Amgen Inc. v. F. Hoffmann-LaRoche LTD et al

Filing 547

DECLARATION of Craig H. Casebeer in Support of Motion for Summary Judgment of No Inequitable Conduct by Amgen Inc.. (Attachments: #1 Exhibit Ex 1#2 Exhibit Ex 2#3 Exhibit Ex 3#4 Exhibit Ex 4#5 Exhibit Ex. 5#6 Exhibit Ex 6#7 Exhibit Ex 7#8 Exhibit Ex 8#9 Exhibit Ex 9#10 Exhibit Ex 10#11 Exhibit Ex 11#12 Exhibit Ex 12#13 Exhibit Ex 13#14 Errata Ex 14#15 Exhibit Ex 15#16 Exhibit Ex. 16#17 Exhibit Ex 17#18 Exhibit Ex 18#19 Errata Ex 19#20 Exhibit Ex 20#21 Exhibit Ex 21-1#22 Exhibit Ex 21-2#23 Exhibit Ex 22#24 Exhibit Ex 23#25 Exhibit Ex 24#26 Exhibit Ex 25#27 Exhibit Ex 26#28 Exhibit Ex 27#29 Exhibit Ex 28#30 Exhibit Ex 29#31 Exhibit Ex 30#32 Errata 31#33 Errata Ex 32#34 Exhibit Ex 33#35 Exhibit Ex 34#36 Exhibit Ex 35#37 Exhibit Ex 36#38 Exhibit Ex 37#39 Exhibit Ex 38-1#40 Errata Ex 38-2#41 Exhibit Ex 39#42 Exhibit Ex 40#43 Exhibit Ex 41)(Gottfried, Michael)

Amgen Inc. v. F. Hoffmann-LaRoche LTD et al

Doc. 547 Att. 42

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EXHIBIT 40

Casebeer Decl to Motion for SJ re IC - Public

Dockets.Justia.com

Case 1:05-cv-12237-WGY
THE JOURNALBIOLOGICAL OF CHEAIIsTRY 0 1987 by The American Society for Biochernietryand Mol&

Document 547-43
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Printed in C.S.A.

Vol. 262, No. 25. Issue of September 5. pp. 12069-12076 1987

Carbohydrate Structure Erythropoietin Expressed in Chinese of Hamster Ovary Cells by a Human Erythropoietin cDNA*
(Received for publication, April 20,1987)

Biochemistry, Imperial College of Science and Technology, London,Great Britain

Hiroshi Sasakis, Brian Bothner, Anne Dell$,and Minoru Fukudall From the Cancer Research Center, La J o b Cancer Research Foundation, La Jolda, California 92037 and the $Department of

The properglycosylation of erythropoietin is essential for its function i n vivo. Human erythropoietins were isolated from Chinese hamster ovarycells transfected with a human erythropoietin cDNA and from human urine. Carbohydrate chains attached to these proteins were isolated and fractionated by anion-exchange high performance liquid chromatography (HPLC) and HPLC employing a Lichrosorb-NHz column. The structuresof fractionated saccharides were analyzed by fast atom bombardment-mass spectrometry and methylation analysis before and after treatment with specific exoglycosidases. Both erythropoietins were found to contain one 0linked oligosaccharide/molof the proteins, and its major component was elucidated to be NeuNAca2+ 3Gal@1*3(NeuNAca24)GalNAcOH (where NeuNAc represents N-acetylneuraminic acid) in both proteins. The N-linked saccharides of recombinant erythropoietin werefound to consist of biantennary (1.4% of the total saccharides), triantennary (lo%),triantennary with one N-acetyllactosaminyl repeat (3.5%), tetraantetraanteanary with one tennary (31.8%), and (32.1%), two (16.5%),or three (4.7%) N-acetyllactosaminyl repeats. All of these saccharides are sialylated by 243-linkages. Tetraantennary with or without polylactosaminyl units are mainly present as d1sialosyl or trisialosyl forms, and these structures exhibit the following unique features. a243-Linked sialic acid and N-acetyllactosaminyl repeats are selectively present in the side chains attached to C-6 and C-2 of 2,6substituted a-mannose and C-4 of 2,4-substituted amannose. We have also shown that thecarbohydrate moiety of urinary erythropoietin is indistinguishable from recombinant erythropoietin except for a slight difference in sialylation, providing the evidence that recombinant erythropoietin is valuable for biological as well as clinical use.

in adult kidney andfetal livercells (2-4). Patients with chronic renal failure are anemic as a result of impaired renal function which leads to a decreased production of erythropoietin (5). Thus, availability of purified erythropoietin in quantity is essential to understand molecular mechanismsof erythropoiesis and for treatment of anemia. However, this has been hampered by the fact that only a very small amount of erythropoietin is present in starting sources, even in such cases as theurine of aplastic anemia patients (6). In order to overcome this problem, cDNA clones for human erythropoietin have beenisolated in several laboratories, and the expression of erythropoietin cDNA clones has been achieved (7-9). Furthermore, the recombinant erythropoietin has been successfully usedto reverse the anemia of patients with endo-stagerenal disease (10,ll).Interestingly, the erythropoietin producedin Escherichia coli or yeast was inactive or very weakly active i n uiuo. On the other hand, the erythropoietin produced in COS cellsor Chinese hamster ovary cells was found to be fully active i n uiuo. In agreement with these results, it has been reported that desialylation of partially purified erythropoietin results in inactivation of erythropoietin activity (12-15). Thus, it is apparent that the proper glycosylation isessential for erythropoietin activity in uiuo. These results prompted us to analyze carbohydrate structures of erythropoietin produced by transfection of recombinant DNA into Chinese hamster ovary cells. In addition, we compared those structures with carbohydrate units present in erythropoietin purified from human urine.
EXPERIMENTALPROCEDURES

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Erythropoietin is a glycoprotein which stimulates proliferation and differentiation of erythroid precursor cells to more mature erythrocytes (1).Erythropoietin is primarily produced

* This work was supported by Grant R01 CA33000 and the Cancer Center Support Grant CA30199 from the National Cancer Institute and a program project grant from the British Medical Research Council. The costa of publication of this article were defrayed in part by the payment of page charges. This article must therefore hereby be marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 On leave of absence from the Central Research Laboratories, Chugai Pharmaceuticals Ltd., Tokyo, Japan. W To whom correspondence should be addressed La Jolla Cancer a Research Found. 10901 N. Torrey Pines Rd., L Jolla, CA 92037.

Erythropoietin-Chinese hamster ovary cells (dihydrofolate reductase-) were transfected with an expression vector which harbors the human erythropoietincDNA as described (7). This expression vector also contains dihydrofolate reductase minigene so that stable transfects can be grown in the presence of methotrexate (16). Erythropoietin was purified from the spent medium of those cells as described (7). The purification procedure was slightly modified from that of Miyake et al. (6), and fractionation on aVydac C, reverse-phase HPLC' column (The Separations Group) was included (7). This erythropoietin will be called recombinant erythropoietin hereafter. Erythropoietin was also purified from the urine of aplastic anemia patients according to Miyake et al. (61,with a similar modification as for purification of recombinant erythropoietin. The erythropoietin purified from urine will be called urinary erythropoietin hereafter. These erythropoietin samples were provided by Chugai Pharmaceutical Co., Ltd. (Tokyo). Isolation of N-Linked Glycopeptides and O-Linked Oligosaccharides from Erythropoietins-Glycopeptides were prepared by Pronase digestion of erythropoietin (5 mg of recombinant erythropoietin and The abbreviations used are: HPLC, high performance liquid chromatography; FAB-MS, fast atom bombardment-mass spectrometry, Hex, hexose; HexNAc, N-acetylhexosamine; NeuNAc, N-acetylneuraminic acid; Lac, N-acetyllactosaminyl repeat.

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Carbohydrate Structure of Human Recombinant Erythropoietin
was 1oO:O at the beginning of the elution and 6040 at the end of elution. This elution system was used because it was preferable to premix acetonitrile and the buffer before the elution started, otherwise, mixing of two solutions causes cooling down of the solvent. The flow rate was 1 ml/min, and each fraction contained 0.5 ml. Structural Analysis of Saccharides-Structures of saccharides were analyzed essentially as described previously for other saccharides. These methods include fast atom bombardment-mass spectrometry of permethylated saccharides (17), analysis of partially 0-methylated monosaccharides after methylation and hydrolysis ("methylation analysis") (17,231, and exoglycosidase digestion combined with methylation analysis (24). Methylation of saccharides and purification of methylated saccharides for FAB-MS and methylation analysis were carried out as described previously (17, 23). Before methylation of saccharides, all samples were desalted by Sephadex G-15 gel filtration eluted with water. Glycosidase digestion of saccharides was carried out as follows. For sequential digestion by @-galactosidaseand @-N-acetylglucosaminidase, the saccharides were incubated with 5 milliunits of Charonia lampas &galactosidase in 40 pl of 0.1 M sodium citrate buffer, pH 4.3, at 37 "Cfor 24 h. After incubation, the incubation mixture was heated in a boiling water bath for 2 min. The sample was then incubated with 100 milliunits of beef kidney @-N-acetylglucosaminidase 140 in p l of 0.1 M sodium citrate buffer, pH 4.3. In order to inhibit apossible contaminating activity of @-galactosidase,100 m (final concentraM tion) galactose was added to this incubation mixture. After further incubation a t 37 "Cfor 24 h, the mixture was heated in a boiling water bath for 2 min. The digests were then purified by Sephadex G50gel filtration followedby HPLC employing a Lichrosorb-NHz column as described above. For extensive digestion by a mixture of the @-galactosidase @-N-acetylglucosaminidase, saccharides were and first incubated with C. lampas @-galactosidaseand then with beef kidney @-N-acetylglucosaminidase without heat inactivation of 8galactosidase or the addition of galactose. After total incubation at 37 "C for 48 h, enzymes were inactivated by heating in a boiling water bath for 2 min, and digested saccharides were purified by anionexchange HPLC. @-Galactosidase from C. lumpas and @-N-acetylglucosaminidase from beef kidney were purchased from Sigma and Boehringer Mannheim, respectively. Determination of Carbohydrate Composition- Sialic acid content was determined by the periodate-resorcinol reaction (25). Neutral sugars and hexosamines were determined after methanolysis in 0.5 N hydrochloric acid in anhydrous methanol at 80 "C for 4 h. Inositol was added as an internal standard. After methanolysis, the products were dried under a nitrogen stream and further in uacw under PB05 and NaOH. The dried products were trimethylsilylated with Tri-Si1 (Pierce Chemical Co.). The trimethylsilylated derivatives were then analyzed by gas-liquid chromatograph-mass spectrometry as described.* In parallel, fetuin was analyzed in order to obtain response factors.
RESULTS

1 mg of urinary erythropoietin, respectively) as described (17). The Pronase digest was applied to asmall column (1.0 X 45 cm) of Sephadex G-15, which was equilibrated and eluted with water. The gylcopeptides, eluted at and near the void volume, where lyophilized and subjected to alkaline borohydride degradation, as described (17). Briefly, the glycopeptides were dissolved in 500 p1 of 0.05 M NaOH, 1 M NaBHI containing 5 mCi of NaB3H4(9 Ci/mmol) and incubated at 45 "C for 24 h. After treatment, 1-2 ml of methanol which contained 1 drop of acetic acid was added to the sample and evaporated under nitrogen. The alkaline borohydride-treated sample was then applied to thesame Sephadex G-15 column for desalting. The glycopeptides and oligosaccharides, which eluted between the void volume and the salt peak, were pooled and applied to a column (1.0 X 140 cm) of Bio-Gel P-4 (200-400 mesh). The column was eluted with 0.1 M NHlHC03 a t a flow rate of 6 ml/h, and each fraction contained 1 ml. Under these chromatographic conditions, N-linked glycopeptides eluted near the void volume,whereas 0-linked oligosaccharides eluted in later fractions (see Fig. 2). Isolation of N-Linked Saccharides from Glycopeptides-The glycopeptide fraction containing N-linked saccharides was digested with Flavobacteriummeningosepticum N-glycanase (peptide-N4-(N-acetyl8-glucosaminy1)aspargineamidase) (18) which was purchased from Genzyme (Boston, MA). The glycopeptides from 5 mg of erythropoietin were dissolved in 500 p1 of 0.1 M sodium phosphate buffer, pH 8.6, containing 20 m EDTA and 20 m 2-mercaptoethanol and M M incubated with 30 m units of the N-glycanase, which corresponds to 30 units of the enzyme expressed by Genzyme, at 37"C for 20h. When the glycopeptides from 1 mgof urinary erythropoietin were digested with N-glycanase, the incubation mixture was scaled down to one-fifth. The digest was desalted by Sephadex G-15 gel filtration, and the saccharides were reduced at room temperature for 30 min with 5 mCi of NaB3H4dissolved in 200 pl of 0.01 M NaOH followed by 3 mg of NaBH4 for 2 h. After reduction, the sample was neutralized with the addition of methanol containing a small amount of acetic acid and evaporated under nitrogen. The dried sample was dissolved in water, and the supernatant obtained after centrifugation was applied to the Sephadex G-15 column for desalting. The saccharides, radioactively labeled at reducing terminals, were applied to a column (1.0 X 45 cm) of Sephadex G-50 (superfine) eluted with 0.2 M NaCl (see Fig. 2B). Each fraction contained 0.5 ml. Fractionation of Oligosaccharidesby Anion-exchangeHigh Performance Liguid Chromatography-Oligosaccharide fractions obtained after Sephadex G-50 gel filtration were subjected to anion-exchange HPLC with a Varian HPLC apparatus (Model 5000, Varian Associates, Inc.). The sample was applied to a Toyo Soda TSK-DEAE column purchased from Kratos Analytical Instruments (Ramsey, NJ). The column (4.6 mmX 24 cm) was equilibrated with 25 m potassium M phosphate buffer, pH 5.0, and after eluting with the same buffer for 10 min, the elution was programed by the linear gradient to 0.4 M potassium phosphate buffer, pH 5.0, over 80 min. The flow rate was constant at 1 ml/min, and each fractioncontained 0.3 ml. The elution was monitored by measuring the absorbance at 206 nm with a Beckman 163detector. Since potassium phosphate has absorbance at 202 nm, the absorbance at 206 nm was measured under these conditions. The base line of the absorbance at 202 nm was increased under these conditions. In order to estimate the elution positions of monosialosyl, disialosyl, trisialosyl, and tetrasialosyl saccharides, IgG (bovine, Sigma), fetuin (19, 20), and nl-acid glycoprotein saccharides (21, 22) were prepared by hydrazinolysis and subjected to HPLC under the same conditions. The saccharides, separated by TSKDEAE, were subjected to methylation analysis to confirm their structures. Mancrl+6(Mancr1+3)Man@l+4GlcNAc@l4(Fucal-+ 6)GlcNAcOH was obtained by extensive digestion of IgG saccharide with @-glactosidase and 8-N-acetylglucosaminidase. Fractionation of Neutral Oligosaccharidesby HPLC-The saccharides were desialylated by mild acid hydrolysis in 0.01 N HCl a t 80 "C for 1 h. The desialylated sample was desalted by Sephadex G-15 gel filtration and evaporated. The neutral saccharides were then fractionated by HPLC with the same apparatus as described above. The saccharides were dissolved in acetonitrile, 10 m potassium phosM phate buffer, pH 4.5 (65:35), and applied to a column (0.4 X 25 cm) of Lichrosorb-NHz (5-pm particle size, Merck). The column was eluted with a linear gradient to acetonitrile, 10 mM potassium phosphate buffer, pH 4.5 (3961), over 60 min. In order to achieve this elution, the first solvent was acetonitrile, 10 m potassium phosphate M buffer, pH 4.5 (65:35, v/v), andthe second solvent was 10 m M potassium phosphate buffer, pH 4.5. The ratio of these two solvents

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Isolation of N-Linked Sacchurldesfrom Recornbinant Etythropoietin-Erythropoietin was purified from the spent mea human dium of Chinese hamster ovary cells transfected with erythropoietin geneand from human urine of aplastic anemic patients as described under "Experimental Procedures." The purified proteins showed a major band with M , 38,000 and a faint band with M , -80,000 (Fig. 1). The latter band is probably the dimer erythropoietin. The carbohydrate comof position of this molecule is shown i n Table I. Glycopeptides were prepared from 5 m g of recombinant erythropoietin by Pronase digestion and isolated by Sephadex G-15gel filtration. Glycopeptides (1.8 mg) were then treated with alkaline and the borohydridetorelease0-linkedoligosaccharides, alkaline borohydride-treated samples were applied to a colu m n of Bio-Gel P-4. As shown in Fig. 2 A , three peaks were to 30detected in addition a salt peak. The second (fractions 35) and the third(fractions 36-40) peakswerefound to M. Fukuda, M. Lauffenburger, H. Sasaki, E. M. E. Rogers, and A. Dell (1987) J. Biol. Chem., in press.

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Carbohydrate Structure Human Recombinant Erythropoietin of

A
kDa
9266.24531-

B

A

VO

+

B

vo

2 1.514.4-

+

"

???

0

10

20

30

40

50

60

FIG. 1. Autoradiogram of sodium dodecyl sulfate gel electrophoresis of recombinant erythropoietin (Zane A ) and urinary erythropoietin (lane B ) . Purified erythropoietins were iodinated with '251 according to Greenwood et al. (26). The radioactively labeled proteins were then applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gel concentration, 15%) according to Laemmli (27), and thegel was directly autoradiographed with Kodak x-ray AR-5 film

TABLE I Carbohydrate compositionof erythropoietin Numbers are exmessed as moles/mole of ervthroDoietin.
Urinary Recombinant erythropoietin Batch 1 Batch 2 Batch 3 Batch 4

FIG.2. Bio-Gel P-4 ( A ) and Sephadex G-SO ( B ) gel filtrations of glycopeptides obtained from recombinant erythropoietin. Glycopeptides were subjected to alkaline borohydride treatment in the presence of NaB3H4and applied to a column of Bio-Gel P-4 ( A ) .The 0-linked oligosaccharides were eluted in fractions 3035 (0-i)and fractions 36-40 ( 0 - i i ) . The glycopeptides, which contained N-linked saccharides (N-Such.), eluted in fractions 24-29 and were digested with N-glycanase. The digest was reduced with NaB3H4 and subjected to Sephadex G-50 gel filtration ( B ) . The fractions, indicated by the horizontalarrows, were pooled and subjected to further analysis. The arrowheads indicate the elution positions of the trisialosyl saccharide from cui-acid glycoprotein (arrowhead 4 ) , the trisialosyl saccharide from fetuin (arrowhead 3 ) , and the asialo form of IgG saccharide (arrowhead 2).

Fractlan Number

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344), 825, 1274, and 1723, which correspond to NeuNAc+, NeuNAc+Hex+HexNAc+, NeuNAc+Hex+HexNAc+ Hex+HexNAc+ and NeuNAc+Hex+HexNAc+Hex+ HexNAc+Hex+HexNAc+. These resultsindicate that Nlinked saccharides contain N-acetyllactosaminyl repeats in the side chains. This conclusion was supported by the detecwhen tion of a fragment ion at mlz 1362 for Hexn.HexNAc3+ contain 0-linked oligosaccharides (see Miniprint): whereas desialylated and methylatedsaccharides were subjected to glycopeptides containing N-linked saccharides eluted t frac- FAB-MS (Fig. 44). Methylation analysis of N-linked saca tions 25-29. charides indicates the following features. 1)Galactose is terFractions 25-29 were pooled and digested with N-glycanase. minal or 3-substituted; 2) all of the N-acetylglucosamine The digest, after reduction with NaBSH4, was subjected to residues except the reducing terminal residue are substituted Sephadex, G-50 gel filtration as shown in Fig. 2B. The sacat C-4; 3) 2-substituted mannose (0.16 mol), 2,4-substituted charides eluting between fractions 29 and 41 were subjected mannose (0.78 mol), 2,6-substituted mannose (1.05 mol), and to further analysis. Methylation Analysis and FAB-MS of N-Linked Sacchar- 3,6-substituted mannose (1.0 mol) were detected (Table 11). After desialylation, a majority of 3-substituted galactose ides from Recombinant Erythropoietin-In order to determine residues were converted to terminalgalactose, indicating that the carbohydrate structures of N-linked saccharides, the saccharides were subjected to FAB-MS and methylation anal- sialic acid is linked to galactose through an a2+3-linkage. yses. As shown in Fig. 3A, FAB-MS of permethylated N- However, 0.82 mol of 3-linked galactose, which corresponds linked saccharides provided fragment ions at mlz 376 (and to 17% of the total galactose derivatives, was still detected after desialylation. This amount of galactose is presumably derived from N-acetyllactosaminyl repeats. The same analysis Portions of this paper (including part of "Results," Figs. 4.6, and also showed that 85% of reducing terminal N-acetylglucosa8-10, and Table I1 and IV) are presented in miniprint at the end of mine is substituted with fucose at C-6, whereas the restof the this paper. Miniprint is easily read with the aid of a standard reducing terminal N-acetylglucosamine contains no fucose. magnifying glass. Full size photocopies are available from the Journal Theseresults suggest that the N-linked saccharides of of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. recombinant erythropoietin are mainly composed of tetraanRequest Document No. 87M-1260, cite the authors, and include a check or money order for $6.80 per set of photocopies. Full size tennary saccharides with or without N-actyllactosaminyl rephotocopies are also included in the microfilm edition of the Journal peats. Isolation of Asialo N-Linked Saccharides-N-Linked sacthat is available from Waverly Press.
~ ~~

Fuc Man Gal GlcNAc GalNAc NeuNAc

2.9 9.2 12.9 16.3 0.9 10.4

4.1 8.7 13.8 17.2 0.9 9.5

2.9 9.4 14.1 18.9 1.4 9.7

2.3 8.2 15.5 17.3 0.8 11.8

3.6 9.1 13.0 19.8 1.4 10.8

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A

931

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I .

1723

F'Ic.3. Fast atom bombardment-mass spectra of permethylated total N-linked saccharides from recombinant erythropoietin (A) and urinary erythropoietin (B).The positive spectra were recorded. A, fragment ions weredetected at m/z 376 (and 344) forNeuNAc', 580 forNeuNAc+Hex+, 825 (and 793) for and 1723 for NeuNAc+Hex+ NeuNAc+Hex+HexNAc+, 1274 for NeuNAc+Hex+HexNAc+Hex+HexNAc+, HexNAc+Hex+HexNAc+Hex+HexNAc+. A small peak was detected also at m/z 464 for Hex+HexNAc+. B, in addition to the ions described above, a minor fragment ion was detected at m/z 913 for Hex+HexNAc+Hex+ HexNAc+.
charides, obtained from 5 mg of recombinant erythropoietin charides (peak 2) was identical to the IgG saccharide which of (Batch 1in Table I), were desialylatedby mild acid hydrolysis is mainly composed Galz GlcNAc, .Man, GlcNAc,. Fuc.In and then desalted by Sephadex G-15 gel filtration. Neutral addition, this saccharide bound to a concanavalin A-SephaM saccharides thus obtained were fractionated by HPLC em- rose and was eluted by20 m methyl-a-glucoside. Those fraction (peak 2) is a typical complex ploying a Lichrosorb-NH2column. As shown in Fig. 5A, N- results establish that this linked saccharides from recombinant erythropoietin provided saccharide withbiantennary side chains. Methylation analysis (Table 11) of the triantennary sacsix peaks which correspond to biantennary (peak 2), triantennary (peak 3), tetraantennary (peak 4), tetraantennary with charides (peak 3) provided 0.65 mol of 2,6-substituted manone N-actyllactosaminylrepeat (peak 5, Lacl), tetraantennary nose ( 3 , 4 - d i - O - m e t h y l r n a ) and 0.35molof 2,4-substiin with two N-acetyllactosaminyl repeats (peak 6, Lacz), and tuted mannose (3,6-di-O-methylmannose) addition to 1 tetraantennary with three N-acetyllactosaminyl repeats (peak mol each of 2- and 3,6-substituted mannose (Table 11). The 7, Lac3). The identical elution profile was obtained when N- saccharides were also digested by a mixture of &galactosidase to linked saccharides were desialylated by clostridial neuramin- and 8-N-acetylglucosaminidase yield Manarl+6(Manal+ as idase. No significant amount of carbohydrate was detected in 3)Man@l4GlcNAc~l4(fFucal+6)GlcNAcOH, judged other fractions. This result indicates that most of the glyco- by HPLC with a Lichrosorb-NH, column followed by methpeptides were digested by N-glycanase since the glycopeptides ylation analysis. These results established the structures of would elute later than saccharideswithout amino acid residues the triantennary saccharides, as shown in Table III. In order to elucidate which the outer a-mannosyl residues of in a Lichrosorb-NHz column. Structures of Biantennary Saccharides and TTiantennary is disubstituted at C-2 and C-4, the N-linked saccharides Saccharides-The elution position of the biantennary sac- (fractions 29-41 in Fig. 2B) were subjected to periodate oxi-

-

-

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Carbohydrate Structure of Human Recombinant Erythropoietin

G O

4

1271

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701

4

1

FIG. 5. HPLC of a i 0 N-linked sd saccharidea derived from erythropoietin. N-Linked saccharides were desialylated by mild acid hydrolysis, applied to a 5-pm Lichrwrb-NH, column, as described under "Experimental Procedures." The effluent was monitored by measuring the absorbance at 202 nm. A and B, total N-linked saccharides from recombinant erythropoietin ( A )and urinary erythropoietin ( B ) ; C-E, N-linked saccharides from recombinant erythropoietin separated by TSK-DEAE ionexchange chromatography as shown in Fig. 7A. Monosialosyl ( C ) ,disialogyl (D!, and trisialosyl ( E ) fractions were deslalylatd, applied to the Lichmrb-NH2 column, and eluted under the same conditions. Ordinate, relative intensity at A= nm; ahwissa, retention time.

4

12

20

28

36

44

I

,

52

,

,

Elution Time (minutes)

04

12

Elutionlime (minutes)

20 28

3 6

44

52

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R

3

2

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Carbohydrate Structureof Human Recombinant Erythropoietin

u )

@a

t :

x

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R

x

x

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Carbohydrate Structure of Human Recombinant Erythropoietin

dation followed byreduction and mild acid hydrolysis (Smith 2,6-substituted mannose, and 3,6-substituted mannose (Table degradation) as described (26). Methylation analysis of the 11), indicating that peak 5 is composed of tetraantennary product provided 2,4,6-tri-O-methylmannose with the con- saccharides. The presence of 3-substituted galactose (0.8 mol) comitant loss of 3,4-di-O-methylmannose. These results inin this asialo form indicates that the saccharides contain one dicate that the 2,4-disubstituted a-mannose is linked to C-3 N-acetyllactosaminyl repeat. FAB-MS of the saccharides of &mannose, as shown in Table 111. after permethylation yielded a signal at m/z 913 for HexStructure of Asialo Tetraantennary Saccharides and Trian- HexNAc-Hex-HexNAc' (Fig. 4C), which is indicative of tennary Saccharides with One N-Acetyllactosaminyl Repeatone N-acetyllactosaminyl repeat. FAB-MS of the saccharides which eluted at peak 4 provided The saccharides of peak 5 were sequentially digested by Pa molecular ion at m/z 3135 corresponding to (Fucl-Hex7. galactosidase and P-N-acetylglucosaminidase and subjected HexNAc6)R (Fig. 423). This ion was associated with A-type to HPLC. As shown in Fig. 623, the major product eluted at ions at m/z 2668 for Hex7-HexNAc; and a t m/z 464 (and 15 min, which corresponds to Gal. GlcNAc' Man3 GlcNAc. 432) for Hex-+HexNAc+. In addition, an ion at m/z913 (+Fuc)GlcNAcOH. The methylation analysis of this product corresponding to Hex-HexNAc-Hex+HexNAc+ was de- provided 0.1 of mol 2-substituted mannose, 0.1 of mol 4tected. The latter result suggests that this peak contains a substituted mannose, 0.8 mol of 6-substituted mannose, and triantennary saccharide with one N-acetyllactosaminyl re- 1 mol each of terminal mannose and 3,6-substituted mannose peat. The presence of a triantennary saccharide was con- as mannose derivatives. These results indicate that 80% of firmed by methylation analysis (Table 11). A small amount the N-acetyllactosaminyl repeat is attached to C-6 of 2,6(0.11 mol) of 2-substituted mannose (3,4,6-tri-O-methylman- substituted mannose, 10%is attachedto C-4 of 2,4-substituted nose) was detected as well as 1 mol of 2,4-substituted and 0.9 mannose, and 10% is attached to C-2 of either 2,6- or 2,4mol of 2,6-substituted mannose. This result indicates that a substituted mannose (Table 111). Structure of Tetraantennary Saccharides with Two N-Acetriantennary saccharide with 2,4- and 2-substituted a-mananalysis of peak nose is present in this saccharide fraction. This triantennary tyllactosaminyl Repeats (Lac&"ethylation saccharide presumably contains one N-acetyllactosaminyl re- 6 in Fig. 5A provided 1.7 mol of3-substituted galactose, 4 mol peat. The presence of 3-substituted galactose supports the of terminal galactose, and 1 mol each of 2,4-substituted, 2,6conclusion that this triantennary saccharide contains one N- substituted, and3,6-substituted mannose, in addition to other derivatives (Table 11).The results indicate that this saccharacetyllactosaminyl repeat (Table 11). The saccharides were sequentially digested with P-galacto- ide fraction consists of tetraantennary saccharides with two sidase and P-N-acetylglucosaminidase and subjected to N-acetyllactosaminyl repeats. FAB-MS of this oligosacchaHPLC. As shown in Fig. 6A, the productsprovided two peaks: ride fraction supported the above conclusion since a fragment 913 corresponding to Hex-HexNAc-Hexthe first peak eluted at 12 min corresponds to Man3. GlcNAc. ion at m/z (+Fuc)GlcNAcOH, and the second peak at 15 min corre- HexNAc' was detected (Fig. 40). The saccharides were digested sequentially by P-galactosponds to Gal. GlcNAc Man3. GlcNAc (+Fuc)GlcNAcOH. The ratio of two peaks was found to be 1.00.19. These results sidase and P-N-acetylglucosaminidase to yield a major peak indicate that about 15% of the saccharides are triantennary at 19 min (peak 2 in Fig. 6C), which corresponds to saccharides with one N-acetyllactosaminyl repeat, whereas (Gal. GlcNAc)2.Mans. GlcNAc-(+Fuc)GlcNAcOH, confirmsample contains tetraantennary sacchar85% of the saccharides are tetraantennarysaccharides. These ing that the starting two molecular species, however, willprovide the same molec- ides with two N-acetyllactosaminyl repeats. Methylation analysis of this product provided 1 mol of terminal mannose, ular ion at m/z 3135 on FAB-MS analysis. In order to elucidate which side chains were elongated to 0.8 mol of 2,6-substituted mannose, and 0.2 mol each of 6form the N-acetyllactosaminyl repeat, the saccharides which and 4-substituted mannose, in addition to 1 mol each of 3,6eluted at 15 min in Fig. 6A (denoted as 1) were methylated. substituted mannose, terminal galactose, and reducing terThe saccharides provided 1 molof 4-substituted mannose minal N-acetylglucosamine and 2 mol of 4-substituted N(2,3,6-tri-O-methylmannose), 3,6-substituted mannose (2,4- acetylglucosamine. However, 2,4-substituted mannose or 3di-0-methylmannose), terminal mannose (2,3,4,6-tetra-O- substituted galactose was not detected. These results indicate methylmannose), terminal galactose (2,3,4,6-tetramethylga- that each side chain arising from 2,6-substituted mannose lactose), 2 mol of 4-substituted N-acetylglucosamine (3,6-di- was elongated by one N-acetyllactosaminyl repeat in 80% of 0-methyl-N-methylacetylglucosamine), 1 mol of 4,6-sub- the saccharides. In addition, 20% of the molecules have Nand acetyllactosaminyl repeats in side chains elongating from Cstituted N-acetylglucosamine (1,3,5-tri-O-methyl-N-methylacetylglucosaminitol). These results indicate that the N-ace- 6 and C-4 (Table 111). Structure of Tetraantennary Saccharides with Three Ntyllactosaminyl repeat is linked to C-4 of a-mannose,as AcetyllactosaminylRepeats (Lac&-The saccharides in peak 7 shown in Table 111. The structure of the saccharide "core" (see Fig. 6A) was eluted at theposition where saccharides containing seven Nconfirmed by methylation analysis and FAB-MS to be acetyllactosaminyl units areexpected to elute. This is because the difference of the elution time between peaks 7 and 6 is Man1+6(Manl+3)Manl+4GlcNAcl+4(Fuc1~6)GlcNActhe same as thatbetween peaks 6 and5. The saccharides were OH (89%) and Man1+6(Manl+3)Man14GlcNAcl--, 4GlcNAcOH (11%). Combining these resultswith exoglycosi- sequentially digested with P-galactosidase and P-N-acetylgludase digestion, the structure of the core was elucidated to be cosaminidase, and the products were analyzed by HPLC. As shown in Fig. 6 0 , a major peak eluted at 24 min, which Mancul+6(Manal+3)Man/31~4GlcNAc~1-4(+6) GlcNAcOH. All of the saccharides including tetraantennary corresponds to (Gal.Gl~NAc)~.Man~.GlcNAc(+Fuc)Glcwith or without N-acetyllactosaminyl repeats were converted NAcOH. However, about 35% of the products eluted at the to this core saccharide when they were digested with a mixture position corresponding to (Gal-GlcNAc)2.Mans-GlcNAc(+ Fuc)GlcNAcOH. of @-galactosidase and P-N-actylglucosaminidase. These results suggest that 65% of the peak 7 saccharides Structure of Asiulo Tetraantennary Saccharides with One N-Acetyllactosaminyl Repeat (Lac&"ethylation analysis of are tetraantennary saccharides with three N-acetyllactosapeak 5 in Fig. 5A provided 1mol of 2,4-substituted mannose, minyl repeats(Lac3), whereas 35% of the saccharides are
I

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tetraantennary saccharides with two N-acetyllactosaminyl repeats (Lac2). These resultswere confirmed by methylation analysis, as shown in Table 11. Methylation analysis of the saccharides provided 2.4 mol of 3-substituted galactose, 4 mol of terminal galactose, and 1 mol each of 2,4-, 2,6-, and 3,6substituted mannose. The same analysis indicates that about 45% of the reducing terminal N-acetylglucosamine was unreduced judging from the amount of 3-O-methyl-N-acetylglucosamine. This compound is produced when the reducing terminal N-acetylglucosamine is incompletely reduced before methylation. In order to elucidate which side chains were elongated to form the N-acetyllactosaminyl repeat, the saccharides which eluted at 24 min (peak 3 in Fig. 6D) were methylated. The saccharides provided 0.6 mol of 2,6-substituted mannose, 0.4 mol of 6-substituted mannose, 0.3 mol of 4-substituted mannose, 0.3 mol of terminal mannose, 0.4 mol of 2-substituted mannose, and 1 mol of 3,6-substituted mannose as mannose derivatives. These resultsindicate that each side chain arising from 2,6-substituted mannose and the side chains arising from C-2 or C-4 of 2,4-substituted mannose were elongated by Nacetyllactosaminyl units. Since FAB-MS of the starting materials afforded a fragment ion at m/z 1723 for NeuNAc-+Hex+HexNAc+Hex+ HexNAc-+Hex-+HexNAc' (see Fig. 3A), it is likely that Lacs saccharides contain three N-acetyllactosaminyl units in one of the side chains which are attached to C-2 or C-6 of 2,6substituted mannose or C-4of 2,4-substituted mannose. This was confirmed by the fact that peak 3 in Fig. 6D provided a small amount (0.1 mol) of 3-substituted galactose on methylation analysis. Structures of Asialo N-Linked Saccharides from Recombinant Erythropoietin-The results obtained above are sum1. marized inTable 1 1 By measuring radioactivity in each fraction, the relative yields of saccharides were calculated. In some cases, it was necessary to obtain the ratio after exoglycosidase digestion. For example, the ratio triantennary with of one N-acetyllactosaminyl repeat and tetraantennary in peak 4 was obtained in Fig. 6A. Fractionation of Intact N-Linked Saccharides by TSKDEAE Ion-exchange Chromatography-In order to determine how these saccharides are sialylated, intact N-linked saccharides were fractionated by HPLC employing a TSK-DEAE column. As shown in Fig. 7A, sialylated saccharides were essentially separated into four fractions: monosialosyl (fraction I) (7% of the total saccharide), disialosyl (fraction 11) (41%),trisialosyl (fraction 111) (48%), andtetrasialosyl (fraction IV) (4%) saccharides. No detectable amount of carbohydrate was present in other fractions. After desialylation, monosialosyl saccharides (fraction I) were found to contain biantennary (12% of the total monosialosyl saccharides), triantennary (17%), tetraantennary (plus triantennary with one N-acetyllactosamine repeat) (28%), tetraantennary with one N-acetyllactosamine repeat (29%), tetraantennarywith two N-acetyllactosamine repeats (1l%), and tetraantennary with three N-acetyllactosamine repeats (3%) (Fig. 5C). Disialosyl saccharides (fraction 11) were found to consist of triantennary (7%of the totaldisialosyl saccharides), tetraantennary (plus triantennary with one N-acetyllactosamine repeat) (43%), and tetraantennary saccharides with one (36%), two (12%), or three (2%) N-acetyllactosamine repeats (Fig. 50). Trisialosyl saccharides (fraction 111), however, contain only tetraantennary (47% of the total tetrasialylatedsaccharides) and tetraantennarysaccharides with one (39%),two (12%), or three (2%) N-acetyhctosaminyl repeats (Fig. 5E). The amount of tetrasialylated

saccharides was significantly low (4.2% of the total). Those saccharides were found to contain tetraantennarysaccharides and tetraantennary saccharides with one or two N-acetyllactosaminyl repeats (data not shown). These results indicate that 1) the biantennary saccharide is almost exclusively in a monosialylated form; 2) the triantennary saccharides are in monosialylated or disialylated forms; 3) the tetraantennary saccharides are mostly in disialylated or trisialylated forms; and 4) thetetraantennary saccharides with one, two, or three N-acetyllactosamine repeats are mostly in disialylated or trisialylated forms. These results suggest that one of the side chains in the saccharides is almost alway terminated without a sialic acid residue (see below). In order to know whether any difference exists among different batches of recombinant erythropoietin, two additional batches (Batches 3 and 4 in Table I) of recombinant erythropoietin were subjected to analysis. Interestingly, these samples contained morehighly sialylated saccharides: the disialosyl form is 18-21% of the total saccharides; the trisialosyl form is 64-67%, the tetrasialosyl form is 10-13%; and the monosialosyl form is less than 6%.However, the relative ratios of asialo biantennary, triantennary, tetraantennary, and tetraantennary saccharides with one, two, or three Nacetyllactosaminyl repeats were almost identical among different samples. These results indicate that sialylation may vary among different batches of recombinant erythropoietin but their backbone structures are the same. Separation of Saccharides with Different Backbone Structure but with the Same Number of Sialic Residues in Side Chains-The results of Fig. 7A suggested to us that each peak in the monosialosyl, disialosyl, or trisialosyl fraction may represent saccharides with different backbone structures but with the same number of sialic acid residues. In order to test this possibility, another 5 mg of recombinant erythropoietin yield N-linked saccharides, (Batch 2in Table I) was treated to and these saccharides were fractionated by TSK-DEAE ionexchange chromatography. This sample provided an elution profile almost identical to that in Fig. 7A. Saccharides were divided into four (fraction 11) or three (fraction 111) fractions, desialylated, and subjected to another HPLC employing a Lichrosorb column. Fraction 11-1, which eluted earliest in TSK-DEAE chromatography, provided tetraantennary saccharides with two or threeN-acetyllactosaminyl repeats (Fig. 8A, see Miniprint), whereas the last peak (fraction 11-4) mainly consists of triantennary and tetraantennary saccharides (Fig. 80). Similarly, the earliest peak in the trisialosyl fraction (fraction 111-1)provided Lac2 and a small amount of Lacl and Lac3 (Fig. 8E), whereas the last peak (fraction III3) provided almost exclusively tetraantennary saccharides (Fig. 8G). These results indicate that the saccharides with higher numbers of N-acetyllactosamine units elute earlier than those with smaller numbers of N-acetyllactosamine units in TSK-DEAE ion-exchange chromatography. Localization of a 2 4 - L i n k e d Sialic Acid in the Side Chains-In order to know whichside chains arepreferentially sialylated, fractions 11-2, 11-3 and 111-3 were digested extensively with a mixture of P-galactosidase and P-N-acetylglucosaminidase, and theproducts were purified by Sephadex G50 gel filtration followedby TSK-DEAE chromatography. The purified products were then subjected to methylation analysis, and theresults are summarized as follows. Disialosyl Tetraantennary Saccharides with One or Two NAcetyllactosaminyl Repeats (Fraction II-2)"Methylation analysis on the exoglycosidase product of fraction 11-2 provided the following mannose derivatives: 0.9 mol each of 2,6-

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(A
l6

t

r

~ r+ n -, r *

Linked oligosaccharide fractions obtained afterSephadex G-50 filtration gel (fractions 29-41 in Fig. 2B) were applied to a column of TSK-DEAE SW2 equilibrated with 25 m potassium phosphate M buffer, pH 5 0 After washing with the .. same buffer for 10 min, the column was eluted with a linear gradient from the same buffer to 400 m potassium phosM phate buffer, pH 5.0. A portion of the fractions indicated by the horizontal arrows were pooled subjected to HPLC and with a Lichrosorb column, as shown in Figs. 5 (C-E) and 8.

FIG. 7. Ion-exchange m L C of Nlinked saccharides obtained from recombinant erythropoietin ( A )and urinary erythropoietin (3). N-

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0

20

40

100

120

140

Fraction Nunbec (0.3ml I Fraction)

of 2,d-substituted mannose and C-4 of 2,4-substituted mannose. B. the saccharides with one N-acetyllactosaminyl repeat(m=l,n=O,ando=O,inW%of the molecules, and o = 1 and m = n = 0 in 10% of the molecules); the saccharides with two N-acetyllactosaminyl repeats ( m = n = 1 and o = 0 in 60% of the molecules, and m = o = 1 and n = 0 in 40% of the molecules); and the saccharides with three N-acetyllactosaminyl repeats ( m+ n o = 3).

FIG. 11. Proposed structures of tetraantennary saccharides(A) and tetraantennary saccharides with Nacetyllactosaminyl repeata ( B ) obtainedfromrecombinanterythropoietin. A, sialylation takes place preferentially at the side chain arising from C-6 and then from the side chain at C-2

A

+NeuNAca2+3Gal81+4Gl

cNAc6l
` 6

+NeuNAcaZ+3Ga161+461cNAc81~ +NeuNAca2+3GalB14Gl cNAc6l 4 `

pllanal\

- a1 +Fuc
Man614Gl cNAc614G1 cNAcdsn
6

Gal 61461cNAc61'

2

Hanal

;

6

I

B

+NeuNAca2+3(Gal614G1cNAc61+3),-Gal61461cNAc61

kManal,
4 `

+Fuc - a1
Man81+4461cNAc614G1cNAc+Asn 3
6

+NeuNAca2+3( Gal614461 cNAc61+3),*Gal614G1 cNAc6l' +NeuNAca2-.3(Ga161+461cNAc61+3);Ga1614G1cNAc61 Gal 814GlcNAc61'

4

+

`Manal' 2

I

substituted andterminal mannose, 0.1 mol 4of and 6- tuted mannose. The results indicate that 60% of the molecules substituted mannose, and 1 mol of 3,6-substituted mannose. are sialylated in both chains elongating from C-2 and C-6 of No other mannose derivatives, including 2,4-substituted man- 2,6-substituted mannose, and 40% of the molecules are sianose, were detected. The results indicate that the 2 sialosyl lylated in the side chain arising from C-4 of 2,4-substituted from C-6 C-2 of 2,6-substituted mannose. or residues are almost exclusively linked tothe side chains mannose and that Trisialosyl Tetraantennary Saccharide (Fraction III-3)arising from C-2and C-6 of 2,6-substituted mannose (see Fig. Methylation analysis after exoglycosidase digestion of frac11). tion 111-3 provided the following mannose derivatives: 1 mol Disialosyl Tetraantennary Saccharide (Fraction II-3)Methylation analysis of the exoglycosidaseproduct of fraction each of 2,6- and 3,6-substituted mannose and 0.5 mol each of 11-3 provided the following mannose derivatives: 0.6 mol of 2,4- and 4-substituted mannose. However, no other mannose terminal mannose, 0.3 mol of 2-substituted mannose, 0.4 mol derivative was detected. The results indicate that the nonsi2,4of 4-substituted mannose, 0.1 mol of 6-substituted mannose, alylated chain almost exclusively arises from C-2 of 0.6 mol of 2,6-substituted mannose, and 1 mol of 3,6-substi- substituted a-mannose.

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Structure of Sialylated Tetraantennary Saccharides with or without N-Acetyllactosamine Repeats-Based on the results described above, the structure of intact tetraantennary saccharides and those with N-acetyllactosaminyl repeats, which represent 85% of the total saccharides, can be proposed as shown in Fig. 11. The tetraantennary saccharides are mainly present as disialosyl or trisialosyl forms, and 2+3-linked sialic acid is attached to the side chains arising from C-6 and C-2 of 2,6-substituted mannose and C-4 of 2,4-substituted mannose. In the tetraantennary saccharides with N-acetyllactosaminyl repeats, sialic acid residues are always present in the side chain which contains N-acetyllactosaminyl repeats. This conclusion was supported by FAB-MS analysis. As shown in Fig. 3A, all of the fragment ions containing polylactosaminyl units are sialylated. In order to delineate this further, trisialylated saccharides (fraction 111) wereextensivelydigested by P-galactosidase and @-N-acetylglucosaminidase. methylation analysis of this The product showed that more than 90% of the side chain attached to C-2 of 2,4-substituted mannose was terminated without sialic acid. These combined results supportthe proposed structures shown in Fig. 11. Structure of Carbohydrate Units of Urinary Erythropoietin-Since only a limited amount of urinary erythropoietin was available, the following experiments were carried out to analyze carbohydrate units of urinary erythropoietin. Glycopeptides, prepared by Pronase digestion of urinary erythropoietin, were subjected to alkaline borohydride treatment. The alkaline borohydride-treated sample was then applied to Bio-Gel P-4 gel filtration. Urinary erythropoietin saccharides provided almost the same elution profile as Fig. 2A. The glycopeptides containing N-linked saccharides (fractions 2429) were digestedby N-glycanase, the digest was subjected and to Sephadex G-50 gel filtration. Again, the elution profile of N-linked saccharides from urinary erythropoietin was almost identical to that from recombinant erythropoietin (see Fig. 2B). Methylation analysis of N-linked saccharides (fractions 2941 after Sephadex (3-50 gel filtration) provided partially 0methylated monosaccharide derivatives, which are almost identical to those produced from highlysialylated Batch 3 of recombinant erythropoietin (Table 11).FAB-MS of permethylated N-linked saccharides provided fragment ions for NeuNAc' (m/z 376 and 344), NeuNAc+Hex+Hex NAc+ ( m / z 825), NeuNAc+Hex+HexNAc-+Hex+HexNAc+ (m/z 1274), and NeuNAc+Hex+HexNAc+Hex+HexNAc+ Hex+HexNAc+ (m/z 1723) (Fig. 3B). These results are essentially the same as those obtained on recombinant erythropoietin (compare Fig. 3, A and B). N-Linked saccharides were then desialylated and subjected to HPLC employing a Lichrosorb-NHz column. As shown in Fig. 5B, urinary erythropoietin saccharides provided triantennary, tetraantennary, and tetraantennary saccharides with one, two, or three N-acetyllactosaminyl units. The relative proportion among these saccharides is almost identical to that obtained on recombinant erythropoietin except that urinary erythropoietin apparently lacks biantennary saccharides. In order to determine the relative amounts of sialylated N-linked saccharides, intact N-linked saccharides were subjected to TSK-DEAE ion-exchange chromatography. Fig.7B shows that N-linked saccharides from urinary erythropoietin contain disialosyl(27%of the total saccharides),trisialosyl(56%), and tetrasialosyl (17%) saccharides. These results indicate that urinary erythropoietin and recombinant erythropoietin have an almost identical set of N-linked saccharide units but

with slightly different sialylation depending upon the batches of recombinant erythropoietin (see above and Table I).
DISCUSSION

This paper reports the detailed structures of the carbohydrate moiety of human erythropoietin produced by recombinant DNA. The protein analyzed was produced in Chinese hamster ovary cells which were transfected with human erythropoietin cDNA (7). As far as we are aware, this is the first report on the detailed carbohydrate structure of a glycoprotein produced by recombinant DNA in comparison with the glycoprotein of natural origin.Although Mutsaers et al.(28) reported the carbohydrate structure of human y-interferon produced in Chinese hamster ovary cells, their studies did not investigate those of naturally occurring human y-interferon. The carbohydrate composition (Table I) showed that erythropoietin contains three N-linked saccharides and one 0linked saccharide, and these conclusions are consistent with the recent report on the amino acid sequence of human urinary erythropoietin (29). The present study revealed that a large proportion of the carbohydrate moiety of recombinant erythropoietin is composed of tetraantennary saccharides with one (32.1% of the total saccharides),two (16.5%),and three(4.7%)N-acetyllactosaminyl repeats. The localization of these polylactosaminyl units was elucidated by sequential exoglycosidase digestion followed by methylation analysis, and the results are summarized as follows (seealso Table 111). When the saccharides contain one N-acetyllactosaminyl repeat, more than 70% of this repeat is preferentially attached to the side chain arising from C-6of 2,6-substituted mannose, and 19% of the repeat is attached to that from C-4 of 2,4substituted mannose. When the saccharides contain two Nacetyllactosaminyl repeats, these repeats are attached to C-2 and C-6 of 2,6-substituted mannose in 80% of the molecules. The rest of the molecule contains N-acetyllactosaminyl repeats in the side chains arising from C-6 of 2,6-substituted mannose and C-4 of 2,4-substituted mannose. These results indicate that N-acetyllactosaminyl repeats are most preferentially added to C-6 of 2,6-substituted mannose and then to C-2 of 2,6-substituted mannose. These conclusions are consistent with previousreports on severalcellular glycoproteins. For example, Cummingsand Kornfeld (30) reported that the mouse lymphoma BW5147 cell line expressed a signX1cant amount of polylactosaminoglycan, whereas mutant, which its lacks the side chain arising from C-6of 2,6-substituted mannose, expresses a minimum amount of polylactosaminoglycan. Li et al. (31) isolated polylactosaminoglycan from Chinese hamster ovary cells in which polylactosaminyl units are attached to C-2 and C-6 of 2,6-substituted mannose. Similarly, polylactosaminyl units were found in triantennary and tetraantennary saccharides of various origins (24,32-34). These results appear to establish that N-acetyllactosaminyl repeats are preferentially added to C-6 of 2,6-substituted mannose and then to C-2 of 2,6-substituted mannose. These 2,6-substituted mannose residues are usually linked to the C-6 side of @-mannose. human erythrocytes, polylactosaminyl elonIn gation can be found in the side chain arising from C-2 of (Ymannose which is linked to C-6 of P-mannose (23, 35). It is likely that human erythroid cells contain very little activity of the N-acetylglucosaminyltransferase which forms a GlcNAcpl4Man branch. As a result, N-acetyllactosamine repeats are formed on the secondary preferable side chain, which is attached to C-2 of a-mannose linked to C-6 of 8mannose, in these erythroid cells. This study showed that human erythropoietin exclusively

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contains a2-3-linked sialic acid. This fact allowed us to mine repeats. It has been shown that ratliver cells uptake the elucidate the locaiization of 2+3-linked sialic acid residues asialo form of glycoproteins which contain tri- or tetraantenasialo among different side chains. This was achieved by extensive nary saccharides (43). It is therefore reasonable that the digestion of intact saccharides with P-galactosidase and P-N- form of erythropoietin is taken up by liver cells through a acetylglucosaminidasefollowed bymethylation analysis of the galactose-binding protein (15). Our preliminary studies products. These results can be summarized as follows. 1) showed that a portionof intact erythropoietinof both recomWhen typical triantennary or tetraantennary saccharides con- binant and urinary origins is takenup by rat liver cells, tain 2 sialic acid residues, they are attached to C-2 and C-6 presumably because of the incomplete sialylation. It will be of 2,6-substituted mannose or C-6 of 2,6-substituted mannose interesting to test if sialylation by a24-sialyltransferase and C-4 of 2,4-substituted mannose. 2) When saccharides elongates the serum concentration of erythropoietin and suswith N-acetyllactosaminyl repeats contain 2 sialic acid resi- tains in vivo activity longer than the startingerythropoietin. dues, they are attached almost exclusively to C-2 and C-6 of Acknowledgments-We thank Dr. Tsutomu Kawaguchi (Chugai 2,6-substituted mannose. This localization is essentially idenPharmaceutical Co., Ltd.)for initiating this joint project, Dr. Friedtical to that of N-acetyllactosamine repeats. Thus, it is ap- rich Piller for useful discussion and Candy Farmer for secretarial parent that polylactosamine is preferably sialylated through assistance. an aB-+S-linkage. These results are consistent with our previous results obREFERENCES tained on polylactosaminoglycans from chronic myelogenous 1. Goldwasser, E., and Kung, C. K. H. (1968) Ann. N. Y . Acad. Sci. leukemia cells;a2-3-linked sialic acid is present on side 149,49-53 chains arising from C-6 and C-2 of 2,6-substituted mannose 2. Jacobson, L. O., Goldwasser,E., Fried, W., and Plzak, L. F. (1957) and C-4 of 2,4-substituted mannose, and those side chains are Nature 179,633-634 3. Fried, W . (1972) Blood 40,671-677 longer than that terminating with 2 4 - l i n k e d sialic acid (24). 4. Zanjani, E. D., Ascensao, J. L., McGlave, P. B., Banisadre, M., Similar results were obtained in human erythrocyte Band 3 and Ash, R. C. (1981) J. Clin. Invest. 6 7 , 1183-1188 polylactosaminoglycans; polylactosaminyl side chains arising 5. Adamson, J. W . ,Eschback, J. W . , and Finch, C. A. (1968) Am. J. from the C-6 side of p-mannose are sialylated through a 2-+ Med. 44,725-733 3-linkage, whereas the shorterchain arisingfrom the C-3side 6. Miyake, T., Kung, C. K.-H., and Goldwasser, E. (1977) J. Bwl. Chem. 262,5558-5564 is sialylated through a 24-linkage (23, 35). Similarly, Ya7. Jacobs, K., Shoemaker, C., Rudersdorf, R., Neill, S. D., Kaufman, Cummings (36) found that mashita et al. (33) and Markle and R. M., Mufson, A., Seehra, J., Jones, S. S., Hewick, R., Fitch, longer polylactosaminyl side chains are almost exclusively . E. F., Kawakita, M., Shimizu, T., and Miyake, T (1985) Nature sialylated through a 2+3-linkage. Interestingly, short poly313,806-810 lactosamine chains in thyroid cell glycoprotein Gp-1 (37) and 8. Lin, F. K., Suggs, S., Lin, C. H., Browne, J. K., Smalling, R., BW5147 (36) are terminated with 2 4 - l i n k e d sialic acid. Our Egrie, J. C., Chen, K. D., Fox, G . M., Martin, F., Stabinsky, Z., Badrawi, S. M., Lai, P. M., and Goldwasser, E. (1985) Proc. results also showed that almost no 2-3-linked sialic acid is Natl. Acad. Sci. U. S. A. 8 3 , 7580-7584 attached to the side chain arising from C-2 of 2,4-substituted 9. Powell, J. S., Berkner, K. L., Lebo, R. V., and Adamson, J. W . mannose. It is noteworthy that this side chain was found to (1986) Proc. Natl. Acad. Sci. U. S. A. 8 3 , 6465-6469 beexclusively sialylated througha 24-linkage in many 10. Winearls, C. G., Oliver, D. O., Pippard, M. J., Reid, C., Downing, glycoproteins including normal and leukemic granulocyte poM. R., and Cotes, P. M.(1986) Lancet ii, 1175-1178 11. Eschbach, J. W., Egrie, J. C., Dowwing, M. R., Browne, J. K., lylactosaminoglycans (19-21, 24, 34, 38-41). and Adamson, J. W . (1987) N. Engl. J. Med. 316,73-78 By using a bovine colostrum a2-&-sialyltransferase, Joziasse et al. (42) have shown that preferential sialylation takes 12. Rambach, W . A., Shaw, R. A., Cooper, J. A. D., and Apt, H. L. (1958) Proc. Soc. Exp. Biol. Med. 99,482-483 place first on C-2 of 2,4-substituted mannose and then on C- 13. Lowy, P. H., Keighley, G., and Borsook, H. (1960) Nature 186, 4 of 2,4-substituted mannose. This branch (or side chain) 102-103 specificity appears to be opposite to the distribution of 2-314. Lukowsky, W.A., and Painter, R. H. (1972) Can. J. Biochem. 60,909-917 linked sialic acid. Thus, it is likely that a2+=3-~ialyltransferase and a24-sialyltransferase have complementary specific- 15. Goldwasser, E., Kung, C. K.-H., and Eliason, J. (1974) J. Biol. Chem. 249,4202-4206 ity toward different side chains. Furthermore, our results raise 16. Simnonsen, C. C., and Levinson, A. D. (1983) Proc. Natl. Acad. the possibility that theside chains containing polylactosamiSci. U. S. A . 8 0 , 2495-2499 nyl units would be preferable sites for 2+3-linked sialylation. 17. Fukuda, M., Carlsson, S. R., Klock, J. C., and Dell, A. (1986) J. Bwl. Chem. 261,12796-12806 This study demonstrated that the carbohydrate moiety of human erythropoietin isolated from human urine is indistin- 18. Plummer, T. H., Jr., Elder, J. H., Alexander, S., Phelan, A. W., and Tarentino, A. L. 10700-10704 guishable from that of recombinant erythropoietin except for 19. Nilsson, B., Nordkn, N. (1984) J. Biol. Chem. 2 5 9 , J. Bwl. Chem. E., and Svensson, S. (1979) a difference in degree of sialylation. Urinary erythropoietin 264,4545-4553 has a similar degree of sialylation as the highly sialylated 20. Baenziger, J. U., and Fiete, D. (1979) J. Biol. Chem. 254, 789795 batch of recombinant erythropoietin (Tables I and 11). The recombinant erythropoietin was produced in Chinese hamster 21. Endo, M., Suzuki, K., Schmid, K., Fournet, B., Karamanos, Y., Montreuil, J., Dorland, L., Van Halbeek, H., and Vliegenthart, ovary cells, and urinary erythropoietin ispresumably derived J. F. G. 267,8755-8760 from human kidney cells. The results therefore suggest two 22. Yoshima, (1982) J. Biol. Chem.Mizuochi, T., Kawasaki, T., and H., Matsumoto, A., possibilities. 1) Chinese hamster ovary and human kidney Kobata, A. (1981) J. Bwl. Chem. 256,8476-8484 cells contain similar glycosyltransferases. 2) The protein ac- 23. Fukuda, M., Dell, A., and Fukuda, M. N. (1984) J. Bwl. Chem. 269,4782-4791 ceptor itself influences glycosylation even when a similar set of glycosyltransferases are not present in two cell types. It 24. Fukuda, M., Bothner, B., Ramsamooj, P., Dell, A., Tiller, P. R., Varki, A., and Klock, J. C. (1985) J. Biol. Chern. 260, 12957will be interesting to see if the carbohydrate moiety of eryth12967 ropoietin produced in other mammalian cells is similar to 5;5. Jourdian, G . W . , Dean, L., and Roseman, S. (1971) J. Biol. Chem. those elucidated in this study. This studyalso demonstrated 246,430-435 that the major carbohydrate units of erythropoietin are te- 26. Greenwood, F. C., Hunter, W . M., and Glove, J. S. (1963) Biochem. J. 89, 114-123 traantennary saccharides with or without N-acetyllactosa-

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Carbohydrate Structure of Human Recombinant Erythropoietin
27. Laemmli, U.K. (1970) Nature 227,680-685 28. Mutsaers, J. H. G. M., Kamerling, J. P., Devos, R., Guisez, Y., Fiers, W., and Vliegenthart, F. G. (1986) Eur. J. Biochem. 166, 651-654 29. Lai, P.-H., Everett, R., Wang, F.-F., Arakawa, T., and Goldwasser, E. (1986) J. Biol. Chem. 261,3116-3121 30. Cummings, R. D., and Kornfeld, S. (1984) J. Biol. Chem. 269, 6253-6260 31. Li, E., Gibson, R., and Kornfeld, S. (1980) Arch. Biochem. Biophys. 199,393-399 32.Eckhardt, A. E., and Goldstein, I. J. (1983) Biochemistry 22, 5290-5297 33. Yamashita, K., Ohkura, T., Tachibana, Y., Takasaki, S., and Kobata, A. (1984) J. Biol. C h m . 2 6 9 , 10834-10840 34. Fukuda, M., Spooncer, E., Oates, J. E., Dell, A., and Klock, J. C. (1984) J. Biol. Chem. 269,10925-10935 35. Fukuda, M., Dell, A., Oates, J. E., and Fukuda, M. N. (1984) J.

Biol. Chem. 269,8260-8273 36. Merkle, R. K., and Cummings, R. D. (1987) J,. Biol. Chem. 2 6 2 , 8179-8189 37. Edge, A. S.B., and Spiro,R. G. (1985) J. Biol. Chem. 260,1533215338 38. Mega, T., Lujan, E., and Yoshida, A. (1980) J. Biol. Chern. 256, 4057-4061 39. Paulson, J. C., Weistein, J., Dorland, L., Van Halbeek, H., and Vliegenthart, J. F. G . (1982) J. Biol. Chem. 2 6 7 , 12734-12738 40. Spik, G., Debruyne, V., Montreuil, J., VanHalbeek, H., and Vliegenthart, J. F.G. (1985) FEBS Lett. 183,65-69 41.Yamamoto, K., Tsuji, T., Irimura, T., andOsawa, T. (1981) Biochem. J. 196,701-713 42. Joziasse, D. H., Schiporst, W. E. C. M., Van den Eijenden, D. H., Van Kuik, J. A., Van Halbeek, H., and Vliegenthart, J. F. G. (1987) J. Biol. Chem. 2 6 2 , 2025-2033 43. Baenziger, J. U., and Fiete, D. (1980) CeU 22,611-620

SUPPLEMENTAL MATERIAL TO CARBOHYDRATE STRUCTURE OF ER'lTHROPOlETlN EXPRESSEO HMSTER OVARY CELLS B Y A HUMAN ERYTHROPOIETIN BY HlroshiSaraki.

TABLE 11. R e l a t i v e r o p o r t i o n s p O f methylated s r i d e s O f r e c m b i n a n t and u r i n a r y e r y t h r o p o i e t i n s .

w a m obtained frm N-linked saccha3

I N CHINESE COW

Methylated Sugars Fudtol -T;5;4Itri-O-Wthyl GalaCtitol 2.3.4.6-retra-O-nethyl 2.4,6-tri-O-mthyl Mami to1 mri-0-methyl 3.6.-di-O-nethyl 3,4-di-O-nethyl 2.1-dl-0-methyl 2-N-methylacetamido2-deoxyqlucitol 1.3,5.6-tetra-O-~thyl 1,3.5-tri-O-rethyl 3.6-di-O-mthyl 3-0-methyl

N-saccharides Recrmbindnt Urinary batch 1 batch3 0.9 1.34 3.52 0.9 0.17 4.30 0.12 0.81 1.07 1.00 0.9 0.38 4.11

4
0.95 3.90 0.11 0.11 1.00 0.90 1.00

5

6

7

B r i a n Bothner. Anne Oell and Minaru FUkVda

l l o l d t i o n O f 0-linkedDligOsaCChmides frm n c n b i n a n t e r y t h r o p o i e t i n The 0 - l i n k e d oligosdcc ar er *ere separ*te Tm n c g ycopep o-Gel P-4 g e lf i l t r a t i o n I s r h a n 'in ' f i g . 2 A . The f r d t T o n s :o"td35 ( d ~ ~ ~ & ~ ~ dand ~those frm 36-40 B -i) (designated 0 - i f ) were separatelypooled and s u b j e c t e dt o TSK-OEAE ?on exchange c h m a t o graphy as I h w n i n F i g . 9

:>d

-

-

0.9 3.0 0 1.00 0.35 1.00

0.9

0.9

0.9 4.0 2.4
0 0.9 1.1 1.0

4.0
0.8 0 1.0 1.0 1.0

4.0
1.7

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The 0 - f r a c t i o p l o v i d e d i s l a l o s y l l i q O s a C C h m i d e s 1 n o and t v l s i a l o s vo l i s o r a c c h a l d;s judged r i d e s , whereas t h e 0 - i i f m t i o n p r o v i d d m i n i y m o n o r i a l o r y l o l i g o s a c c h a 6 d e s . by FAB-MS. The f T a C t i O nc a r r e r p a n d i n go n o ~ i a l o s y l -d l r i a l o r y l - . l tm , and t r l l i a l o s y l 0-1 and (I-H f r a c t i o n s were pooled and subjected t o e t h y l a t i o n m s a c c h w i d s sr o mh e ft a n a l y s i s and FAR-MS maly611.

0.78 1.05 1.00

0.16

0.76 0.98 1.00

0.26

0.65

1.0 1.0 1.0

0

0.10 0.80 5.82 0.10

0.12 0.81 5.76 0.07

0.14 0.90.81

troce
4.0 0.1

5.54 0.05

0.9 5.0 0.05

0.05

t r a c e 0.05 0.9 0.85 6.0 6.7

0.05 0.10

0.10 0.55 7.4 0.35

TmLE IY. R e l a t i v e p r o p o r t i o n s of methylatea sugar obtained frm 0-linkedsaccherfdts o f r e c m b i n a n t and u r i n a r y e r y t h r o p i o e t i n r . molecule (The d e t a i l e d mechaniimr f oB ~ c l e a v a g e r and 6 - e l i m i n a t i o w i l l n be der;ribed elraher;.21. 01igoSdCChalide The was r e g u c n t i a l l yd i g e s t e d by c l o s t r i d i a ls i a l i d a s e and E s h e r i c h i ac o l i6 - g a l a c t 0 l i d d l e a s describedpreviously(17). These r e w l t s . t o g e t h e r w i t h mthylation-iiiilysis (Table 1V) i n d i c a t et h a tt h ed l 1 i m l o l y lo l i q o s a c c h a r i d e 1 1 NeuNAeoGaldctitola 2.4.6-tn-O-wthyl 2-N-rethylacetamidoZ-de~xyqalactit~l trace 0.8 1,4,5,6-tetra-O-methyl 0.1 1,4,5-tn-O-methyl 4-mono-O-mthylb
1.0

Recmbinant Mo".ali.lolyl O i r T l l oiuyO l a rl y i a l l. l il n Y r

1.0

1.0

1.0

1.0

0 0.9

0.2 0.7 0.1

frm u r i n a r y e r y t h r o p o i e t i n The 0 - l i n k e d S t r u c t w e s of 0-linked OligosaCChdridel oligosaccharides f m u r i n a r ye v y t h r o p o i c t i n were recovered i n f r a c t i o n 30-40 i n Blo-Gel P-4 g ef li l t r a t i o n !5*e Fig. ZA). I n i t i aL t t m p ttl o l fwther f r a c t i o n a t0 - l i n k e d e O l i g o s a