Proteolytic processing of theAplysia A peptide precursor in AtT-20 cells

BRAIN RESEARCH ELSEVIER

Brain Research 633 (1994) 53-62

Research Report

Proteolytic processing of the Aplysia A peptide precursor in AtT-20 cells Paolo Paganetti, Richard H. Scheller * Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305, USA (Accepted 10 August 1993)

Abstract

When the Aplysia ELH precursor is expressed in AtT-20 cells, the carboxyterminal derived peptides are packaged and stored in secretory vesicles, while the aminoterminal region of the precursor is constitutively secreted. In contrast, when the highly homologous A peptide precursor is transfected into AtT-20 cells, both aminoterminal and carboxyterminal derived peptides are packaged in storage granules. We propose that this is due to the fact that the initial cleavage of the A peptide precursor occurs more slowly, and perhaps later in the secretory pathway, than the ELH precursor. We further suggest that in the A peptide precursor, the first cleavage occurs after the sorting site resulting in co-packaging of the multiple products derived from a single precursor protein. To determine the structural features of the prohormones responsible for this differential sorting, we made chimeric precursors and determined the rates of the initial cleavage as well as the efficiency of storing the peptide products. From these studies, we conclude that the differential sorting is regulated both by the amino acid sequence of the first processing site, and by more global aspects of the precursor structure.

Key words: Neuropeptide; Protein trafficking; Proteolytic processing; Exocrine gland; Egg-laying behavior

I. Introduction

Proteins destined for the regulated secretory pathway are sorted from constitutively secreted molecules, packaged in secretory granules and stored until an appropriate stimulus triggers their release [12,30]. Various lines of evidence suggest that regulated secretory proteins are actively sorted to granules, whereas some fraction of the transport through the constitutive secretory pathway may occur by bulk flow [25]. The targeting and packaging into regulated secretory granules appears to be tightly linked to the formation of the granule. Prohormone proteins destined for regulated secretion may condense into large aggregates in response to environmental changes, such as an increase in calcium concentration or a decrease in pH, and in this way exclude other proteins [8,17,32,31]. Identification of the specific signals that mediate sorting to the regulated pathway has been difficult [32,40].

* Corresponding author. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 1 5 7 - X

Many proteins are covalently modified as they traverse the secretory pathway. Neuropeptide precursors are often proteolytically processed into several peptide fragments. This processing is closely coordinated with packaging into secretory vesicles [2,21,43]. Recently, three enzymes involved in processing neuropeptides have been identified [33,38,37,42,41]. P C 1 / 3 , PC2 and furin are calcium-dependent serine-proteases each having a catalytic domain related to bacterial subtilisins. P C 1 / 3 and PC2 are restricted to specialized endocrine and neuronal tissues and appear to be sorted into secretory granules. For instance, P C 1 / 3 is expressed at a high level in the anterior lobe of the pituitary, whereas PC2 is found in the intermediate lobe. Interestingly, POMC is processed to A C T H and /3-1ipotropin in the anterior lobe but to a - M S H and CLIP in the intermediate lobe, suggesting a differential function of these enzymes in vivo [3,46]. In addition to the proteolytic processing in secretory granules, some precursor proteins are proteolytically cleaved in the constitutive secretory pathway [4,6,7,9,14,23,49]. The amino acid sequence of these precursors suggests that cleavage usually occurs at a

54

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Fig. 1. Domain structures and processing schemes of the precursors of A peptide or egg-laying hormone ( E L H ) in Aplysia californica. A peptide precursor is synthesized as a 174 amino acid protein starting with a signal sequence (filled squares). The five internal processing sites of A peptide precursor used in the atrial gland are indicated by vertical solid bars. Two peptides are biologically active, A peptide (zig-zag shading) and A-ELH (cross-hatched shading). The carboxytermini of A peptide and A-ELH are amidated. In AtT-20 cells, the single arginine cleavage site on the aminoterminus of A peptide may not be processed. The ELH precursor is synthesized in the bag cells. At least eight internal sites are used during processing. The first cleavage of ELH precursor occurs at a unique tetrabasic site in the trans-Golgi network. Further processing in two distinct secretory granule populations leads to the formation of a-, /3- and y-bag cell peptides and ELH [16]. All peptides and intermediates are labeled.

conserved motif Arg-Xaa-Lys/Arg-Arg [5,18,50]. Mutated proinsulin containing this consensus site is cleaved to biologically active insulin in the constitutive secretory pathway when expressed in COS-7 cells [48]. A good candidate for the enzyme with this activity is the ubiquitously expressed furin, a mammalian homolog of the S. cerevisiae KEX2 gene product [11]. This enzyme appears to be localized in the Golgi and exhibits a preference for the Arg-Xaa-Lys/Arg-Arg consensus sequence [7,18,24]. Thus, it is likely that furin processes a variety of growth factor precursors that contain the consensus site. The processing and packaging of the E L H precursor in bag cell neurons of Aplysia has been studied in some detail (for review see Jung and Scheller, 1991). Following translocation into the endoplasmatic reticulum and removal of the signal sequence [47], proteolytic processing is initiated in the Golgi or in the T G N at a unique tetrabasic site Arg-Arg-Lys-Arg [29]. The so derived aminoterminal and carboxyterminal intermediates (F2 and I3, respectively, Fig. 1) are then segregated from each other and packaged into different classes of secretory granules [10,44]. Following packaging, the intermediates are further processed to

the mature physiologically active bag cell peptides (BCPs) and ELH [19]. The relative level of these peptides is tightly regulated in the bag cells. While the BCPs and ELH are synthesized in equimolar amounts, these peptides are recovered in a 1:5 ratio [10]. In addition to differing in their peptide content, the two populations of secretory granules are also differentially distributed in anatomically and functionally distinct processes of the same cell [45]. Thus, it appears that the first cleavage in the TGN is mandatory for the correct sorting of E L H precursor derived peptides. To understand the mechanisms regulating this complex processing of the ELH precursor, the tetrabasic sitc was mutated and the outcome of this manipulation was analyzed in AtT-20 cells [16,15]. Mutation of the tetrabasic site to a dibasic site or complete deletion of this site did not dramatically impair sorting of the two portions of the precursor. Only after deletion of the tetrabasic site and of an additional tribasic site (Fig. 1), was differential sorting strongly retarded. This resulted in the co-segregation of amino and carboxyterminal derived peptides. To further understand the processing and packaging of prohormones, we have examined the A peptide precursor expressed in the exocrine atrial gland of Aplysia. This precursor is highly homologous to the ELH prohormone, however, it is cleaved in a different pattern and all of the peptide products are packaged in a single vesicle (Fig. 1; [1,13,22,36]). The A peptide precursor does not contain a furin consensus sequence [39] and, therefore, we proposed that proteolytic processing initiates later than the ELH precursor [28,27]. Our results demonstrate that the initial proteolytic cleavage of the A peptide precursor occurs more slowly than the ELH precursor, suggesting cleavage does indeed occur later in the secretory pathway. The data obtained by expressing in AtT-20 cells the A peptide precursor, E L H precursor deletion mutants and three different ELH p r e c u r s o r / A peptide chimeric proteins further support this working hypothesis. Studies of the processing of the chimeric precursors also suggest that structure of the prohormone is an important determinant of the processing.

2. Materials and methods 2.1. Antibodies Rabbit A peptide antiserum was raised against A peptide purified from the Aplysia californica atrial gland [13] as described previously [19]. Rabbit ELH and F3B-C antisera were raised against synthetic peptides and affinity purified as described previously [15]. F3B-C antiserum recognizes an epitope in the ELH precursor aminoterminal intermediate F2. A peptide antiserum was affinity purified as follows: A peptide was isolated from 3-4 atrial glands by acid acetone extraction and by high pressure liquid chromatography

P. Paganetti, R.H. Scheller / Brain Research 633 (1994) 53-62 on a preparative C18 reverse phase column [10]. 2 mg of purified A peptide were coupled to activated agarose (Amino Link Immobilization Kit 1, Pierce 44890, Rockford, IL). 6 ml crude A peptide antiserum in 18 ml of phosphate buffered saline (PBS) were batchincubated with the A peptide resin for 12-15 h at 4°C. The 1 ml column was washed sequentially with 5 vol PBS, 10 vol 0.35 M NaCl in PBS and finally 5 vot of PBS. The specifically bound antibody was eluted with 0.1 mM glycine, pH 2.5 in 1 ml fractions and the adsorption at 280 nm measured. Fractions enriched in antibody were pooled, neutralized with 1.5 M Tris-HC1, pH 8.8 and stored at -20°C in 10% glycerol, 0.5% BSA, 0.05% natrium azide. Approximately 0.2 mg antibody per run were recovered.

2.2. Cell culture AtT-20 cells [25] were cultured in DMEM H-21 (UCSF cell culture facility) supplemented with 10% fetal calf serum and penicillin/streptomycin at 100 units/ml, in an incubator at 12.5% CO2 and 37°C.

2.3. In vitro mutagenesis, plasmid construction and transformations A 1.29 kb cDNA encoding the ELH preprohormone [15] and a 0.98 kb cDNA encoding the A peptide preprohormone (clone M, [39]) were subcloned into the EcoRI site of the Bluescript (pBS-KS-) plasmid polylinker (Stratagene, La Jolla, California), and used to construct deletion mutants and chimeric precursors. /3- and y-bag cell peptide deleted ELH precursor (/3yA) was constructed by inserting two new HpaI sites at position 438 and 681 of the ELH precursor cDNA (the nucleotide or amino acid positions refer to the sequence M29345 deposited in the GenBank) by site-directed mutagenesis [20] using the following oligonucleotides: 5'GCAGTAAAATCGTI'AACACCTI'FCG-3' and 5'-GCAGTVI'CTATGTTAACAGATGAAAACTC-3'. The resulting ELH(HpaIHpaI) cDNA was cut with HpaI and religated to produce an in-frame deletion (81 amino acids) that corresponds to the natural deletion found at the same site in the cDNA for the A peptide precursor (Fig. 1; [22]). The amino acid sequence of /3ya precursor flanking the deletion site is 41RAVKSLTDENSp52. The underlined leucinethreonine amino acids were serine-serine in the ELH precursor and leucine-serine in the A peptide precursor. The truncated ELH precursor (CA) cDNA was obtained by mutating lysine 243 into a stop codon (243 K ~stop) using the oligonucleotide 5 ' - C G T C T C T T G G A A A A G G G C T A G C G G A G T T C T G GCGGC-3'. This produced a shorter ELH precursor terminating at the carboxy end of the ELH peptide. The last amino acid is a glycine residue and, therefore, carboxyterminal amidation of ELH may still occur during its maturation through the regulated secretory pathway, All cDNA constructs obtained through site-directed mutagenesis were sequenced using the dideoxy-chain termination method ([34] Sequenase 2.0 kit, US Biochem. Corp., Cleveland, OH, USA). A peptide/ELH chimeric precursors were constructed using two conserved sites in the A peptide- and ELH precursors (PstI at position 790 and XhoI at position 893 of the ELH precursor cDNA, Fig. 6) and the corresponding conserved PstI and XhoI sites in the pBS-KS- polylinker. Briefly, the pBS-KS-A peptide precursor was cut with PstI and the resulting 460 base pair insert substituted with the 473 base pair Pst I insert isolated from the pBS-KS-ELH precursor to construct the cDNA for AlE1. A l E 2 precursor was obtained by replacing the 615 base pair XhoI insert of pBS-KS-A peptide precursor with the 615 base pair XhoI insert of pBS-KS-A/E1 precursor. A / E 3 precursor was constructed by replacing the 920 base pair XhoI insert of pBS-KS-ELH precursor with the 615 base pair XhoI insert of pBS-KS-A peptide precursor. For expression in AtT-20 cells all cDNA constructs were subcloned in the HindIII and XbaI sites of the p R C / C M V vector

55

(Invitrogen Corp., San Diego, CA, USA) downstream of the cytomegalovirus (CMV) promoter/enhancer and upstream of the bovine growth hormone polyadenylation signal. Transfections were performed by the lipofectin method following the instructions of the manufacturers (DOTAP reagent, Boehringer Mannheim, Germany). 50 p~g plasmid were mixed with 70 ~zg DOTAP, incubated 10 min and diluted in 6 ml D M E M / 1 0 % FCS. This solution was used to feed a 60-80% confluent AtT-20 cell layer in a 10 cm culture plate (Primaria/Falcon #3801, Becton Dickinson, Basel, Switzerland). Geneticin (G418, Gibco BRL, Grand Island, NY, USA) resistant clones were selected with 0.5 m g / m l active drug for 3 weeks. Clones were collected separately and screened for Aplysia californica neuropeptide expression by immunohistochemical staining with affinitypurified antisera followed by a rhodamine-labelled secondary antibody [15].

2.4. Pulse-chase experiments and immunoprecipitations For each construct, we selected transfected AtT-20 cell clones expressing similar levels of the desired precursor protein. In order to demonstrate similar levels of expression, pulse chase experiments were performed in triplicate as follows. 80-90% confluent cell layers in 10 cm culture plates were washed with methionine and cysteine deficient DMEM (Sigma, St. Louis, MO, USA), incubated for 1 h in 3.0 ml of this medium supplemented with 2% dialyzed fetal calf serum (pulse medium), and pulsed for 10 min with 2.5 ml pulse medium/0.1 mCi/ml [35S]methionine and [35S]cysteine (Expre 35S Protein Labelling Mix, NEG-072, NEN-Dupont, Delaware). The pulse was stopped by transferring the culture plates on ice and lysing the cell layers in lysis buffer (1% Nonidet 40, 0.15 M NaCI, 10 mM EDTA, 3 mM PMSF, 50 mM HEPES, pH 7.0) for 30 min on ice. Cell extracts were used for immunoprecipitations as described below. For all other experiments, the cells were prestarved for 30-60 min in pulse medium and pulsed for the times indicated in the figure legends and under the conditions described above. At the end of the pulse, the cells were washed with 3.0 ml of regular DMEM, supplemented with 2% fetal calf serum and 1 mM methionine and 1 mM cysteine (chase medium), and incubated for the times indicated. At the end of the chase, conditioned medium and cell extracts were collected separately and used for immunoprecipitations. In a separate series of experiments, we found that maximal incorporation of radioactive amino acids into neuropeptides occured with a delay of 5 min into the chase period. This is likely to be the time necessary for complete exhaustion of the label from internal stores. Immunoprecipitations, SDS polyacrylamide gel electrophoresis, fluorography, densitometry and quantification were performed as described previously [15]. To obtain the absolute amount of processing intermediates distributed between cells and culture medium during the chase, the value obtained from densitometry analysis was divided by the number of methionine and cysteine residues contained in each fragment.

3. Results 3.1. Expression o f A peptide precursor in A t T - 2 0 cells The anterior pituitary tumor cells AtT-20 were used to express the A peptide precursors from the atrial g l a n d o f Aplysia californica. A c D N A e n c o d i n g t h e A p e p t i d e p r e c u r s o r [22] w a s i n s e r t e d i n t o t h e p R c / C M V v e c t o r w h i c h is d e s i g n e d f o r h i g h l e v e l e x p r e s s i o n i n m a m m a l i a n c e i l s ( s e e S e c t i o n 2). T r a n s f e c t i o n s o f A t T 20 cells w e r e p e r f o r m e d w i t h l i p o f e c t i n . S t a b l e t r a n s -

56

t'. Paganetti, R.tt. Scheller / Brain Research 033 (1994~ 53-62

fectants were selected under restrictive conditions in the presence of the geneticin analog G418. After two to three weeks in culture (7-8 cell divisions), well isolated clones were separately collected and tested for expression of neuropeptides by indirect immunofluorescence with a specific antiserum against ELH which cross-reacts with A-ELH [10,15]. ELH-like immunoreactivity was observed in about 50% of the clones transfected with A peptide precursor cDNA (AP-cells). In these clones, punctate staining was found distributed in the perinuclear region and concentrated in the tips of the neurites (Fig. 2A). A similar staining pattern was observed for the endogenously expressed ACTH (not shown, [26,15]), and in cells expressing ELH precursor (ELH-cells, Fig. 2B and [15]). Thus, this pattern of staining is characteristic of neuropeptides packaged and stored in secretory granules. No ELH-like immunoreactivity was detected in non-transfected or

mock-transfected AtT-20 cells (results not shown). In an attempt to detect peptides derived from the aminoterminus of the precursor, we probed AP-cells with an affinity antiserum raised against purified A peptide [10]. A peptide-like immunoreactivity was strongly localized in the perinuclear region, and again in the tips of the neurites (Fig. 2C). In contrast to this, no A peptide-like immunoreactivity was observed in ELH precursor transfected cells (Fig. 2D) [28]. Jung et al., 1993 reported an identical result for ELH transfected AtT-20 cells using an antiserum against another aminoterminal epitope.

3.2. Intracellular targeting of A-ELH and A peptide To characterize in more detail the posttranslational processing and targeting of A peptide precursor in AtT-20 cells, p u l s e / c h a s e experiments were per-

Fig. 2. Cells expressing A peptide precursor are immunoreactive with A-ELH and A peptide antisera. Fluorescence micrographs of AtT-20 cells stably expressing the A peptide precursor (a, c) and the egg-laying precursor (b, d). Formaldehyde fixed cultures were extracted with the detergent saponin and processed for immunostaining with rabbit antisera raised against ELH (a, b) or A peptide (c, d) and a secondary rhodamine-labelled antibody to rabbit immunoglobulins. ELH- and A peptide immunoreactivity is observed with a punctate staining in the cell body and concentrated in the process tips. ELH-cells did not stain above background levels with the A peptide antiserum (d). Bar = 30 ~zm.

P. Paganetti, R.H. Scheller / Brain Research 633 (1994) 53-62

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Fig. 3. AtT-20 ceils process the A peptide precursor and target peptides to stored secretory granules. AP cells (A) or ELH cells (B) were pulsed for 15 min with radiolabelled amino acids and chased for 5 (lanes 1 and 4) or 120 min (lanes 2 and 5) as indicated. Cell extracts (lanes 1, 2, 4, and 5) and conditioned media (lanes 3 and 6) were immunoprecipitated with ELH antiserum (lanes 1-3) or aminoterminal specific antisera against A peptide (A lanes 4-6) or F3B-C (B lanes 4-6), separated on 20% SDS polyacrylamide electrophoresis and autoradiographed. At 5 rain chase, cells express predominantly precursor proteins (L1 and P), whereas at 120 min of chase, cells contain A-ELH, ELH or 5 kDa A peptide (A2). Partially processed carboxy- and amino-intermediates (A1, F2, I3, and I4) are constitutively secreted in the medium. Bands are as labeled in Fig. 1.

formed. AP-cells were metabolically labelled for 15 min with [35S]methionine and [35S]cysteine and chased for 5 rain or 2 h in the presence of excess unlabelled amino acids. Cell extracts were then prepared and immuno-precipitated with E L H antiserum and protein A sepharose. Again, E L H precursor expressing cells were used for comparison. Fluorography of a representative experiment is shown in Fig. 3. At the beginning of the chase (i.e. 5 rain time point, see methods), the cells produced A peptide precursor migrating with an apparent molecular weight of 19 kDa. After a 2 h chase, A peptide precursor was completely processed to a smaller 4 kDa product. This latter product comigrates on SDS polyacrylamide electrophoresis with E L H (see below), hence, we conclude that this product is A - E L H (Fig. 3A, lanes 1-3). AtT-20 cells synthesized E L H precursor as a 30 kDa protein that is processed to mature 4 kDa E L H during the chase (Fig. 3B, lanes 1-3; [15]). The processing of A peptide precursor and particularly E L H precursor occurs rapidly, since already during the 15 min pulse, some of the precursor proteins were recovered as partially processed intermediates (see 5 min chase). In addition to

57

A-ELH, an aminoterminal A peptide-immunoreactive derivative was found stored in AP-cells. This low molecular weight protein (5 kDa) is likely to have the A peptide carboxyterminus and extend to the aminoterminus of the precursor. In the atrial gland, cleavage on the aminoterminus of the A peptide occurs at a single arginine residue, this cleavage may not take place in AtT-20 cells (Fig. 3 and Fig. 1). Some A peptide precursor escaped targeting to the regulated secretory pathway and was recovered in the medium as a kDa 11 aminoterminal-intermediate. Transfected AtT-20 cells also constitutively secrete the unprocessed precursor of insulin [26] and the amino and carboxyterminal intermediates I3 and F2 of the E L H precursor [16]. Further biochemical evidence that A-ELH is targeted to the regulated secretory pathway and stored in secretory granule was obtained by stimulating AP-cells with secretagogues. AP cells were pulsed for 60 min, chased for 90 rain; these cells were treated with fresh chase medium in the absence or presence of 5 mM 8-bromo-cAMP for 30 min. Cell medium was then immunoprecipitated with E L H antiserum and analyzed by SDS polyacrylamide gel electrophoresis. A - E L H was released from the intracellular stores only upon cAMP-stimulation of intact cells. No aminoterminal products were precipitated from release media (not shown). A set of three independent AP- or ELH-cell clones were used for quantification of stored and released products. The percent of A peptide precursor and E L H precursor processing products recovered at the end of the chase were calculated for each clone with respect to initial counts, present in each of the respective precursors and intermediates. 11 _+ 3% (averaged + S.E.M.) of the initially synthesized A peptide precursor was recovered as stored A-ELH, whereas 56 _+ 10% was constitutively secreted as partially processed carboxyterminal intermediate. In contrast, E L H was more efficiently targeted to secretory granules: 43 _+ 16% of the initial pool of E L H precursor was stored in the cells as mature E L H and 45 + 6% was constitutively secreted as I3. A low, but significant amount of 5 kDa A peptide product was detected at the end of the chase in AP-cell extracts (5_+ 2%). On the basis of these results, we concluded that AtT-20 cells processed A peptide precursor to A - E L H and 5 kDa A peptide which are then stored in secretory granules.

3.3. Subcellular localization and order of A peptide precursor processing The initial cleavage in the T G N appears to play a crucial role for the correct processing and targeting of E L H precursor in AtT-20 cells [15]. Since in the A peptide precursor no furin-consensus sequence was

P. Paganetti, R.ft. Scheller / Brain Research 633 (1994) 53-62

58

identified, we sought to determine if any cleavage of the A peptide precursor occurs prior to the exit from the TGN. To characterize the time course of cleavage of the A peptide precursor, AP cell cultures were pulsed for 5 min, and chased for various times up to a total of 90 min (Fig. 4). Interestingly, the rate of initial cleavage of A peptide precursor is slower than that of E L H precursor. At the beginning of the chase, only 15-30% of A peptide precursor was cleaved to amino and carboxy-intermediates, whereas during the same time period 70-80% of E L H precursor was cleaved to I3 and F2. This is likely a result of early cleavage of the E L H precursor, not later than in the T G N . The initial cleavage of A peptide precursor was virtually completed after 60 min chase. In contrast, cleavage of the E L H precursor was nearly complete after less than 20 rain. In another set of experiments, we examined the first 30 min of chase in detail, these studies established that at 37°C, 50% of A peptide precursor was cleaved in 25 min, whereas 50% of the E L H precursor was cleaved in ~ 7 min. Since the rate of constitutive release of E L H and A peptide precursors are the same, it is unlikely the different rates of initial cleavage are due to different transport times through the secretory pathway. To further investigate the processing of these precursors, we inhibited protein trafficking through the secretory pathway by incubation at 20°C, a temperature known to strongly reduce or block protein exit from the T G N [35]. Under these conditions, proteolytic processing of A peptide precursor is dramatically slowed while processing of the E L H precursor is not altered. The amount of A peptide precursor recovered during the 20°C chase is given in Fig. 4 as a function of time. As much as 52 + 12% of the initial pool of A peptide precursor was still unprocessed after 45 min of chase, and 22 + 2 after 90 min; in contrast, only 22 _+

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8% of E L H precursor remains uncleaved after 5 min chase and the precursor is almost completely processed at 45 min. Thus, cleavage of the A peptide precursor may occur in the T G N , but at a strikingly slower rate than in the case of the E L H precursor. Incubation at 20°C or in the presence of monensin also blocks the further maturation to A - E L H and A peptide (not shown).

3.4. Processing of sorting of E L H deletion mutants and A peptide/ELH precursor chimeras The rate of initial cleavage of A peptide precursor is slower than that of E L H precursor in transfected AtT20 cells. This could be due to the absence of an optimal furin- (or a related TGN-resident enzyme) consensus sequence in the A peptide precursor a n d / o r differences in the structural features of the carboxy and aminoterminal domains. To further understand these issues, we constructed a variety of mutant prohormones. Firstly, we constructed two deletion mutants of the E L H precursor (Fig. 5)/37A precursor lacks /3and 7-BCPs as well as the rest of the 81 amino acid insert excluded from the A peptide precursor. CA precursor lacks the acidic peptide at the carboxy end of ELH. Secondly, we used two conserved restriction enzyme sites flanking the tribasic site of the A peptide precursor as well as the tetrabasic site of the E L H precursor, to construct three chimeric proteins (Fig. 5). The A / E 1 precursor consists of A peptide precursor with the carboxyterminal half, including the tetrabasic site, of the E L H precursor. A / E 2 precursor differs from A peptide precursor only in the region centered around the initial cleavage site and differs by only 3

P. Paganetti, R.H. Seheller / Brain Research 633 (1994) 53-62

Table 2 Targeting of ELH-like immunoreactivity to stored secretory granules. Construct Cell extract Cell m e d i u m

Table 1 Time dependency of initial precursor cleavage at 20°C. Construct

5 min

45 min

90 min

ELH /37/1 CA A/E1 A/E2 A/E3 AP

22 51 69 74 97 97 80

2 12 10 24 55 62 52

0 8 2 10 28 35 27

ELH

43_+ 16 17+ 3 18-+ 3 20_+ 5 12-+ 2 9_+ 1 11-+ 1

flyA CA A/El A/E2 A/E3 AP

Protein export from the trans-Golgi network was blocked by incubation at 20°C. The AtT-20 cell clones expressing the constructs indicated were pulsed for 30 min at 20°C and chased for the indicated times as described in Section 2. Values represent the% of uncleaved precursor with respect to initial counts (i.e. the total initial counts present in each of the respective precursors and intermediates) as a function of chase time. Each construct was analyzed in two to three independent clones, values varied by approximatly 25%.

amino acid substitutions in a stretch of 35 residues. In the A / E 3 precursor a shorter carboxyterminal domain excluding the initial cleavage site was substituted. AtT-20 cell lines expressing these constructs were obtained as described earlier. The level of precursor expression was established by a short pulse (see Section 2). Clones expressing similar levels of protein were chosen to test the rate of initial cleavage at 20°C. At this temperature the cleavages presumably occur in the Golgi or trans-Golgi network (Table 1). The initial cleavage occurred for all constructs with slower kinetics than those of the E L H precursor (2 + 1% intact precursor at 45 min). E L H precursor deletion mutants /3yA and C were cleaved at a relatively rapid rate (12 + 3% and 10 + 3, respectively). A / E 1 was cleaved at an intermediate rate (24 + 3%). A / E 2 and A/E3 chimeric precursors were cleaved at a slow rate (55 + 4% and 62 + 14%, respectively) more similar to the A

5 120

5

59

120

45_ 6 25-+ 2 96-+ 5 20-+ 6 6-+ 1 15_+ 2 56_+10

Cells expressing the construct indicated were tested as described in Section 2. For each construct three independent clones were used. Given are the average v a l u e s + S . E . M , of the % of ELH-like immunoreactivity recovered at the end of the chase in cell extracts or cell media.

peptide precursor. Thus, the introduction of a tetrabasic site in the A peptide precursor (as in A / E 2 ) was not sufficient to accelerate processing in the TGN. Next, we investigated the efficiency of targeting of A - E L H and E L H to the regulated secretory pathway. Cells were pulsed for 15 min and chased for 5 min or 120 min. Cell extracts and cell media were then analyzed by immunoprecipitation with E L H antiserum. Intact precursor proteins were detected at the beginning of the chase, whereas after 120 min, A - E L H or E L H was stored in the cells, however, at different levels (Fig. 6 and Table 2). Maximal efficiency of targeting was observed in ELH-cells (43 _+ 16). flyza-, CA- and A / E l - c e l l s stored E L H about half as efficiently as ELH-cells, whereas A / E 2 - , A / E 3 - and APcells stored A - E L H or E L H at about 1 / 4 the efficiency of ELH-cells. Interestingly, complete recovery of initial immunoreactivity at the end of the chase was found only for the CA precursor, whereas for the other constructs recovery varied considerably. In A / E 2 and A / E 3 transfected cells only about 20% of the initial

5

120

5120

5 120

I

43

m

29 18 - i ~ 14 --II--

m

6.2

I

3.0 - - ~

i

~yA

cA

A/E1

A/E2

A/B3

Fig. 6. Processing and targeting of ELH-like immunoreactivity in E L H precursor deletion mutants and A p e p t i d e / E L H precursor chimeric proteins. Transfected AtT-20 cells expressing the constructs indicated were pulsed and chased as described as in Fig. 3 and the methods. Shown are cell extracts immunoprecipitated with E L H antiserum at 5 min- or 120 min-chase. Apparent molecular weights of the precursors were: flyA - 20 kDa, CA - 27 kDa, A / E 1 - 22 kDa, A / E 2 - 19 kDa, A / E 3 - 22 kDa. The amount of A - E L H or of E L H targeted to storage granules are tabulated in Table 2.

60

P. Puganetti. R.If. Scheller/ Brain Research 633 (1994) 53 ~2

immunoreactivity was recovered. The amount of aminoterminal-like immunoreactivity recovered at the end of the chase was similar for all clones (20-30%), with the exception of the CA-clone (60%). This suggests that varying amounts of the precursors may be degraded, perhaps due to incomplete or partially inaccurate folding of the proteins.

4. Discussion

In this report we investigated the processing of the Aplysia A-peptide precursor. This prohormone is almost 90% identical to the E L H precursor, however, many of the differences between these molecules are specifically localized in regions which effect the posttranslational proteolytic processing of the precursors. For instance, A peptide precursor contains only five potential processing sites compared to nine sites in the E L H precursor. Moreover, a 81 amino acid insert is deleted from the A peptide precursor, so that /3- and T-BCPs are replaced by A peptide. In addition, all of the products of the A peptide precursor are copackaged and co-secreted. In the E L H precursor, the peptide products are differentially packaged in distinct classes of vesicles. The close sequence homology and the large number of differences in processing, packaging, and secretion make these proteins ideal for studies of prohormone processing and sorting. Here, we present evidence that AtT-20 cells also process the exocrine A peptide precursor. After translocation into the endoplasmatic reticulum and transport through the Golgi complex, an initial proteolytic cleavage of A peptide precursor ensued, possibly in the trans-Golgi network, yielding two fragments. These amino- and carboxyterminal intermediates were then both sorted to the regulated pathway and further processed to 5 kDa A peptide and A-ELH. P u l s e / chase experiments showed that sorting efficiency for A - E L H was 11 _+ 1%. Thus, sorting of the A peptide precursor was a factor of 3-4-fold less efficient than the E L H precursor (Table 2). 5 kDa A peptide is likely to be co-segregated with A - E L H in secretory granules. In sharp contrast to this, no aminoterminal derivatives of E L H precursor were stored suggesting sorting to the regulated secretory pathway [15]. As is the case with all peptide precursors studied to date, some of the products resulting from the transfected peptide A precursor gene are constitutively secreted from AtT-20 cells. A significant delay in the proteolytic processing of A peptide precursor compared to that of the E L H precursor was observed. Whereas 50% of this latter precursor was cleaved after about 7 min chase, cleavage of 50% A peptide precursor required approximately 25 min (Fig. 5). As both precursors are constitutivly secreted at the same rate, we propose that this is not due

to a slow rate of transit of A peptide precursor along the secretory pathway. When protein transport was blocked in the T G N by incubation at 20°C or in the presence of monensin, 50% cleavage to amino and carboxyterminal intermediates was completed in about 45 rain for A peptide precursor and in approximately 5 rain for E L H precursor. Thus, the 20°C block significantly slows processing of the A peptide precursor, but had no effect on the ELH precursor. Cleavage of A peptide precursor after a 20°C block of AtT-20 cells was unexpected since in the exocrine cells of the atrial gland of Aplysia californica no proteolytic processing of this precursor occurred in the presence of monensin for up to 20 h [44]. The data suggest that the enzyme responsible for the first cleavage of the A peptide precursor is localized later in the secretory pathway than furin which is thought to produce the first cleavage of the E L H precursor. Perhaps enzymes which are normally most active in granules process the A peptide prohormone at a relatively slow rate in the Golgi region. The level of immunoreactivity r e c o v e r e d / d e c l i n e d during the chase to reach a stable level of 40% (Fig. 5). Accordingly, we assumed that a share of A peptide precursor was degraded, perhaps in lysosomes. Degradation of the aminoterminal derivatives of E L H precursor was observed in Aplysia californica bag cells [10] and possibly also in AtT-20 cells [16]. In order to identify the domains that may either promote the correct processing and sorting of the E L H precursor or delay these events in the A peptide precursor, we deleted the 81 amino acid insert in one construct and the weakly conserved acidic peptide in a second construct (CA- and /3yA-precursors). The processing of these deletion mutants in the T G N was only slightly retarded compared to the wild type precursor (Table 1). On the other hand, the sorting of E L H to secretory granules was reduced to about 50% (Table 2). Thus, deletions affecting domains distant to the E L H precursor were equally potent in delaying processing and reducing targeting efficiency. Interestingly, full recovery of CA-precursor was observed at the end of the chase, suggesting that this construct was not degraded in lysosomes. Finally, we constructed and expressed in AtT-20 cells A peptide and E L H precursor chimeric proteins. We investigated the effect of substitution of the tribasic site in the A peptide precursor for the tetrabasic site along with a 15-20 amino acid region on both its sides of the E L H precursor (A/E2-precursor). Introduction of a furin consensus sequence affected neither the cleavage rate nor the sorting to secretory vesicles (Tables I and II). Thus, the slower initial proteolytic processing of A peptide precursor was not due solely to the absence of a furin-consensus sequence. Substitution of the carboxyterminus of A peptide precursor for

P. Paganetti, R.H. Scheller / Brain Research 633 (1994) 53-62

that of the ELH precursor did not improve the sorting efficiency to secretory granules (Table II; A/E3 precursor). This suggests that neither ELH nor the acidic peptide were alone sufficient to drive an efficient sorting. Moreover, If we included in the A / E 3 construct also the tetrabasic site, the cleavage in the TGN was accelerated and sorting of mature ELH to secretory granules improved considerably (Tables I and II; A / E 1 precursor). All these data appear to favor a scenario where sorting of ELH or A-ELH to the regulated secretory pathway can be governed by a combination of the rate and intracellular location of precursor cleavage. This is most likely determined by higher order structural features of the precursors. These data are generally consistent with the hypothesis that proteolytic cleavages are coordinately regulated along with prohormone packaging into secretory granules. Further, the specific processing site sequences along with overall structural features of the prohormone are important in determining the efficiency of processing and sorting to the regulated secretory pathway. Acknowledgements. The authors wish to thank Dr. Linda Jung for support throughout this study and Harvey Fishman for help with HPLC analysis. This work is supported by a grant to P.P. from the Swiss National Science Foundation Grant NR 823A-028352, and the Julius Klaus-Stiftung Foundation and by a grant to R.H.S. from the National Institutes of Mental Health.

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