calclum concentration (mM)

FIG. 7. Calcium dependence of enzymatic activity of PCls against RYKR-AMC. Solid line with solid circles, 74166-kDa PC1; dashed line with open circles, 87-kDa PC1.

TABLE I Effect of proteinase inhibitors on different forms of PC1

Inhibitors Final Activity Concentration 87-kDa PC1 74/66-kDa PC1

Control EDTA pCMS

5.0 mM 1.0 mM

1,lO-Phenanthroline 1.0 mM

Pepstatin A 0.1 mM

PMSF 1 mM

TPCK 1.0 mM

TLCK 0.3 mM

0.15 mhf STI" 0.15 mg/ml n-Tyr-Ala-Lys-Arg-CH,CI 0.1 mM

E-64

olo 100

0 0

96 99 98 96 99 98

109 46

%

100 0 0

94 101 97 56 88 93

120 15

" STI, soybean trypsin inhibitor.

pothesis appears to be less likely, since the biochemical properties of the carboxyl-terminal converting activity completely overlapped with those of the catalytic activity of recombinant 87-kDa PC1 throughout purification on hydrophobic interac- tion, ion exchange, and gel exclusion chromatography. In addition, many enzymatic properties of the carboxyl-terminal PC1 cleavage event were similar to those of the RYKR-AMC cleavage reaction of 87-kDa PC1 (Zhou and Lindberg, 19931, such as the calcium dependence and proteinase inhibitor profiles. Moreover, carboxyl-terminal conversion could be inhibited by PC1 substrates. Taken together, these results strongly support the idea that 87-kDa PC1 can be autocatalytically cleaved at its carboxyl terminus to generate 74- and 66-kDa forms. It is un- clear at this time if there is an obligatory precursor-product relationship between the 74- and 66-kDa forms of PC1. In the time-dependent experiment, 74-kDa PC1 is present as an in- termediate form, appearing prior to the 66-kDa form (Fig. 1);

74/66 kDa PC1 87 kDa PC1 FIG. 8. Cleavage of proenkephalin by different PC1 forms. Re-

combinant proenkephalin was cleaved by 0.75 pg of either 74/66-kDa or 87-kDa PC1 obtained though Mono Q chromatography. The products were analyzed by Western blotting using polyclonal antiserum against peptide F. The incubation time (in hours) is indicated above each lane. S, Rainbow molecular weight markers (Amersham Corp.) represent (from top to bottom): 30,000, 21,500, 14,300, and 6,500 Da.

however, we have observed that the generation of 66-kDa PC1 was less affected by variations in calcium concentration, proteinase inhibitors, and even low temperature than was the generation of 74-kDa form. We have detected slow generation of 66-kDa, but not 74-kDa, PC1 in unfrozen (Le. glycerol-containing) samples of 87-kDa PC1 stored at -20 "C. These results indicate that different mechanisms might be involved in the generation of these two forms. It is possible that conformational changes may play a role in the various cleavage events through alteration of the favored cleavage site or by affecting interac- tions between PC1 forms.

Chymotrypsin and trypsin were also able to cleave 87-kDa PC1 at its carboxyl terminus to generate 74- and 66-kDa-like forms, suggesting that the cleavage sites at PC1 carboxyl terminus are located in exposed regions that can readily be at- tacked by other proteinases. Therefore, autocatalysis may not represent the sole mechanism of PC1 carboxyl-terminal conversions in vivo. Further identification of the physiologic mechanism of PC1 carboxyl-terminal cleavage must await site- directed mutagenesis experiments.

We found that carboxyl-terminal cleavages altered the enzymatic properties of PC1 in several respects. First, carboxyl- terminal cleavage initially increased PC1 enzymatic activity against fluorogenic peptide substrates, but decreased long term enzyme stability, as demonstrated using carboxyl-terminally truncated PC1 generated either by limited digestion with chymotrypsin or by spontaneous conversion. This conclusion was also supported by the experiment involving in vitro cleavage of proenkephalin. Second, carboxyl-terminal cleavages of PC1 altered optimal conditions for PC1 activity. 74/66-kDa PC1 required more than 20 mM calcium and a pH between 5.0 and 5.5 for maximal enzymatic activity, as compared to the 87-kDa form, which required only 2 mM calcium and exhibited a broader range between pH 5.0 and 6.5. Third, the susceptibility of PC1 to certain proteinase inhibitors was altered by carboxyl- terminal cleavages.

Although the physiological significance of carboxyl-terminal processing of PC1 is not yet clear, the results described above suggest that it may represent a critical event for PC1 enzymatic activity. Carboxyl-terminal cleavage of 87-kDa PC1 occurs mainly in cells possessing a regulated secretory pathway, and 66-kDa PC1 is the predominant PC1 form within secretory granules (Christie et al., 1991; Vindrola and Lindberg, 1992). Recent data suggest that the carboxyl-terminal processing event occurs after exit from the trans-Golgi network, probably within secretory granules (Milgram and Mains, 1994). It is believed that secretory granules contain about

Carboxyl-terminally Truncated PC1 18413

10-50 mM calcium and a pH of about 5.0-5.5 (discussed by Hutton (1990)). Therefore, secretory granules may not only represent the major locus for PC1 carboxyl-terminal processing, but may also, according to our in vi tro study, provide the optimal conditions for enzymatic activity of carboxyl-terminally truncated PC1. However, our data indicate that the enzymatic activity of carboxyl-terminally cleaved PC1 does not appear to be as stable as that of 87-kDa PC1 under such secretory granule conditions. This lability of recombinant 66-kDa PC1 is in agreement with our observations of endogenous 66-kDa PC1. We have found that the majority of 66-kDa PC1 released from AtT-20 cells by stimulation with secreta- gogues was inactive, while the 87-kDa form appeared to be still active.' We were also unable to use the purification meth- ods described in this paper to purify enzymatically active 66- kDa PC1 from bovine adrenal chromaffin granules,' and other investigators have also failed to demonstrate high levels of calcium-activated enzyme activity within chromaffin granules (Azaryan and Hook, 1992). In line with our kinetic data on the in vitro cleavage of proenkephalin by both forms of PC1, we speculate that within secretory granules, carboxyl-terminally cleaved PC1 (i.e. the 66-kDa form) might play a more important role than the 87-kDa form in total proteolytic processing due to its initially higher specific activity. However, because of the relative lability of this carboxyl-terminally cleaved PC1, the timing of carboxyl-terminal conversion of 87-kDa PC1 to the 66-kDa form may also be important in determining the amount of cleaved prohormone. During in vitro cleavage of proenkephalin, 87-kDa PC1 is concurrently converted to 74- and 66-kDa forms'; this may represent the in vitro counter- part of an actual event occurring in vivo. At this moment, no direct evidence exists to show whether carboxyl-terminal cleavage of PC1 is absolutely required for proper prohormone cleavage. Transfection of mutant forms of PC1 at carboxyl-terminal regions coupled with analysis of prohormone processing will be helpful in determining the relative contributions of the various forms of PC1 to total processing. Such studies are now in progress.

the PC1 RdCMV expression vector, Dr. Elliott Shaw for supplying the Acknowledgments-We thank Dr. Nabil Seidah for initially providing

dibasic chloromethyl ketone peptide, and Dr. David Christie for providing antiserum to peptide F. We also thank Dr. Christopher Batie for many helpful suggestions during the course of this project, Chad Donaldson for help with Western blotting, and Joelle Finley for assist- ance with tissue culture.

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