0013-7227/92/1304-2052$03.00/O Endocrinology Copyright Q 1992 by The Endocrine

Vol. 130, No. 4 Printed in U.S.A.

Society

Carbohydrate Structures Chorionic Gonadotropin Individual* TAMAO ENDO, RYUICHIRO NISHIMURA, KEIICHI NOMURA, MAKOTO KATSUNO, KANCHAN MUKHOPADHYAY, SHIGEAKI

of &Core Fragment of Human Isolated from a Pregnant SHIGEKI SAITO, KENJI KANAZAWA, KOUZUI SHII, BABA, AND AKIRA KOBATA

Department of Biochemistry, Institute of Medical Science, University of Tokyo (T.E., S.S., K.K., A.K.), Minato-ku, Tokyo 108; the Hyogo Institute of Clinical Research (R.N., K.S., K.M., S.B.), Akashi, Hyogo 673; Japan Chemical Research, Ltd. (K-N.)., Nishi-ku, Kobe 673-02; and Wake Pure Chemical Industries, Ltd. (M.K.)., Matsumoto, Nagano 390, Japan

ABSTRACT. Highly purified P-core fragment was obtained from urine of a pregnant woman with use of an immunoaffinity column. The amino acid sequence of &core fragment indicated that it is composed of two polypeptides linked by a disulfide bond. The two polypeptides correspond to the 6-40 and 55-92 portions of hCG b-subunit. Both As# and Asnm residues were glycosylated. The N-linked sugar chains of &core fragment were quantitatively released as radioactive oligosaccharides by hydrazinolysis, followed by IV-acetylation and NaBSHI reduction. The radioactive oligosaccharides were fractionated by serial lectin

H

CG IS produced by trophoblasts of placenta and composed of two subunits, CYand ,f3. Each of the two subunits of this glycohormone contains two N-linked sugar chains.’ The @-subunit also contains four O-linked sugar chains in cluster close to its C-terminal. Structures of the N-linked sugar chains of hCG were elucidated independently by Endo et al. (1) and Kessler et al. (2). An interesting and important finding is that the four asparagine sites of hCG are glycosylated in a strict manner (3); nonfucosylated monoantennary oligosaccharides are linked to the one asparagine site, and nonfucosylated biantennary oligosaccharides are linked to the other site of the a-subunit, while fucosylated and nonfucosylated biantennary oligosaccharides are linked to the two asparagine-sites of p-subunit. Received November 4,1991. Address all correspondence and requests for reprints to: Dr. Akira Kobata, Department of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-l Shirokanedai, Minato-ku, Tokyo 108, Japan. * This work was supported in part by a Grant-in-Aid for Specially Promoted Research from the Ministry of Education, Science, and Culture of Japan. 1 Subscript OT indicates NaB”Hr-reduced oligosaccharides. All sugars mentioned in thii paper are of D configuration, except for fucose, which is of L configuration.

column chromatography and Bio-Gel P-4 column chromatography, and their structures were investigated by sequential exogly cosidase digestion and periodate oxidation. The results indicated that they were a mixture of the four oligosaccharides: Manal-+ 6(~Manal+3)Man&+4GlcNAc @1-~4(iFucal+6)GicNAc. The structural characteristics of the sugar chains of d-core fragment are quite different from those of the &subunit of hCG whose structures were typical biantennary sugar chains containing the Neu5Aca2-*3Gali?1+4GlcNAc@l+2 group as their outer chains. (Endocrinology 130: 2052-20531992)

In addition to hCG, its uncombined subunits (free asubunit and free P-subunit) and its fragments are also found in the urine of pregnant women. The structures of three major P-subunit fragments were elucidated. They are the C-terminal peptide portion of P-subunit (@CTP) (4), @-subunit with a cleavage between Gly4’ and Va148 (nicked &subunit) (58), and the 6-40 and the 55-92 portions P-subunit linked by a disulfide bond (o-core fragment) (9, 10). /l-Core fragment appears as a major immunoreactive component in the urine of pregnant women (9, 10) and is also found in the urine of patients with trophoblastic and nontrophoblastic tumors (8, 11, 12). Two research groups who analyzed the sugar chains of &core fragment in pregnancy urine (10, 13, 14) reached different results. One of the reasons for this discrepancy is considered to be due to contaminants in their final preparation of @core fragment. Therefore, we highly purified p-core fragment from pregnancy urine and performed a structural study of its iv-linked sugar chains. Materials

and Methods

Enzyme immunoassay (EIA) and immunoabsorbent

Monoclonal antibodies were obtained by immunization of isolated from hCG. The antibodies

mice with hCG P-subunit

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/3-CORE FRAGMENT in ascites fluids were then purified with the use of protein-Acellulofine (Seikagaku Kogyo Co. Ltd., Tokyo, Japan). Monoclonal antibody 229 (MoAb 229), which recognizes both free hCG o-subunit and p-core fragment, was used for EIA to monitor the immunoreactivity of &core fragment and for immunosorbent. EIA was performed by the sandwich method, using MoAb 229 for capture antibody coated on a polystyrene bead and polyclonal anti-hCG P-subunit for second antibody conjugated with horseradish peroxidase. After MoAb 229 (5 mg) was conjugated to 1 ml CNBractivated Sepharose (Pharmacia LKB Biotechnology, Tokyo, Japan), 3 ml 1 M ethanolamine, pH 8.0, were added to block the remaining reactive groups. The immobilized antibody was packed in a column (0.6 x 8 cm) and washed sequentially with 10 ml each of 0.1 M NaHCOs-0.5 M NaCl (pH 8.3) and 0.1 M acetate buffer-O.5 M NaCl (pH 4.0). After the sample was applied to this immunoaffinity column, the column was washed with 0.5 M NaCl and distilled water. @-Core fragment, which was retained in the column, was then eluted with 1 N acetic acid. The eluate was immediately neutralized with ammonium nydroxide and then lyophilized. Purification

of

&core fragment

Acetone (20 liters) was added to the urine (10 liters) collected from a first trimester pregnancy woman. The precipitate was collected by centrifugation and redissolved in a minimum volume of 0.1 M ammonium acetate. The solution obtained after centrifugation at 10,000 X g was subjected to gel filtration on a Sephadex G-100 column (superfine; 2.6 x 100 cm) equilibrated with 0.1 M ammonium acetate. The effluent was fractionated in 3.75 ml, and the amounts of proteins and p-core fragment in each tube were determined by absorption at 280 nm and by MoAb 229-EIA, respectively. The eluate containing p-core fragment detected by MoAb 229 was pooled and lyophilized. This material was dissolved in 0.05 M PBS, pH 7.4, and applied to the immunoaffinity column, as described above. Reduction

and S-carboxymethylation

Before S-alkylation, 100 pg of the p-core fragment were reduced in 0.1 M Tris-HCl buffer, pH 8.0, containing 8 M guanidine HCl, 5 mM EDTA, and 50 mM 2-mercaptoethanol for 2 h under a nitrogen atmosphere. The reduced @-core fragment was S-carboxymethylated by adding sodium monoiodoacetate to the final concentration of 50 mM and left for 1 h under a nitrogen atmosphere in the dark. The reaction mixture was subjected to a Vydac C4 HPLC column (4.6 x 150 mm; Hesperia, CA) with a Gilson model 305 apparatus (Oberlin, OH) to remove excessreagents and to separate reduced and S-carboxymethylated @core fragment. The following solvent systems were used for elution: solvent A, 0.1% trifluoroacetic acid in water; and solvent B, 0.1% trifluoroacetic acid in 80% acetonitrile-HzO. The elution was carried out with a linear gradient of the solvent B from O-100% at a flow rate of 1 ml/ min. Peptides were detected by measurement at 220 nm. Amino

acid analysis and sequence analysis

Peptides (CM-l and CM-2) purified by HPLC were hydrolyzed with a Pica Tag Work Station apparatus (Waters, Mil-

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ford, MA) using 6 N HCl containing 1% phenol at 150 C for 1 h. The hydrolysates were analyzed with a Beckman 6300 amino acid analyzer, employing protocols supplied by Beckman (Tokyo, Japan). Since peptide CM-1 was assumed to contain Nlinked sugars, peptide CM-l (3 nmol) was digested with almond glycopeptidase-A (GPas-A; EC 3.5.1.52; Seikagaku Kogyo Co. Ltd.) in 0.1 M citrate-phosphate buffer, pH 5.5, at 37 C for 24 h. The GPase-A cleaves N-linked oligosaccharides and converts the asparagine residue to aspartic acid (15). The ratio of enzyme to substrate was approximately equimolar. Amino acid sequence analysis was performed on an automated Applied Biosystems model 473A protein/peptide gas phase sequencer (Foster City, CA) with the recommended reagents and sequencing cycles. Chemicals

and methods

NaB3H4 (341 mCi/mmol) was purchased from New England Nuclear (Boston, MA). Concanavalin-A (Con-A)-Sepharose was purchased from Pharmacia LKB Biotechnology. Aleuria aurantiu lectin (AAL)-Sepharose (16) was prepared according to the cited reference. /3-N-Acetylhexosaminidase and cu-mannosidase were purified from jack bean meal according to the method of Li and Li (17). Amp&aria a-L-fucosidase and pmannosidase were kindly supplied by Tokyo Zouki Chemical Co. (Tokyo, Japan). Analytical

methods

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed in 18% acrylamide slab gel as described by Laemmli (18). 2-Mercaptoethanol (final concentration, 20%) was used for the analysis under reducing conditions. Proteins were visualized by silver staining. Sequential exoglycosidase digestion, Bio-Gel P-4 column chromatography (Bio-Rad Laboratory, Richmond, CA), paper chromatography, high voltage paper electrophoresis, and other analytical procedures used in this study have been described previously (19-21). Oligosaccharides

Manal-+6(ManLul-+3)Man~l-r4GlcNAc~1-+4GlcNAco~I (Mans. GlcNAc . GlcNAcor) and Manal+6(Mannl+3)Man~1-&GlcNAc/31~4(Fuccul-&)GlcNAcoT (Man,. GlcNAc. Fuc. GlcNAcoT) were obtained from human placental alkaline phosphatase, as described previously (20). GlcNAc~l+4GlcNAco~ and [“H]N-acetylglucosaminitol were prepared by NaB3H4 reduction of N,N’-diacetylchitobiose and N-acetylglucosamine, respectively. Glycosidase digestion

Sugar samples (l-10 X lo4 cpm) were incubated with one of the following mixtures at 37 C for 18 h: jack bean p-N-acetylhexosaminidase, 0.5 U enzyme in 0.1 M sodium citrate buffer, pH 5.0, (50 ~1);jack bean a-mannosidase, 0.8 U enzyme in 0.05 M sodium acetate buffer, pH 4.5, containing 1 mM ZnCh (50 ~1); Ampuliuria @-mannosidase, 10 mU enzyme in 0.1 M sodium acetate buffer, pH 5.5 (50 ~1); and Ampulluria Lu-fucosidase,0.1 U enzyme in 0.1 M sodium citrate buffer, pH 5.0 (50 ~1). One drop of toluene was added to each reaction mixture to inhibit bacterial growth. The reaction was terminated by heating the reaction mixture in a boiling water bath for 3 min.

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2054

B-CORE FRAGMENT

Periodate oxidation

Radioactive oligosaccharide (2 x lo4 cpm) was dissolved in 100 ~10.05 M sodium acetate buffer, pH 4.5, containing 0.04 M sodium metaperiodate, and the mixture was kept at 25 C for 16 h in the dark. Ethylene glycol (10 ~1) was added, and the mixture was kept at 25 C in the dark for 1 h. Then, 10 mg NaBH4 in 0.1 M sodium borate, pH 9.5, were added, and the mixture was kept at 30 C for 3 h. The reaction was stopped by adding 100 ~1 acetic acid, the mixture was passed through a mix bed column (0.5 x 3 cm) of Bio-Rad AG-50 (H’ form) and AG-3 (OH- form), and the column was washed with 3 bed vol distilled water. The eluate and washing were combined and evaporated repeatedly with methanol to remove boric acid. The residue was dissolved in 0.3 ml 0.05 N H2S04 and heated at 76 C for 1 h. The reaction mixture was passed through a column of AG-3 (OH- form), and the radioactive products in the effluent were analyzed by Bio-Gel P-4 column chromatography. Liberation of the N-linked sugar chains of p-core fragment

&Core fragment (400 pg) was subjected to hydrazinolysis for 9 h, followed by N-acetylation, as described previously (18-20). The oligosaccharides in the reaction mixture were reduced with NaB3H, (400 &i) in 100 ~1 0.05 N NaOH at 30 C for 4 h to obtain tritium-labeled oligosaccharides. The radioactive oligosaccharides were purified by paper chromatography using ethylacetate-pyridine-acetic acid-water (5:5:1:3, vol/vol) as a solvent. Results Purification

of p-core fragment

The P-core fragment was purified sequentially by acetone precipitation, Sephadex G-100 column chromatography, immunoaffinity chromatography, and fast protein liquid chromatography gel filtration on Superose 12, as described in Materials and Methods. The purified p-core fragment, thus obtained, gave a single broad band with a mol wt between 12,000-16,000 under nonreducing conditions. After reduction with 2-mercaptoethanol, the pcore fragment was dissociated into two bands with mol wt between 8,000-12,000 and 5,000-6,000, respectively (data not shown). Amino acid analysis and sequence analysis

After reduction and S-carboxymethylation, the P-core fragment was subjected to a reverse phase HPLC (Fig. 1A). The P-core fragment was separated into two fractions, CM-l and CM-2, by this treatment. The amino acid compositions of CM-1 and CM-2 were virtually the same as those of the 6-40 and 55-92 portions of the hCG P-subunit peptide (22), respectively. When the amino acid sequences of CM-l and CM-2 were analyzed, CM-2 could be sequenced completely. In contrast, the amino acid residues at the 8th and 25th positions of CM-l could not be determined, suggesting that these residues were modified by glycosylation. To

Rndo’ 1992

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obtain the complete amino acid sequence, CM-l was digested by almond GPase-A. After the digestion, a new peak (CM-l-l) appeared before the undigested CM-l (Fig. 1B). The amino acid sequence of CM-l-l was totally identical to that of CM-l, and the 8th and 25th residues could be identified as aspartic acid. These results indicated that CM-l contains two N-linked sugar chains attached to the 8th and 25th asparagine residues. Complete amino acid sequences of the two peptides making up the p-core fragment were exactly the same as those previously reported by Birken et al. (10). It should be noted that although they purified P-core fragment from crude urinary hCG preparation obtained from Organon (Oss, Holland), we did it from a single donor. High voltage paper electrophoresk

analysis

Paper electrophoresis (pH 5.4) of the N-acetylated and tritium-labeled oligosaccharides mixture obtained by hydrazinolysis of the p-core fragment gave only neutral component. In contrast, mono- and disialylated oligosaccharides were detected in the @-subunit of hCG in addition to neutral oligosaccharides, as reported previously (3). This result suggested that the p-core fragment contains a different set of iv-linked oligosaccharides. Fractionation of the neutral oligosaccharides by AALand Con-A-Sepharose column chromatography

When the radioactive oligosaccharide fraction was subjected to chromatography on a column containing AAL-Sepharose, which binds the oligosaccharides with fucosylated trimannosyl core (16), 59% of the fraction passed through the column without any interaction. The remainder was retained by the column and eluted with the buffer containing 1 mM L-fucose. The fractions retained and not retained by the AAL-Sepharose column were named AAL’ and AAL-, respectively. Oligosaccharides in both fractions were then separated into two fractions by affinity chromatography on a Con-A-Sepharose column. As shown in Fig. 2, part of oligosaccharides retarded in the column, and the remainder bound strongly to the column and eluted with the buffer containing 100 mM methyl cY-mannopyranoside. The retarded and the bound fractions were named Con-A’ and Con-A+, respectively. As reported by Ogata et al. (23), oligosaccharides with one cy-mannosyl residue interacting with Con-A retard in a Con-A-Sepharose column, and those with two such Lu-mannosyl residues bind to the column and elute with the buffer containing 100 mM methyl a-mannopyranoside. The percent molar ratios of the oligosaccharides in the four fractions, calculated on the basis of their radioactivities, are as follows: AAL-Con-A’, 33.6%; AAL-Con-A+, 25.4%; AAL+ConA’, 25.0%; and AAL+Con-A+, 16.0%.

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P-CORE

FIG. 1. Purification of the reduced and S-carboxymethylated @-core fragment by reverse phase HPLC. The experimental conditions were described in Muteriala and A4ethd.s. A, The ,!I-core fragment was separated into two groups of peptides (designated CM-1 and CM-2), and these groups were separately pooled and lyophilixed. The chromatogram in B was obtained by subjecting fraction CM1 in A to GPase-A digestion. The black arrow in B indicates the elution position of GPase-A.

FRAGMENT

ELUTION

2055

1

0 E E :: N 0.

..--a0

2

-/

,’

.’

d-2

10

RETENTI

5

URINE

_. I

j/l

0

l,i,

FROM

10

15

VOLUME

TIME

I 30 (min)

-60

s

-40; v -20

r” V

E z 0.

-60

3

-40”

5 g 0. a

-202

r^ 5 V

-0

-0 f

40

I

10

1

L

20

RETENTION

TIME

30

(min)

J 40

20 (ml)

FIG. 2. Con-A-Sepharose column chromatograms of the two fractions obtained by affinity chromatography on an AAL-Sepharose column. A, Fraction AAL-; B, fraction AAL’. Black arrows indicate the positions where the buffer was switched to that containing 100 mM methyl (Ymannopyranoside. The white arrow indicates the elution position of authentic oligosaccharide Man~1+4GlcNAc~l+4G1cNAcoT, which does not show any interaction with the Con-A-Sepharose column. ELUTION

Gel permeation chromatography exoglycosidme digestion

and sequential

Each of the four radioactive oligosaccharide fractions thus obtained contained a single component, as revealed by Bio-Gel P-4 column chromatography (Fig. 3). To determine the anomeric configurations and sequence of monosaccharides of the oligosaccharide in each peak in Fig. 3, they were subjected to sequential exoglycosidase digestion, and the radioactive products were analyzed by Bio-Gel P-4 column chromatography. Upon incubation with a-mannosidase, fractions AAL+Con-A+ and AAL+Con-A’ released two and one mannose residues, respectively (Fig. 4A, solid line). Fractions AALCon-A+ and AAL-Con-A’ released two and one mannose residues by the same treatment, respectively (Fig. 4A, dotted line). Both radioactive products in Fig. 4A released another mannose residue by /?-mannosidase digestion (Fig. 4B). The solid line peak in Fig. 4B released an N-acetylglucosamine residue by jack bean /3-N-acetylhexosaminidase digestion (Fig. 4C). It was then converted to radioactive N-acetylglucosaminitol by tu-fucosidase digestion

VOLUME

(InI)

FIG. 3. Bio-Gel P-4 column chromatography of oligoeaccharide fractions obtained by serial lectin column chromatography. Black arrows indicate the elution positions of glucose oligomers (the numbers indicate the glucose units). White arrows indicate the elution positions of authentic oligosaccharides: I, Mana. GlcNAc . Fuc . GlcNAc,; and II, Mana. GlcNAc . GlcNAcWr. A, Fraction AAL-Con-A’; B, fraction AAL-Con-A+; C, fraction AAL’Con-A’; D, fraction AAL+Con-A’.

with the release of one fucose residue (Fig. 4D). On the other hand, the dotted line peak in Fig. 4B was converted to radioactive N-acetylglucosaminitol with release of an N-acetylglucosamine residue by jack bean /3-N-acetylhexosaminidase digestion. These data together with the binding specificity of a Con-A-Sepharose column indicated that the structures of the oligosaccharides in the four fractions were as follows: Fraction AAL+Con-A+ Fuccrl Manlvl \6 Man&

i Manpl+4GlcNAcpl-+4G1cNAcoT

P3

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,&CORE FRAGMENT

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210

240 ELUTION

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Ihdo. I992 Vol130. No 4

4(Fucal + 6)GlcNAco~ + Man/31 + 4GlcNAcSl + 4GlcNAcoT. When the radioactive oligosaccharide in Fig. 3A was subjected to periodate oxidation and analyzed by a BioGel P-4 column, a single radioactive product corresponding to GlcNAr$?l+4XylNAco~ was produced (Fig. 4E). XylNAcoT was detected as the only radioactive material after jack bean b-N-acetylhexosaminidase digestion of the peak in Fig. 4E (data not shown). On the other hand, periodate oxidation of the oligosaccharide in Fig. 3C produced a single radioactive product corresponding to GlcNAcj31+4GlcNAco~ (data not shown). Based on these results, it was concluded that the cu-mannosyl residues of both oligosaccharides in Fig. 3, A and C, occur as the Manal+ group only. In Fig. 5, the structures and the percent molar ratio of the oligosaccharides proposed on the basis of the analytical data so far described are summarized.

270 (ml)

4. Sequential exoglycosidaae digestion of each radioactive peak in Fig. 3. Biuck arrows are the same as described in Fig. 3, and white arrows indicate the elution positions of authentic oligosaccharides: I, iV,N’-Diacetylchitobiitol; II, N-acetylglucosaminitol. A, The oligosaccharides in Fig. 3, C and D (solid line), and in Fig. 3, A and B (dotted line), after digestion with jackbean a-mannosidase; B, the solid line and dotted line represent, respectively, the solid line component and the dotted line components in A incubated with Ampulhria fl-mannosidase; C, the radioactive solid line component in B after digestion with jackbean fl-Nacetylhexoaaminidase; D, the component in C after digestion with Ampulluria a-fucosidase; E, the radioactive product obtained by periodate oxidation of the radioactive peak in Fig. 3A.

Discussion

FIG.

Fraction AAL+Con-A Fuccul

Based on the monosaccharide composition analysis and the behavior of the ,&core fragment on lectin column chromatography, Blithe et al. (13, 14) concluded that oligosaccharides II and IV are the sugar chains of this glycoprotein. Although lectin column chromatography is an effective method to obtain information on the sugar chain structure, it is hard to interpret the data obtained for a glycoprotein with more than two sugar chains, such as the p-core fragment. If the sugar chain of one glycosylation site could bind to a Con-A-Sepharose column, all such glycoproteins will bind to the column even if the remaining sugar chains cannot bind to the column. Because of this situation, each glycosylation site must be separated before being analyzed by a lectin column. This

B (“4

Mancul+6(3)Man@1+4GlcNAcfll+4GlcNAc0~ Fraction AALCon-A’ Mantrl \6

I

Manal

II

Manal.

Mancul P3 To determine whether the Con-A’ fractions are mixtures of both isomers listed above, the two radioactive fractions were subjected to periodate oxidation. By this treatment, the isomeric oligosaccharides should be converted to different radioactive products: ManLvl + 6 Mar@1 -+ 4GlcNAcfll+ 4GlcNAcoT -+ GlcNAc/31+ 4XylNAcoT; Mancul + 3Manal + 4GlcNAcPl -+ 4GlcNAco~ -+ Man/31+4GlcNAc~l+4XylNAcoT; Mancul-&Man@l-, 4GlcNAc/31+ 4(Fuccrl+ 6)GlcNAcoT + GlcNAcPl -+ 4GlcNAcor; and Mancul + 3ManPl + 4GlcNAcPl +

-+6Manpl+4GlcNAc~l

-+rlGlcNAc

33.6

6 25.4

Manpl+4GlcNAc~1-t4GlcNAc Manalx3 Fucal 1 6 III

Manal

-t6Manpl-+4GlcNAc~1-+4GlcNAc

25.0 Fucal

IV

.L 6

Manalx6 ~3 Mar@1 -+4GlcNAc~l-+4GlcNAc

10.0

Manal FIG. 5. Proposed structures and percent molar ratio of oligosaccharides liberated from p-core fragment by hydrazinolysis.

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B-CORE

FRAGMENT

is the reason why Blithe et al. could not detect oligosaccharides I and III, which are the major sugar chains of the P-core fragment. Birken et al. (10) reported that the &core fragment contains a significant amount of galactose. Our data, however, completely denied the presence of galactose in this glycoprotein. This discrepancy may indicate the presence of structural variations in p-core fragments. However, the data reported by Blithe et al. (13, 14) also indicated the absence of a galactose residue. Therefore, the galactose residues detected by Birken et al. (10) may originate from contaminating glycoprotein. Although the mechanism of the excretion of p-core fragment in urine is not known, detection of Mancul-, GMan/31+4GlcNAc~l+4(~Fuc~l+6)GlcNAc in &core fragment suggests the route of this glycoprotein from the B-subunit. These oligosaccharides have already been found in some lysosomal glycoproteins, such as cathepsin-B from porcine spleen (24) and rat liver (25), 1(3glucuronidase from human spleen (26), a-mannosidase from porcine kidney (27), acid phosphatase from Tetrahymena (28), and activator protein-l from human liver (29). Interestingly, it was revealed by these studies that only the oligosaccharides with the Manal-&Man linkage, not those with the Manal+3Man linkage, were present in the two possible isomeric forms of the linear oligosaccharides. These data suggested the possible occurrence of an a-mannosidase that cleaves only the Mancul+3Man linkage of the trimannosyl core portion of N-linked sugar chains. Therefore, structures of the sugar chains of b-core fragment were similar to these intralysosomal glycoproteins, indicating that P-core fragment might be the degradation product of hCG and/or hCG P-subunit within the lysosomes. Structural changes were found in the N-linked sugar chains of urinary hCGs of patients with choriocarcinoma and invasive mole, but not in those with hydatidiform mole (19, 30, 31). By making use of the alteration, a diagnostic method to discriminate malignant hCG from normal hCG in urine samples has been developed (32). The occurrence of structural changes in the sugar chains of ectopic hCG (33) and ectopic free a-subunit (34) have also been reported. In view of the evidence that some choriocarcinoma hCGs contain nicked P-subunit and become more susceptible to dithiothreitol treatment (5, 7,8, 34), structural alteration of sugar chains may affect the processing of hCG and hCG-related molecules. Since P-core fragment is also found in the urine of patients with trophoblastic and nontrophoblastic tumors (5, 7, 11, 12), it is of interest not only as a normal metabolite, but also as a potential marker for the diagnosis of malignancy. Study of the sugar chains of the P-core fragment purified from the urine of patients with trophoblastic

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and nontrophoblastic cation.

tumors is essential for such appli-

Acknowledgment We wish to thank Ms. Yumiko preparation of the manuscript.

Kimizuka

for her excellent

References 1. Endo Y, Yamashita K, Tachibana Y, Tojo S, Kobata A 1979 Structure of the asparagine-linked sugar chains of human chorionic gonadotropin. J Biochem 85669-679 2. Kessler MJ, Reddy MS, Shah RH, Bahl OP 1979 Structures of Nglycosidic carbohydrate units of human choriogonadotropin. J Biol Chem 254:7901-7908 3. Mizuochi T, Kobata A 1980 Different asparagine-linked sugar chains of the two polypeptide chains of human chorionic gonadotropin. Biochem Biophys Res Commun 97:772-778 4. Amr S, Wehmann RE, Birken S, Canfield RE, Nisula BC 1983 Characterization of a carboxylterminal peptide fragment of the human choriogonadotropin &subunit excreted in the urine of a woman with choriocarcinoma. J Clin Invest 71:329-339 5. Nishimura R. Utsunomiva T. Ide K. Kitaiima T. Chen HC. Strobe1 JL, Hussa RO, Mochizuki ‘M 1987 Characterization of human chorionic gonadotropin in urine of patients with trophoblastic diseases by Western blotting using specific antibodies. Jpn J Cancer Res 78833-839 6. Bidart JM, Puisieux A, Troalen F, Foglietti MJ, Bohuon C, Bellet D 1988 Characterization of cleavage product in the human chorionic gonadotropin P-subunit. Biochem Biophys Res Commun 154~626-632 7. Nishimura R, Ide K, Utsunomiya T, Kitajima T, Yuki Y, Mochizuki M 1988 Fragmentation of the &subunit of human chorionic gonadotropin produced by choriocarcinoma. Endocrinology 123: 420-425 8.

9.

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