THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 250, No. 13, Issue of July 10, pp. 5247-5258, 1975

Printed in U.S.A.

The Amino Acid Sequence of Human Chorionic Gonadotropin THE a SUBUNIT AND 6 SUBUNIT*

(Received for publication, July 15, 1974)

FRANCIS J. MORGAN,+ STEVEN BIRKEN, AND ROBERT E. CANFIELD

From the Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032

The amino acid sequences of both the cy and p subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the 01 subunit is: Ala Pro Asp - Val Gln Asp - Cys Pro - Glu

- Ck! - Thr Leu Gln Glu - Asp - Pro Phe - Phe - Ser G?i - Pro - Gly Ala - Pro - Ile - Leu - Gln Cys - 30

Met - Gly Cys - Cys - Phe - Ser Arg Ala Tyr Pro Thr - P% Leu - Arg Ser - Lys - Lys Thr - Met - 50

Leu Val - Gln Lys Asn - Val - Thr - Ser - Glu Ser Thr - Cys C$! - Val - Ala Lys - Ser Tyr - Asn

Arg Val - Thr 4: - Met - Gly Gly Phe Lys - Val - Glu Asn - His TEy Ala - Cys His - Cys - Ser -

Thr - Cys Tyr - Tyr - F?ii Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH,-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The

amino acid sequence of the p subunit is: Ser Lys - Glu Pro Leu Arg Pro - Arg - Cys Ai; - Pro Ile - 20 30

Asn Ala - Thr - Leu - Ala - Val - Glu Lys Glu Gly - Cys - Pro Val Cys Ile - Thr - Val - Asn - Thr - 50

Thr - Ile - Cys - Ala - Gly - Tyr Cys - Pro - Ttt Met Thr - Arg - Val - Leu - Gln Gly Val - Leu - Pro -

Ala - Leu Pro Gln - Val - Val Cys Asn Tyr A!: - Asp Val - Arg Phe - Glu - Ser Ile Arg - Leu - 80

P?g Gly - Cys - Pro - Arg - Gly - Val - Asn Pro Val Val - Ser - Tyr Ala - Val - Ala - Leu Ser Cys -

Gln - C$ - Ala - Leu - Cys - Arg - Arg - Ser Thr Thr - Asp ?$ - Gly - Gly Pro - Lys - Asp His Pro - 110 120

Leu Thr Cys Asp - Asp Pro Arg Phe - Gln - Asp Ser Ser - Ser Ser - Lys Ala Pro - Pro - 130 140

Pro - Ser - Leu - Pro - Ser - Pro - Ser - Arg Leu Pro - Gly Pro - Ser Asp - Thr - Pro - Ile Leu - Pro Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the p sequence.

Human chorionic gonadotropin is a glycoprotein hormone secreted by the placenta and excreted in the urine; the highest levels are found in the first trimester of pregnancy. Estimation of hCG’ by various means is the basis for diagnostic tests of pregnancy. The physiological role of hCG is still not completely

* This work was supported by National Institutes of Health Grant AM 09579, NIH Contract NOl-HD-O-2251, and a grant from the Population Council of New York. The proposal for the amino acid sequence of hCG 01 was presented initially at the IV International Congress of Endocrinology, Washington, D.C., June 18 to 24, 1972 (1).

$ Present address, St. Vincent’s School of Medical Research, Mel- bourne, Victoria 3065, Australia.

‘The abbreviations used are: h, human; CG, chorionic gonadotro- pin; LH, luteinizing hormone; KM, reduced, S-carboxymethylated; PTH, 3-phenyl-2.thiohydantoin; TSH, thyroid-stimulating hormone.

understood, although it appears to be important in prolonging the life of the corpus luteum as a steroid-producing entity until the placenta is established sufficiently to perform this function (2-4). The hormone actions of hCG appear to be the same as those of pituitary LH, and hCG and LH are bioassayed by the same methods (5).

hCG consists of two nonidentical subunits (6) designated the cy and p subunits. The subunits can be isolated from the native hormone by ion exchange chromatography in urea-containing buffers (7, 8). The isolated subunits possess essentially none of the native biological activity in conventional bioassays, but they can be recombined with substantial restoration of this activity (8, 9).

In this paper we present the details of our proposal for the

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amino acid sequences of the hCG N subunit and the hCG /3 subunit. While this paper was in preparation another detailed proposal for the amino acid sequence of hCG appeared (10, 11). There is essential agreement between the two proposals for the hCG (Y primary structure. However, there are significant discrepancies between our proposal for the hCG B amino acid sequence and that of Carlsen et al. (11). Portions of these structures (8, 12, 13) and a preliminary report of the complete sequences of hCG N and hCG p (14) have been published.

EXPERIMENTAL PROCEDURE

Hormone Preparation-The hCG cy and hCG p subunits were prepared from purified hCG (15) as previously described (8, 16). The amino acid analysis of each subunit is given in the supplement to this paper.

Materials and Methods-Details of the materials and methods used are given in the supplement to this paper.

Peptide Nomenclature-The following prefixes are used to denote the origin of various peptides: T-, tryptic digest; Thr-, thrombic digest; TM-, tryptic digest of maleyl derivative; CNBr-, cyanogen bromide cleavage; C-, chymotryptic digest. Tryptic and thrombic peptides are numbered according to their order in the final sequence proposal. For other peptides, an arbitrary numbering is used.

RESULTS

The summaries of the method of elucidation of the amino acid sequences of asialo RCM-hCG LY and RCM-hCG /3 are illustrated in Figs. 1 and 2. The remainder of the details of the results from which the sequences of hCG LY and hCG p subunits were deduced are given in the supplement to this paper as Tables I to XIV.’ Figs. 3 through 8, which illustrate chromato- graphic separations, are in the supplement

Amino Acid Sequence of hCG o(

Isolation and Analysis of Tryptic Peptides of Asialo RCM hCG a--A series of unique tryptic peptides, whose total analysis accounted for the composition of hCG a, was isolated by a combination of ge! filtration, ion exchange chromatogra- phy, and high voltage paper electrophoresis (Fig. 3). The purification sequence for each peptide is summarized in Table I. The amino acid composition of each peptide is shown in Table II. The majority of the peptides were subjected to manual Edman degradation and the results are shown in Fig. 1. Identifications of residues were made two or more times either on the same peptide or on peptides isolated from other methods of cleavage.

Degradation of Asialo RCM-hCG a--Automated Edman degradation of asialo RCM-hCG o( yielded a partial sequence up to residue 20 (Fig. 1). A second minor underlying sequence was observed for the first 10 residues of the degradation; this

*Some of the data are presented as a miniprint supplement immediately following this paper. Figs. 3 through 8 and Tables I through XIV will be found on pp. 5255 to 5258. Material published on miniprint form can be easily read with the aid of a large-field reading glass of a type readily available at most opticians. If desired, full sized photocopies are available as Document No. 74M-1076A at a cost of $4.65. Further data concerning the identification and yields of PTH amino acids for individual peptides are available in the form of microfiche or full size photocopies: Document 74M-1076B for the hCG a subunit, $1.00 for photocopy; Document 74M-1076C for the hCG (3 subunit, $1.20 for photocopy. A single microfiche including both documents can be ordered for $2.50. Orders for supplementary material should specify the title, authors, the document number, the form (microfiche or full sized photocopy), and the numbers of copies desired. Orders should be addressed to the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014, and must be accompanied by a remittance to the order of the journal in the amounts specified above.

corresponded to the major chain with the first 3 residues lacking, Gas chromatographic determination of the NH,-ter- minal PTH derivative showed a molar ratio PTH-Ala/PTH- Val = 66:33. This NH,-terminal amino acid determination was repeated on several preparations and in each case the ratio, Ala:Val, remained approximately the same. In some instances a small amount of PTH-Asp, approximately 5 to lo%, was observed on thin layer chromatography, which was interpreted as representing a small proportion of molecules lacking the initial 2 residues of the major chain.

Cyanogen Bromide Cleavage of Asialo RCM-hCG D-The cyanogen bromide cleavage mixture was subjected to gel filtration on Sephadex G-50 (Fig. 4), giving three main peptide peaks. &NBrI was pooled as indicated and rechromato- graphed under the same conditions. NH,-terminal amino acid analysis gave a single derivative, PTH-Leu. Automated Edman degradation resulted in a partial NH,-terminal amino acid sequence of 19 residues (Fig. 1). The amino acid composi- tion is given in Table III. This analysis showed a single methionine residue and no homoserine, and indicated a total content of approximately 45 residues. As the NH,-terminal sequence of otCNBr1 is not that of intact hCG a, and since the other cyanogen bromide peptide isolated (cuCNBrII1, see below) contains a homoserine residue, aCNBr1 therefore results from failure of CNBr to cleave at the 3rd methionine residue in the molecule and represents the COOH-terminal portion of hCG a.

cvCNBrII1 was pooled as shown (Fig. 4) and rechromato- graphed under identical conditions. A single NH,-terminal amino acid, glycine, was identified. The composition of &N- BrIII is given in Table III; it contains a homoserine residue. The partial amino acid sequence as determined by manual Edman degradation is given in Fig. 1. Since this peptide is not the NH,-terminal region of hCG N, it must precede cZNBr1.

The remaining component in Fig. 4, cuCNBrI1, which con- tained three peptides (including the NH,-terminal fragment) as determined by sequence analysis, was not further studied.

Tryptic Digest of Maleyl Asialo RCM-hCG a-The tryptic digest of‘maleyl asialo RCM-hCG o( was chromatographed on Sephadex G-50 (Fig. 5). Peptide cvTMII1 was isoiated in a pure state from the gel filtration and was identical with tryptic peptide cuT2. Since hCG (Y possesses 3 arginine residues, one would predict the existence of three additional maleyl tryptic peptides. These remaining maleyl tryptic peptides were not completely purified, but were utilized for the purposes of identifying overlaps of the tryptic peptides. Pool aTMIA was rechromatographed under identical conditions and was sub- jected to automated Edman degradation after removal of the maleyl groups. It proved not to be homogeneous, but a major NH,-terminal sequence Ser-Lys-Lys-Thr-Met- (Fig. 1) was readily identified. Pool aTMIB was rechromatographed on Sephadex G-50 and subjected to automated Edman degrada- tion. A partial NH,-terminal sequence of 13 residues was determined (Fig. 1).

Chymotryptic Digestion of Asialo RCM-hCG a-Chymo- tryptic peptides were separated on a column of Dowex 50-X4 and assessed for purity by paper electrophoresis. In many cases the peaks from ion exchange chromatography contained com- plex mixtures, and only the three chymotryptic peptides which were used in the final sequence proposal are noted in Table III. Sequence determinations on these three peptides are given in Fig. 1; these enabled completion of the sequence of aT1 and LuT9.

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IO 20 H2N-Ala- Pro-Asp-Val-Gln-Asp-Cys-Pro-Glu-CyS-Thr-Leu-Gln- Glu-Asp-Pro-Phe-Phe-Ser-Gln- Pro-Gly- Ala-

d(C6B 7-7-717 CtC6A 7-7-7

30 40 Pro-Ile-Leu-Gln-Cys-Met-Gly-Cys-Cys-Phe-Ser-Arg-Ala-Tyr-Ro-Thr-Pro-Leu-Arg-Ser-Lys-Lys-Thr-

50 CHO 60 Met-Leu-Val-Gln - Lys-Asn-Val- Thr-Ser-Glu -Ser-Thr-Cys-Cys-Val-Ala -Lys-Ser -Tyr-Asn-Arg-Val - Thr-

70 CHO a0 90 Val-Met-Gly-ay- Phe-LyS-Val-Gl~-Asn-HlS-Thr-Ala- Cys-HIS -Cys-Ssr-Thr- Cys-Tyr-Tyr-Hl~-~ys-~er-coo~

4

FIG. 1. Summary of the data used to determine the amino acid methods (see text); 5, identification of the COOH-terminal free sequence of the hCG LY subunit. Peptides are designated by the method arginine following Edman degradation of the peptide was made by high that was employed for cleavage according to the following prefixes: T, voltage paper electrophoresis or by amino acid analysis. Use of tryptic; TM, maleyl-blocked tryptic; CNBr, cyanogen bromide; C, carboxypeptidase A for assignment of residues is designated by -. The chymotryptic. Method of determination of amino. acid sequence is amide assignment for peptide nT7 was made following digestion with designated as follows: +, automated Edman degradation and direct leucine aminopeptidase. Peptide &NBr I was digested with trypsin to identification of PTH derivative; -, manual Edman degradation produce peptides cuT7, cuT8, and LuT9, as well as a fragment containing and direct identification of PTH derivative; broken arrows ( .--+ or residues 48 to 51; these were isolated and identified by their amino acid ---T ), direct identification of PTH derivative was not possible or compositions. was ambiguous and assignment of that position was based on other

Carboxypeptidase A Digestion of RCM-hCG a-Carbox- corresponded to the NH, terminus 01 hCG o( and provided a

ypeptidase A digestion of RCM-hCG LY for 24 hours at 37” fragment through residue 35. The NH,-terminal sequence of released 0.5 residue of serine per mol of o( subunit. Smaller otCNBrII1 provided the overlap otTl-cuT2, and allowed aT quantities of lysine, histidine, and tyrosine were also released; (3+4) to be placed on the basis of residual composition. The however the data clearly indicated that serine was the COOH- partial sequence of aTMIA gave the overlap between cvT4 and terminal residue. cuT5. The NH,-terminal sequence determination of LvCNBrI

Alignment of Tryptic Peptides of hCG cu-Peptide cvT1 extended from the COOH terminus of (uT5 through aT6 into

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I IO (CHO) 20 (CHO) H2N- S~-Lys-Glu-~o-~-Arg-Ro-Arg-Cys-Arg-Ro-Ile-A~-Alo-Thr-Leu-Ala-Val-Glu-Lys-Glu-Gly-Cys-Ro-Vol-Cys-Ile-Thr-Val -Asn

l-+++++++-+++--+j I-

bTI dT2 JT3 / 7 7 ---;, 7 7 7 7 7 7 7 7 7 7 7 7 7 7 ---7

# THR I j + j -3 j -> + -3 -3 + + -3 -3 -3 -3 --:+ -+ $T32, w

977 BCNBr Jl JCNBrll

40 50 60 Thr-Th-lle- Cys-Ala-Gly-Tyr-Cys-Pro-Thr- Met-Thr-Arg-Val-Leu-Gln-Gly-Vol-Leu-Ro-Ala-Leu-Pro-Gln-Val-Val-Cys-Asn-Tyr-Arg

-4 J3T3 bT4

7777777777 777-777-7-7777777-7

4 THR 2 j5 CNBr Ii 77

BTHR 2 --

----_-__------ BCNBr 1

70 80 90 Asp-Val-Arg-Phe-Glu-Ser-Ile-Arg-Leu-Pro-Gly-Cys-Ro-Arg-Gly-Val-Asn-Ro-Vol-Val-Ser-Tyr-Ala-Val- Ala-Leu-Ser-Cys-Gin-Cys

8~6 - dT7 - 8T8 BTS 777-7 77771 -77-77-7 7777777

p THR 3 +‘THR (4t5) ++++--++--3-3+-+~+---?~+-+

7 7 7 7 7 ---,’ 7 7 7 7 7 $THR 4

/ /

100 I IO 120 Ala- Leu-Cys.Arg-Arg-Ser-Thr-Thr-Asp-Cys-Gly-Gly-Pro-Lys-Asp-H~s-Pro-Leu-Thr-Cys.A~-Asp-~o~Arg- Phe-Gln-Asp-Ser-Ser-Ser

I 4 TS ITlO 8 TII ,3 T I2 - BT I3

7-771 77-7777 77777777r 77777

,JT(II t 12) I

7 7 7 7 7 7 ---, ---7 7

4 THR (4 +5) ,5 THR (4 t 5) j -> ---I. j -> ---3 j j

(CHO) (CHO) 130 @HO) (CHO) 140 I45 ~-Lys-Alo-Ro-Pro-Pro-Ser-Leu-Ro-Ser-Ro-Ser-Arg-Leu-Pro-Gly-Ro-Ser-Asp-Thr-Ro-lle-Leu-Pro-Gln-COOH

bT I3 ---, 1

d’T(l4 + IS) 7 7 7 -‘-7 7 7 7 7 -‘-, 7 7 7 7 7 T---T--

l-7 +9T I5 --7--77---7777777

pTHR(4 t 5)

~THR 5

FIG. 2. Summary of the data used to determine the amino acid same as those employed in Fig. 1. The COOH-terminal lysine of PT13 sequence of the hCG p subunit. Peptides are designated by the method was identified by back hydrolysis after Edman degradation of the that was used for cleavage according to the following pref’ixes: T, preceding residues. Carboxypeptidase C digestion (-) was used for tryptic; Thr, thrombic; CNBr, cyanogen bromide. The symbols desig- the determination of the COOH-terminal residue of pT(14+15); nating the method of determination of the amino acid sequence are the carboxypeptidase A was employed with @l%.

cuT7. Free serine, the only tryptic product not containing an arginine or lysine, was placed at the COOH-terminal end of the molecule (otTlO). The chymotryptic peptide uC26 corre- sponded to the COOH-terminal3 residues of cuT9 (this peptide being the only histidine-containing tryptic peptide) together with cuTlO. ~uT8 was therefore placed as the remaining peptide; this arrangement of (uT8 is supported by the finding of cuT8 at the NH, terminus of aTMIB. This alignment was confirmed by a tryptic digestion of aCNBr1. The tryptic peptides were

isolated by high voltage paper electrophoresis. Peptides corre- sponding to aT7, aT8, and otT9 were isolated, together with a peptide whose composition (Lys 1.15, Glu 1.00, Val 0.81, Leu 0.79) corresponded to the NH,-terminal sequence of olCNBr1. The complete amino acid sequence of hCG N based on these arrangements is given in Fig. 1. The amino acid composition of the LY subunit accounting for the total number of residues shown in Fig. 1 is shown in Table IV.

Sites of Carbohydrate Attachment-Total hexose analysis,

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case of COOH-terminal lysine, the last residue was back hydrolyzed for identification. Since /3Tl was not isolated preparatively from the tryptic digest, the sequence was deter- mined by automated degradation of asialo RCM-hCG p.

Isolation and Amino Acid Composition of Thrombic Pep- tides of Asialo RCM-hCG P-Digestion with thrombin at 37” for 8 hours produced five peptides, four of which were isolated by gel filtration (Fig. 7A) and paper electrophoresis, as summarized in Table VIII. No further peptides were formed when digestions were continued up to 24 hours at 37”. The amino acid compositions of the thrombic peptides are given in Table IX. flhrl is identical with pT1, based upon amino acid analysis and the results of automated Edman degradation of asialo RCM-hCG p. A peptide of relative mobility 0.95, with respect to arginine was eluted from analytical paper electro- phoresis of Fraction IV (Fig. 7A) and its composition corre- sponded to peptide PThrl (or PTl). PThrl was not prepara- tively isolated and sequenced. There is general agreement between the sum of the peptide analyses and the total amino acid composition of hCG /3 (Table XI). The results of partial amino acid sequence determinations of these peptides are given in Fig. 2.

A more limited digestion occurred when asialo RCM-hCG fi was incubated with thrombin at 23” for 4 hours. When the gel filtration pattern of this digest on Sephadex G-50 (Fig. 7B) is compared with that of the complete digest (Fig. 7A), Fraction III (representing PThr4) is seen to be absent. The most rapidly eluting region (Fraction I, Fig. 7B), composed of two poorly separated peaks, was rechromatographed on DEAE-Sephadex A-50. Two fractions were obtained (Fig. 7C). Fraction 1 was identical with /YI’hr2. The results of partial amino acid sequence analysis of Fraction 2 are consistent with the assign- ment of this peptide as @Thr(4+5) (Table IX).

Cyanogen Bromide Cleavage-Asialo RCM-hCG p was re- acted with CNBr as outlined in the supplement. Only the NH,-terminal /3 subunit sequence was found in the reaction product, indicating absence of cleavage at the single methio- nine residue. Since the Edman degradation results for peptide ~uT3 indicated that methionine, threonine, and arginine were its 3 COOH-terminal residues and since Met-Thr bonds are known to be resistant to cleavage by cyanogen bromide (19), the observed failure to cleave was most consistent with a Met-Thr-Arg sequence for residues 41 to 43.

A second sample of asialo RCM-hCG p (50 mg) was treated with a significantly greater molar excess of CNBr (2000-fold) in an attempt to cleave the resistant Met-Thr bond, based upon the observations of Liao et al. (20), where employment of excess CNBr successfully cleaved a Met-Ser bond. When the reaction product was passed over Sephadex G-100, most of the protein material emerged in the position of RCM-hCG /3. However, two peaks containing smaller molecular weight fragments were observed (Fig. 8). The results of manual Edman degradation of the material obtained from the larger of these two fragments (PCNBrI) revealed a clear Thr-Arg-Val sequence. The degradation procedure was performed twice and no contaminating residues were detected on either occasion. Manual Edman degradation of the smaller fragment (/3CNBr- II, Fig. 8) showed a clear Ser-Lys-Glu sequence. Amino acid composition data for PCNBrI were consistent with a peptide composed mostly of residues 42 to 145, while PCNBrII contained mostly residues 1 to 40, homoserine and its lactone (Table X). These data support the assignment of Met-Thr-Arg for residues 41 to 43.

using the phenolsulfuric acid reaction of the effluent from the gel filtration of the tryptic digest of hCG LY showed that Fraction II (Fig. 3A) alone contained carbohydrate. Amino acid analysis of RCM-hCG a, after treatment with NaOH (Table XIV), showed no significant loss of serine or threonine, indicating that the carbohydrate can be expected to be attached to the protein chain by an asparaginyl-N-acetyl- glucosamine linkage (17, 18). The two peptides purified from Fraction II, Fig. 3A, namely aT6 and otT9, contained glucosa- mine (Table II); no other peptides from the tryptic digest contained amino sugars. The amino acid sequence of aT6 was determined by manual Edman degradation of the tryptic peptide as well as automated degradation of &NBrI, which encompasses cuT6. No PTH derivative was recovered at the step corresponding to the NH, terminus of aT6, but the residue could be assigned as Asx on the basis of a subtractive analysis. Since peptide cuT6 contains 1 Asx residue and the sequence Asx-Val-Thr is of the type associated with carbohydrate attachment, an oligosaccharide side chain was placed at this position, and the amide assignment made by analogy with accepted structural determinations for this type of linkage (17, 18). Similar considerations led to the placement of the other oligosaccharide chain at Step 3 of peptide otT9.

Amino Acid Sequence of hCG /3

Isolation and Analysis of Tryptic Peptides of Asialo RCM- hCG P---A complete set of tryptic peptides was isolated (Fig. 6) by a combination of gel filtration, ion exchange chromatogra- phy, and high voltage paper electrophoresis. A summary of the purification steps for each peptide is given in Table V. The amino acid composition of each peptide, together with the estimated integral value for each residue, is given in Tables VI and VII; the sum of the individual amino acid compositions is consistent with the composition of hCG p (Table XI).

The NH,-terminal peptide PTl was not isolated by chroma- tography of the tryptic digest. Its composition and sequence were determined by automated Edman degradation of asialo RCM-hCG p (Fig. 2). A rapidly migrating peptide (relative mobility 0.95 with respect to arginine) was eluted in low yield from the analytical paper electrophoresis of Fraction VI (Fig. 6A). Its composition corresponded to the initial 8 residues determined by automated degradation of asialo RCM-hCG p.

Peptide PT8 appears to result from chymotryptic-like cleav- age between residues 82 and 83. The fact that PT9 was isolated in reasonable yield is consistent with this interpretation and supported by the observation that peptides PT(8+9) occur together as PThr4.

The peptide bond between residues 104 and 105 was not cleaved completely under the conditions employed, and the composite peptide, @T( 11+12) was purified by ion exchange chromatography in addition to peptides PTll and PT12. Similarly, the bond 133-134 was hydrolyzed incompletely and the major peptide obtained from this region was /~“I’(14+15). Pep,ide PT15 was purified independently, but the peptide corresponding to PT14 was not isolated.

NH,-terminal Amino Acid Sequences of Tryptic Pep- tides-The extent of the amino acid sequence as determined by duplicate manual degradation is shown in Fig. 2. In most cases, the semimicro technique described in the supplement allowed extensive degradation of the tryptic peptides. The COOH-ter- minal residue was identified in some cases as free arginine after cleavage of the phenylthiocarbamyl dipeptide remaining after degradation of the NH,-terminal portion of the peptide. In the

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Alignment of Thrombic Peptides-The following series of observations allowed us to deduce the order of thrombic peptides in the /3 subunit. Automated Edman degradation of asialo RCM-hCG p permitted the assignment of PThrl adja- cent to /3Thr2 (Fig. 2). Sequential degradation of flhr (4+5) established the order flhr4-mhr5, since the NH,-terminal structure of flhr5 was identified in /YI’hr(4+5).

Of the tryptic peptides only ,0T8 and pT15 did not contain lysine or arginine, and therefore they were candidates for the COOH-terminal region of the molecule. PT8 had been shown to be a fragment from the NH, terminus of PThr4 by sequence analysis. This left pT15 to be assigned as the COOH terminus of the p subunit. The amino acid composition and galactosa- mine content of pT15 and PThr5 were consistent with the conclusion that /3T15 was a component of PThr5.

Since pThr1 and @Thr2 were established as the NH,-termi- nal end of the subunit, and by the above reasoning @Thr(4+5) had been identified as the COOH terminus of the subunit, it was then possible to place the remaining fragment, pThr3, in the center. This reasoning permitted the ordering: PThrl- PThr2-@Thr3-/3Thr4+Thr5.

Alignment of Tryptic Peptides-$Tl and PThrl are identi- cal. PThr2 contains three tryptic peptides; /3T2 and PT3 were ordered by NH,-terminal sequence analysis (Fig. 2) and the remaining composition placed PT4 as the next peptide. Se- quential analysis of pThr3 placed PT5, PT6, and /3T7. PT8 is the NH,-terminal peptide of /3Thr4. Edman degradation of pThr(4+5) extended from PT8 into PTll. Degradation of PThr5 gave the ordering PTlO-PTll-PTlB-PTl~~. PT( 14+ 15) was isolated as a single peptide, and PT15 had already been placed at the COOH terminus. This ordering scheme is summarized in Fig. 2 where the combination of sequence data and ordering of the tryptic peptides resulted in the proposal for the complete linear amino acid sequence of the hCG p subunit. The amino acid composition of the p subunit accounting for the total number of residues shown in Fig. 2 is shown in Table XI.

COOH Terminus of Asialo RCM-hCG /.-No free amino acid was identified on hydrazinolysis of asialo RCM-hCG p. Neither carboxypeptidase A nor carboxypeptidase B released any free amino acids from asialo RCM-hCG p or from the COOH-termi- nal peptides PT15 or PT(14+15). Carboxypeptidase C diges- tion of /JT(14+15) showed unequivocally that glutamine was the COOH-terminal amino acid, and that the probable COOH- terminal amino acid sequence was -Ile-Leu-Pro-Gln- COOH (Table XII). This sequence is consistent with the Edman degradation and amino acid analysis data for PT15. The glutamine residue is consistent with the negative result from hydrazinolysis. The penultimate proline accounts for the lack of attack by carboxypeptidases A and B and the susceptibility to carboxypeptidase C (21).

Sites of Carbohydrate Attachment-Amino acid analysis of peptides /3T2 and fiT3 showed the presence of glucosamine (Table VI). No PTH derivative was detected at Step 5 of /3T2 (Fig. 2) and the existence of aspartic acid (or asparagine) at this step was indicated by subtractive Edman degradation (Table XIII). This is the probable site of carbohydrate attach- ment in this peptide; PT2 does not contain serine and there are no alkali-labile threonine residues in hCG p (Table XIV). Asparaginyl-N-acetylglucosamine can form a PTH derivative (22). In the case of Step 5 or peptide PT2, the thiazolinone was presumably formed but not extracted from the reaction mix- ture by diethyl ether. Analogous reasoning places a carbohy-

containing glucosamine are therefore found at residues 13 and 30 of the intact molecule (Fig. 2). In both cases the sequence at these positions is of the type Asn-X-Thr, which appears to be a common recognition site for carbohydrate attachment of poly- peptide chains by glycosyltransferases (17). Residues 13 and 30 have been considered as asparagine rather than aspartic acid by analogy with the detailed structural determination of the peptide-carbohydrate linkage by other workers (18).

Peptides PT(14+15) and pTl5 contain galactosamine, but no glucosamine (Tables VI and VII). These peptides as well as asialo RCM-hCG B were subjected to alkali treatment to determine the number of alkali-labile serine or threonine residues (Table XIV). Four alkali-labile serine residues were found in hCG /J. Peptides ,0T13, PT(14+15), and @Tl5 possess 1, 3, and 1 residues of alkali-labile serine, respectively. The region of PT14 which was not isolated independently should therefore possess 2 alkali-labile serine residues. Threonine was not lost from @T(14+15), from pT15, or the entire subunit on alkali treatment. Aspartic acid was identified directly as its PTH derivative at position 3 of pT13 and position 6 of PT15, indicating that these residues were not sites of carbohydrate attachment.

The PTH-serine derivative is partly degraded by /I elimina- tion in the Edman degradation, but a sufficient quantity remains to permit identification (23). However, when the hydroxyl group of serine is substituted, PTH-serine is not seen and the PTH derivative of the modified serine is so unstable that it breaks down completely (24). pT13 possesses 4 serine residues, one of which is alkali-labile. In the Edman degrada- tion of pT13, PTH-serine was clearly identified at Steps 4, 5, and 6, and therefore the carbohydrate side chain was assigned to serine 7 as the most likely position. However, the difficulty in quantitating seryl residues in the degradation of a triseryl sequence renders this assignment less than certain, and a re-examination of this region by other methods may be necessary for the location of the carbohydrate moiety. In the degradation of peptide PT(14+15) PTH-serine was clearly identified at Step 8, whereas no PTH derivative was seen at Step 5 or Step 10. Serine was identified at these steps by subtractive degradation (Table XIII). Carbohydrate chains were therefore positioned at Steps 5 and 10 of this peptide. There is only 1 serine residue in peptide /3T15 and this must be the position of carbohydrate attachment. No PTH derivative was seen in Step 5, and serine was identified at this position by subtractive degradation (Table XIII). Carbohydrate side chains attached to serine residues were therefore assigned to residues 121, 127, 132, and 138 of the hCG p subunit (Fig. 2).

DISCUSSION

hCG (Y Subunit-The hCG CY subunit is a glycoprotein with a polypeptide chain of 92 residues. Oligosaccharide side chains are attached at residues 52 and 78, and the amino acid sequences in these positions are of the type Asn-X-Thr, commonly associated with carbohydrate attachments. Our preparations of hCG cy are heterogeneous at the NH,-terminal region, with a proportion of the chains lacking either 2 or 3 residues (12). Virtually the same type of heterogeneity has been reported to occur in the human follicle-stimulating hormone u subunit (25). There are other proteins in which NH,-terminal heterogeneity has been recognized. Ovine LH a exhibits variations in the 8 residues at the NH, terminus (26). Human fibrinopeptide A (24), human placental lactogen (27),

drate side chain at Step 10 of pT3. Carbohydrate side chains and bovine growth hormone (28, 29) possess minor variants

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PTH derivative. At residue 55 we find valine rather than leucine and this discrepancy is reflected in the different amino acid analysis reported for the corresponding peptide (PT4). However, in this case the data of Carlsen et al. (11) concerning the thermolytic and chymotryptic products of the pT4 region are equally consistent with our proposal. We find an extra serine residue at position 121 and the analysis of PT13 supports the presence of 4 serines in that peptide (Table VI). One serine residue disappears from the peptide after alkaline elimination (Table XIV), and this site of carbohydrate attachment has been assigned at position 121 because the other three PTH- serine derivatives were identified by thin layer chromatog- raphy. However, as pointed out previously, some doubt re- mains about the position of the carbohydrate at 121, although it is certain that a site of carbohydrate attachment exists in the region 119 to 121. While the tryptic peptide pT13 contained only 3 serines by analysis in the proposal of Carlsen et al. (ll), their results reported for a chymotryptic product, pC19,T-2, are more consistent with the 4 serines that we have proposed. As indicated in Fig. 2, we find a serine at residue 138 instead of proline. We find no evidence for the existence of the COOH-terminal tripeptide Ser-Leu-Pro-OH described by Carlsen et al. (II).

Additional support for the possibility of ultimate agreement between the two proposals for the primary structure of the COOH-terminal region can be found in the data of Carlsen et al. (11) since their reported compositions of peptides @19,T-3, PC19,T-3b, and PTh21,T-3 could be interpreted as consistent with the structural proposal presented in this paper. No details of direct COOH-terminal amino acid determinations are given by Carlsen et al. (11). Their assign- ment of a COOH-terminal proline is not consistent with our negative finding on hydrazinolysis. We made the assignment of a COOH-terminal glutamine on the basis of carboxy- peptidase C digestion of peptide PT( 14+ 15).

We find 4 alkaline-labile serine residues in hCG /3 indicating four sites of carbohydrate attachment rather than the three proposed by Carlsen et al. (11). The differences in placement of the carbohydrates on serine residues are largely due to the amino acid sequence differences discussed above for the COOH-terminal region.

It is evident from the differences in the two proposals for the COOH-terminal portion of hCG p that this region represents a particularly difficult amino acid sequence problem and merits further study. The structure of the COOH terminus is of particular interest because antibodies raised against synthetic polypeptides containing portions of its sequence could theoreti- cally be employed to measure plasma hCG by radioimmunoas- say in the presence of hLH since this region represents the only structure of hCG that does not contain a high degree of homology with either the 01 or fl subunit of hLH. Such discriminating radioimmunoassay methods using antisera against unique hCG /I subunit structures would improve the capability of the early detection of pregnancy (44), the treat- ment of choriocarcinoma (45), and the recognition of ectopic hormone-producing malignancies (46). Initial difficulties in the determination of the structure of glycoprotein hormones also occurred with ovine LH and these have been discussed in detail (47).

Bovine thrombin has had limited use in sequence studies (48, 49), but was of value in this work. Thrombin is a serine protease (50). In its natural substrate, fibrinogen, it cleaves two pairs of Arg-Gly bonds (51) and studies using synthetic

lacking some NH,-terminal residues. The significance of these findings is not established. The effect may be an artifact of the purification process, but it could be an in vivo catabolic event or result from a nonspecific conversion of a precursor into the circulating product.

The amino acid sequence presented here agrees with the proposal for the o( subunit of Bellisario et al. (10). However, we have made an additional 11 amide assignments.

The hCG CY subunit is closely related to the cy subunits of other glycoprotein hormones, in accordance with the predic- tions from the finding of Pierce and co-workers (30) that bovine TSH N and bovine LH w were identical in polypeptide structure. The polypeptide chain of hCG o( is probably identi- cal with that of hLH cy (31) and hTSH a (32) except that the proposal for hLH (Y lacks the 3 NH,-terminal residues of hCG a. This may merely reflect a difference in the proportions of the various modified chains, with hLH having the majority of chains lacking the 3 NH,-terminal residues. There is also significant structural homology with the cy subunits of ovine (26), bovine (30), and porcine (33) glycoprotein hormones. Immunological studies have suggested that the cy subunits are less closely related between species than are the /3 subunits, while the reverse is true within species (34). However, immuno- logical data derived from native proteins must be interpreted cautiously as evidence for assigning evolutionary relationships (35).

The close relationships of placental and pituitary hormones have previously been well documented in the case of placental lactogen and growth hormone (27, 36). Almost total homology was seen to be preserved between hCG 01 and hLH N, although this was not the case with the p subunits (11, 14, 37, 38). Whether the individual cy or p subunits of glycoprotein hormones have any significant physiological action has not been clarified, and the reason for the apparent restraint on the evolution of the cy subunit structure remains unclear. Follicle- stimulating and thyrotropic activity have also been reported to be produced by the placenta (39, 40). Although the hormones responsible for these activities might be predicted to be structurally similar to the analogous pituitary hormones, as is the case for hCG and hLH, preliminary immunological studies did not detect the presence of an cy type subunit in preparations of chorionic thyrotropin (41). There is evidence that purified hCG possesses thyrotropic activity (42).

hCG /3 Subunit-Our sequence determination of the hCG p subunit, based on tryptic, thrombic, and carboxypeptidase C digests, shows hCG p to have 145 amino acid residues. The hCG /3 sequence bears a marked homology with hLH p (37, 38), which possesses 115 residues of which approximately 80% are identical with those of hCG /3 when the two protein structures are aligned from their NH,-terminal ends without the interpolation of any gaps in either sequence. HCG /3 thus possesses an additional 30 residues at the COOH terminus not found in hLH p or in other reported glycoprotein hormone @subunits.

The alignment of tryptic peptides of the p subunit outlined in Fig. 2 is in general agreement with the proposal of Carlsen and co-workers (11). However, there are significant differences in the details of the amino acid sequence. Some of the differences between the earlier communication of the hCG /3 structure by Bahl and co-workers (43) and the initial reports of our findings (13, 14) have now been resolved (ll), but some disagreements remain. We find residue 3 to be glutamic acid not glutamine by direct identification of the

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peptides indicate that its specificity may be limited to a cleavage COOH-terminal to arginine residues (52). Our results tend to confirm this suggestion. Excepting two Arg-Pro se- quences which would not be expected to be cleaved, hCG /I possesses 10 other arginyl bonds, of which four are cleaved by the thrombin preparation. We obtained no evidence that the other arginyl bonds were cleaved on prolonged digestion, nor was there evidence of any cleavage at lysine residues. This thrombin preparation proved useful here in producing a small number of large peptide fragments, and may be helpful in other sequence determinations, especially as the use of auto- mated degradation favors the use of larger fragments. All our data are consistent with the conclusion that the cleavages observed are caused by thrombin. However, although the thrombin purification procedure used removes clotting Factor X from the preparation (53), further studies would be neces- sary to exclude completely the possibility that the cleavages reported were due to minute amounts of activated Factor X remaining in the enzyme preparations.

Acknowledgments-The technical assistance of Mrs. G. M. Agosto and Messrs. M. Schindler, R. Robbins, and J. O’Connor throughout this work is gratefully acknowledged. We express our gratitude to Dr. E. J. Harfenist for useful discussions.

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F J Morgan, S Birken and R E Canfieldbeta subunit.

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and

1975, 250:5247-5258.J. Biol. Chem. 

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