expression of human interlewkin-2 in recombinant baby hamster

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 264, No. 29, Issue of October 15, pp. 17368-17373, 1989 Printed in U. S . A

Expression of Human Interlewkin-2 in Recombinant Baby Hamster Kidney, Ltk-, and Chinese Hamster Ovary Cells STRUCTURE OF 0-LINKED CARBOHYDRATE CHAINS AND THEIR LOCATION WITHIN THE POLYPEPTIDE*

(Received for publication, January 3, 1989)

Harald S. ConradtS, Manfred Nimtzs, Kurt E. J. Dittmar, Werner Lindenmaier, Jiirgen Hoppe, and Hansjorg Hauser From the Department of Cell Biology and Genetics and §Molecular Structure Research Group, Gesellschaft fur Biotechnologische Forschung mbH, Mascheroder Weg 1, 0-3300 Braunschweig, Federal Republic of Germany

The similarity or identity of 0-glycosylation in glycoproteins from natural sources or produced in heterologous cell lines, a central problem for the develop- ment of many biotechnologically relevant production processes, was examined using interleukin-2 (IL-2) as a model.

Human interleukin-2 was constitutively expressed in several mammalian cell lines in high amounts. The recombinant proteins were purified to homogeneity and their carbohydrate structures were analyzed. Only the NeuAca2-3Gal~l-3[NeuAca2-6]GalNAc oligosaccharide structure or the NeuAca2-3Gal~l-3GalNAc were found in all IL-2 preparations secreted from recombinant Ltk-, Chinese hamster ovary, and baby hamster kidney cell lines. The 0-linked chains were exclusively linked to Thr in position 3 of the polypeptide chain which is the carbohydrate attachment site in natural human IL-2. The proportions of O-glycosylated versus nonglycosylated forms of the protein secreted by each recombinant cell line were independent of productivity or of cell culture conditions.

Our results show that 0-glycosylated human IL-2 can be produced by applying recombinant DNA technology in heterologous cell lines with the same type of post-translational modification that is observed for the protein secreted from natural T lymphocytes.

Many human proteins with clinical potential can be obtained from natural sources in only limited amounts. Recom- binant DNA technology is now widely used to produce such proteins, e.g. interferons, lymphokines, clotting factors, and other growth and differentiation factors in microorganisms in high amounts. In many cases these polypeptides are post- translationally modified by N - and/or 0-glycosidically linked carbohydrates. For a complete description of their in uitro and in vivo biological properties it is necessary to obtain these proteins in their glycosylated form. In principle, the manipu- lation of heterologous mammalian cell lines with defined DNA fragments encoding the desired polypeptide should lead to production of glycoproteins. However, it is not clear whether or not a given mammalian host cell line is capable of glyco-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 To whom correspondence and reprint requests should be ad- dressed Dept. of Genetics, G. B. F., Mascheroder Weg 1, D-3300 Braunschweig, Federal Republic of Germany.

sylating in a fashion which will yield the natural protein form. For N-glycosylated recombinant human interferon-p expressed in several mammalian cell lines, considerable differ- ences in oligosaccharide structures have been reported (1). These differed also from those found in interferon-p from human foreskin fibroblasts.

In contrast to N-glycosylation where carbohydrate is always attached to the amide nitrogen of an asparagine residue in the tripeptide sequence Asn-X-Thr/Ser (2, 3), a recognition peptide sequence for the attachment of 0-glycans to Thr or Ser side chains of proteins could not so far be identified. Secondary structure predictions from amino acid sequences found adjacent to glycosylated Ser or Thr residues in proteins, applying the method of Chou and Fasman (4), do not reveal unique structural features (for review, see Ref. 5).

Natural human interleukin-2 (IL-2)’ is a protein of 133 amino acids which is secreted by activated human T lymphocytes. This polypeptide contains a hydroxyamino acid cluster at the amino terminus (NH2-Ala-Pro-Thr-Ser-Ser-Ser-Thr- Lys-Lys-Thr . . . ) in which, exclusively, Thr in position 3 is post-translationally modified by an 0-linked NeuAca2- 3Galpl-3GalNAc chain or by the disialylated derivative thereof (6). This glycosylation has been suggested as playing a role in clonal outgrowth and long-term propagation of allo- activated human T cells in the presence of IL-2 (7).

In the production of a 0-glycosylated human protein in heterologous cell lines it was questionable if the carbohydrate moieties were identical with those found in the natural prod- uct, and if they were attached at the same amino acid residues. To investigate this, several mammalian cell lines were trans- fected with a plasmid containing the human IL-2 gene. Cell lines which constitutively secreted human IL-2 were obtained and the purified recombinant proteins were analyzed with respect to their carbohydrate structure(s) and the carbohydrate attachment site. A preliminary account of this work has been presented (8).

EXPERIMENTAL PROCEDURES AND RESULTS~

DISCUSSION

Apart from disadvantages associated with high expression of mammalian proteins in genetically manipulated bacteria,

The abbreviations used are: IL-2, interleukin-2; BHK, baby hamster kidney; CHO, Chinese hamster ovary.

Portions of this paper (including “Experimental Procedures” and “Results,” Tables I-IV, and Figs. 1-10) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

17368

0-Glycosylated Human IL-2 from Recombinant Mammalian Cells 17369

e.g. lack of disulfide bond formation and accumulation in dense inclusion bodies, a major consideration in choosing mammalian host cells for production of clinical important human proteins has been their capability of carrying out protein glycosylation.

Structural analysis of N-linked carbohydrate constituents from several human secretory proteins expressed in different heterologous cell lines (1,37,39) indicate that N-glycosylation is host cell-specific and oligosaccharides from recombinant glycoproteins may differ considerably from those observed for the natural proteins (1). In contrast, the present study shows that 0-glycosylated human interleukin-2 can be produced in genetically engineered mammalian cell lines; biochemical analysis of the IL-2 protein purified from supernatants of BHK, Ltk-, and CHO cells revealed that: (i) the same type of 0-linked oligosaccharide(s) are attached to the IL-2 protein derived from the different cell lines indentical to those found on IL-2 secreted by natural human T lymphocytes (6); (ii) the oligosaccharide chains are exclusively linked to Thr in position 3 of the polypeptide chain, the same site that is modified in natural human T cell-derived IL-2 (6).

Each recombinant cell line characteristically secretes glycosylated IL-2 N and nonglycosylated IL-2 M protein at a constant ratio that is not affected by culture conditions like cell density or age of culture. Similarly, high producer cell clones secrete both IL-2 forms at an identical ratio when compared to low producer clones obtained from the same cell line.3 Ltk- and BHK (and in transient expression experiments also HeLa cells) secrete 40-60% of the total IL-2 protein in the glycosylated form, whereas CHO cells as well as the mutant CHO cell line Lec 2 secrete preponderantly (greater than 90%) the glycosylated IL-2 N species. Similarly, a mix- ture of IL-2 N and M forms is secreted also by stimulated peripheral human T lymphocytes (6); their final ratio has been shown to depend on the induction protocol used to stimulate the cells (9). The reason why in each cell type a characteristic proportion of the IL-2 protein obviously escapes from the cellular glycosylation machinery is not clear. How- ever, once a GalNAc residue is attached to the polypeptide, galactose is transferred into position 3, forming the classical “core disaccharide” Galpl-3GalNAc structure most frequently found in 0-glycosylated proteins (5). This latter oligosaccharide is quantitatively sialylated by a cellular GalP1- 3GalNAca2-3 sialyltransferase and (at least in the case of CHO cells) by a R-GalNAca2-6 sialyltransferase completing the final NeuAca2-3Gal@l-3(NeuAc~u2-6)GalNAc structure, since no intermediates like Gal@l-3GalNAc-Ol or GalNAc-01 were detected after P-elimination of the [3H]sugar-labeled IL- 2 protein secreted by the recombinant cells. The CMP-NeuAc transport-defective mutant CHO cell line Lec 2 secretes only the Galpl-3GalNAc form of IL-2.

Therefore, a key role in regulating 0-glycosylation of at least the IL-2 protein can be ascribed to the polypeptide: ( d - 0)GalNAc-transferase. The enzyme operating in all tested cell lines exhibits a striking substrate specificity towards IL-2 as a substrate in that it recognizes specifically Thr at position 3 of the IL-2 polypeptide chain; none of the adjacent Ser or Thr side chains serve as an acceptor site for 0-linked carbohydrate.

The substrate specificity of the enzyme from bovine sub- maxillary glands has been studied in some detail (10). Small peptides with a blocked NH2 terminus of the Thr acceptor residue (R1-Thr-Pro-Pro-Pro-R,) containing a COOH-termi- nal triprolyl sequence were found to be most active. However, such proline-rich sequences adjacent to Thr/Ser are not es-

H. S. Conradt and H. Hauser, unpublished results.

sential for this type of post-translational modification to occur. The human erythrocyte membrane protein glycophorin A, e.g. contains hydroxyamino acid clusters (Leu’-Ser-Thr- Thr-Gly-Val‘ . . . . and Met8-His-Thr-Ser-Thr-Ser-Ser-Ser-

. . . ), where all Ser/Thr residues are modified by 0- linked carbohydrate chains (11); on the other hand, human chorionic gonadotropin contains a sequence Glull‘j-Asp-Ser- Ser-Ser-Ser-Lys’** . . . , where only Ser”’ bears an 0-linked oligosaccharide; a hydroxamino acid cluster (residues 51-55) in human glycophorin C is not glycosylated at all (11).

The recognition signal for 0-glycosylation of a given gly- coprotein may depend on the overall conformation, rather than on any linear amino acid sequence since no well defined primary structure common to all glycoproteins in the neigh- borhood of 0-glycosylation sites has been detected (5).

Our finding that a polypeptide: (al-0)N-acetylgalactosa- minyltransferase is present in BHK, Ltk-, CHO, and human T cells exhibiting an identical specificity towards the human IL-2 protein as a substrate (Thr in position 3 of the polypeptide chain) is a prerequisite for further studies to explore the structural requirements of a protein to serve as an acceptor for 0-linked carbohydrate(s), e.g. using site-directed mutagen- esis of the IL-2 gene and the expression of corresponding variants in heterologous mammalian cell lines.

Acknowledgments-We thank Walter Sebald for receipt of a cDNA clone containing the coding sequences for human IL-2 and Pamela Stanley for providing us with the CHO Lec 2 mutant cell line. The expert technical assistance of K. Schaper, B. Kornack, and M. Aus- meier is highly appreciated. We thank John Collins for linguistic advice and Sabine Peters for typing the manuscript.

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supp1enentary Haterla1 TO :

EXPRESSION OF HUMNI IMTERLLUKIN-2 I N RECOnBlWT BHX, Ltk- U I O CHO CELLS: STRUCTURE OF 0- LINKED CARBOHYDRATE CHIINS MID THEIR LOCATIMI YlTHlM THE POLVPEPTIDE

H A l t R l A L S EXPERIMENTAL PROCEDURES

H E I H O O S '

. L Y .

na tura l human IL-2 (6). Serum free It-2 EOntainlng medwn was clanfled by centrifugLtlon Purification of recomblnant 11-2 was performed rlm?larily 1s dercrlbed prevloully for

a t 10.000 xg for 10 manutes a t 4.C adjusted to pH 6.5 and adsorbed onto rontrolled.pore g1a11 beads (mesh size 120/100 Si&~a] The column was washed with phosphate buffered 5111ne (5 volumer) and I t - 2 "3; eluted w t h 30 X ethylene glycol in phosphate buffered rallne ( v / v ) containing 0.7 H rDd>um chloride. 11-2 activity was obtained ~n greater than 90 X yleld and was further fractronated by gelf!ltratlon an 1 2.5 x 100 cm column of Ultrogel AcA 51. I L ~ Z x t i v l t y was eluted I n the region of myoglobin Corresponding fractlenr were pooled and loaded anta 3 Spherlrarb 55 hexyl column (2.2 x 25 c m ) . After washing of the column with 2 5 r# potassium phorphate/l?O nW NaCl pH 4.3 (buffer A) Untll base line adrorbance 11% reached a linear gradlent vas applied from 0-35 X n-propanol ~n buffer A I t a flow rate of 2 ml/iin for 60-90 minuter. subsequent t o an lrocratrng run for 30-60 alnuter I recood gradtent from 35 % - 45 X pr&l in buffer A War applied fa? elution of IL:2 protein. 2 m1 fractlonr were collected and It-? was detected by SDS-PAGE and by analyr~r of bleactlvity DI described above. By this procedure up t o 1 ng m o u n t s of recomblnant human It-2 could be purified Ylthln 3 days.


T U L E I

L.".lS O f IL-2 .xpr.rrlon fro. recc&ln.nt c.11 1lrnS')

+I0 z fCS -FCS

LTK- 2200t462 2100t566 BHK 4500x620 3600x121

CHO 650x 85 41Or136 Lec 2 550x112 400t124

Pur i f icat ion of human 11-2 frm the medium o f recombinant c e l l l i n e s

TABLE I 1

P u r l f l c a t l o n o f r o c o m b l n a n t human 11-2 secreted b y I H K c e l l s

............................................................. s t e p : v o l u m e t o t a l 1 1 - 2 r c c a v e r y

I. c e l l free c u l t u r e

( .I ) ( ""Its 1 I z 1 ............................................................. medium 2000 8 . 5 x 106 100

11. controlled-porn

Ill. AcA 54 e l u a t e 8 0 1 . 3 x 106 8 5 . 9

glass e l Y l t C 30 1 . 5 x 106 88.9

I V . RP-HPLC a l v l t r I2 6 . 9 I I O 6 81.2 .............................................................

c e l l s w e r e s u b j e c t e d to the same p u r i f i c r t l o n procedure. Almost C u l t u r e supernatants f r o m r e c o m b l n l n t CHO. LTK- and Lec 2

ldentlcal v a l u e s for r e c o v e r y o f I t - 2 b i o a c t i v i t y were o b t a l n e d . The f l n a l s p e c l f l c a c t i v i t y o f 1 1 - 2 p r e p a r a t i o n s from a l l r e c o m b i n a n t c e l l Ilnrr w a s 1 . 5 x IO7 u n l t r l m g o f protcln I S d e t e m l n e d b y CO.partsOn w l t h t h e BRMP-standard 11-2 prcparatlon

c e l l s w e r e s u b j e c t e d to the same p u r i f i c r t l o n procedure. Almost C u l t u r e supernatants f r o m recombinant CHO. LTK- and Lec 2

ldentlcal v a l u e s for r e c o v e r Y o f I t - 2 b i o a c t i v i 1 ) r were o b t a l n e d .

M - + 67 60 - 44 0

36 - 30 -

S

- LO c

3s

a ] no 11-2 bioactivity was detected i n supernatants of c e l l s p v i o r to transfectlon.

b) confluent cells *ere g r m In 15 cd t l ssue cu l tu re f l asks f o r 24 hours i n the presence or absence of fetal c a l f serum; c e l l s were counted and It-2 bioactivity was determined as described under 'Methods'.

17372 0-Glycosylated Human IL-2 from Recombinant Mammalian Cells

I

TMLE 111

mt-tenlnal s q w w o f recmbinblnlnt (n f o r a ) a d h-n ~ P ~ Y F Y T-cell der1v.d 11-2: ..................................................................................... cycle BHU Ltk . CW PEL N - f o n VEL M - f O n

PTH-amino x l d ........................

-b -b -b

yield') ...............................................

470 412

b 123 98

104 76 87

201 222

79 304 389 297 312 289

"'. I

(panel C )

a ) vie161 are expressed I S -1 of PTH m h o acld recovered In each cycle for the VEL 11.2 )I (on. Similar yields fov PTH amino acids were obtained for vecrmbinant nonglycarylated and glycosylated It-2 f o m s (except for cycle NO. 31.

b) no residue identified

250C . ........ r:,.

, .

2000-. I

I I1


Methylated 1~911 m l a r ratw in ollqorrccharlder:

BHK I BHK I I L t k ' I L t k ' 11. CHO I

2.3.4.-tri-O-&lhyl. galacite1 . .

2.4.6.trI.O.methyl- 9ala<1101 1.0 1.0 1.0 1.0 1.0

2.N-methylacetamldo- I.4.5.6.tetrl-O-~thyl

2.deory 9alaot01 0 0.65 0 0.12 0.69

1,4.5.-lrt-O-methyl 2.N-methylacctm~do~ 2.deox~-9a11c1t01 0.69 0 0.59 0 0.73

1,

expression of human interlewkin-2 in recombinant baby hamster

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