Studies on the disulfide bonds in human pituitary follicle-stimulating hormone

428

Biochimica et Biophysica Acta, 6 2 4 ( 1 9 8 0 ) 4 2 8 - - 4 3 5 © Elsevier/North-Holland Biomedical Press

BBA 38488 STUDIES ON THE DISULFIDE BONDS IN HUMAN PITUITARY FOLLICLE-STIMULATING HORMONE

YUKIO FUJIKI *, PREMILARATHNAMand BRIJ B. SAXENA** Division of Endocrinology, Department of Medicine, and Department of Obstetrics and Gynecology, CorneU University Medical College, 1300 York Ave., New York, N Y 10021 (U.S.A.)

(Received January 2nd,

1980)

Key words: Follicle-stimulating hormone; Disulfide bond

Summary Human follicle-stimulating hormone (FSH) was digested with subtilisin, thermolysin, cyanogen bromide, pronase and trypsin to isolate the cystine-containing peptides. These peptides were purified by gel filtration through Sephadex G-50 column and by high-voltage paper electrophoresis at pH 6, 3.5 and/or 2. The location of the cystine~containing peptides in human FSH a- and ~-subunits was established by amino acid composition, end-group analysis and deterruination of the amino acid sequence by Edman degradation. The results indicate that the disulfide bonds are present between half~ystine residues located between positions 7 and 10, 28 and 87 and 82 and 84 in the a-subunit, and between positions 3 and 28, 17 and 51 and 32 and 104 in the ~-subunit of human FSH. Introduction The amino acid sequences of the a- and ~-subunits of human FSH have been established [1,2]. The a- and ~subunits of human FSH contain 10 and 12 residues of half-cystine, respectively. Since no free sulfhydryl groups are present in the subunits, all the cysteine residues must be linked as disulfide bridges. The Supplementary data to this article are deposited with, and can be obt a i ne d from, Elsevier/North-Holland Biomedical Press B.y., BBA Data Deposition, P.O. Box 1345, 1000 BH A ms t e rda m, The Netherlands. Reference should be made to No. B B A / D D 1 5 1 / 3 8 4 8 8 / 6 2 4 (1980) 428. The s u p p l e m e n t a r y i n f o r m a t i o n includes: isolation of cystine-contalnlng peptides from the chemical and e n z y m a t i c digests of h u m a n FSH (Figs. 2 and 3) and the identification of each cystlne-contain/ng peptide. * Present address: The Rockefeller University, New York, NY 10021, U.S.A. ** To w h o m correspondence should be addressed.

429

determination of the disulfide bridges is essential in the elucidation of the conformation of the subunits of FSH, which may have important bearing on the interaction of the molecule at the receptor site to express its biological activity. Partial determination of the disulfide bridges in luteinizing hormone (LH) [3--9] and in thyroid-stimulating hormone (TSH) [5,9] has been reported. We describe here studies on the partial characterization of the disulfide bonds of the a- and ~-subunits of human pituitary FSH. Materials and Methods Highly purified human pituitary FSH (113 mg, equivalent to approx. 3.5 #mol) was used for enzymatic degradation as shown in Scheme I. All enzymHuman FSH, 113 mg Digestion with subtilisin and thermolysin0 pH 6.5 S e p h a d e x G - 5 O l ( s u p e r f i n e ) ( F i g . 2) * I

II + I l I

IV

V + VI

VII

VIII

IX

X-XII

CNBr Digestion Digestion with pronase and trypsin, pH 6.5 S e p h a d e x G - 5 O l ( S u p e r f i n e ) (Fig. 3) * I

II

IIl

IV

V + VI

VII

VIII

IX

X + XI

H i g h - v o l t a g e p a p ~ e l e c t r o p h o r e s i s a t p H 6, 3 . 5 , a n d / o r 2 ¢~ : 8 2 - 8 4 : 3-28

a : 7-10 a : 28-87 : 17-51

a : 31, 32-59, 60 ~ : 32-104

S c h e m e I. I s o l a t i o n o f c y s t i n e - c o n t a i n i n g p e p t i d e s f r o m h u m a n F S H .

atic digests were carried out under nitrogen. The FSH was dissolved in 5 ml 0.1 M pyridine acetate buffer (pH 6.5) and digested with 1.9 mg subtilisin in 0.1 ml buffer (Protease Carlsberg, Type VIII, Sigma), at 37°C for 9 h (FSH/ subtilisin = 1 : 50, M/M). The digest was made 6 M in urea and 1 mM in CaC12 and was again incubated with an additional aliquot of 4.35 mg subtilisin (M/M = 1 : 15) for 16 h at 37°C. The incubate was further digested at 40°C with 6.1 mg thermolysin ( M / M = 1 : 20) (Calbiochem) in the presence of 8 M urea and 2 mM CaC12. Another aliquot of 6.1 mg thermolysin was added after 6 h and the incubation was continued for 24 h at 40°C. At the end of the incubation, the digest was adjusted to pH 3 with 1 M acetic acid.

Gel filtration o f sub~ilisin-thermolytic digest o f FSH on Sephadex G.50 superfine (Fig. 2) * The enzyme digest was applied to a 1.8 X 250 cm column of Sephadex G-50 superfine, previously equilibrated with 0.1 M acetic acid. The column was * See footnote

on p. 428.

430

eluted with 0.1 M acetic acid with a flow rate of 6.8 ml/h. Fractions of 3.4 ml each were collected. The eluate was monitored simultaneously at 280 as well as at 206 nm (Uvicord III, LKB Instrument, Stockholm, Sweden) to locate the peptides. The content of cystine-containing peptides in each of the pooled fractions was determined by means of performic acid oxidation followed by amino acid analysis. (The performic acid was prepared by mixing 1 vol. 30% H20~ and 9 vols. 98% HCOOH. The mixture was kept for 1 h at room temperature, then chilled in an ice bath and 1 vol. CH3OH and 0.1 vol. 80% phenol were added.) Aliquots of the fractions to be analyzed were oxidized by reaction with 50 pl of the performic acid solution for 2.5 h at 0°C; the reagent was removed by drying the reactants in vacuo in a dessicator [10]. The performic acid-oxidized fractions were then subjected to acid hydrolysis in 5.7 N glassdistilled HCI at 105°C for 24 h. The acid hydrolysates were dried in vacuo and analyzed by an automatic amino acid analyzer (Model D-500, Durrum Instruments).

Digestion of fractions H and III from the Sephadex G-50 (Fig. 2) column with cyanogen bromide, trypsin and pronase Fractions II and III, containing large molecular weight material, were pooled, lyophilized and dissolved in 1.5 ml 70% HCOOH. 66 mg CNBr in 60 ~l 70% HCOOH was added to the pooled fractions and the mixture was incubated under N2 for 24 h at room temperature with constant stirring. The incubate was suspended in 13.5 ml distilled H20 and lyophilized. The material was resuspended in 2 ml H:O and relyophilized. The CNBr digest was then dissolved in 2 ml of 0.1 M pyridine acetate buffer, pH 6.5, containing 5 mM CaCI: and digested with 0.2 mg pronase (Calbiochem) at 40°C. After 24 h, another aliquot of 0.2 mg pronase was added and the digestion was continued for an additional 24 h at 40°C. An aliquot of 20 #1 of a 1% solution of L-(1-tosylamido2-phenyl)ethyl chloromethyl ketone-treated trypsin (Worthington) in 4 mM HC1, containing 20 mM CaC12, was then added to the solution and the incubation was extended for an additional 24 h at 37°C. Gel filtration of CNBr.pronase-tryptic digest on Sephadex G-50 (Fig. 3) * The digest was centrifuged and the supernatant was gel-filtered through a 1.8 X 250 cm column of Sephadex G-50 (superfine) equilibrated with 0.1 M acetic acid. The column was eluted at a flow rate of 7.2 ml/h to collect peptidecontaining fractions.

Purification of the peptide fractions The fractionsIV--XII from gel-filtrationof the subtflisin-therrnolyticdigest on Sephadex G-50 column (Fig.2) and peptide-containingfractions obtained from the gel-filtrationof fractionsII and III afterfurthertreatment with CNBr, trypsin and pronase (Fig.3), were individuallypurified by high-voltagepaper electrophoresis at pH 6 and at pH 3.5 [I], to isolate peptides. After elution from the paper with 30% acetic acid, known aliquots of the isolated peptides

* S e e f o o t n o t e o n p. 4 2 8 .

431

were oxidized with performic acid [10], in order to convert cystine to cysteic acid prior to hydrolysis with 5.7 N HC1 and amino acid analysis.

Identification of the cystine-containing peptides In order to cleave the disulfide bonds and to separate and identify the peptide pairs linked in the isolated cystine-peptides, aliquots of the peptides were oxidized with performic acid, or reduced with 2-mercaptoethanol and then carboxy-methylated [11]. The resultant peptide pairs were separated by highvoltage paper electrophoresis at pH 2, or at pH 3.5, and were eluted from the paper. The peptides were identified by amino acid composition. The •amino acid sequences were determined whenever the peptides were obtained in quantities sufficient for Edman degradation. Aliquots of the cystine-containing peptides were also subjected to Edman degradation with and without prior oxidation with performic acid. If Edman degradation were performed without prior oxidation, the peptides were oxidized with performic acid prior to the amino acid analysis. Cystine~ontaining peptides were also analyzed for amino- and carboxylterminal amino acid residues by the use of leucine aminopeptidase and carboxypeptidases A and B [12]. Results

Isolation of the peptides The gel filtration of the subtilisin and thermolysin digest of human FSH is shown in Fig. 2. The fractions IV--XII did not contain any cystine-containing peptides. Fraction IV yielded the a78 glycopeptide on further purification by high-voltage paper electrophoresis. Fractions II and III contained large molecular weight material, representing undigested portion of the FSH and were, therefore, subjected to chemical degradation with CNBr followed by digestions with pronase and trypsin. The gel filtration pattern of the CNBr-pronase~trypsin digest of fractions II and III on Sephadex G-50 is shown in Fig. 3. Fractions I and II contained large molecular weight or undigested material. The cystine~containing glycopeptides were separated from fractions III and IV as shown in Scheme I. The other cystine-containing peptides were isolated from fractions V, VI and VII. No cystine-containing peptide was recovered from fractions VIII--XI.

Identification of the disulfide pairs in the cystine-containing peptides The details of the experimental data for the identification of each cystinecontaining peptide are given in the Supplementary information *. From human FSH~, the disulfide pairs of a 7 ~ 1 0 , (a31, a 3 2 ~ 5 9 , a60), a 8 2 ~ 8 4 and a28a87, were identified (Fig. 1). A cystine-containing peptide, glycine
432

r_~S'S---~ 20 Ala-Pro-Asp-Val-Glu-Asp-~ -Pro-Glu-~ -Thr-Leu-Gln-Glu-Asn-Pro-Phe-Phe-Ser-Gin-Pro-Gly3O Ala-Pro-Ile-Leu-Gln-~ -Met-Gly-~-~ - [ ~ -Phe-Ser-Arg-Ala-Tyr-Pro-Thr-Pro-Leu°A 40 rg-Se~Lys50 60 Lys-Thr-Met-Leu-Vai-Gln-LysoAsn(CHO)-Val-Thr-Ser-Glu-Ser-Thr- [ ~ - [ ' ~ -VAI-Ala-Lys-Ser-

70

so

~1:::s-s~

Tyr-Asn-Arg-Val-Thr-Val-Met-Gly-Gly-Phe-Lys-Val-Glu-Asn(CHO)-His-Thr-Alac~C_~-His-[Cys~-Ser9O Thr- ~--~-s]-Tyr-Tyr-ttis-Lys-Ser human

FSH.~

lO ;o -G|u-Leu-Thr-Asn(CHO)-Ile-Thr-ne-Ala-lle-Glu-Lys-Glu-Glu-Arg-Ph -Leua9 Asn-Ser& 30 [ Thr-Ile-Asn(CHO)oThr-Thr-Trp-~y-~-Ala-Gly-Tyr-~-~-Tyr-Thr-Arg-Asp-Leu-Val-ryr-Lys-Asn~50 ~

"~

. . . .

Pro-Ala-Arg-Pro-Lys-ile-Gln-Lys-Thr- [Cys]-Thr-Phe-Lys-Glu-Leu~V~l-Ty~Glu-Thr-Val-Arg-ValPro-Gly- ICys]-Ala-His-His-Ala-Asp-Ser-Leu-Tyr-Thr-Tyr-Pro-Val-Ala-Thr-~n-~ ~ys I-His-ICys]-GIF90 100 Lys-V~-Asp-Ser-Asp-Ser-Thr-Asp- ~ - ~ -Thr-Val-Arg-Gly-Leu-Gly-Pro-Ser-Tyr-~-~-Ser-Phe-Gly110 118 Glu-Met-Lys-Gln-Tyr-Pro-Thr-Ala-Leu-Ser-Tyr human FSH-~ Fig. 1. A m i n o acid s e q u e n c e s of h u m a n F S H - ~ and j3-subunits s h o w i n g the disulfide linkages d e t e r m i n e d in this s t u d y . *o data d e p o s i t i o n .

phoresis. Two peptides were identified as Gly-Cys-Cys in sequence a30--32 and Thr-Cys-Cys in sequence a58--60, respectively, i.e., two disulfide bonds linking residues a31, a32 to either a59 and a60, or a60 and a59. Thus from a total of six disulfide pairs in human FSH-/~, so far the three pairs, viz.,/33-~28, {317-/351 and ~32-~104 could be identified (Fig. 1). Discussion The human FSH preparation used in these studies contained a biological activity of 9000 IU of 2nd International Reference Preparation of Human Menopausal Gonadotropin/mg as determined by ovarian augmentation method [13]. The high biological activity of the FSH was considered as evidence for little change in the conformation of the molecule during purification. Since amino acid sequences of the subunits of human FSH have already been established, we have used intact FSH in this study, to avoid the losses of material during isolation of the subunits and thus obtain maximum yields of cystine-containing peptides. The possibility of the disulfide interchange in this study was minimized by keeping the pH at or below 6.5 during the experimental procedures. Minor disulfide exchange occurring during the purification procedures, however, could not be completely ruled out. Subtilisin and thermolysin, used in the digestion of human FSH, retain their protease activity in 6 M and 8 M urea solutions, respectively. The subtilisin and thermolysin do not contain any half-cystine residues, thus there was no possibility of contamina-

433 tion with cystine-containing peptides from the enzymes used for proteolytic degradation. Following the digestion of FSH with subtilisin and thermolysin, a large amount of material remained undigested as shown in fractions II and III obtained by gel filtration on Sephadex G-50 (Fig. 2). Hence fractions II and III had to be further digested to obtain cystine peptides. Similar to our findings, the sequential enzymatic digestion of porcine LH-a with pepsin, trypsin, chymotrypsin and the digestion of bovine LH-a with subtilisin had also resulted in undigested material containing the cystines [4,14]. These observations suggest a highly globular nature of glycoprotein hormones as indicated by earlier physicochemical studies. The subunits of FSH also appear to be of tightly folded configuration even in the presence of 8 M urea, which is further supported by the observations that the CD spectra of the ~-subunit of ovine LH were not affected by the presence of denaturants [15]. In this study, all the peptides containing the five disulfide pairs of the a-subunit were recovered. However, only three disulfide pairs of the /~-subunit, namely, ~3-28, ~17-51 and ~32-104, could be identified. The inability to recover peptides containing the remaining three pairs in the ~-subunit may indicate incomplete digestion of the ~-subunit or the paucity and excessive losses of the material during purification by high-voltage paper electrophoresis and especially during elution of the peptides from the paper. There are two cysteine residues at positions 7 and 10 in the first ten residues at the N-terminal of the human FSH-a subunit. Equine FSH-a subunit contains cysteine residues homologous to the human FSH-a subunit, except that it lacks the f~st ten amino acid residues at the N-terminal. The equine FSH~ subunit, however, does not contain a free sulfhydryl group [16]. This also suggests that the cysteines at positions 7 and 10 are linked together in the human FSH-a subunit, since a deletion of the first ten amino acids will not create a free sulfhydryl group. The assignment of the disulfide linkage between residues a82 and a84 in Cys-His-Cys, is in agreement with that between residues a86 and a88 of bovine LH~ [5] (Table I). Even though this disulfide bond represents an unusual linkage, it is stereochemically possible. When the peptide bonds are in trans configuration, the imidazole ring of the histidine must be projected completely away from the peptide backbone. The unusually low pK of His-aS7 in porcine LH-a(compatible to Hiss83 in human FSH~) might be due to the above steric property [17,18]. A comparison between the a-subunit vs. the ~-subunit (Fig. 1) suggests that the ~-subunit may be more tightly coiled. This is in concurrence with the greater accessibility of the disulfide bonds of the a-subunits in human chorionic gonadotropin, human LH and human TSH to reduction, where the rate of reduction of the a-subunit also exceeded that of the ~-subunits [19--21]. The similarities found in the sequences of the subunits of human FSH, LH, TSH and human chorionic gonadotropin on the alignment of cysteine residues [19,22,23] may suggest that the disulfide bridges in each hormone may be the same. Although the amino acid sequences of a- and ~-subunits from LH contain some similarities to human FSH, a comparison of the disulfide bridges is not always in agreement with either those of FSH or within those of the subunits of LH as reported from different investigators (Table I) [ 3--9].

434 TABLE I THE LOCATION OF DISULFIDE BONDS IN HUMAN FSH AND LH Cysteine residues o f disulfide linkage in LH are c o m p a t i b l e w i t h t h o s e o f h u m a n FSH. Dim~flde linkage in p a r e n t h e ~ is d e t e r m i n e d b y elimination Human FSH-~

7-10 28-87 31 ~ r5 9 32~-~60~ 82-84

Ovine LH-~ (3)

7-60 10-82 28-84 31-59 (32-87)

Porcine LH-~ (4)

7-84 10.60 28-31 59-82 (32-87)

Bovine LH-~ (5,6)

Human FSH-~

7-31 10-32 28-60 59-87 82-84

3- 28 17- 51 32-104

Ovlne LH-~ (7)

3- 32 17- 66 20-104 51- 82 87- 94 (28-84)

Ovine LH-~ (8)

3- 84 17- 66 20-104 28- 82 87- 94 (32-51)

Bovine LH-~ (9)

87-94

The three disulfide bonds in ovine LH-~ (cysteine residues 23 and 72, 26 and 110, 93 and 100, i.e comparable to cysteine residues at positions 17 and 66, 20 and 104, 87 and 94 in human FSH-fl) obtained by the conventional procedures without partial acid hydrolysis (Table I) are in good agreement between two laboratories [7,8]. The localization of the S-S bonds in human FSH-~ in the present study are not exactly those in ovine LH-~ and bovine LH-~ in terms of the number of the residue [7--9], however, it should be noted that they exist in the same vicinity. Table I also shows species differences in the disulfide bonds of ovine, porcine and bovine LH~ and LH-~ subunits. It is not clear at this time whether these differences are real, especially those in hormonespecific ~-subunits, or caused by disulfide interchanges during experimental procedures. These studies, however, provide the first insight into the disulfide bonds of FSH. Acknowledgements This study was supported by the National Institute of Health (Grant No. HD06543) and The Ford Foundation (Grant No. 670-0455A). Thanks are due to Ms. Inessa Hakker for excellent technical assistance and Mr. Anthony Tolvo for able assistance with the amino acid analyses. References 1 2 3 4 5 6 7 8 9 10 11 12 13

R a t h n a m , P. and S a x e n a , B.B. (1975) J. Biol. Chem. 250, 6735--6746 Saxena, B.B. and R a t h n a m , P. (1976) J. Biol. Chem. 2 5 1 , 9 9 3 - - 1 0 0 5 Chung, D., Salram, M.R. and IA, C.H. (1973) Arch. B i o c h e m . Biophy$. 159,678---682 Combarnous, Y. and H e n n e n , G. (1974) Biochem. Soc. Trans. 2, 915---917 CorneB, J.S. and Pierce, J.G. (1974) J. Biol. Chem. 249, 4 1 6 6 - - 4 1 7 4 Giudiee, L.C. and Pierce, J.G. (1979) J. Biol. Chem. 254, 1164--1169 Chung, D., Saizam, M.R. and I.A, CM. (1975) Int. J. Peptide Protein Res. 7 , 4 8 7 - - 4 9 3 Tsunasawa, S., Liu, W.K., Burleigh, B.D. a n d Ward, D.N. (1977) B i o c h i m . B i o p h y s . A c t a 4 9 2 , 3 4 0 - 356 Reeve, J.R., Chen, K.W. and Pierce, J.G. (1975) B i o c h e m . B i o p h y s . Res. Commun. 6 7 , 1 4 9 - - 1 5 5 Liao, T.H., Sainikow, J., Moore, S. and Stein, W.H. (1973) J. Biol. Chem. 248, 1 4 8 9 - - 1 4 9 5 Crestfleld, A.M., Moore, S. and Stein, W.H. (1963) J. Biol. Chem. 238, 622--627 Salnikow, J., Liao, T.H., Moore, S. and Stein, W.H. (1973) J. Biol. Chem. 248, 1480---1488 Steelman, S.L. and P o h l e y , F.M. (1953) Endocrinology 53, 604---616

435 14 Giudice, L.C. and Pierce, J.G. (1978) iJn Structure and Function of the Gonadotropins (McKerns, K.W., ed.), pp. 81--110, Plenum Press, New York 15 Pernollet, J.C. and Gamier, J. (1971) FEBS Lett. 18,189--192 16 Rathnam, P., Fujiki, Y., Landefeld, T. and Saxena, B.B. (1978) J. Biol. Chem. 253, 5355--5362 17 Maghuin-Rogtster, G., Degelaen, J. and Roberts, G.C.K. (1978) FEBS Lett. 87, 247--250 18 Maghuln-Rogister, G., Degelaen, J. and Roberts, G.C.K. (1979) Eur. J. Blochem. 96, 59--68 19 Pierce, J.G., Faith, M.R., Giudice, L.C. and Reeve, J.R. (1976) in Polypeptide Hormones: Molecular and Cenul~r Aspects (Ciba Foundation Symposium 41, new series), pp. 225--250, FAsevier/Excerpta Medlca fNorth-Holland, Amsterdam 20 Holmgren, A. and Morgen, F.J. (1976) Eur. J. Biochem. 70, 377--383 21 Pierce, J.G., Gludlce, L.C. and Reeve, J.R. (1976) J. Biol. Chem. 251, 6388--6391 22 Stewart, M. and Stewart, F. (1977) J. Mol. Biol. 116, 175--179 23 Saxena, B.B. and Rathnam, P. (1978) in Structure and Function of the Gonadotroplns (McKerns, K.W., ed.), pp. 183--212, Plenum Press, New York