Bioscience Reports i, 843-849 (1981) 843 Printed in Great Britain

P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of a m i n o - t e r m i n a l p r o - o p i o c o r t i n p e p t i d e s f rom human p i t u i t a r y glands

Charles McLEAN, 3ames HOPE, Philip SALACINSKI, Fernando ESTIVARIZ,* and Philip 3. LOWRY

Department of Chemical Pathology, St. Bartholomew's Hospital, 51-53 Bartholomew Close, London, ECIA 7BE, U.K.

(Received 12 October 1981)

Two cyst ine-containing peptides isolated from human pituitaries were partially sequenced. The peptides are f r a g m e n t s der ived from the amino-terminal of pro- opiocortin (NPOC) and have been chemically charac- t e r i z e d as N P O C 1-28 (which Jacks y-MSH) and NPOC 2-~9. Their availability enables us to investigate new p u t a t i v e biological roles for the amino-terminal conserved sequence of pro-opiocortin.

The hormones corticotropin (ACTH) and 6-1ipotropin (6-LPH) and a l a rge g l y c o p e p t i d e are c l e aved enzymatically from their common p recu r so r , p r o - o p i o c o r t i n ( see Fig. 3), in the cort icotrophes and melanotrophes of the pituitary gland (Eipper & Mains, 1980). Further processing may occur in the intermediate lobe producing small peptides having distinct biological activit ies in some species, e.g. ~-melano- cyte-st imulat ing hormone (~-MSH) from ACTH, and 6-endorphin from 8-LPH. These biologically active peptides are derived from two of the regions of pro-opJocortin which have been highly conserved between species during evolution (Eberle, t981); a third such region is the amino terminal nf pro-opiocortin (NPOC; see Fig. 3) (Nakanishi et al., 1979; Drouin & Goodman, 1980; 5eidah et al., 1980; Chang et al., 19g0). We have shown that NPOC t-76 (see Fig. 3) which we purified from acid extracts of human pituitaries (Estivariz et al., 1980) is one of the molecular forms which circulates in the human (Hope et al., 1981), is released with ACTH and 8-LPH from adenoma cells by h y p o t h a l a m i c e x t r a c t s ( E s t i v a r i z et al. , 1981), and potentiates ACTH-induced steroidogenesis in isolated adrenal ceils (AI-Dujaili et al,., 1981). As a third MSH core sequence occurs at residues 53-58 in this peptide, at tention has naturally been focused on the biological act ivi ty of synthetic peptides derived from this region (Pederson et al., 1980). The sequence NPOC 1-48 (lacking MSH) however, is also highly conserved and is separated from y-MSH by a pair of dibasic

Abbreviations used: NPOC-peptide, amino-terminal pro-opiocortin peptide; i-NPOC, immunoreactive amino-terminal pro-opiocortin; HPLC, high-performance liquid chromatography; TFA, tri-fluoro- acetic acid; PMSF, phenylmethylsulphonylfluoride; EDTA, ethylene- diaminotetra-acetic acid. *Present address: Facultad de Ciencias Medicas, Universidad Nacional de la Plata, Castilla de Correo 455, 1900 La Plata, Argentina.

�9 The Biochemical Society

844 McLEAN ET AL.

amino acids known to divide the precursor into functional domains (Chang et al. , 1980). The existence of NPOCt-#s as a possible product of physiological processing in the intermediate lobe of sheep (Hope & Lowry, 1981) and rat (3ackson & Lowry, 1981) further suggests a biological role for this peptide. This communication reports the purification and partial chemical characterization of two peptides, NPOC t-28 and NPOC 2-59, from human pituitary glands.

Mater ia l s and Methods

Human pituitary glands were collected post-mortem and stored at -20~ Solvents used for high-pressure liquid chromatography (HPLC) and automated sequence analysis were purchased from Rathburn Chemicals (Walkerburn, Scotland). #-Vinyl-pyridine was from Aldrich Co. (Dorset, U.K.), while leucine aminopeptidase (EC 3.#.11.1) and carboxypeptidase C ('carboxypeptidase Y') were from Sigma Chemical Co. (Poole, Dorset, U.K.). All other chemicals were as previously descr ibed (Es t i va r i z et al., 1980). Extraction, fractionation, and centri fugation procedures were carried out at #~ All centrifugations were performed in an MSE 18 centrifuge at 10 000 g for I h.

Frozen human pituitary glands (800 glands; 400 g wet wt.) were homogenized in 0.05 M Tris-HCI, pH 8.5, containing 0.01 M Na2HPO~, 0.005 M EDTA, and 0.0015 M phenylmethyl sulphonylfiuoride. Growth hormone was precipitated as described by 3ones et al. (1979) and ammonium sulphate (120 g) was dissolved in the resulting supernatant D (2.2 l i tres) prior to the addition of ethanol (2.2 l i t res). The precipitate was removed by centrifugation and further ethanol added to the supernatant to a final concentration of 75% (v/v). The resulting p rec ip i t a te was collected by centrifugation, re-dissolved in 0.01 M NH#HCO3, pH 8.5 (1 l i t re) , and centrifuged, and (NH~)250 # (450 g) was dissolved in the supernatant. The precipitate recovered by centrifugation of this 80%-saturated (NH,,)2SO ~ solution was dissolved in 0.01 M NH~HCO 3 (100 ml) and subjected to gel f i l t rat ion.

Chromatography

The glycoprotein extract was loaded onto a column (5 x 90 cm) of Sephadex G-100 gel eJuted at 25 ml h -I with 0.01 M NH#HCO 3, pH 8.5. Fractions were assayed by the fiuorometric method of Hakanson and Sundler (1971) for N-terminal tryptophan peptides (Estivariz et al., 1980) and by a radioimmunoassay for N-terminal pro-opiocortin peptides (Hope et al., 1981). Both the major N-terminal tryptophan peak and the immunoreactive peptide peak co-eluted (between KD = 0.33-0.5) and were pooled and chromatographed on DEAE-cellulose. This column (1.5 x 30 cm) was developed with a linear salt gradient ( total volume g00 ml) from 0.01 M to 0.# M NH~HCO3, pH 9, at a flow rate of 20 ml h-l, and fractions were assayed as before. A main peak containing both N-terminal tryptophan and immunoreactive peptide was recovered (between 0.23 M and 0.33 M NH#HCO 3) and pooled, and the pH of the pool was adjusted to # with 25 M formic acid. The pool was desaited on a column of Sephadex G-15 (5 x 45 cm) equ i l ib ra t ed in 0.01 M ammonium formate (pH 3.0) and the pH adjusted to 7 by the addition of 1 M NH 3 prior to freeze-drying.

P R O - O P I O C O R T I N P E P T I D E S 845

0.6

I o 0.4

<

0.2

I

50 70 90

FRACTIONS

~0

10

30

10

20

o r_

15 E o

E

o_ z .•

g

Fig. I. Ion-exchange chromatography on CM-cellulose of fractions obtained after gel filtration and ion-exchange chromatography of a fractionated Tris-HCl (pH 8.5) extract of human pituitarie s . Absorbed protein was eluted from the column (1 cm x 90 cm) by a linear gradient of 0.01 M to 0.4 M ammonium formate (pH 3.2); the arrow indicates the start of the gradient; buffer flow-rate~ i0 ml h -I. 30-min fractions were collected; a total gradient volume of 800 ml was used. Alternate fractions were assayed for i-NPOC (-O-) and N-Trp peptides (-I-); the u.v. absorbance of the eluted protein is given

by A280 ( ).

The f reeze-dr ied powder was dissolved in 0.005 M ammonium formate, pH 3 (80 ml), and applied to a column (t x 90 cm) ol CM-cellulose equilibrated in 0.0l M ammonium formate, pH 3.2. The column was washed with the equilibration buffer (100 ml) before the application of a linear salt gradient (total volume 800 ml) from 0.01 M to 0.# M ammonium formate, pH 3.2 (Fig. 1). Fractions 80-93 were pooled, mannitol (100 rag) was added, and the mixture was freeze-dried.

Resolution of the mixture of peptides in the freeze-dried powder was achieved by chromatography on a column of Sephadex G-50 (1.5 x 80 cm) eluted with 0.25 M formic acid (Fig. 2).

Chemical characterization

Peptides ! and II (Fig. 2) were analysed for their hexose content and subjected to amino acid analysis after both acid (Estavariz et al., I980) and enzyme (Bennett et al., 197#) hydrolysis. Amino acid analyses were also performed after treatment of each peptide with #-v iny l -pyr id ine in the presence of dithiothreitol (Friedman et al., t970). The pyridyl ethyl cysteinyl (PEC) peptides were desalted on a column ( i x 90 cm) of Biogel P6 eluted with 12.5 M acetic acid and

8#6 McLEAN ET AL.

0.6

I 0.4

<

0.2

30

o 20

0- ~0

h#LPH

I I I

50 60 70

FRACTIONS

I I

80 90

r

40 -5 _E

20 ~

z

0

Fig. 2. Gel filtration on Sephadex G-50 of fractions obtained after cation-exchanged chroma- tography. The column (1.5 x 80 cm) was developed with 0.25 M formic acid at a flow rate of 6 ml h-l; 20-min fractions were collected and alternate fractions were assayed for i-NPOC (-O-) and N-Trp peptides (-m-); the u.v. absorbance of the eluted protein is given by A280 ( ). Two pools I and II were derivatized with PEC and subjected to final purification on HPLC as described in 'Methods'.

purified by HPLC on a column (0.4 x 10 cm) of octadecylsilylsilica (Hypersil ODS, 5 IJm) developed with a linear gradient oi 0-80% aqueous methano l con ta in ing TFA (1%, v /v ) . The amino acid sequence of each of the pur i f ied PEC-pep t ides (30 nmol) was d e t e r m i n e d by Edman degradation (Edman, 1950) of their phenyl-

i so th iocyanate derivatives in a Jeol (U.K. Ltd.) 47-K 6AH automated spinning cup sequencer using the normal protein programme, with 0.5 M Quadrol as coupling buffer and 3 mg Polybrene carrier, as described by McLean and Lowry (1981).

Resu l t s and Discussion

If0 mg of immunoreactive N-terminal pro-opiocortin was extracted irom 800 glands; 30 mg was recovered after Sephadex G-100 in a pool which also contained 1.4 pmol oi N-terminal tryptophan peptides and had an apparent molecular weight between that of 6-LPH (10 000) and f ree a lphasubuni t (22 000) used to ca l i b r a t e the column. Anion-exchange chromatography resolved this pool into two N-terminal tryptophan peptide peaks, each of which contained immunoreactivity. The more acidic and major peak contained 9.1 mg of immunoreactive pep t ide and 0.6 iJmol of / / - t e rmina l tryptophan and was iurther

PRO-OPIOCORTIN PEPTIDES 847

processed by cation-exchange chromatography. The partial resolution of N- te rmina l t r yp tophan peptides from those containing immuno- react ivi ty accomplished by CM-cellulose (Fig. 1) indicated that at least two peptides were present at this stage. Separation of these peptides was achieved by gel filtration on Sephadex G-50 (Fig. 2). A total of 5.6 mg of immunoreactive peptide and 0 .7 IJmol of N-terminal t r y p t o p h a n was loaded onto the G-50 column and the ~.8 mg of immunoreactive peptide recovered (Fig. 2) chromatographed exclusively as the larger-molecular-weight peptide (Peak I, fractions 52-60). The sma l l e r peptide (Peak If, fractions 6g-73) contained 0.~l Mmol of N- te rmina l t r yp tophan , while the remain ing t ryp tophan ac t iv i ty chromatographed with Peak I.

The amino acid analysis of peptide II af ter derivatization with 4-vinyl-pyridine and purification by HPLC was: Asx3, Thr2, Ser3, Glxs, Prop Alal, PECk, Ile[, Leus, Lysl, TrPl , Arg 1. Amino acids absent were- glycine, valine, methionine, tyrosine, phenylalanin% and histidine. The tryptophan content of this peptide calculated from amino acid analysis fully accounted for the 0.41 tJmol of N-terminal tryptophan equ iva l en t s r e c o v e r e d in Peak II. Similarly, peptide I gave the analysis: Asx7, Thr~, Ser6, Glx7, Pros, GIy3, Ala2, PECk, Vall, Met2, Ile i, Leu 6, Tyr 1, Phe 2, His 1, Lys2, Trpp Arg 3. (Each amino a d d value was within 10% of a whole number integer af ter purification on HPLC.)

N e i t h e r p e p t i d e was h o m o g e n e o u s a t t he /7 t e r m i n a l . Aminopeptidase digestion Ior # h at 37~ of samples of each PEC- peptide ind ica ted that for peptide I (Peak I) 23% of the molecules contained N-terminal tryptophan, while the remainder had pyridyl ethyl cysteine at the amino terminus; similarly for peptide II (Peak II) 64% ol the molecules had //-terminal tryptophan and again the remainder h a d d e r i v a t i z e d c y s t e i n e as an N - t e r m i n a l a m i n o a c i d . Carboxypeptidase C digestion Ior 4 h at 37~ released leucine as the unique C-terminal amino acid from peptide II. In contrast, no amino ac ids were r e l ea sed under these conditions from peptide I. The apparent mixture of peptides obtained af ter DEAE-cetlulose chroma- tography did not bind to Con-A-Sepharose; similarly, purified samples of peptides I and II did not bind to Con-A-Sepharose or give an indication of glycosylation in the anthrone test, in contrast to that found for the larger-molecular-weight glycosylated peptide obtained from acid extracts (Estivariz et al., 1980).

From the amino acid analyses, the presence of /~-terminal trypto- phan (and PEC), and the fact that peptide I fully displaced iodinated N-terminal pro-opiocortin from its antibody (Hope et al., 1981), we surmised that peptides I and II were structurally related to each other and to the N-terminal pro-opiocortin glycopeptides we have previously i so l a t ed from acid extracts of human pituitaries (Estivariz et al., 1980). This belief was substantiated by sequence analysis. Both peptides gave the expected tandem sequencing pattern owing to their N-terminal heterogeneity; the first 25 amino acids of peptide II and the f i r s t 10 ol p e p t i d e I were identified. The sequences were identical to each other and agreed substantially with the predicted corresponding bovine sequence (Nakanishi et al., 1 9 7 9 ) . In the amino acid sequence ol peptide II we find glutamic acid at residue 1 9 - instead of alanine predicted in the bovine s e q u e n c e - and arginine at

8#8 McLEAN ET AL.

Trp 1 I , ' 11

Tyr 511,y-MSH62H65 177Gin 3,3-MSH

Trp 1 I 28 Leu Peptide ]1[

cys 21 ' iVMs. I vs0 Pep,.

Zr0 1 I MSH~ '~ NPOCl '~

Fig. 3. The structural relationships between pro-opiocortin (top), and the peptides isolated by acid (NPOC 1-76" Estivariz et al., 1980), and alkaline (peptides I and II) extraction of frozen human pituitary glands~ Solid vertical bars indicate known sites of pairs of dibasic residues (e.g. Lys, Arg); cross hatching is the region bound by a specific NPOC antibody (Hope et al., 1981). The attachment site of a carbohydrate chain is indicated (CHO).

res idue 22 in p lace of g lyc ine predicted from the partial DNA sequence of a human pro-opiocortin gene (Chang et al., 1980), in agreement with Seidah et al. (1980). The amino acid analyses of peptides I and II and of the l- to-76 fragment of /C-terminal pro- opiocortin (Estivariz et al., 1980) are consistent with the substitution of methionine (human) ior valine (bovine) at residue 34 as found by Seidah et al. ( I980).

The two peptides isolated in this study comprise residues 1 to 28 (peptide II) and 2 to 59 (peptide I) (allowing for N-terminal hetero- geneity) of /C-terminal pro-opiocortin (Fig. 3). They are probably derived from the fl-terminal tryptophanyl glycopeptides obtained using acid extraction (Estivariz et al., 1980), by proteolytic cleavage during fractionation and chromatography in mild alkaline conditions, since the major stored form in the adult human pituitary as assessed by our radioimmunoassay (Hope et al., 1981; see also Fig. 3) is NPOCt-76; whether this cleavage takes place physiologically, af ter secretion, is not known and is the subject of further investigation. None of the peptides we have previously isolated (Estivariz et al., 19g0) and those described here have demonstrable hypophysiotrophic activity (Hope & Lowry , 1981) desp i t e the p r e sence of pro-opiocortin peptides in cerebral nerves, especially in the hypothalamus (Bugnon et al., 1979).

PRO-OPIOCORTIN PEPTIDES 8/49

Nei ther does NPOC1-76 potentiate the adrenal weight maintenance act iv i ty of ACTH in hypophysectomized rats (Estivariz et al., 19g0). Known physiological properties of NPOC 1-76, e.g. its roles in steroid- ogenesis and as a weak melanotropin, can be ascribed to the y-MSH region and its carboxyl-terminal extension; no biological role(s) has so far been reported for the conserved amino terminal. We believe that the two peptides isolated here used in conjunction with our specific antiserum (Fig. 3) will help to determine such a physiological role.

R e f e r e n c e s

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Bugnon C, Bloch B, Lenys D & Fellmann D (1979) Cell Tissue Res. 199, 177-196.

Chang ACY, Cochet M & Cohen SN (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 4890-4894.

Drouth J & Goodman HM (1980) Nature 288, 610-613. Eberle AN (1981) in Peptides of the Pars Intermedia, pp 13-31,

Pitman Medical, London. Edman P (1950) Acta. Chem. Scand. 4, 277-278. Eipper BA & Mains RE (1980) Endocrine Reviews I, 1-27. Estivariz FE, Hope J, McLean C & Lowry PJ (1980) Biochem. J. 1919

125-132. Estivariz FE, Gillies G & Lowry PJ (1981) Pharmac. Ther. 13,

61-67. Friedman M, Krull LH & Cavins JF (1970) J. Biol. Chem. 245, 3868-

3871. Hakanson R & Sundler F (1971) Biochem. Pharmacol. 20, 3223-3225. Hope J & Lowry PJ (1981) Front. Horm. Res. 89 44-61 (Greidanus

Tj B van W, ed) Karger, Basel. Hope J, Ratter SJ, Estivariz FE, McLoughlin L & Lowry PJ (1981)

Clinical Endocr. 15, in press. Jackson S & Lowry PJ (1981) Europ. Soc. Comp. End. XI. Abst. 4.5,

54. Jones RL, Benker G, Salacinski PR, Lloyd TJ & Lowry PJ (1979) J.

Endocr. 82, 77-86. McLean C & Lowry PJ (1981) Nature 290, 341-343. Nakanishi S, Inoue A, Kita T, Nakamura M, Chang ACY, Cohen SN &

Numa S (1979) Nature 278, 423-427. Pederson RC, Brownie A & Ling N (1980) Science 208, 1044-1045. Seidah NG, Benjannet S, Routhier R, De Serres G, Rochemont J, Lis

M & Chretien M (1980) Biochem. Biophys. Res. Commun. 95, !417-1424.