The posttranslational modification of β-endorphin in the intermediate pituitary of the toad, Bufo marinus, includes processing at a monobasic cleavage site

Peptides, Vol. 15, No. 8, pp. 1497-1504, 1994 Copyright© 1994ElsevierScienceLtd Printed in the USA.All rightsreserved 0196-9781/94$6.00 + .00

Pergamon 0196-9781(94)00132-4

The Posttranslational Modification of/3-Endorphin in the Intermediate Pituitary of the Toad, Bufo marinus, Includes Processing at a Monobasic Cleavage Site R O B E R T M. D O R E S , z K A R E N G I E S E K E R A N D T A M I C. S T E V E S O N

University o f Denver, Department o f Biological Sciences, Denver, C O 80208 Received 16 J u n e 1994 DORES, R. M., K. GIESEKER AND T. C. STEVESON. The posttranslational modification ofl3-endorphin in the intermediate pituitary of the toad. Bufo marinus, includes processing at a monobasic cleavage site. PEPTIDES 15(8) 1497-1504, 1994.-Fractionation of an acid extract of 15 B. marinus intermediate pituitaries by a combination of gel filtration chromatography and cation exchange chromatography revealed one major and five minor forms of/3-endorphin in this tissue. Based on reversed-phase HPLC and immunological properties, as well as amino acid composition and primary sequence analysis, it was deduced that the sequence of the major form of B. marinus/3-endorphin is N-acetyl-YGGFMTPE.Overall, the steady-state analyses of the minor forms of ~-endorphin indicated that the posttranslational processingof/3-endorphin in the toad intermediate pituitary includes endoproteolytic cleavage at both paired basic and monobasic cleavage sites. fl-Endorphin

Intermediate pituitary

Amphibian

IN the intermediate pituitary of mammals, the posttranslational processing of proopiomelanocortin (POMC) results in the generation of N-acetylated forms of c~-MSH and /3-endorphin (7,47). In addition to the N-acetylation reaction, f3-endorphin also undergoes a series of proteolytic cleavages that yield Cterminally truncated forms of this peptide (33). This set of reactions yields N-acetyl-/3-endorpbin(l-27) and N-acetyl-/3endorphin(1-26) as major end-products in the intermediate pituitaries of several mammalian species (48). Generally, in mammals, forms ofl3-endorphin smaller than 13-endorphin(126) are produced only as minor end-products in the intermediate pituitary (33). /3-Endorphin-specific C-terminal cleavage reactions are not restricted only to mammals. Similar processing events have been observed in the intermediate pituitary systems of reptiles (14,15), ray-finned fishes (16,20,26), a lobe-finned fish (17), and cartilaginous fishes (21,32). For the aforementioned groups of vertebrates, the generation of forms of ~-endorphin with molecular weights less than 2 kDa occurs as a minor processing event. The anuran amphibians are an exception to this generalization. Studies on representatives from three families of anuran amphibians (18,19,42) reveal that the major form of /3-endorphin produced in the intermediate pituitary of these

N-Acetylation

Monobasiccleavage

amphibians is N-acetylated and has an apparent molecular weight of 1.2 kDa. When considering a possible mechanism for generating /3-endorphin-related products from/3-1ipotropin (/~-LPH) in the anuran intermediate pituitary, it would be reasonable to assume that the mechanism would involve the amphibian equivalents of the prohormone convertases, PC1 and PC2, and carboxypeptidase H (1,11,23). The actions of these enzymes should yield /3-endorphin(1-27) and/3-endorphin(1-26) in the anuran intermediate pituitary. To generate the 1.2 kDa form of anuran /3-endorphin, there are at least two possible mechanisms. A carboxypeptidase could trim the larger forms of/3-endorphin to yield the 1.2 kDa form. This mechanism should generate several intermediate forms of/3-endorphin that differ in apparent molecular weight. Alternatively, because an arginine is located at position nine in the sequence of/3-endorphin in at least two species of anuran amphibians (25,34), the generation of the 1.2 kDa form could be due to an endopeptidase with specificity for a monobasic cleavage site. To establish how many steps are needed to accomplish the/3-endorphin-related events observed in the anuran intermediate pituitary, it is essential to characterize the 1.2 kDa form of/3-endorphin, and to identify any intermediate forms of/3-endorphin that may be present.

t Requests for reprints should be addressed to Robert M. Dores.

1497

1498

DORES, GIESEKER AND STEVESON METHOD

Animals and Extraction Procedure Adult male and female Bufi~ marinus (average length 13 cm) were purchased from Charles Sullivan Co., Inc. (Nashville, TN). The animals were held in terraria on a dark background with a 12 h:12 h light:dark cycle at a temperature of 27°C, and an average ambient humidity of 47%. The toads were fed daily. The animals were quickly sacrificed by decapitation, and dissection of the intermediate pituitary was performed as described previously (42). After the dissection, pools of 15 or 20 intermediate pituitaries were separately extracted in 2 ml of 5 N acetic acid that contained 0.3% phenylmethylsufonyl fluoride and mechanically homogenized. These pools were then centrifugated at 12,000 X g for 15 min at 4°C; the supernatant was concentrated under vacuum (Savant Speed Vac) in the presence of 0.2% 2-/3-mercaptoethanol.

Isolation Procedure Each Bufo intermediate pituitary extract was resuspended in 0.5 ml of 50% acetic acid and fractionated by gel filtration chromatography on a Sephadex G-25 column (1 × 40 cm) equilibrated in 10% formic acid. The flow rate was 6 ml/h and the fraction size was 0.6 ml. The column was calibrated with the following synthetic standards, all purchased from Bachem (Torrance, CA): dynorphin A( 1-17), ACTH( 1-13)-NH2, and methionine enkephalin. Aliquots of column fractions were screened with an N-acetylspecific ~3-endorphin RIA and a C-terminal-specific/3-endorphin RIA. Antisera for both RIAs were produced by Hazelton Research Products (Denver, PA). The N-acetyl-speciflc/3-endorphin antiserum was used at a final concentration of 1:20,000. This antiserum fully recognizes human N-acetyl-/3-endorphin(1-31 ), camel N-acetyl-/3-endorphin(1-27), camel N-acetyl-13-endorphin(1-26), and salmon N-acetyl-/3-endorphin(1-16)II, but has less than 0.1% molar cross-reactivity with the nonacetylated forms of these peptides (all peptides were purchased from Bachem) (15). The C-terminal 13-endorphin antiserum was used at a final dilution of 1:10,000. This antiserum fully recognizes human/3-endorphin(1-31 ), camel ¢3-endorphin(1-27), and camel B-endorphin(l-26), but has less than 0.1% molar cross-reactivity with human a-endorphin(1-16) and methionine enkephalin (15). Following the gel filtration analysis, the Bufo/3-endorphinrelated material could be separated into a high molecular weight pool (2.0-3.0 kDa) and a low molecular weight pool (<2.0 kDa). The low and the high molecular weight pools of/3-endorphinrelated material were separately fractionated by cation exchange chromatography on a Bio-Sil CM 2SW column (4.6 mm X 25 cm). The column was eluted with a linear ammonium formate/ 30% acetonitrile gradient (buffer A: 40 m M ammonium formate/ 30% acetonitrile; buffer B: 400 m M ammonium formate/30% acetonitrile). The flow rate was 1 ml/min and fractions were collected at 1-min intervals. Aliquots of each column fraction were screened with the N-acetyl-specific and the C-terminal-specific/3-endorphin RIAs. The cation exchange column was calibrated with the following synthetic standards: Xenopus laevis N-acetyl-13-endorphin(l-8) (synthesized by Macromolecular Resources, Colorado State University, Department of Biochemistry), human N-acetyl-a-endorphin(1-16), mammalian a-MSH, mammalian ACTH( 1-13)-NH2, and human/3-endorphin(1-31) (all purchased from Bachem).

change chromatography was further analyzed by reversed-phase HPLC on a Beckman C-18 ODS column (4.6 mm × 25 cm) [Fig. 3(A)]. The column was equilibrated in 0.1% trifluoroacetic acid and eluted with a nonlinear acetonitrile gradient (buffer B: 0.1% trifluoroacetic acid/80% acetonitrile). Aliquots of column fractions were screened with the N-acetyl-specific/3-endorphin RIA. The synthetic standards, Xenopus laevis N-acetyl-/3-endorphin(l-7), Xenopus laevis N-acety143-endorphin(l-8), and Xenopus laevis N-acetyl-C3-endorphin(1-9) (all synthesized by Macromolecular Products), were chromatographed separately. The major peak of immunoreactivity isolated in Fig. 3(A) was further fractionated by reversed-phase HPLC on a Toyosota 120T column (4.6 mm X 25 cm) equilibrated in 0.1% trifluoroacetic acid and eluted with a linear isopropanol gradient (buffer B: 0.1% trifluoroacetic acid/80% isopropanol) [Fig. 3(B)]. The flow rate was 1 ml/min and fractions were collected at 0.5-rain intervals. The synthetic standards, X. laevis N-acetyl-C3-endorphin(1-8) and X. laevis N-acetyl-~-endorphin(l-9), were chromatographed separately. The major peak ofimmunoreactivity isolated following fractionation on the TFA/isopropanol HPLC system was concentrated for amino acid composition analysis by the Pico-TagT M method (2). The PTH-labeled amino acids were analyzed on a Waters HPLC system equipped with a WISP autosampler system. The amino acid analysis was performed by Macromolecular Resources (Fort Collins, CO). A second extract of 20 intermediate pituitaries also was fractionated as described in the preceding paragraphs, and primary sequence analysis was done on the 1.2 kDa form of~-endorphin. Because the N-terminal of this form is acetylated, it was necessary to remove the blocking group to perform N-terminal sequence analysis. For this procedure, 600 ng of the purified 1.2 kDa form was solubilized in 200 ul of concentrated trifluoroacetic acid, heated to 85°C for 1 h, and concentrated under vacuum (Speed Vac, Savant). The sample then was neutralized with n-methylmorpholine (25 ul), evaporated to dryness, resolubilized in 20 ~1 of MES buffer, pH 5.0, and spotted onto a Sequelon-Aryl amine membrane (Sequelon AA Attachment Kit, Millipore Corp., Bedford, MA). The membrane was initially washed by flowing ethyl acetate over the membrane for 30 s, and then dried by flowing argon over the membrane for 2 min. Using this procedure, the C-terminal amino acid of the sample was covalently linked to the membrane. The membrane was installed into the Blot Cartridge on an ABI 473A Protein Sequencer (Applied Biosystems, Inc.). The sample was then analyzed by Macromolecular Products (Fort Collins, CO) using the Edman degradation procedure and a modified version of the Blot Cycle program.

Further Analysis of the High Molecular Weight Pool of l3-Endorphin The apparent molecular weights of peaks 2, 3, 4, and 5 [Fig. 2(B)] were determined by separately chromatographing an aliquot of each peak on the calibrated Sephadex G-25 column. Peak 6 was chromatographed on a calibrated Sephadex G-50 column. Both columns were equilibrated in 10% formic acid. The Sephadex G-50 column was calibrated with the following synthetic standards, all purchased from Bachem: human ACTH(I-39), human /3-endorphin(l-31), porcine dynorphin A(1-17), and mammalian c~-MSH. RESULTS

Characterization of the 1.2 kDa Form of Bufo 13-Endorphin

Sephadex G-25 Column Chromatograph),

The single peak of immunoreactivity isolated following the fractionation of the low molecular weight pool by cation ex-

An extract of 15 Bujo marinus intermediate pituitaries was fractionated by gel filtration chromatography on a Sephadex G-

AMPHIBIAN N-ACETYLATED /3-ENDORPHIN

1499

25 column as shown in Fig. 1. This procedure yielded two pools of B-endorphin-related material that were designated the low molecular weight pool (fractions 41-50) and the high molecular weight pool (fractions 25-33). The high molecular weight immunoreactive material eluted in a molecular weight range between 2.9 kDa and 1.8 kDa, and was detected with both the N-acetyl-specific fl-endorphin RIA and the C-terminal-specific fl-endorphin RIA. At least two peaks of immunoreactivity were present in the high molecular weight pool at Kay values of 0.09 and 0.24, respectively. The low molecular weight pool of material eluted at a K,v value of 0.69. This immunoreactive material was only detected with the N-acetyl-specific fl-endorphin RIA, and had an apparent molecular weight of 1.2 kDa. The low molecular weight pool and the high molecular weight pool then were separately concentrated for further analysis by cation exchange chromatography.

Characterization of the Low Molecular Weight fl-Endorphin

5OO

ii o E cL

i

4o

300

30

200

20

100

I0

0~

100

m

0 5

--

The low molecular weight pool of B. marinus N-acetyl-/3endorphin-related material isolated by gel filtration chromatography (Fig. 1) eluted as a single peak of immunoreactivity following fractionation by cation exchange chromatography [Fig. 2(A)]. This immunoreactive peak eluted with a retention time similar to synthetic Xenopus laevis N-acetyl-/3-endorphin(1-8) and synthetic human N-acetyl-c~-endorphin(1-16). At pH 2.75, these synthetic peptides have net charges of 0 and +1, respectively. Hence, it appears that neither the Bufo N-acetyl-/3-endorphin-related material nor the two synthetic standards were retained on the cation exchange column. Following cation exchange chromatography, the Bufo N-acetyl-fl-endorphin-related material was further fractionated by reversed-phase HPLC on a Beckman C-18 ODS column equilibrated in 0.1% trifluoroacetic acid and eluted with an acetonitrile gradient [Fig. 3(A)]. Approximately 92% of the immunoreactive Bufo N-acetyl-fl-endorphin eluted with the same retention time (23.5 min) as synthetic Xenopus N-acetyl-/3-endorphin(1-8). No immunoreactive forms were detected with retention times similar to either Xenopus N-acetyl-fl-endorphin(1-7)or Xenopus N-acetyl-fl-endorphin(1-9). The absorbance profile, measured at 220 nm for this chromatogram, revealed several contaminants with

5O

A)

10 15 20 25 30 35 40 45 50 55 60 65 70 50

8)

60

30

40

20

20

10

II.

co

0

5

5

10 15 20 25 30 35 40 45 50 55 60 65 70

Retentlon Tlme (mln.)

FIG. 2. Cation exchange chromatography of the low molecular weight pool and the high molecular weight pool. Fractions from Fig. 1 were pooled, concentrated, and fractionated by cation exchange chromatography on a Bio-Sil CM 2SW column. The column was equilibrated in a 40 mM ammonium formate/30% acetonitrile buffer (pH 2.75). Buffer B was 400 mM ammonium formate/30% acetonitrile.Aliquotsof column fractions were screened with the N-acetyl /3-endorphin RIA (O). (A) Fractionation of the immunoreactive 13-endorphin-relatedmaterial detected in fractions 41-50 of Fig. 1 (low molecular weight pool). (B) Fractionation of the immunoreactive/3-endorphin-relatedmaterialin fractions 25-33 from Fig. 1(high molecular weight pool). The followingstandards were chromatographed separately: (A) Xenopus laevis N-acetyl-B-endorphin(l-8) (net charge, 0) and human N-acetyl-c~-endorphin(l-16) (net charge, + 1); (B) mammalian c~-MSH (net charge, +3); (C) mammalian ACTH(I-13)-NH2 (net charge, +4); and (D) human /3-endorphin(l-31) (net charge, +6).

3OO HighMole~lmrWe~M I:ool

250

= o

200

o.

11111

Low Moluul~ Welg~ Pm~

0=4

t

o.es

0.-A

15

20

25

30

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35

40

45

50

55

60

Fraction Number

FIG. 1. Sephadex G-25 column chromatography. An acid extract of 15 intermediate pituitaries from adult Bufo marinus were fractionated by gel filtration column chromatography on a Sephadex G-25 column. AIiquots of column fractions were screened with the N-acetyl-specific/3endorphin RIA (©) and the C-terminal-specific/3-endorphin RIA (111). The void volume (Vo) and total volume (Vt) were marked with bovine serum albumin and 2-/3-mercaptoethanol. Both internal markers were detected spectrophotometricallyat A28o.

retention times between 22 and 26 min (data not shown). Hence, it was not possible to perform amino acid composition analysis on the major form of Bufo N-acetyl-fl-endorphin following this procedure. In an effort to separate these contaminants from the major form of Bufo N-acetyl-~-endorphin, the immunoreactive peak with a retention time of 23.5 min was chromatographed on a Toyosota 120T column equilibrated in 0.1% trifluoroacetic acid and eluted with an isopropanol gradient [Fig. 3(B)]. Following this step, a single peak of immunoreactivity was detected that eluted with a retention time of 46.5 min. The immunoreactive B. marinus N-acetyl-fl-endorphin eluted with a retention time identical to the retention time of X. laevis N-acetyl-/3-endorphin(1-8), and clearly distinct from the retention time of X. laevis N-acetyl-f/-endorphin(1-9). Two additional extracts of B. marinus intermediate pituitaries were analyzed as described above, and identical reversed-phase HPLC profiles were obtained. The fractions corresponding to the immunoreactive peak in Fig. 3(B) then were pooled for amino acid composition analysis. As shown in Table 1, the amino acid composition of the major

1500

DORES, G I E S E K E R A N D STEVESON 400

40

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ANALYSIS

OF

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Xenopus N-Acetyl-

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15

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Bz([b 1.2 kDa N-Acetyl-

Amino Acid Detected

o 0

ACIDS

BU[O N - a c e t y l - / ~ - e n d o r p h i n

0

tO

TABLE 1 AMINO

Glutamic acid Glycine Threonine Proline Tyrosine Methionine Phenylalanine

fl-Endorphin

~'-Endorphin (1-8) (34)

1.1 ( l ) 1.6 (2) 0.8 ( 1) 1.0 (1) 0.3 ( 1)* 0.2 ( 1)* 2.4 (2)

1 2 1 1 1 1 1

* Estimated.

40

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E

20

11111 5O

m

10

~

0

0 0

5

10

15

20

25

30

35

40

45

50

55

60

Retention Time (mln.)

FIG. 3. Reversed-phase HPLC analysis of the major form of Bujo/3endorphin. (A) The immunoreactive fl-endorphin-related material detected in fractions 4-7 in Fig. 2(A) was concentrated for further fractionation by reversed-phase HPLC on a Beckman C-18 ODS column. The column was equilibrated in 0.1% trifluoroacetic acid. Buffer B was 0.1% trifluoroacetic acid/80% acetonitrile. Aliquots of column fractions were screened with the N-acetyl-specific/3-endorphin RIA (©). The following synthetic standards were chromatographed separately: (arrow A) Xenopus laevis N-acetyl-/3-endorphin( 1-7); (arrow B) Xenopus laevis Nacetyl-~-endorphin( 1-9); and (arrow C) Xenopus laevis N-acetyl-/%endorphin(1-8). (B) The major peak of immunoreactive ~-endorphin detected in (A) was further fractionated by reversed-phase HPLC on a Toyosota 120T column equilibrated in 0.1% trifluoroacetic acid. Buffer B was 0.1% trifluoroacetic acid/80% isopropanol. Aliquots of column fractions were screened with the N-acetyl-specific ~-endorphin RIA (e). The following synthetic standards were chromatographed separately: (arrow A) Xenopus laevis N-acetyl-~-endorphin(1-9): and (arrow B) Xenopus laevis N-acetyl-~-endorphin(1-8).

form of BuJo N-acetyl-/3-endorphin is very similar to the amino acid composition of X. laevis N-acetyl-/3-endorphin(l-8). The Bufo N-acetyl-fl-endorphin contains two glycine residues, one proline residue, one threonine residue, and one glutamic acid residue. Threonine, proline, and glutamic acid residues are located, respectively, at positions 6, 7, and 8 ofX. laevis N-acetyl/3-endorphin(1-8) (Table 4). Although tyrosine was detected, the yield of this amino acid was low. It would appear that the acid hydrolysis procedure did not efficiently deacetylate the N-acetyltyrosine of Bufo ~-endorphin. In addition, the yield of methionine was also low. It appears that a high percentage of the methionine residues were oxidized to the methionine sulfoxide form during the preparation of this sample for acid hydrolysis. The major anomaly in the composition analysis was the apparent detection of two phenylalanine residues. At this point, it was unclear whether a minor contaminant was present in the BuJo N-acetyl-/3-endorphin sample, or whether the spurious phenylalanine peak could be due to a salt artifact generated during the

reversed-phase isolation procedure (C. Miles, Macromolecular Resources, personal communication). In an effort to confirm the primary sequence of the major form ofBt(lb N-acetylated ~-endorphin, an extract of 20 B. marinus intermediate pituitaries was purified as described above. First, an aliquot of the HPkC-purified Bufo N-acetylated ~-endorphin (600 ng) was covalently linked to a Sequelon-Aryl amine membrane. Next, hydrolysis was performed as described in the Method section in an effort to remove the acetyl group from the tyrosine residue at position 1, and still maintain the peptide linkages in the immobilized peptide. As indicated in Table 2, this hydrolysis strategy was partially successful. Beginning with the second cycle of the primary sequence analysis, it was possible to unambiguously detect a single residue at each cycling step, which revealed the sequence G F M T P . It was expected that glutamate, the C-terminal residue of the purified material, would remain attached to the Sequelon-Aryl amine membrane and would not be recovered. In addition, it appeared that the hydrolysis conditions resulted in the removal of the tyrosine residue at position 1 and the glycine residue at position 2 prior to primary sequence analysis. Because two earlier attempts to remove the N-terminal acetyl group had resulted in the complete hydrolysis of the purified Bttfi) ~-endorphin, the partial sequence obtained by the current procedure was considered a success. Collectively, the primary sequence analyses and the reversed-phase HPLC analyses support the conclusion that the sequence of the major form of Bt!lb/3-endorphin is N-acetyl-YGGFMTPE. Given the apparent high degree of primary sequence conservation among amphibian ~3-endorphins (Table 4), at least three

TABLE 2 N-TERMINAL HYDROLYSIS PROCEDURE: PRIMARY SEQUENCE ANALYSIS Cycle l

2 3 4 5 6 7

Amino Acid Detected m

Glycine Phenylalanine Methionine Threonine Proline --

Recovered (pmol) --

0.23 1.25 0.40 0.48 0.91 --

AMPHIBIAN N-ACETYLATED /3-ENDORPHIN

1501

chromatography yielded six distinct peaks of immunoreactivity [Fig. 2(B)]. Peaks 3 and 4 were detected with both the N-acetylspecific and the C-terminal-specific fl-endorphin RIAs, and had net positive charges of +3 and +4, respectively. Peaks 2, 5, and 6 appeared to contain predominantly nonacetylated forms off3endorphin. Overall, these peaks eluted with net positive charges of +2, +6, and +7, respectively. Aliquots of peaks 2, 3, 4, and 5 [Fig. 2(B)] then were separately chromatographed on a Sephadex G-25 column to obtain an estimate of the apparent molecular weight of each peak. An aliquot of peak 6 also was separately chromatographed on a Sephadex G-50 column. The results of these gel filtration analyses are summarized in Table 3. As expected, there was a direct correlation between the net positive charge of the immunoreactive peak and the apparent molecular weight of the immunoreactive peak. The molecular weight for the major form of Bufo/3-endorphin [peak 1; Fig. 2(A)] also is included in Table 3, and is based on the predicted primary sequence of this peptide.

TABLE 3 SUMMARY OF THE FORMS OF Bufo B-ENDORPHIN ISOLATEDBY CATION EXCHANGECHROMATOGRAPHY

Low molecular weight peak High molecular weight peak 2 3 4 5 6

Net Charge (pH 2.75)

Apparent Molecular Weight

0

1.0 kDa ~*

+2 +3 +4 +6 +7

1.8 kDa2i2.3 kDa2t 2.5 kDa2-t 2.9 kDa2t 3.5 kDaS~t

* Based on primary sequence. i" Based on Sephadex G-25 column chromatography. :~Based on Sephadex G-50 column chromatography.

DISCUSSION

mechanisms are proposed that could generate Bufo ~3-endorphin(l-8): a) endoproteolytic cleavage at both the paired basic amino acid processing site (K27,R28)and the putative monobasic processing site (R9) in the primary sequence of amphibian #endorphin could yield a minimum of four intermediate immunoreactive forms; b) cleavage at position 9 to directly yield /3-endorphin(1-8) and 13-endorphin(10-31) could occur; and c) cleavage by a nonspecific carboxypeptidase could yield many C-terminally truncated intermediate forms. The steady-state analysis of the high molecular weight pool ofBufo/3-endorphinrelated material should be useful to resolve these possibilities.

Previous analyses of the POMC system in the intermediate pituitaries of the archeobatrachian anuran amphibians, Bornbina orientalis and Xenopus laevis (18,19), and the neobatrachian anuran amphibian, Bz(fo marinus (42), indicated that the major form of ¢3-endorphin present in these species was N-acetylated and had an apparent molecular weight of 1.2 kDa. Recent studies utilizing HPLC chromatographic criteria (35) and FAB tandem mass spectroscopy (46) support the conclusion that the 1.2 kDa form of/3-endorphin in the intermediate pituitary of X. laevis is N-acetyl-/%endorphin(1-8) [N-acetyl-YGGFMTPE (34)]. The results of the current study also indicate that the sequence of the major form of ~-endorphin in the intermediate pituitary of the toad, B. marinus, is also N-acetyI-YGGFMTPE. Because X. laevis is an archeobatrachian anuran amphibian and B. marinus

Analysis of the High Molecular Weight Pool Fractionation of the high molecular weight pool of/3-endorphin-related material detected in Fig. 1 by cation exchange

TABLE 4 COMPARISON OF B-ENDORPHIN SEQUENCES 1

Human (29) Bovine (30) Ostrich (37) Turkey (9) Xenopus (34) Rana (25) Dogfish (32) Salmon II (26) Salmon I (26)

10

20

30

YGGFMT_SEKSQTPLVTLFKNAIIKNAYKKGE YGGFMT_SEKSQTP_LVTLFKNAIIKNAHKKGQ YGGFMS_SERGRA_PLVTLFKNAIVKSAYKKGQ YGGFMT_SEHSQMP_LLTLFKNAIVKSAYKKGQ YGGFMT_PERSQTP_LMTLFKNAIIKNSHKKGL YGGFMT_PERSQTP_LMTLFKNAIIKKNAHKKGQ YGGFMKSWDERGQKPLLTLFRNVIVKDGEH YGGFMKSWNERSQKP_LLTLFKNVIIKDGQQ YGGFMKPYTKQSHKP_LITLLKHITLKNEQ

PARTIALSEQUENCEOF THE CLEAVAGESITESOF OTHERMONOBAS1C CLEAVAGESUBSTRATES* Dynorphin B Somatostatin-28 PROSRIF-I PROSRIF-II Caerulein Megainin Levitide * From(12).

-RQFKVVT_RSQED_PNAY-EMRLELQRSANSNPAM-AHADLERAASGGPRE-GRMNLERSVDSTNNL-EEVNDREVR_GFGSFLGK-DELEDRDV_RGIGKFLHS-NEDVDRYVR_GWASKIGQ-

1502

DORES, GIESEKER AND STEVESON TABLE 5 COMPARISON OF MONOBASICCLEAVAGEENZYMES

Dynorphin convertase (13) Thiol protease Requires substrates with a basic amino acid located -3, -5, or - 7 position N-terminal to the cleavage site pH optimum: 7.5 Substrate: dynorphin-29 Found in secretory granules Intestinal somatostatin cleavage enzyme (4) Serine protease Cannot cleave substrate ifArg is located at the - 3 position Nterminal to the cleavage site pH optimum: 7.4 Substrate: prosomatostatin Found in secretory granules Yeast YAP3 (3) Aspartyl protease Will cleave prosomatostatin to yield somatostatin-28 Frog skin Arg-X-Val-Arg-Glyendoprotease (28) Inhibited by EDTA Strict requirement for the sequence: Arg-X-Val-Arg-Gly Assay performed at pH 7.4 Found in secretory granules Frog skin endopeptidase (39) Metalloprotease Inhibited by EDTA Cleaves on the N-terminal side of lysine No apparent consensus sequence; recognizesan amphipathic ahelix motif at the cleavage site pH range 6-8 Frog skin XSCEP l endopeptidase (10) Cysteine protease (activated by EDTA) Prefered substrate: Val-Arg-Gly pH optimum: 5.5 (appears to be related to cathepsin B & L) Found in secretory granules

is a neobatrachian anuran amphibian, it would appear that: a) the primary sequence of the N-terminal region of anuran amphibian/3-endorphin has been conserved, and b) the sequence ofposttranslational processing events that results in the cleavage of anuran amphibian/3-endorphin(1-31 ) to yield f3-endorphin( 18) evolved prior to the radiation of the anuran amphibians over 150 million years ago (8). In addition to N-acetyl-/3-endorphin(1-8), at least five other forms of~-endorphin also were detected in extracts ofB. marinus intermediate pituitaries (Fig. 2, Table 3). Given the number of intermediate immunoreactive forms detected in Fig. 2(B), it does not appear that the potential cleavage of Bt~['o/3-endorphin( 131) to yield both ¢/-endorphin(1-8) and/5-endorphin(10-31) as major end-products is a major processing event in this species. However, pulse/chase analyses should be used to confirm that /5-endorphin( 10-31 ) is not a major end-product in this pathway. In addition, the results of the reversed-phase HPEC analysis for peak 1 (Fig. 2) do not support the hypothesis that a nonspecific carboxypeptidase is involved in the processing of Bz¢fo/3-endorphin. Therefore, given the steady-state picture presented in Fig. 2, it would be reasonable to conclude that Bulb/3-endorphin(131) first would be converted to Bz~fi) N-acetyl-~-endorphin(127), and then to Bufo N-acetyl-/3-endorphin(1-26) through the

combined action of the amphibian equivalents of prohormone convertase 2 (PC2), carboxypeptidase H, and a /3-endorphinspecific N-acetyltransferase. These enzymes have been implicated as well in the processing of ~-endorphin in mammalian melanotropes (11,23,24,33,41,45). In support of this hypothesis, PC2 has been cloned from a X. laevis intermediate pituitary cDNA library (5). However, in mammalian melanotropes, the proteolyric cleavage of/3-endorphin is essentially completed with the generation of N-acetyl-/5-endorphin(1-26), whereas in anuran amphibians the major proteolytic cleavage events have yet to happen. Hence, the most parsimonious explanation for the steady-state picture presented in Fig. 2 is that the processing of (5-endorphin in the melanotropic cells of B. marinus includes endoproteolytic processing at a monobasic cleavage site. Cleavage of/5-endorphin at a single arginine residue to generate low molecular weight forms of this peptide is not limited to anuran amphibians. Studies on the processing of~5-endorphin in the intermediate pituitary of the dogfish, Squalus acanthias, indicate that ~-endorphin(1-10) (Table 4) accounts for approximately 15% of the total/3-endorphin isolated from melanotropes (32). Similarly, approximately 10% of salmon/3-endorphin II is processed to a low molecular weight form [Table 4; (42)]. However, the presence of an arginine residue in the N-terminal region of/3-endorphin does not always mandate cleavage at this site. For example, truncated forms of/5-endorphin have not been detected in the pituitary of birds [Table 4; (9,37)]. In addition, an arginine residue is present in the N-terminal region of the ¢~-endorphin isolated from the intermediate pituitary of the squamate reptile, Anolis carolinensis (14), yet low molecular weight forms of/3-endorphin are not detected in this tissue. A comparison of the/3-endorphin sequences in Table 4 suggests that the sequence -E-R-S/G-Q-may define the /5-endorphin monobasic cleavage site. This sequence is not found in avian/5endorphins (Table 4), and it is predicted that this sequence would not be present in reptilian /3-endorphins. Furthermore, the/% endorphin monobasic cleavage reaction appears to have a strict requirement for the presence of an arginine residue at the cleavage site because the substitution of a lysine residue, as seen in mammalian ¢/-endorphins, does not result in cleavage (31). A review of the literature indicates that monobasic cleavage reactions play a significant role in the processing of at least 18 neuropeptides and polypeptide hormones (12). These endoproteolytic reactions fall into two general categories: proline directed (proline residue located C-terminal to the cleavage site), and nonproline directed (40). As seen in Table 4, the ~5-endorphinspecific cleavage reactions would be categorized as proline-directed monobasic cleavage events. When the cleavage sites of a limited number of peptides that undergo monobasic cleavage reactions are compared (Table 4), it is difficult to identify a consensus sequence that explains the processing of all these peptides. Usually, monobasic cleavage reactions require the presence of a basic amino acid located at either the - 3 , - 5 , or - 7 position N-terminal to the cleavage site, as seen in the sequences of dynorphin B, prosomatostatin, and several frog skin peptides (12). However, the anuran amphibian/3-endorphinslack basic amino acids at any of these positions. In addition, there is no apparent sequence homology between the monobasic cleavage sites in the anuran amphibian f3-endorphin and the frog skin peptide sequences presented in Table 4. A similar spectrum of diversity also is seen in the properties of the monobasic cleavage enzymes that have been characterized from a variety of tissues. The unifying feature of all the enzymes listed in Table 5, apart from monobasic cleavage activity, is that these enzymes are found in secretory granules. Overall, several different classes of enzymes appear to be able to perform mono-

A M P H I B I A N N - A C E T Y L A T E D /3-ENDORPHIN

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basic cleavage reactions (i.e., thiol, serine, aspartyl, metalloprotease, and cysteine). Furthermore, these enzymes function over a wide pH range from 5.5 to 8.0. Surprisingly, the enzymes associated with neuroendocrine cells (dynorphin-converting enzyme, intestinal somatostatin endopeptidase) have pH optima at 7.4. Because the pH of secretory granules is acidic (38), it is possible that the monobasic cleavage reaction may occur prior to packaging into secretory granules or early in the maturation of secretory granules. One hypothesis that reconciliates the apparent lack of a clear consensus sequence at monobasic cleavage sites is to propose that monobasic cleavage sites share a c o m m o n secondary structure. Resnick et al. (39) have isolated a monobasic cleavage enzyme from frog skin that cleaves substrates in which the secondary structure around the putative monobasic cleavage site is in an amphipathic a-helix. In fact, an amphipathic a-helix also is found in human/3-endorphin (44). However, this a-helical region is restricted to residues 14-31 of human ¢/-endorphin, well outside the putative monobasic cleavage site (position 9). Based on these observations, it would appear that there are subcategories of monobasic cleavage reactions. Hence, the

~-endorphins appear to define a unique subcategory ofprolinedirected monobasic cleavage reactions. If the degree of processing is a good indicator of the concentration of the 13-endorphinspecific monobasic cleavage enzyme, then the anuran amphibian intermediate pituitary appears to be an excellent model system for studying this reaction. In this regard, a final possibility to consider is that in the intermediate pituitary of anuran amphibians, prohormone convertases may be involved in mediating monobasic cleavage reactions. Mbikay et al. (36) suggest that PC I could cleave some growth factors at monobasic cleavage sites. Furthermore, Brenner and Fuller (6) have observed that the yeast endoprotease, Kex2, has weak monobasic cleavage activity. Finally, Dupuy et al. (22) have observed that coexpression of PC1 and prodynorphin in rat pheochromocytoma cells resuited in the cleavage of prodynorphin at a monobasic cleavage site. Although m a m m a l i a n PC2 appears to lack monobasic cleavage activity (1,11), it is possible that amphibian PC2 (5) or the putative amphibian PC1 (27) have evolved this activity. ACKNOWLEDGEMENTS This research was supported by NIH Grant RR06565 and NSF Grant IBN 9412115.

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41. Seidah, N. G.; Marcinkiewicz, M.; Benjannet, S.; et al. Cloning and primary sequence of a mouse candidate prohormone convertase PC I homologous to PC2, furin, and Kex2: Distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2. Mol. Endocrinol. 5:111-122; 1991. 42. Steveson, T. C.: Jennett, C. L.; Dores, R. M. Detection of N-acetylated forms of ~-endorphin and nonacetylated a-MSH in the intermediate pituitary of the toad, Bt~/~9marinus. Peptides 11:797803; 1990. 43. Takahashi, A.: Kawauchi, H.; Mouri, T.; Sasaki, A. Chemical and immunological characterization of salmon endorphins. Gen. Comp. Endocrinol. 53:381-388; 1984. 44. Taylor, J. W.; Miller, R. J.; Kaiser, E. T. Characterization of an amphiphilic helical structure in /3-endorphin through the design, synthesis, and study of model peptides. J. Biol. Chem. 258:44644471; 1983. 45. Thomas. k.; Leduc, R.; Throne, B. A.; Smeekens, S. P.: Steiner, D. F.; Thomas, G. Kex2-1ike endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: Evidence for a common core of neuroendocrine processing enzymes. Proc. Natl. Acad. Sci. USA 88:5297-5301; 1991. 46. van Strien, F. J. C.; Jenks, B. G.; Heerma, W.: Versluis, C.; Kawauchi, H.: Roubos, E. W. a,N-acetyl /3-endorphin [1-8] is the terminal product of processing of endorphins in the melanotrope cells of Xenopus laevis, as demonstrated by FAB tandem mass spectrometry. Biochem. Biophys. Res. Commun. 191:262-268; 1993. 47. Zakarian, S.; Smyth, D. G. Distribution of active and inactive forms ofendorphins in rat pituitary and brain. Proc. Natl. Acad. Sci. USA 76:3972-3976; 1979. 48. Zakarian, S.: Smyth, D. G. Distribution of/3-endorphin related peptides in rat pituitary and brain. Biochem. J. 202:561-571: 1982.