Glycobiology Vol. 2 no 5 pp. 419-427. 1992

Characterization of recombinant murine interleukin 5 expressed in Chinese hamster ovary cells

Shiho Kodama, Tamao Endo1, Masafumi Tsujimoto and Akira Kobata1'2 Suntory Institute for Biomedical Research. Shimamoto-cho, Mishima-gun, Osaka 618 and 'Department of Biochemistry, Institute of Medical Science. University of Tokyo, 4-6-1, Shirokanedai, Minato-ku. Tokyo 108. Japan 2 To whom correspondence should be addressed

We have purified recombinant murine interleukin 5 (rmIL-5) from the supernatant of Chinese hamster ovary cells. Each peptide fragment of the purified rmIL-5 generated by Achromobacter protease I digestion was characterized and glycosylation sites were determined. Although rmIL-5 contains three potential sites of N-linked glycosylation (Asn-26, Asn-55 and Asn-69), Asn-69 is not glycosylated. The oligosaccharides released from the protein by hydrazinolysis were fractionated by paper electrophoresis, lectin column chromatography and gel permeation chromatography, and their structures were analysed by sequential exoglycosidase digestion in combination with methylation analysis. The results indicated that they are a mixture of bi-, tri- and tetraantennary complex-type sugar chains with and without a fucose at the C-6 position of the proximal /V-acetylglucosamine residue and high-mannosetype sugar chains. Although > 80% of the sugar chains are neutral oligosaccharides similar to recombinant human IL-5 (rhIL-5; Kodama,S., Endo,T., Tsuruoka,N., Tsujimoto,M. and Kobata,A. (1991) J. Biochem., 110, 693-701), rmIL-5 has more tetraantennary oligosaccharides than rhIL-5. A site differential study revealed that Asn-55 has more tetraantennary oligosaccharides than Asn-26. Key words: interleukin 5/N-linked sugar chain/recombinant glycoprotein

Introduction Interleukin 5 (IL-5) is a glycoprotein generated by T cells and has various biological functions, including enhancement of the growth and differentiation of B cells, and induction of eosinophil differentiation and cytotoxic T cell differentiation (Takatsu et al., 1988). Although purified IL-5 is essential for understanding the molecular mechanism of this important cytokine, it has been extremely difficult to obtain IL-5 in sufficient quantities from natural sources. In order to overcome this problem, cDNA clones for murine and human IL-5s have been isolated (Azuma et al., 1986; Kinashi et al., 1986), and the recombinant IL-5s have been successfully produced by transfection of Chinese hamster ovary (CHO) cells with cDNA (Tsujimoto et al., 1989; Tsuruoka et al., 1990). Recently, many glycoproteins have been produced by recombinant techniques and their carbohydrate structures have been analysed in detail. A comparative study of the sugar moieties of these glycoproteins revealed that both quantitative r

- Oxford University Press

and qualitative differences are found in their sugar patterns (Cumming, 1991). Elucidation of this regulatory mechanism of /V-glycosylation on each protein is very important. We have recently analysed the N-linked carbohydrate structures of recombinant human IL-5 (rhIL-5) produced in CHO cells (Kodama et al., 1991). Our studies showed that rhIL-5 contains mainly non-sialylated biantennary sugar chains. Since human IL-5 showed —70% amino acid sequence homology with murine IL-5 (Azuma etai, 1986; Kinashi et al., 1986), it is quite interesting to determine whether both IL-5s contain a similar set of sugar chains. It is a good model to investigate the contribution of the primary amino acid sequence to protein glycosylation.

Results Identification of N-glycosylation sites Murine IL-5 contains three potential /V-glycosylation sites: Asn-26, Asn-55 and Asn-69 (Kinashi et al., 1986). To identify the /V-glycosylation sites of recombinant murine IL-5 (rmIL-5), we first performed peptide map analysis of the molecule. Purified rmIL-5 was digested with Achromobacter protease I (API), which cleaved the C-terminal side of lysine residue, and the digested peptides were separated by reversed-phase HPLC column (Figure 1). When the amino acid sequence of each peptide in Figure 1 was determined, all the peptides which were expected from the murine IL-5 cDNA sequence were identified (Figure 2). It should be noted that heterogeneity of peak 5 and peak (3 + 6) was observed. This was due to the differential cleavage by API at Lys74 -Lys 75 (peak 5), Lys 81 -Glu-Lys 83 [peak (3 + 6)] sequence, and some deletion of Gly-113, locating at the C-terminal end of the molecule, respectively (Kinashi et al., 1986). Table I shows the results of amino acid sequence analysis of the peptides which contain potential N-glycosylation sites. We could not detect Asn-26 (peak 2) and Asn-55 (peak 4) residues, suggesting that these asparagine residues are glycosylated. On the other hand, Asn-69 in peak 4 could be detected, indicating that this residue is not glycosylated. Fractionation of oligosaccharides by paper electrophoresis Tritium-labelled oligosaccharide fraction obtained from rmIL-5 was subjected to high-voltage paper electrophoresis. As shown in Figure 3A, the oligosaccharide mixture was separated into a neutral (N) and two acidic (Al and A2) fractions. The percent molar ratios of N, Al and A2 on the basis of their radioactivities were 88, 11 and 1, respectively. The pooled acidic components (A 1 plus A2) were completely converted to neutral components (named AN in Figure 3B) by Newcastle disease virus (NDV) sialidase digestion, which cleaves the Siaa2-*3Gal linkage (where Sia is sialic acid and Gal is galactose), but not the Siaa2->-6Gal linkage (Paulson et al., 419

Recombinant murine IL-5

CH3CN(%)

A215

60

Fig. 1. HPLC peptide mapping of nnIL-5 on a C-18 reversed-phase column. After API digestion, rmIL-5 was loaded onio a column and fractionated as described in Materials and methods.

1 10 20 H-Met-Glu-lie-Pro-Met-Ser-Thr-Val-Va1-Lys-Glu-Thr-Leu-Tfir-Gln-Leu-Ser-Ala-His-Arg-

30 40 Ala-Leu-Leu-Thr-Ser-Asn-Glu-Thr-Met-Arg-Leu-Pro-Val-Pro-Thr-His-Lys-Asn-Hia-Gln-

50 60 Leu-Cys-Ile-Gly-Glu-Ile-Phe-Gln-Gly-Leu-Asp-Ile-Leu-Lys-Asn-Gln-Thr-Val-Arg-Gly-

eo Gly-Thr-Val-Glu-Met-Leu-pne-Gln-Asn-Leu-Ser-Io"-'

s-' ys-Lys-Tyr-Ile-Asp-Arg-Gln-

4

I

5a

90 100 Lys-Glu-Lys-Cys-Gly-Glu-Glu-Arg-Arg-Arg-Thr-Arg-Gln-Phe-Leu-Asp-Tyr-Leu-Gln-Glu6a —» 6b 6c 110 113 Phe-Leu-Gly-Val -Met -Ser-Thr-Glu-Trp- Ala-Met -Glu-Gly-OH

Fig. 2. Primary structures of rmIL-5 produced by CHO cells. The peptide fragments obtained in this study are numbered according to their separation, as shown in Figure 1 and Table 1. Dashed arrows indicate the amino acids not detected by sequence analysis.

420

S.Kodama et al.

1982). These results indicated that the acidic nature of the oligosaccharides in fractions Al and A2 can be totally ascribed to sialic acid residues, and the sialic acid linkage is only the Siaa2-»-3Gal linkage based on the specificity of NDV sialidase. Upon mild acid hydrolysis (0.01 N HC1 at 100°C for 3 min) for partial removal of sialic acid residues, it was revealed

Table I. Sequence analysis of API generated peptides (peak 2 and peak 4) from rmIL-5 Cycle number

Peak ~

1 2 3 4 5 6 7 8 9 10

E (11) T L T

11

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

.

L S A H R

n.d (55) Q T V R G G T V E

A

M

L L T .S jtJ.

L F Q N

.1 T

S L I

q

M R L P

L

K(74)

•y

P

that fractions Al and A2 contain mono- and disialyl oligosaccharides, respectively (data not shown). Fractionation of the neutral oligosaccharides by serial lectin—Sepharose column chromatography The oligosaccharides in fractions N and AN were subjected to sequential chromatography on columns containing immobilized Aleuria aurantia lectin (AAL), concanavalin A (Con A) and Datura stramonium agglutinin (DSA). Since the carbohydrate binding specificities of these lectin columns have been reported previously (Ogata et al., 1975; Yamashita etai, 1987; Fukumori etai, 1990), they are briefly described here. All complex-type N-linked sugar chains containing an a-fucosyl residue linked at the C-6 position of the proximal AZ-acetylglucosamine residue of their trimannosyl core bind to an AAL-Sepharose column, but those without the fucose residue do not (Fukumori et al., 1990). The presence of at least two a-mannosyl residues, either free or substituted only at the C-2 position, is required for binding to a Con A-Sepharose column (Ogata etai., 1975), and biantennary oligosaccharides were eluted with 5 mM a-methylglucoside solution and highmannose-type oligosaccharides with 100 mM a-methylmannoside (Merkle and Cummings, 1987). All oligosaccharides which contain the Gal/3 1—4G1CNACJ31 -^4(Gal/31 ^4GlcNAc/31-*2)Man group (where GlcNAc is AZ-acetylglucosamine and Man is mannose) are retarded in the DSA—Sepharose column and those which contain either the Gal/3 l^-4GlcN Ac/31 -*6(Gal/31-*4GlcNAc,81^2)Man group or the Gal/31 -*4GlcNAcj31-*3Gal/31-*4GlcNAc group are all recovered in the bound fraction eluted by buffer containing 1% A'-acetylglucosamine oligomers mixture (Yamashita etai., 1987). The fractionation scheme is summarized in Figure 4. Fraction N and fraction AN were first applied to a column containing AAL-Sepharose. A portion of the radioactive oligosaccharide passed through the column (AAL~ fraction) and the remainder was bound and eluted with buffer containing 1 mM

T H

K(37) n.d., not detected. Numbers in parentheses indicate the amino acid number.

Fraction N (AN) |AAL-column|

73.3 (10.5)

14.6 (1.5)

|Con A-column|

ICon A-columnl

r 5.1

7.0 E

19.4 (6.8)

2.5 F

lDSA-column|

IDSA-columnl

0 10 20 30 DISTANCE FROM ORIGIN (cm) Fig. 3. Paper electrophoresis of the radioactive oligosaccharides obtained from rmIL-5 by hydrazinolysis (A). The electrophoretogram in (B) was obtained by subjecting the mixture of fractions Al and A2 in (A) to NDV sialidase digestion and then to paper electrophoresis. Arrows indicate the migration positions of authentic oligosaccharides and dye. (1)

+ 5.1

54.0 (3.7) D



r 2.4(1.1) B

17.0(5.7) A

Fig. 4. Fractionation of fractions N and AN obtained from rmIL-5 by serial affinity chromatography on immobilized AAL, Con A and DSA columns. The numbers in the figure represent the percent molar ratio of total radioactive oligosaccharides released from rmIL-5 (those in parentheses indicate the amount from fraction AN). The capital letters are used to indicate the final fractions used for structural study, r represents the fraction which was retarded in a DSA-Sepharose column.

421

Recombinant murine IL-5

fucose (AAL + fraction). Because of the limited amount of the sample, further study of the fraction AN (AAL~) could not be performed. By affinity chromatography on a Con A column, fraction N (AAL") was separated into the Con A" fraction, Con A + fraction (eluted with 5 mM a-methylglucoside solution) and Con A + + fraction (eluted with 100 mM a-methylmannoside solution) (Figure 4). In contrast, the other two oligosaccharide fractions were separated into the Con A~ fraction and Con A + fraction. The Con A~ fraction was further separated into the DSAr fraction (retarded with buffer) and DSA + fraction (eluted with buffer containing 1 % 7V-acetylglucosamine oligomers mixture) upon affinity chromatography on a DSA-Sepharose column. The fractions thus obtained were named A - F and the amount of oligosaccharide in each fraction was expressed as the percent molar ratio to total oligosaccharides released from rmIL-5 (Figure 4 and the numbers in parentheses for fraction AN). Bio-Gel P-4 column chromatography of fractions A—F Fractions A - F in Figure 4, obtained from rmIL-5, were subjected to Bio-Gel P-4 column chromatography (Figure 5). In order to determine the anomeric configuration and sequence of monosaccharides of oligosaccharide in each peak in Figure 5, they were subjected to sequential exoglycosidase digestion and analysed by Bio-Gel P-4 column chromatography.

Structures of oligosaccharides a-g When digested with diplococcal /3-galactosidase, which cleaves the Gal/3l—4GlcNAc linkage only (Paulson etal, 1978), radioactive oligosaccharide a released four galactose residues (Figure 6A, solid line). Upon diplococcal /3-N-acetylhexosaminidase digestion, the radioactive peak in Figure 6A (solid line) released only one N-acetylglucosamine residue (Figure 6A, dashed line), whereas it was converted to Man3 • GlcNAc • Fuc • GlcNAcOT (where Fuc is fucose and the subscript OT indicates NaB3H4-reduced oligosaccharides) with the release of four jV-acetylglucosamine residues by jack bean /3-7V-acetylhexosaminidase digestion (Figure 6A, dot-dashed line). The structure of this radioactive component was elucidated as Man3 • GlcNAc • Fuc • GlcNAc0T by the same procedures as described in the previous paper (Endo et ah, 1988). Since the analytical data utilized to make the structural assignments were the same as those reported in that paper, they are not described here. These results indicated that oligosaccharide a is a typical tetraantennary complex-type oligosaccharide with a fucosylated trimannosyl core, as shown in Figure 7.

18 16

14

12 11 10

9

A

8

7

I II

6

III

/\ / \

J |\

B

C

/ D

A E

F

f\

/ \ '

A

\ .

-• ~ , ^*

A

A i

G

A_

//

\ \

A

J

150 200 ELUTION VOLUME (ml) 100

150 200 ELUTION VOLUME (ml)

Fig. 6. Sequential glycosidase digestion of oligosaccharides in Figure 5 The arrowheads at the top are the same as those in Figure 5. and the white arrows indicate the elution positions of authentic oligosaccharides: Fig. 5. Bio-Gel P-4 column chromatography of oligosaccharide fractions I. Man 5 GlcNAcGlcNAc OT , II, Man3-GlcNAc-Fuc GlcNAc0T. from rmIL-5. Panels A - F represent the data obtained from fractions A - F III. Man 3 • GlcNAc GlcNAcOT. Solid lines, dashed lines and dot-dashed in Figure 4. respectively. Black arrowheads indicate the elution positions of lines in panels A - F are the Bio-Gel P-4 column chromatograms of oligoglucose oligomers and the numbers indicate glucose units. The white arrows saccharides a—f sequentially digested with diplococcal /3-galactosidase. indicate the elution positions of authentic oligosaccharides: I, Gal 4 diplococcal j3-/V-acetylhexosaminidase and jack bean ^-W-acetylGlcNAc 4 Man 3 -GlcNAc-Fuc GlcNAcOT: II. Gal3• GlcNAc3• Man3•hexosaminidase. respectively. Panel G is the Bio-Gel P-4 column GlcNAc • Fuc • GlcNAcOT: III. Gal, • GlcNAc, • Man3 • GlcNAc • Fuc • GlcNAcOT: chromatogram of oligosaccharides g after digestion with A.saiioi IV. Gal, • GlcNAc, • Man3 • GlcNAc• GlcNAcOT. a-mannosidase I

422

S.Kodama et al.

Radioactive oligosaccharides b and c released three galactose residues by diplococcal /3-galactosidase digestion (Figure 6B and C, solid lines) and three A'-acetylglucosamine residues by subsequent jack bean /J-iV-acetylhexosaminidase digestion. The radioactive products at this stage moved to the same positions as authentic Man3 • GlcNAc • Fuc • GlcNAc0T (Figure 6B, dot-dashed line) and Man3 • GlcNAc • GlcNAcOT (Figure 6C, dot-dashed line). These results indicated that they are triantennary oligosaccharides with fucosylated and nonfucosylated trimannosyl cores. When digested with diplococcal j3-A'-acetylhexosaminidase, the solid line peaks in Figure 6B and C released only one A'-acetylglucosamine residue (Figure 6B and C, dashed lines). These results indicated the presence of a 2,6-branched outer chain moiety because diplococcal /3-/V-acetylhexosaminidase can not cleave the GlcNAc/31-*2Man linkage in the GlcNAc/31-*6(GlcNAc/31-*2)Man group (Yamashita et al., 1981). This conclusion was also supported by the evidence that oligosaccharides b and c bound to a DSA—Sepharose column.

(Figure 6E and F, solid lines). Further digestion with diplococcal /3-A/-acetylhexosaminidase converted the solid line peaks in Figure 6E and F to Man3 • GlcNAc • Fuc • GlcNAc0T and Man3 • GlcNAc • GlcNAcOT with the release of two A'-acetylglucosamine residues, respectively (Figure 6E and F, dashed lines). Based on these results, the structures of oligosaccharides e and/were concluded to be typical biantennary complex-type oligosaccharides with fucosylated and non-fucosylated trimannosyl cores as shown in Figure 7. The radioactive oligosaccharides g were completely resistant to diplococcal /3-galactosidase, /3-A/-acetylhexosaminidase and endo-/3-A'-acetylglucosaminidase D digestion (data not shown). However, both peaks in Figure 5F released one and two mannose residues upon Aspergillus saitoi a-mannosidase I digestion, which cleaves only the Manal-*2Man linkage (Yamashita et al., 1980), and were converted to a radioactive oligosaccharide with the same mobility as authentic Man5 • GlcNAc • GICNACQT (Figure 6G). The structure of this radioactive component was elucidated as Man5 • GlcNAc • GlcNAc 0T by the same procedures as described in the previous papers (Wold et al., 1990; Kodamaet al., 1991). Furthermore, it was converted to radioactive A'-acetylglucosaminitol by digestion with endo-/3-A'-acetylglucosaminidase D (data not shown). These results indicated that oligosaccharides g are high-mannose-type sugar chains, which have at least one Manal-»2 residue linked to the Manal^-3 arm of their trimannosyl core, as shown in Figure 7. The molar ratio of these sugar chains of rmIL-5 is summarized in Table II. For comparison, the previously reported data on rhIL-5(Kodama et al., 1991) are also included in Table II.

Oligosaccharide d also released three galactose residues by diplococcal /3-galactosidase digestion (Figure 6D, solid line). The solid line peak in Figure 6D was then converted to Man3 • GlcNAc • Fuc • GlcNAc0T (Figure 6D, dot-dashed line) with the release of three A'-acetylglucosamine residues by jack bean /3-A'-acetylhexosaminidase treatment. These results indicated that it is also a triantennary oligosaccharide. When digested with diplococcal /3-ALacetylhexosaminidase, the solid line peak in Figure 6D released two A'-acetylglucosamine residues (Figure 6D, dashed line). Considering the substrate specificity of diplococcal /3-A'-acetylhexosaminidase (Yamashita et al., 1981), its structure was proposed as shown in Figure 7. By incubation with diplococcal /3-galactosidase, radioactive oligosaccharides e and / released two galactose residues

Galp1-»4GlcNAcp1Gaipi->4GlcNAcpi Gaipi-»4GlcNAcpi • Galp1->4GlcNAcp1-

4

_Mana1

Galp1-*4GlcNAcpi^ 6 _ManaK f i Gaipi->4GlcNAcpi -* !? Gaipi -»4GlcNAcpi ^2Mana1 •*

b R2

c

Gaipi ^4GlcNAcpi ->2Mana1 ^ R Galp1^4GlcNAcp1^ 4 g Gaipi->4GlcNAcpi^

±Mana1->2

Methylation analysis of fraction N plus fraction AN obtained from rmIL-5 (Table III) revealed that the mannose residues occur as M a n l ^ , -^Manl-*, Z^Manl^"-, I^Manl-*-, and ^fManl-*. Detection of 1, 3, 5, 6-tetra- and 1,3, 5-tri-Omethyl 2-A'-methylacetamido-2-deoxyglucitols supported the above estimation that both fucosylated and non-fucosylated trimannosyl cores are included among the oligosaccharides. Detection of only 3,6-di-O-methyl 2-N- methylacetamido-2deoxyglucitol as the di-0-methyl derivative of A'-acetylglucosamine indicated that complex type sugar chains contain only the Gal)31-*4GlcNAc group in their outer chain moieties. These results support the oligosaccharide structures proposed in Figure 7. Table n. Characteristics of N-linked sugar chains of rmIL-5 and rhIL-5'

Gaipi -^4GlcNAcpi -»2ManaK °Manp1->4 Galp1^4GlcNAcpi-^2Manai^ O

Methylation analysis

R2

ManaV ,Manp1->4R 2 Mana1->2Manai'

Hi • GlcNAcpi->4(Fuca1 ^6)GlcNAc OT , R2 GlcNAcpi ->4GlcNAc 0T Fig. 7. Structures of oligosaccharides a—g in Figure 5.

Oligosaccharides

rmIL-5

rhIL-5b

Asn-26

Asn-55

a b c d e

9.9 12.8 5.1 3.5 57.7 7.0 2.5

1.0 8.7 n.d. 5.1 80.7 2.6 1.0

4.0 11.7 3.9 5.3 62.8 8.1 4.1

17.5 12.9 6.8 1.7 51.9 6.6 2.5

f

••Values in the table were calculated from the radioactivity incorporated into each oligosaccharide. 'The data were taken from the previous paper (Kodama el al., 1991). n d., not detected.

423

Recombinant murine IL-5

Table i n . Methylation analysis of fraction N plus AN obtained from rmIL-5 by hydrazinolysis Methylated sugars

Molar ratio"

Futitol 2,3,4-Tri-0-methyl(l,5-di-0-acetyl)

08

Galactitol 2,3,4,6-Tetra-0-methyl(l,5-di-O-acetyl)

2.3

Mannitol 2,3,4,6-Tetra-0-methyl(1.5-di-0-acetyl) 3,4,6-Tri-0-methyl(l,2,5-tri-O-acetyl) 2,4-Di-0-methyl(l,3,5,6-tetra-0-acetyl) 3,6-Di-O-methyl(l,2,4.5-tetra-0-acetyl) 3,4-Di-O-methyl(l,2,5,6-tetra-0-acetyl)

tr" 15 1.0 0.1 0.3

2-A'-Methylacetamido-2-deoxyglucitol 1.3,5,6-Tetra-O-methyl(4-mono-0-acetyl) l,3,5-Tn-O-methyl(4,6-di-O-acetyl) 3,6-Di-O-methyl( 1,4,5-tri-O-acetyl)

0.1 0.8 3.0

•The numbers were calculated by taking the value of 2.4-di-O-methyl mannitol as 1.0. b < 0 1.

Distribution of the N-linked sugar chains in rmIL-5 In order to determine the distribution and heterogeneity of the N-linked sugar chains in the rmIL-5 molecule, peaks 2 and 4 in Figure 1 were collected and subjected to hydrazinolysis, and structural studies of the released oligosaccharides were performed. The radioactive oligosaccharide mixture obtained from each peak was fractionated by lectin column chromatography after sialidase treatment and Bio-Gel P-4 column chromatography. As summarized in Table II, Asn-55 had more tetraantennary sugar chains than Asn-26.

Discussion Recently, many glycoproteins have been produced by recombinant techniques and some of them have already been applied to human therapy (Cumming, 1991; Takeuchi and Kobata, 1991). Among the cells used as hosts, CHO cells have most frequently been used for the expression of various recombinant glycoproteins. A comparative study of the sugar moieties of these glycoproteins produced in CHO cells revealed that both qualitative and quantitative differences are found in their sugar patterns (Mutsaers et al., 1986; Kagawa etal., 1988; Takeuchi etal., 1988; Cumming, 1991). These differences must be produced by the different controls of the peptide moieties, because CHO cells have the same sets of glycosylation machinery. In this study we demonstrated that rmIL-5 and rhIL-5, which show 70% amino acid sequence homology, have similar but slightly different sets of sugar chains (Table II). Non-sialylated biantennary sugar chains are major components in both samples. However, tetraantennary complex-type sugar chains are more abundant in rmIL-5. Although both IL-5s showed high sequence homology, they are not identical. Differences include the number of potential glycosylation sites, which is three for rmIL-5 (Kinashi et al., 1986) and two for rhIL-5 (Azuma etal., 1986). Since the primary amino acid sequence is considered to be a factor which specifies protein-linked sugar chain structures, rmIL-5 has similar but not identical sets of N-linked sugar chains to rhIL-5. 424

There are many examples indicating that distinct sites of a glycoprotein are glycosylated differently. The sugar chains at Asn-117 of human tissue plasminogen activator expressed exclusively high mannose type, but those at Asn-184 and Asn-448 were complex type (Pohl et al., 1987; Spellman et al., 1989; Cumming, 1991). Human chorionic gonadotrophin is composed of a- and /3-subunits, both of which have two //-glycosylation sites. The /3-subunit contains 1 mol each of biantennary sugar chains with fucosylated and non-fucosylated trimannosyl cores, while the a-subunit contains 1 mol each of monoantennary and biantennary sugar chains with nonfucosylated trimannosyl cores (Mizuochi and Kobata, 1980). Therefore, we analysed the sugar chain structures of each glycosylation site of rmIL-5. The present study demonstrates the heterogeneity of sugar chains between two glycosylation sites of rmIL-5 (Table II). The sugar chains attached at Asn-26 are composed of biantennary (71%), triantennary (21%) and tetraantennary (4%) chains. On the other hand, those at Asn-55 are composed of biantennary (58%), triantennary (21%) and tetraantennary (18%) chains. Interestingly, the complex sugar chains with the GlcNAc|31-*6Man group are more abundant at the site close to the C-terminal region. It is tempting to speculate, therefore, that 7V-acetylglucosaminyltransferase V can be more accessible to Asn-55 than Asn-26. These results indicated that the peptide structure surrounding the glycosylation site is clearly an important factor influencing the structures of the sugar chains formed. It is interesting that the sugar chains are formed on Asn-26 and Asn-55, but not on Asn-69 of rmIL-5. The phenomenon is similar to the case of rhIL-5 in which glycosylation occurs on Asn-28, but not on Asn-71 (Minamitake et al., 1990; Kodama et al., 1991). Intensive studies have revealed that the tripeptide, Asn-X-Ser/Thr, is the minimal peptide structure required for glycosylation (Hubbard and Ivatt, 1981). However, the finding that the tripeptide sequence is not glycosylated is not unprecedented. Although actual rules for this mis-glycosylation are not well understood, it is thought that the accessibility of the tripeptide site to oligosaccharide transferases is a most plausible factor that determines whether glycosylation occurs or not because denaturation or protease cleavage of some proteins induces in vitro glycosylation of the unused glycosylation site. In some cases, the primary amino acid sequence seems to influence glycosylation. We drew hydropathy plots for both IL-5s according to the method of Kyte and Doolittle (1982). As shown in Figure 8, hydropathies of the amino acid sequences after the glycosylation sites (Asn-26 and Asn-55 of rmIL-5, and Asn-28 of rhIL-5) show rather a hydrophilic property. On the other hand, those of non-glycosylated sites (Asn-69 of rmIL-5 and Asn-71 of rhIL-5) show a hydrophobic property. These results suggested that, at least in part, the hydrophilicity of the primary amino acid sequence of IL-5s after the potential glycosylation site may be important in accepting or preventing glycosylation of an asparaginyl residue. This tendency coincides with Asn-80 in the case of human /3-interferon, and Asn-38 and Asn-83 of erythropoietin, but not with Asn-24 of erythropoietin. Accordingly, the hydrophilic character of the primary amino acid sequence after the potential glycosylation site can apply as a regulatory factor of glycosylation to the limited potential sites. It is necessary to define additional structural features around the potential glycosylation sites with and without sugar chains. The accumulation of data on recombinant protein glycosylation will no doubt contribute to a better understanding of the rules governing glycosylation.

S.Kodama el al.

Sequence

position

28 rhlL-5 rmlL-5

TLLIANETLRIPVALLTSNETMRLPV26

71

ERLFKNLSLIKKY LDILKNQIVRGGT55

EMLFQNLSUKKY

69

Fig. 8. Comparison of the hydropathy plots of rmIL-5 (A) and rhIL-5 (B). The hydropathic scale is given on the left according to Kyte and Doohttle (1982) with (-) indicating relative hydrophihcity and ( + ) relative hydrophobicity. The sequence of the amino acids around each potential glycosylation site is represented by a one-letter code. Arrows and underlines indicate the potential /V-glycosylation sites.

Materials and methods Isolation of rmIL-5 rmIL-5 expressed in CHO cells was purified by the procedures previously reported and rmIL-5 employed in this study was of the same lot as that reported previously (Tsuruoka etal., 1990). Briefly, rmIL-5 was purified by serial chromatographic separations on Matrix Blue-A. DEAE-Sepharose, phenylSepharose and Sephacryl S-200 columns. The preparation was shown to be biologically active in an in vitro assay for IgM secretion with a specific activity of 5 x 10* units/mg. Chemicals, enzymes and lectins NaB3H4 (600 mCi/mmol) was purchased from New England Nuclear, Boston, MA. NaB2H4 was from Nacalai Tesque, Inc., Kyoto. (3-Galactosidase and /3-A'-acetylhexosaminidase were purified from the culture fluid of Diplococcus pneumoniae according to the method of Glasgow el al. (1977). Another /3-N-acetylhexosaminidase and a-mannosidase were purified from jack bean meal by the method of Li and Li (1972). a-Mannosidase I from A.saitoi (Yamashita et al , 1980) and sialidase from NDV (Paulson el al., 1982) were

purified according to the cited references. Con A-Sepharose was purchased from Pharmacia LKB Biotechnology. Recombinant AAL was prepared by the method of Fukumori etal. (1990) and coupled to Sepharose 4B. DSASepharose was prepared as described previously (Yamashita etal., 1987). 3'-Sialyllactose was obtained from human milk as described previously (Kobata, 1972). API (lysyl endopeptidase) was purchased from Wako Pure Chemical Industries, Osaka.

Peptide mapping Digestion of rmlL-5 was performed using API at a substrate to enzyme ratio of 1000:1 (w/w) at 37°C for 2 h. The typical procedure is as follows. To a solution of rmIL-5 (2 nmol) in 50 /A of 0.1 M phosphate-buffered saline (PBS) (pH 7.2) containing 5 M urea was added API. The reaction was allowed to proceed for 2 h at 37°C. After acidification with 1 M HC1, the mixture was subjected to a C-18 HPLC column (YMC-Pack A-302, 5 urn, 4.6 x 150 mm) previously equilibrated with 0.1 % trifiuoroacetic acid (TFA), and the peptides were eluted with a linear acetonitrile gradient from 0 to 80% in 0.1 % TFA over 50 min at a flow rate of 1 ml/min.

425

Recombinant murine IL-5 Ammo acid analysis The peptides were hydrolysed in 6 N HC1 containing 0.1 % phenol at 110°C for 24 h in an evacuated and sealed tube. The amino acid analysis was carried out with a Hitachi amino acid analyser, Model 835.

recombinant human IL-5; rmIL-5, recombinant murine interleukin 5; Sia, sialic acid; TFA, tnfluoroacetic acid. The subscript OT is used to indicate NaB3H4-reduced oligosacchandes. All sugars mentioned in this paper are of D-configuration, except for fucose which is of L-configuration

Ammo acid sequence analysis Sequence analysis of the peptides obtained from API digestion was performed using an Applied Biosystem 477A gas-phase sequencer according to the manufacturer's instructions. The resulting phenylthiohydantoin derivatives were identified by reversed-phase HPLC with an Applied Biosystem 120A on-line system.

References

Azuma.C, Tanabe.T., Konishi,M., Kinashi.T.. Noma.T., Matsuda,F., Yaoita.Y., Takatsu,K., Hammarstrom.L.. Smith,C.I.E., Sevennson.E. and Honjo,T. (1986) Cloning of cDNA for human T-cell-replacing factor (interleukin 5) and comparison with the murine homologue. Nucleic Acids Res , 14, 9149-9158. Cumming.D.A (1991) Glycosylation of recombinant protein therapeutics: conCarbohydrate analysis trol and functional implications. Glycobiology. 1. 115-130 Endo.T., Ohbayashi.H., Hayashi.Y., Dcehara.Y., Kochibe.N. and Kobata.A. Sequential exoglycosidase digestion, Bio-Gel P-4 column chromatography. (1988) Structural study on the carbohydrate moiety of human placental paper chromatography, paper electrophoresis and other analytical procedures alkaline phosphatase J. Biochem., 103, 182-187. used in this study have been described in the previous paper (Kodama et al., Fukumori.F., Takeuchi,N., Hagiwara.T., Ohbayashi,H., Endo,T., Kochibe, 1991). Methylation analysis of ohgosaccharides was performed as described N., Nagata,Y. and Kobata,A. (1990) Primary structure of a fucose-specific previously (Endo et al., 1988). lectin obtained from a mushroom, Aleuria aurantia. J. Biochem., 107, 190-196. Oligosaccharides Glasgow.L.R., Paulson.J.C. and Hill.R.L. (1977) Systematic purification of Gal/31 -*4GlcNAc/31 — 2Mana 1 —6(Gal/31 —4GlcNAc/31 —2Manc* 1 —3)Man/? 1 - five glycosidases from Streptococcus (Diplococcus) pneumoniae. J. Biol. Chem.. 252, 8615-8623. —4GlcNAc/31 — 4(Fuca 1 — 6)GlcNAcOT (Gal, • GlcNAc, • Man3 • GlcNAc • Fuc • Hubbard,S C. and Ivatt.R J (1981) Synthesis and processing of asparagineGlcNAcOT) and Gal/3 l-4GlcNAc/31-2Manal-6(Gal/3 l-4GlcNAc/31-2Iinked oligosaccharides. Annu. Rev. Biochem., 50, 555—583. Mana 1 —3)Man/31—4GlcNAc/31—4GlcNAc0T (Gal2 • GlcNAc, • Man3 • GlcNAc Kagawa,Y., Takasaki,S., Utsumi,J., Hosoi.K , Shimizu.H., Kochibe,N. and •GlcNAc0T) were obtained from human placental alkaline phosphatase by Kobata,A. (1988) Comparative study of the asparagine-linked sugar chains of hydrazinolysis followed by reduction with NaB3H4 (Endo et al , 1988) Both natural human interferon-/31 and recombinant human interferon-/3 1 produced [Gal/31 -4GlcNAc/31 -6(Gal/31 -4GlcNAc/31 -2)Mana 1 - 6](Gal/31-4GlcNby three different mammalian cells. /. Biol. Chem., 263, 17508-17515 Ac/31 - 2 M a n a 1 -3)Man/31 -4GlcNAc/31 -4(Fucc* 1 -6)GlcNAc OT (Gal3 • GlcKinashi,T., Harada,N., Sevennson,E., Tanabe,T., Sideras.P., Konishi.M., NAtyMan 3 - GlcNAc- Fuc -GlcNAcOT) and [Gal/31—4GlcNAc/31 — 6(Gal/31 — Azuma.C, Tominaga,A., Bergstedt-Lindqvist,S., Takahashi.M., 4GlcNAc/31 —2)Mana 1—6] [Gal/31 —4GlcNAc/31 —4(Gal|31—4GlcN Ac/31—2)Matsuda.F., Yaoita,Y., Takatsu,K. and Honjo.T. (1986) Cloning of comMana 1 -3]Man/31 -4GlcNAc/31 -4(Fuca 1 -6)GlcNAc 0T (Gal., • GlcNAc4 • plementary DNA encoding T cell-replacing factor and identity with B cellMan3 • GlcNAc • Fuc • GlcNAc0T) were obtained from human placental fibronecgrowth factor II. Nature, 324, 70-73. tin (Takamoto el al., 1989). Manal—6(Mano:l—3)Man/31—4GlcNAc/31—4 (Fucal—6)GlcNAc0T (Man3-GlcNAc-Fuc-GlcNAc0T) and Manal—6(ManKobata,A. (1972) Isolation of oligosaccharides from human milk. Methods

Characterization of recombinant murine interleukin 5 expressed in Chinese hamster ovary cells.

We have purified recombinant murine interleukin 5 (rmIL-5) from the supernatant of Chinese hamster ovary cells. Each peptide fragment of the purified ...
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