THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 23, lsaue of August 15, pp. 11504-11510,1988 Printed in U.S.A.

Effect of Amino Acid Analogs on the Processing of the Pancreatic Polypeptide Precursor in Primary Cell Cultures*

(Received for publication, January 20, 1988)

Thue W. SchwartzS From the Laboratory of Molecular Endocridom. University Department of Clinical Chemistry, Rigshospitalet 6321, DK-2100 Copenhagen, Denmark

Amino acid analogs, which can be incorporated into nascent peptide chains were used in cultures of endo- crine cells from canine pancreas to study the effect on processing of the metabolically labeled precursor for pancreatic polypeptide. Analogs for basic amino acids, canavanine, and aminoethylcysteine prevented the di- basic processing of the prohormone. The polar leucine analog, B-hydroxyleucine, only partially perturbed the function and cleavage of the signal peptide but effi- ciently and unexpectedly blocked the dibasic cleavage of the prohormone. Other nonbasic amino acid analogs, B-hydroxynorvaline and azetidine-2-carboxylic acid, which only could be incorporated into the prohormone at a distance from the processing site, also prevented dibasic cleavage of the prohormone. Although there are no phenylalanine residues in the prohormone, an- alogs for this amino acid, fluoro-phenylalanine and particularly phenylserine, could also block the proc- essing of the prohormone at the dibasic site. This effect was prevented by addition of a small quantity of phen- ylalanine. It is concluded that amino acid analogs can interfere with precursor processing through altering both the primary and the secondary structure of the precursor but also through incorporation into cosyn- thesized protein(s) which are necessary for the precur- sor processing.

Amino acid analogs, i.e. analogs of amino acids which normally occur in proteins, are found in large quantities in certain plant seeds (1). They are believed to serve both as nitrogen stores and as protective agents against herbivores (1, 2). Certain amino acid analogs can be incorporated into nascent protein chains in competition with the normal amino acid, e.g. canavanine for arginine, leading to structurally al- tered proteins and eventually death of the herbivore (1, 2). Normally, aminoacyl-tRNA synthetases are highly selective as they only charge the corresponding tRNA with the in- tended amino acid. However, this specific mechanism can be overridden by amino acid analogs. Apparently, these analogs have not been important in the general evolutionary process to ensure correct protein synthesis, although certain strains of herbivores have actually developed aminoacyl-tRNA syn- thetases which are able to discriminate better between cana- vanine and arginine. Such animals are not affected by the

*This work was supported in part by grants from the Danish Natural Science Research Council, The NOVO Foundation, and the Carlsberg Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertkement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a professorship in molecular endocrinology from the Danish Medical Research Council and the Weimann Foundation.

presence of canavanine in the seeds they eat (3). Amino acid analogs were used as tools in the study of

protein synthesis in the early work on cellular processing of viral proteins (4, 5). A long series of both naturally occurring and synthetic amino acid analogs can compete with common amino acids for incorporation into nascent protein chains (1, 2,643). Direct demonstration of incorporation has been shown both by amino acid analysis and by metabolic labeling with radioactive analogs (5,9-12). Indirect evidence for the incor- poration of analogs in proteins, as reflected in altered chro- matographical and electrophoretical characteristics, have been demonstrated in several studies (6, 8). Within recent years amino acid analogs have proven useful in studies on post-translational modifications of secretory proteins, espe- cially collagen (reviewed in Ref. 13) and regulatory peptides (reviewed in Ref. 14). For example N-linked glycosylation at Asn-Xaa-Thr sequences has been prevented both by the as- paragine analog p-fluoroasparagine (15,16) and the threonine analog p-hydroxynorvaline (17-20).

In the present paper the effect of a series of different amino acid analogs is studied in primary cultures of endocrine pan- creatic cells (21). These cells synthesize the common precur- sor for pancreatic polypeptide and pancreatic icosapeptide, shown in Fig. 1, and process it through both monobasic and dibasic-specific mechanisms (21-23). Inhibition of prohor- mone processing by incorporation of analogs for basic amino acids has been described in several systems (24-29). In pan- creatic polypeptide cells canavanine has previously been used to determine the signal peptide cleavage site of the precursor. This was achieved by amino-terminal sequence determination of the prohormone which accumulated in the presence of the basic amino acid analog (30). As demonstrated in the present paper, not only basic amino acid analogs but also several other amino acid analogs inhibit the dibasic processing of the pre- cursor, even in instances where this cannot be explained by their incorporation in the precursor per se. It is concluded that the analogs can interfere with the cellular processing both through altering the structure of the precursor and the structure of cosynthetized proteins.

EXPERIMENTAL PROCEDURES

Amino Acids L-Canavanine, &hydroxylysine, S-2-aminoethyl-~-cysteine (thi-

alysine), DL-P-hydroxynorvaline (DL-3-hydroxy-2-amino-pentanoic acid), ~-azetidine-2-carboxylic acid, 4-fluoro-~~-phenylalanine, and phenylserine (2-hydroxyphenylalanine) were all purchased from Sigma. 8-Hydroxy-DL-leucine was purchased from U. S. Biochemicals (Cleveland, OH). The purity of all amino acid analogs was confirmed by high performance liquid chromatography of their phenylthiocar- bamyl derivatives as described previously (32). Individual standard amino acids were taken from the Pierce Chemical Co. kit.

11504

Amino Acid Analogs and Peptide Precursor Processing 11505

Met Ala Ala Ala C y s A r g C y s Leu Phe Leu Leu L e u L e u Ser Ala C y s Val Ala L e u L e u 1 10 20

Signal pe Iidase P Leu Gln Pro Pro L e u Gly Thr Arg Gly Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp Asp

30 40

Ala Thr Pro Glu Gln Met Ala Gln Tyr Ala Ala Asp Leu Arg Arg Tyr Ile Asn Met Leu 50 60

Dibasic cleavage

Thr Arg Pro Arg T y r / G l y [ L y s A r g ] A ~ p A r g Gly Glu Met A r g Asp Ile Leu Glu Trp Gly 70

Monobasic cleavage

Ser Pro His Ala Ala Ala Pro Arg Glu Leu Met Asp Glu +

90

t H3N NH

'yff I,.. ..I.

NH

: 0,; F H 2

CH2 CH / \

H 2 N COOH

CANAVANINE

Arg - 'CH2: ,-.

'. ..,

t y 3 F H 2

F H z

: S , , -C.H2

,.I-,

/ \ CH

H 2 N COOH

ETHYL CYSTEINE S-2-AMINO-

I , , , I ,

H , C - . C H ~

N-CH I 1

H COOH I 1

2-CARBOXYLIC ACID AZETlDlNE

F H 2 C H

H 2 N COOH

p- FLUOR0 PHENYLALANINE

/ \

P h e - ' ~ H , j ..

H3C CH3 \ /

:HO,XH ." I CH

H 2 N COOH / \

p -HYDROXY

, \ F H ..

LEUCINE

.~ Leu -I H

80

: ,-- HO-C Q H

I . . I

/ \ C H

H 2 N COOH

PHENYL SERINE

"

Phe-: H ~...,

FIG. 1. The structure of the common precursor for pan- creatic polypeptide and pancreatic icosapeptide and struc- tures of the amino acid analogs used. At the top is shown the amino acid sequence of the canine precursor, as deduced from the cDNA structure (31). The sites for post-translational events are indicated based on previous work (21-23, 30). The "prohormone" indicated in the following figures is the peptide from Ala3' to Gius3. Pancreatic polypeptide, Ala3" to Tyrffi, is carboxyamidated in the secreted form in accordance with the following combined cleavage and amidation site Gly-Lys-ArgW" of the prohormone. The other peptide product of the prohormone, pancreatic icosapeptide, Asp6' to A r c , is cleaved from its intermediate form by a monobasic endopep- tidase activity (23), as indicated by the arrow. In the structural formula for the amino acid analogs, the part differing between the analog and the corresponding normal amino acid is indicated in a broken ring. Below each analog, the structure found in the normal amino acid is shown in a similar ring.

Metabolic Labeling

Preparation of Isolated Cells Single cells were isolated from the duodenal part of canine pancreas

by tryptic digestion in Ca2+-free medium as previously described (21). The endocrine cells were purified on a discontinuous Percoll@gradient (Pharmacia, LKB Biotechnology Inc., Uppsala, Sweden) as described (22). After washing twice, aliquots of approximately 2 X 10' cells were each incubated in 0.5 ml of basal medium Eagle, with Hanks salts, 15% fetal calf serum (GIBCO) minimal essential medium vitamins (GIBCO), 5 mM of glutamine, and 0.1 mM of the following amino acids: Arg, Cys, His, Ile, Leu, Lys, Phe, Thr, Trp, Tyr, and Val. The incubation was performed in an atmosphere of 5% CO2 in 0 2 at 37 'C in flat-bottomed, plastic scintillation counting vials of 5 ml (BN Plastics, Helsinge, Denmark) placed in a metabolic shaker.

Labeling Experiments Cells were washed and preincubated for 15 min in 0.5 ml of medium

lacking both methionine and the normal amino acid corresponding to the analog to be tested. The amino acid analog was then added at

different concentrations, usually at a few millimolars. Control label- ing experiments were performed in the medium lacking the normal amino acid but with no analog added. After 30 min of incubation, ~-[~'S]methionine, 0.25 mCi (Sj 235, Amersham Corp.) was added to each vial. The label was reconstituted in 0.05 ml of medium after removal of the manufacturers' preserving solvent under Nz. The labeling experiments were terminated after 3 h by the addition of 1 ml of ice-cold medium. The cells were then washed twice with 1.5 ml of medium before extraction with 1 ml of 3 M acetic acid in a boiling water bath for 1.5 min. The extract was frozen on dry ice and stored at -20 "C prior to analysis.

Peptide Analysis

Gel Filtration Biosynthetically labeled peptides were characterized on 1.6 X 95-

cm Bio-Gel P-30 columns (Bio-Rad) eluted with acetic acid, 0.5 M, containing 10 mg/liter bovine serum albumin, A4503 (Sigma), and a constant flow of 7.5 ml/h at 4 "C.

Polyacrylamide Gel Electrophoresis SDS-polyacrylamide gel electrophoresis was performed according

to Laemmli (33) using 16% acrylamide, 0.25% bisacrylamide, and 2.5- mm spacers in a Protean I1 cell (Bio-Rad). After trichloroacetic acid precipitation of peptides, the gel was stained with Coomassie Blue G, washed in 5% acetic acid, and dried prior to autoradiography. This was performed at -80 "C for 4 days using XAR-5 film (Kodak).

Enzymatic Digestion of Peptides This was performed on biosynthetically labeled prohormone follow-

ing purification by gel filtration. Tryptic Digestion-The dried peptide and the carrier protein from

the gel filtration eluant were dissolved in 0.25 ml of N-ethylmorpho- line (Pierce Chemical Co.), 0.1 M, containing CaClZ, 0.01 M, adjusted to pH 8.0 with glacial acetic acid (Merck). Tosylphenylalanyl chlo- romethyl ketone-treated trypsin (Worthington), 0.3 nmol, was dis- solved in 0.02 ml of buffer and incubated with the peptide solution at room temperature for 2, 12, 30, and 60 min. The tryptic fragments were purified by gel filtration as described above. The results were calculated by integration of the elution profile of the undigested prohormone and the tryptic fragments.

Endoproteinuse Lys-C-Endoproteinase Lys-C digestion of biosyn- thetically labeled, gel-filtered prohormone was performed at 37 "C for 30, 60, and 120 min in 0.1 M N-ethylmorpholine (sequanal grade, Pierce Chemical Co.) adjusted to pH 8.65 with glacial acetic acid (Merck). The dried prohormone was dissolved in 0.2 ml of buffer and 1.5 nmol, 5 pl, of the enzyme (Boehringer Mannheim) was added. The digestion was terminated by addition of 0.1 ml of aprotinin, 2000 KI units (Bayer, Leverkusen, Federal Republic of Germany). Acetic acid, 0.7 ml, 0.5 M containing 10 mg/liter of bovine serum albumin, was added prior to characterization and quantitation by gel filtration as described above. Digestion of the prohormone by this enzyme results in two well-defined fragments as described previously (23).

Immunoprecipitation Gel filtration fractions were divided into two aliquots and the

solvent removed under vacuum. Labeled peptides were reconstituted in 0.2 M Tris buffer, pH 7.6, containing 2.5% bovine serum albumin, and 1 pg of bovine pancreatic polypeptide, a generous gift from Ronald E. Chance (Eli Lily) was added to one of the aliquots. Immunoprecip- itation was performed by preformed, washed immunocomplexes as described previously (21). The amount of label specifically immuno- precipitated was calculated as the difference in radioactivity precipi- tated in the absence and presence of pancreatic polypeptide.

Calculation of the Effect of Amino Acid Analogs

Inhibition of Protein Synthesis The gel filtration radioactivity profile was integrated corresponding

to the prohormone, the pancreatic polypeptide peak, and the icosa- peptide peak, respectively. The effect of the amino acid analog on protein synthesis was calculated by comparing the total radioactivity in all three peaks in the analog experiment with the total radioactivity in the control experiment. The dose of amino acid analog was calcu- lated as the concentration of L form, assuming that the mixtures contained equal amounts of the D and L forms.

11506 Amino Acid Analogs and Peptide Precursor Processing

[35d Met

Y radio- activity (CPm)

4

2

0 :. + AminoethykCys

40 50 60 70 00 90 100 Gelfiltration fraction, number

100

70 of control

50

0- I I

0.2 0.5 1 2 5 10 20

Amino acid analog,mmol/l

FIG. 2. The effect of lysine analogs on the processing of the prohormone in the pancreatic polypeptide cells. The upper double panel indicates the gel filtration profile, Bio-Gel P-30, of radiolabeled peptides extracted from endocrine cells after incubation for 3 h in the absence (-Lys control) and presence of aminoethyl- cysteine (2.5 mM). The elution positions of the prohormone, pan- creatic polypeptide (PP), and the pancreatic icosapeptide (PI) are indicated. In the lower panel is shown the effect on protein synthesis of different doses of aminoethylcysteine (open circles), 6-hydroxyly- sine (open squares), and the effect on the conversion of prohormone (solid symbols). Details on calculation of the effect of the analogs are given under “Experimental Procedures.”

Inhibition of Prohormone Conversion In each experiment the amount of label in the products, the

pancreatic polypeptide peak, and the icosapeptide peak, were ex- pressed as a fraction of the total radioactivity in all three peaks. The fraction of radioactivity found in the products in experiments with amino acid analogs were then compared with the fraction found in the control experiment.

RESULTS

The present study describes mainly the effect of amino acid analogs on the dibasic processing mechanism which cleaves

100-

Percent of . prohormone. digested . by trypsin

50-

0 control 0 +canavanine

0- 0 2 12

100- Percent of . prohormone. digested by endo-Lys-C

50-

0-

0 30 60 120

Time, minutes

FIG. 3. Time course of digestion of prohormones by endo- peptidases with specificity for basic residues. In the upperpanel, biosynthetically radiolabeled prohormone isolated by gel filtration from control cells and from cells treated with 5 mM canavanine was digested with trypsin at room temperature. In the lower panel pro- hormones from control cells and cells treated with 2.5 mM aminoeth- ylcysteine were digested by endoproteinase-Lys-C at 37 ‘C. For details in enzymatic digestion and calculations see “Experimental Proce- dures.”

the pancreatic polypeptide from its precursor (21-23, 30). It has previously been substantiated that the dibasic cleavage can be followed by gel filtration, as shown in Fig, 2 (23). In this system the prohormone, residues 30-93, is separated from the pancreatic polypeptide, residues 30-65, and from the pancreatic icosapeptide and its intermediate form, residues 69-88 and 69-93, respectively (the two latter peptides coelute during gel filtration).

Effect of Basic Amino Acid Analogs-The rationale for these experiments was to alter the structure of the dibasic cleavage site. Arginines occur at several sites in the precursor, whereas the single lysine of the precursor, L Y S ~ ~ , is found in the cleavage site. Dose-response experiments were performed with the two lysine analogs, aminoethlycysteine and hydroxylysine and the results compared to experiments performed in control medium with no lysine added. Both analogs inhibited the dibasic processing. For aminoethylcysteine the half-maximal inhibitory dose for effect on conversion was 0.5 mM, which had only a minimal effect on protein synthesis. At 2.5 mM prohormone processing was abolished, and protein synthesis was inhibited by approximately 60%, Fig. 2. The ID50 for hydroxylysine was approximately 15 mM. The effective doses of the lysine analogs are similar to those described in experi- ments on bone marrow cells (36). The arginine analog cana- vanine inhibited prohormone conversion at similar doses as aminoethylcysteine and with a similar effect on protein syn- thesis (data not shown) (30). The radiolabeled prohormone which accumulated in the presence of the amino acid analog has previously been shown by radiosequence analysis to have

Amino Acid Analogs and Peptide Precursor Processing 11507

[35S] Met

x 4

radio- activity (CPm)

2

0

d

-Leu control

\ Prohormone

+p-hydroxy-Leu

(5 mmol/l)

% $ I I Prohormone

i o $0 i o 80 90 loo i o Gelfiltration fraction, number

'"1

p-hydroxy-Leu ,mmol/l

FIG. 4. The effect of the polar leucine analog, &hydroxy- leucine, on post-translational processing of the pancreatic polypeptide precursor. The upper double panel indicates the gel filtration profile, Bio-Gel P-30, of radiolabeled peptides extracted from control cells (-Leu control) and cells treated with 5 mM of 0- hydroxyleucine. Total radioactivity (0--0) and radioactivity spe- cifically immunoprecipitated with pancreatic polypeptide antiserum (0--0) are shown. The elution position of the prohormone, pan- creatic polypeptide (PP) and pancreatic icosapeptide (PI) are in&- cated. The arrow points to a minor peak of immunoprecipitable radioactivity found in cells treated with the analog which may rep- resent preprohormone. The lower panel demonstrates the effect of different doses of p-hydroxyleucine on protein synthesis (0) and prohormone processing (0).

an identical amino-terminal sequence to the normal prohor- mone (21,30).

The susceptibility of the prohormone to enzyme digestion was tested in vitro with biosynthetically labeled peptide. Pro- hormone, purified from cells with abolished processing due to canavanine treatment, was at least as susceptible to tryptic digestion as the prohormone isolated from cells in the control experiment, Fig. 3. The prohormone isolated from cells treated with aminoethylcysteine could be cleaved by endopro-

-Prohormone

} PP and PI

FIG. 5. Characterization of radiolabeled proteins from en- docrine pancreatic polypeptide cells treated with amino acid analogs. Autoradiography was performed on a sodium dodecyl sul- fate-polyacrylamide slab-gel of biosynthetically labeled proteins ex- tracted from cells incubated with [36S]methionine. The position taken by the prohormone, pancreatic polypeptide (PP), and pancreatic icosapeptide (PI) in this system are indicated. The small arrows point to some of the radiolabeled proteins which have most markedly changed position or to the position where a protein band is absent in the cells treated with amino acid analogs.

teinase-Lys-C although at a slower rate than the control precursor, Fig. 3. The two fragments generated by digestion of the prohormone from the aminoethylcysteine experiment were similar to the two fragments generated from the control prohormone (data not shown). These experiments show that the prohormone from analog-treated pancreatic polypeptide cells can be cleaved by some endoproteases with specificity for basic residues. In the case of the aminoethylcysteine- labeled prohormone, the cleavage apparently occurs in the dibasic processing site despite the presence of the lysine analog.

The Effect of a Polar Leucine Analog-p-Hydroxyleucine was tested on the basis of its ability to inhibit the transloca- tion and signal-peptidase activity in cell-free translation sys- tems (37-40). Immunoprecipitation of gel filtration fractions from cells treated with 0-hydroxyleucine did show the pres- ence of a biosynthetically labeled molecular form of pancreatic polypeptide which was larger than the prohormone, presum- ably the preprohormone, a peptide which was not found in control cells, Fig. 4. However, even at the highest doses tested, some prohormone was produced indicating that it was not possible in these intact cells to totally prevent the transloca- tion and signal-peptidase activity. The prohormone made in the presence of hydroxyleucine behaved normally both in gel filtration and sodium dodecyl sulfate-gel-electrophoresis, Fig. 5. Unexpectedly, further processing of the prohormone was inhibited by the 0-hydroxyleucine, and in a dose-dependent manner, Fig. 4. At a concentration of 5 mM, the analog was only partially able to inhibit the function of the signal-peptide. However, it could totally abolish the prohormone processing. This is surprising since the 29-residue signal peptide contains

11508 Amino Acid Analogs and Peptide Precursor Processing

Lj"d Met

radio- activity (C Pm)

X

5

0

I $ 1 U Azetidine 2-COOH

I

40 50 60 70 80 90 100

Gelfiltration, fraction number

FIG. 6. The effect of the proline analog, azetidine-2-carbox- ylic acid on prohormone processing in pancreatic polypeptide cells. The gel filtration profile, Bio-Gel P-30, of radiolabeled peptides extracted from control cells (0--0) and cells treated with 10 mM of azetidine-2-carboxylic acid (0--0) are shown. The elution po- sitions of the prohormone, pancreatic polypeptide (PP), and by pancreatic icosapeptide (PI) are indicated.

p5s] Met

rad io - activity

10'~x

(CPm) 10

5

0

40 50 60 70 80 90 100 Gelfiltration, fraction number

FIG. 7. The effect of the phenylalanine analog, 4-flUOrO- phenylalanine, on prohormone processing in pancreatic poly- peptide cells. The gel filtration profile, Bio-Gel P-30, of radiolabeled peptides extracted from control cells (0--0) and from cells treated with 10 mM of 4-fluoro-phenylalanine, parafluoro-Phe (0--0) are shown. The elution positions of the prohormone, pancreatic polypep- tide (PP), and pancreatic icosapeptide (PI) are indicated. Note that although the analog inhibits the incorporation of radiolabel into the prohormone it inhibits the appearence of label in the products to a larger extent.

9 leucine residues of presumed crucial importance for its function, whereas the prohormone contains only 5 leucine residues, and none of these are in close proximity to the dibasic cleavage site, Fig. 1.

The Effect of Other Nonbasic Amino Acid Analogs-The proline residues are important for folding of the pancreatic polypeptide molecule (47) and therefore, presumably also for the folding of the precursor. As shown in Fig. 6, the four- atom-ringed analog of proline, azetidine-2-carboxylic acid, did inhibit prohormone processing. Also the threonine analog, /3- hydroxynorvaline had a similar effect (data not shown). In neither case is the effect of the analog on the dibasic cleavage likely to be caused by the changes in primary structure of the prohormone.

The Effect of Phenylalanine Analogs-The lack of phenyl- alanine residues in the prohormone sequence offers a possi- bility to test whether the inhibition of dibasic processing by

Met X

radio- activity (CPm)

la

5

0

10

5

0

-Phe Control + 5 mmol/l , Phs

I I I I I

+ 20 mmol/l, Phs

n ti

A - Prohormone

, 1 1 1 ,

t ZOmmol/l, Phs t 1 mmoI/t, Phe

I I I I I

70 80 90 100 110 70 80 90 100 110

Gelfiltration, fraction number

FIG. 8. The effect of the polar phenylalanine analog, phen- ylserine (Phs), on the processing of prohormone in pancreatic polypeptide cells. The gel filtration profiles, Bio-Gel P-30, of radi- olabeled peptides extracted from cells in four parallel experiments are shown: control cells (-Phe control), cells treated with 5 mM of phenylserine, with 20 mM of phenylserine, and with 20 mM of phen- ylserine plus 1 mM of phenylalanine. Peptides with an apparent M, = 10,000-1,000 only are shown. The elution position of the prohor- mone, pancreatic polypeptide (PP) and pancreatic icosapeptide (PI) are indicated in one of the profiles.

nonbasic amino acid analogs is mainly caused by changes in secondary structure of the prohormone or changes in the structure of other proteins. Fluorophenylalanine was tested at doses of 10 and 20 mM and did inhibit prohormone proc- essing, but only to a rather small extent, as shown in Fig. 7. However, the more polar phenylalanine analog, phenylserine, tested at doses from 5 to 40 mM, showed a more clear inhibi- tion of the dibasic cleavage, Fig. 8. Inhibition could be seen with 5 mM of phenylserine and an almost total arrest of dibasic cleavage was obtained with 20 mM. When 1 mM of phenylalanine was added on top of the 20 mM of phenylserine, nearly normal processing was observed, Fig. 8. This indicates that the inhibition is not merely a toxic or unspecific effect but is due to competition with phenylalanine. Since there are no phenylalanine residues in the prohormone, the effect of the analogs must be due to alteration of the structure and function of some other protein which is synthetized in parallel with the prohormone.

DISCUSSION

The objectives of the present study were to disturb the structure of newly synthetized prohormone by incorporation of amino acid analogs and to study the effect of this on the processing of the prohormone. As reviewed by Hortin and Boime (14), several problems should be addressed in order to interpret the results of cellular experiments with amino acid analogs. Most importantly incorporation of the analogs into newly synthetized proteins will eventually perturb the folding and function of many cellular proteins and will eventually cause multiple and serious problems for the cells. To prevent

Amino Acid Analogs and Peptide Precursor Processing 11509

this the primary cell cultures were only exposed to the analogs for a rather short period of 3-4 h in the present investigation.

Only certain amino acid analogs are useful in these studies. The analog should be sufficiently similar to its normal coun- terpart to fool the specificity controling elements of the pro- tein synthesis machinery to be incorporated into the nascent protein chains. On the other hand, the analog should also be sufficiently dissimilar to cause notable changes in the struc- ture of the final protein. Hydroxyleucine and phenylserine are examples of analogs which can cause a considerable dis- turbance in the protein structure by introducing a polar group in an apolar side chain. In all cases the experiments with amino acid analogs were performed in medium holding 0.1 mM of the other essential amino acids but lacking the normal amino acid corresponding to the analog. In the case of the phenylalanine analogs, tyrosine was present in the medium whereas phenylalanine was left out. To further investigate the specificity of the analogs, control experiments were per- formed in which a small dose of the normal amino acid, in this case phenylalanine, was added on top of the effective dose of the analog (Fig. 8). That phenylalanine is able to reverse the effect of phenylserine is taken as strong evidence that the analog acts as a specific phenylalanine analog.

The major observation in the present study, that not only basic but also nonbasic amino acid analogs can inhibit the cellular processing of a prohormone at a dibasic cleavage site, can have three possible explanations. 1) The incorporation of the nonbasic amino acid analog could inhibit the processing through the structural changes it creates in the primary sequence of the prohormone at a distance from the dibasic processing site. 2) The analog could disturb the secondary structure of the prohormone. 3) The analog could perturb the structure of some cosynthesized protein which is necessary for the dibasic processing of the prohormone.

Altered Primary Structure of the Prohormone-In previous studies on post-translational modifications of regulatory pep- tides in which amino acid analogs have been used, attention has mainly been directed toward inducing changes directly in known recognition sites in the primary structure of the pro- hormone. In this way basic amino acid analogs have success- fully been used to stop processing at dibasic cleavage sites (24-29). Analogs for asparagine and threonine have been used to inhibit glycosylation at Asn-Xaa-Thr sequences (15-20). Also, an analog for threonine has been used to stop signal- peptide cleavage at a Thr-Leu sequence in the precursor for prolactin (41-43). Finally, the function of the hydrophobic core of the signal peptide has been disturbed by incorporation of the polar leucine analog, P-hydroxyleucine (37-40). In the stated examples, most of the effects of the analogs were conceivably mainly a consequence of the anticipated changes in the primary structure of the prohormone. For example it is assumed that the processing enzyme with specificity for pairs of basic residues cannot cleave the prohormone when the analog is substituted at the processing site. However, this may not be the complete explanation. In the present study the prohormone from the pancreatic polypeptide cells treated with arginine and lysine analogs was still susceptible to cleav- age by endopeptidases with specificity for basic residues (Fig. 3). Apparently this susceptibility may vary among prohor- mones since precursors from anglerfish islets treated with similar analogs are resistant to both trypsin digestion and digestion with proteases from lysed granules (24), whereas pro-opiomelanocortin in similar experiments could be di- gested with trypsin.' In the present study it was also found that several analogs which can be incorporated only at a

P. Loh, personal communication.

distance from the dibasic cleavage site nevertheless can in- hibit the processing. There is no apparent concensus sequence for the amino acids surrounding a dibasic processing site, and it is generally accepted that the processing enzymes do not recognize much more than the pair of basic amino acids (44). Thus, it is unlikely that an analog substituting for leucine more than 6 residues away from the processing site inhibits the cleavage merely by its presence in the primary structure of the prohormone.

Alteration in Secondary Structure of the Prohormone-It has been suggested that the secondary structure of the pre- cursor is important for the processing and that the dibasic processing sites are generally situated in turns in the prohor- mone structure (45, 46). In the case of the prohormone for pancreatic polypeptide, it is indeed very likely that the mole- cule has an ordered three-dimensional structure based on the well-characterized pancreatic polypeptide fold (47). It would, therefore, be tempting to suggest that analogs like hydroxy- leucine, azetidine-2-carboxylic acid, and hydroxynorvaline in- hibit the dibasic processing by altering the secondary struc- ture of the prohormone and thereby rendering the dibasic cleavage site inaccessible to the processing enzyme. It has been shown that incorporation of hydroxyleucine and 4-thia- isoleucine into immunoglobulin light chains prevents, not the glycosylation, but the final processing of the asparagine- linked oligosaccharide moiety to the complex oligosaccharide form (48). This effect could only be explained by alteration of the peptide configuration, as the author had evidence that the carbohydrate-modifying enzymes were not impaired in the system.

Alteration in Cosynthesized Proteid4 Necessary for Proc- essing-In the present study the effect of the phenylalanine analogs cannot be explained by an induced change in either the primary or the secondary structure of the prohormone itself, since there are no phenylalanine residues in the pro- hormone (31). Thus, the phenylalanine analogs, which are incorporated into proteins (5,49), must disturb the structure of some crucial cosynthesized protein(s). The processing en- zymes would be the most likely candidates for such proteins, Although very little is known about the synthesis and turn- over of these enzymes, there is some evidence in favor of the notion that enzymes are synthesized and packed in parallel with the prohormones. The dibasic processing is thought to occur in the newly formed secretory granule (50), and other secretory granule enzymes, the amidating enzyme and the carboxypeptidase-E, are known to be secreted from endocrine cells together with the regulatory peptides (51). The analogs could also interfere with the sorting of the prohormone leading to impaired processing secondary to mis-sorting. However, other prohormones and peptides from amino acid analog- treated cells are secreted almost normally (27, 28). Unfortu- nately, in the present investigation extracellular degradation of secreted peptides in the primary cell cultures prevented similar studies.

It is concluded that amino acid analogs can interfere with the processing of prohormones by both altering the structure of the precursor and that of cosynthesized proteins. In in- stances where the analogs are incorporated into the prohor- mone, the mechanism by which they inhibit the dibasic cleav- age cannot be ascertained with certainty, as the analog could very well affect both the structure of the prohormone and the structure of cosynthesized proteins. The inhibition of prohor- mone processing induced by the phenylalanine analogs, which cannot be incorporated into the prohormone itself, indicates a possible cohort mechanism in the later biosynthetic proc- esses; i.e. that prohormones are synthesized and packed in

11510 Amino Acid Analogs and PC

parallel with proteins necessary for their processing, among these conceivably the processing enzymes.

Ackrwwkdgments-Bente Fisher Friis, Bente Jbrgenson, and Lone Bredo Petersen are gratefully thanked for technical assistance and Henny Jensen for secretarial help. Professor Peder Olesen Larsen is thanked for helpful discussions and Mairead O’Hare for critical reading of the manuscript.

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