1. IN’IXODUCTION
2. MATERIALS
The accumulating cvidcIlcc concerning the biologicnl rclcvancc of the carbohydrate part of glycoprotcins has highly stimulated the intcrcst in biochemical and pharmaccutical aspects of these biopolymcrs [l-S]. In particular, it has activated the discussion with respect to genetically engineered proteins cxprcssed in hetcrologous cell types with or without glycosylation machinzrics [4,5]. Because the cellular glycosylation machinery can vary among different cell !ines, different glycosylation patterns of glycoproteins can be expected. Based on the present knowlecige about primacy carbohydrate structures of recombinant human glycoproteins, the Chinese hamster ovary (080) cells seem to be preferred [6]. In the framework of our program on the microanalysis of (recombinnnr.; glyzoprotein glycans, we have discovered that 0X0 cells can generate asparaginelinked carbohydrate chains containing N-glysolylneuraminic acid (NeuSGc), as illustrated here for 4 recombinant human glycoproteins.
Conmcrcially ;lWililblc rccoiiibinanr him11n tissue pl:l!illliIirlgcll astivntor (IPA), cxprcssc‘d in CHO cells (Astilysc), ws obtained from ‘II~o~t~ac (Bihurilrh 011dcr Hiss, FRO). CK-lO.ccll dcrivcd reconlbinailt I~uII~:I~~rhiincric pl;~smincgcn activator (kzt~PA) was a gift of Cibn-
Correspondence
address:
J.P. Kamerling, Bijvoet Center, Department of Bio-Organic Chemistry, Utrccht University, P.O. BOX 80.095, NL.3508 TB Utrccht, The Netherlands
AbDrev/arions: NeuSAc, N-acetylneuraminic acid; NeuSGc, N-glycolylneuraminic acid; Fuc, fucose; CHO, Chinese hamster ovary; tPA, tissue plasminogen activator; katuPA, recombinant chimeric plasminogen activator; EPO, erythropoietin; FSI-I, follicle stimulating hormone, follitropin; hCG, human chorionic gonadotropin; PNGase-F, peptide-N’.(N-acetyl-P-glucosaminyl)asparagine amidase F; DMB, 1,2-diamino-4,5-methylene-dioxybsnzcne. Published
by Eiscvier
00145993/90/$3.50
Science Publishers
B. V. (Biomedical
AND METHODS
Geigy Ltd (Bnscl, Switscrlnnd),
dcrivcd recombinant Tckniklr 13V(Ilox. [cl, The Ncthcrlnnds), iuld CHO~ccll dcrivcd recombinant human foi]iIropin (FSH) WIS a gift of Diosynth BV (Oss, ‘Ihc Netherlands), Human scrotrnnsfcrrin and b0vii.e fibrinoycn wcrc obtnincd from
hWal1
cryt]lro])oictin
CHO.scll
(EPO) was u gift of Organon
Sigma. Pcptide.N’-(N-acetyl-~~-~lucosnrTlinyl)ns]~ara~i~~c (PNGascaF) from Nuvobacfcriun~ nleningouepliclrnl
alnidasc I: wns purchased
from Bochringcr Mannheim, 2.2. Liberation of N4~tkcd carbollydmtc ctlmins The N-linkedcarbohydrate chains of kztuPA and EPO were relensed by treatment with non-immobilized PNGase.F cssentialiy as described [9,8]. In case of EPO a complete liberation of oliposaccharides was obtained without the addition of Non-idet P-40, as indicated by SDS-PAGE. Released carbohydrate chains were collected by gel-permeation chromatography on Bio-Gel P-100 (BioRad) using 50 mM (k2tuPA) or 25 mM (EPO) NHdHCO,, pH 9,0, as cluent. In each case, the pooled carbohydrate fraction was scparatcd according to charge by FPLC on Mono Q (Pharmacia), followed by HPLC of the subfractions on Lichrosorb-NH2 (Chrompack). 2.3. Analysis of sialic acids The analysis of sialic acids was carried out essentially as described 191. Briefly, aliquots of sialoglycoproteins (0.25-I mg) in 0.2 ml of 2 M acetic acid were heated for 3 h at 80°C, and released sialic acids wereconverted with 1,2-diamino-4,5-methylene-dioxybenzene (DMB) into fluorescent derivatives. WAcetylneuraminic acid (NeuSAc) and N-glycolylneuraminic acid (NeuSGc) were used as standards. HPLC analysis was carried out on a Kratos Spectroflow 400 HPLC system equipped with a Chromspher Cls (Chrompack) reversed-phase CO]t!mn (25 x 0.46 cm) and Kratos 9lsO fluorescence detector operating at an excitation wavelength of 393 nm, detection emission at wavelengths > 448 nm using a cut-off filter. Elutions were performed
Division)
0 1990 Federation of European Biochemical Societies
9
NCUSAC iruscc
NcuSAc
3.2. Identjficarion of NeuSGc-containing chains in kztuPA md EPO
NeuSGc
-0
_A
IO (ml)
r
0
,
IO (ml)
0
IO (1111)
Fig. 1. HPLC Profile of DMB derivatives of sialic acids, released from different glycoproteins, on a Chromspher CI~ column (25 x 0.46 cm). The elution was carried out with acetonitrile : methanol : water (9 : I : 84, v/v/v) at a flow rate of 1 ml/min. (A) human serotransferrin; (B) bovine fibrinogen; (C) recombinant human FSPI, expressed in CM0 cells,
10
1C the HPLC profile of the DMEi sialic acids derived from recombinant human FSH is presented, showing peaks at clution positions corresponding witfa NeuSCc and NeuSAc in a ratio of 3:97. Similar patterns were found for recombinant kztuPA, EPO, and PA. These findings strongly suggest that the occurrence of NcuSGc as a constituent of carbohydrate chains in rl;lcombinanr glycoprotcins expressed in not:nal CHO cells, is a general feature. N-linked
The pool ot N-linked carbohydrate chains of kztuPA, released by PNGase-F, was separated on Mcrlo Q, yielding 4 carbohydrate-positive peaks (K l-N4) having the same retention volumes as mono-, di-, tri-, and tetra-sialo N-type antennary structures, respectively [8], Each FPLC fraction was iurther separated by HPLC on Lichrosorb-NHz. Tn the context of this paper, the WPLC subfractions N2.2 and N2.3 of FPLC fraction N2 (Fig. 2) will be discussed in more detail. The major fraction N2.2 represents the following conventional disialo diantennary compound:
The ‘I-I-NMK
spccrrum shows the characteristic structural reporters for the cclnstituting Gal., GlcNAc, Man and Fuc residues, being identical to those of 1~2.2 (see also [7]). However, as compared to N2.2, thcaintensity of the NAc signal of NcuSAc at 6 = 2.031 ppm is halvcd, whcrcas a new singlet at 6 = 4.121 ppm is observed, diagnostic for the N-glycolyl group of NcuSGc [8]. In addition, two sialic acid H-3e double doublets arc observed at d = 2.958 pprn (NeuSAc) and 6 = 2.775 ppm (NeuSGc), whereas in the H-3a chemical-shift region 4 triplets occur at 6 = 1.797 ppm (Neu5Ac, Manaul-3 branch), 6 = 1.801 ppm (NcuSAc, Manor l-6 branch), f = 1.813 ppm (NcuSGc, Manoll-3 branch), and 6 = 1.820 :?pm (NeuSGc, Mancxl-6 branch), The assignments of the H-3 signals arc based 0;1comparison with the related signals in N2,2, and on shift increments going from NeuSAc to NeuSGc in ~~2-6linked sialicacid-containing diantennary str\ictures (As = + O.OlV
0.019 ppm) [8] and in CI-!inkcd ,~iecv2-3Gnl~~l-3GalNAcc\l (H-3c, AS = i-O.013 ppm; I-l-3& A& = -i-O.017 ppm) [13]. The rclarivc rctcntion volume of N2,3 with rcspcct to N2.2 is in accordance with the rcplaccmcnt of only one Ncu§Ac rcsiduc by NcuSGc (compnrc with the disialo(a2-6) diantcnnacy carbohydrate chains from bovine fibrinogen [8]), The pool of N-linked carbohydrate chains of EPO released by PNGasc-F VW fractionated on Mono Q, yielding 4 carbohydrate-positive peaks (Al-A4) at the same retention positions as mentioned for k2tuPA (see above). Each FPLC fraction was further subfracrionatcd by HYLC on Lichrosorb-NHz. In the context of this report, the HPLC subfractions A4.4 and A4.5 of FPLC fraction A4 (Fig. 3) will be discussed in more der.all. ‘H-NMR ana!gsis of subfraction 144.4 demonstrated the presence of the fo!l;;wing tetrasialo tetraantennary compound:
NeuS Accr%-3Gal/3 l-4GlcNAcP l-6 NeuS AcaZ-3Galp l-4GlcNAcP I -&anol l-6 , ,
FllCCXll-6
Manp l-4GlcNAcp I-4&cNAc
[A&4]
Neu5Acc&3Gal/31-4GlcNAc~l-~Mancy1-3 MeuSAcrxZ-3Galpl-4GlcNAc@l-4
11
FEBS LETTERS
Volume 275, number 1,2
,’ A.4.4
--
v,
60
0
120 (ml)
Fig. 3. HPLC Fractionation pattern at 205 nm of the EPO FPLC fraction A4 on a Lichrosorb-NI-iz 10 pm column (25 X 0.46 cm). The elution was carried out isocraticaliy wirh 30 mM K~HPO+/KHZPO~, PI-I7.0 : acetonitrile (37.5 : 62.5, v/v) at a flow rate of 2 ml/min at ambient temperature. Fractions were collected as indicated.
The NMR data of A4.4 are in agreemknt with those reported for the tetrasialo(Neu5Ac(u2-3) tetraantennary
November 1990 .
oligosaccharide from recombinant human FSH [14]. The four ~~2-3linked NeuSAc residues give rise to the characteristic set of structural reporters:at 6 = 2.031 ppm (4x ‘NAc), 6 = 1.804 ppm (4 x H-3a), and 6 = 2.756 ppm. (4 x H-3e). Comparison of the ‘H-YMR spectra of the subfractions A4.4 and A4.5 shows that the niajor component in subfraction A4.5 has the same characteristic structural reporters for the constituting Gal, GlcNAd, Man and Fuc residues as those found for A4.4 (see, also [14]). Howeyer, in case of A4.$ the 6region covering the H-2 signal for al-6 linked Man, the H-3 signal of Gal (4 x), and the H-5 signal of Fuc, contains an additional singlet at 6 = 4.121 ppm,‘typical for the NGc signal of ‘NeuSGc [g]. As corhpared 90 the relative intensity of the NAc singlet of NeuSAc in the NAc region of the NMR spectrum of A4.4, the intensity of the NAc singlet in the spectrum of A4;S is reduced to about 75%. Furthermore, additional signals are piesent in the sialic acid H-3 regions, Double doublets for H-3e are observed at 6 = 2.757 ppm (major, Neu5Ac) and 6 = 2.794 ppm (minor, Neu5G@, whereas’ triplets .are detected for H-3a at S = I.804 ppm (major, Neu§Ac) and d = 1.821 ppm (minor, NeuSGc). Based on the : NMR, data and the relative retention volume of A45 as compared to 84.4 (&.s = 1.3 x 1p~d.4)[a], it can be concluded that overall one of the NeuSAc residues has been replaced by NeuSGc in the tetrasialo tetraantennary structure. Whether or not a specific antenna is involved needs to be clarified.
Gal0 14GlcNAcBl-ZMancul -6 [A4.5]
(NeuSAc&3)sNeuSGcc&3 GalP II-4GlcNAcfi l -2F
l-3
@alp14GlcNAcBl-4
4. DISCUSSION In view of the literature data on the recombinant variants of human interferon-y [15], hCG f6], human FSH [14], human interferon-@1 [16-183, bovine lutropin [ 191, human EPO [%O-221, human #A [23-2>]; human transforming growth factor-@1 precursor [26], human immunodeficiency virus envelope glycoprotein gp120 [%7,28], human CD4 [29,30], and human interleukia-5 [311pit can be concluded that CM0 cells can synthesize glycoproteins with (phosphorylated) oligomannose, hybrid, as well as IV-acetyllactosamine type of structures, depending on the- protein involved. 12
The last type compris& mono-, di-; tri-, tri’-, and tetraantennary glycans. Also pslj+V-acetyllactosamin@ sequences can occur. The terminal NeuSAc residue is always exclusively linked in (~2-3 .position to Galpl4GlcNAc, indicating the absence of O-Gal a%-6-sialyltransferase activity. It has been shown that the terminal, sialylation of N-linked glycans in CHQ cells can be altered by expresBion df this transferase [32]. Structures with bisecting GlcMAc 1141, the antennary element GalNAc~l-4GlcNAc [ 191, peripheral Fuccul-3GlcNAc [33] and the antennary element Galm1-3Gal@l-4 [4] are not produced. Recently, also the transfection of a human al-3 fucosyltransferase gene
Volume 275, number 1,2
PEBS LETTERS
into CHO cells has been reported [33]. Sialylated Olinked oligosaccharides as NeuTAca2-3Galpl-3GalNAc and NeuSAccr2-3Galp1-3[NeuSAccr2-6)GalNAc are synthesized in a normal.vir&y, .includitig’ a2-3 as well as ~2-6 linked NeuSAc, as found for the recombinant variants of hCq [6], human EPO 1203, human interleukin-2 [34], and human granulocyte-colonystimulating factor [35]. This means that the CHO cell ’ has normal: a-GalNAc ,rr2-&sialyltransfeiase activity. Cbmparison of the carbohydrate chains in those cases in which the native and recombinant glycoproteins have both been analyzed, has made it clear that from a carbohydrate’point of view, the CHO cell line is highly attractive. In this report we have furnished data that recombinant glycoproteins expressed in CHQ cells contain carbohydrate chains with a small percentage (-3%) of 1~2-3linked Neu5Gc. The occurrence of Neu5Gc in norma1 human tissue and soluble glycoproteins has not been established conclusively 136,371. Preliminary investigations ‘have shown that glycoconjugate-bound NeuSGc is present regularly in a&mounts lower than 0.01% of the total sialic acid fraction in human tissues and in blood plasma glycoproteins [38]. However, there are indicatibns from studies with mice that low amounts of Neu§Gc in glycosonjtigates might be of nutritional origin [39]. So far, the occurrence of?+acetglneuramirrate mono-oxygenase (EC 1.14.99.18) activity, responsible for the conversion of CMP-NeuSAc into CMPNeuSGc [39,40]; has not been demonstrated to come to expression ifi normal ad.ult human tissue [+Il]. Extensive studies Erave shown that when normal adult humans are exposed to sera of animal species, irnmunogenic responses may occur. The so-called Hanganutziu-D&her (serum-sickness) antibodies are directed, towards glycoconjugates containing terminal NeuSGc, c&3 linked to @-Gal [$2,44]. Wanganutziu-Deicher antigens have alsd been detected in sera and tissues of patients suffering from various diseases including cancer, having never received sera from animals [43,45]. Literature data have indicated the occurrence of Neu5Gc in fetal human tissue 1463, in certain tumors [47,48], and in various human tumor cell lines 149,501. lin case of human colon cancer, NeuSGccontaining gangliosides were demonstrated to occur in amounts < i %I l&j. The demonstration of NeuSGc,as an oricofetal antigen in humans suggests that postnatally the expression of Neu:jGc is completely suppressed, but that re-expression of NeuSGc can take place in certain disease states.
This investigation was supported by the A cknowledgemetils: Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Orgallization for Scientific Research (NWO), and the Netherlands Program for Innovation Oriented Carbohydrate Research (IOP-kj with financial aid from the Ministry of Esoriomis Affairs and the Ministry of Agriculttir~,
Novembei
1990
REFERENCES [l] Kobata, A. (1984) in: The Carbohydrates of Glycoproteins,‘v~l. 2 (Ginsbbrg, V. and Robbins, P.\Y. cds) pp. k7-161, Wiley and Sons, New York. [2] Rademacher, T.W., Parekh, R.B, and Dwek, R.A. (L989) A;;nu. Rev. Biochem. 57, 785-838. [3] Paulson, J.C. (1989) Trends Biochem. Sci. 14, 272-276. [4] Parekh, R.B., Dwek, R.A., Edge, C.J. and Rademachw, ‘F.W. (1989) Trends Bioteshnoi. 7, 117-622. [S] Knight, P. (1989) Biotechnology 7, X5-40. [6] Kamerling, J.P.. H&d, K. and ‘Vliegentha.rr, J.F.G. ;lPPo) Biotherapy, in press. [71 Dam~m, J.&L., Kemerling, J.P., Van Dedcm, G.W.E, aad :‘iiegenthart, J.F.G. (1987) Glysoconjugate J. 4, 229-144, [S] Damm, J.B.L,, Bergwerff, AA., HBrd, K.: Kamerling, J.P. and Vliegenthart, J..F.G. (1989) Resl. Trav. shim. Pays-Bas 108, 351-.359. [9] Hera, S., Yamagushi, M., Takemori, Y ., Furuhata, I!., Ogura, I-K.and Nakamura, M. (1989), Anal, Biochcm. 179, 16%166, [IO) Vliegenthart, J.F.G., Dorland, L. and Van Halbeek, H, (19h3) hdv. Carbohydr. Chem. Biochem. 41, 209-374. [i 11 Kamerling, J.P. and Vliegendlaut, J.F.G. .(1989) is: Clinical tiioshemistry - Principles; Methods, &@ications (Curt&, N.Ch. ani! Roth, M,‘eds), vol. 1? Mass Spectrometry (Lawson, A.M. ed), pp. 175-263, F?!altsr de c’lruyter, Berlin. /12] Spik, G., Bayard, R., Fournet, R., Strecker, O., BouquClet, S. and Montreuil, J. (1975) FE&S Lett. 50, 296-29X [13] Savage, A.V.,, iCoppen, F.L., Schiphorst, W33.C.M., Trippelvite, E.iO.‘w.,Van Halbeek, H., yliegenthart, J.F.G. aud Van den Bijnden, D.H. (1986) Eur. J. Biochem. 160, 123-129. f14,]Hhrd, K., Mekking, A.,, Qamm, J.U.L., Kamerling, J.P., De Boer, W., Wijnands, R.A. and Vliegenthart,J.P.0. (1990) Eur. J. Biostiem., in press. [15] Mutsaers, J.H.G.M., Kamerling, J.P., Dcvos, R., Guisez, Y., ‘Fiers, ‘W. and Vliegenthart, J.F.G. (19861 Imon, J ,, ih~.a.
i.~'q
I~}I C'arr, ,~,A,, llclldil)~, .~01,1~.,l,'.l~ll:l.Wa,~cCm:Uh (i., S~,.¢cl.
{,:ol Mu~.til1'~Ofc,I!,A., Mile~ki, M,, Varkh A, ;~,;
2. MATERIALS
The accumulating cvidcIlcc concerning the biologicnl rclcvancc of the carbohydrate part of glycoprotcins has highly stimulated the intcrcst in biochemical and pharmaccutical aspects of these biopolymcrs [l-S]. In particular, it has activated the discussion with respect to genetically engineered proteins cxprcssed in hetcrologous cell types with or without glycosylation machinzrics [4,5]. Because the cellular glycosylation machinery can vary among different cell !ines, different glycosylation patterns of glycoproteins can be expected. Based on the present knowlecige about primacy carbohydrate structures of recombinant human glycoproteins, the Chinese hamster ovary (080) cells seem to be preferred [6]. In the framework of our program on the microanalysis of (recombinnnr.; glyzoprotein glycans, we have discovered that 0X0 cells can generate asparaginelinked carbohydrate chains containing N-glysolylneuraminic acid (NeuSGc), as illustrated here for 4 recombinant human glycoproteins.
Conmcrcially ;lWililblc rccoiiibinanr him11n tissue pl:l!illliIirlgcll astivntor (IPA), cxprcssc‘d in CHO cells (Astilysc), ws obtained from ‘II~o~t~ac (Bihurilrh 011dcr Hiss, FRO). CK-lO.ccll dcrivcd reconlbinailt I~uII~:I~~rhiincric pl;~smincgcn activator (kzt~PA) was a gift of Cibn-
Correspondence
address:
J.P. Kamerling, Bijvoet Center, Department of Bio-Organic Chemistry, Utrccht University, P.O. BOX 80.095, NL.3508 TB Utrccht, The Netherlands
AbDrev/arions: NeuSAc, N-acetylneuraminic acid; NeuSGc, N-glycolylneuraminic acid; Fuc, fucose; CHO, Chinese hamster ovary; tPA, tissue plasminogen activator; katuPA, recombinant chimeric plasminogen activator; EPO, erythropoietin; FSI-I, follicle stimulating hormone, follitropin; hCG, human chorionic gonadotropin; PNGase-F, peptide-N’.(N-acetyl-P-glucosaminyl)asparagine amidase F; DMB, 1,2-diamino-4,5-methylene-dioxybsnzcne. Published
by Eiscvier
00145993/90/$3.50
Science Publishers
B. V. (Biomedical
AND METHODS
Geigy Ltd (Bnscl, Switscrlnnd),
dcrivcd recombinant Tckniklr 13V(Ilox. [cl, The Ncthcrlnnds), iuld CHO~ccll dcrivcd recombinant human foi]iIropin (FSH) WIS a gift of Diosynth BV (Oss, ‘Ihc Netherlands), Human scrotrnnsfcrrin and b0vii.e fibrinoycn wcrc obtnincd from
hWal1
cryt]lro])oictin
CHO.scll
(EPO) was u gift of Organon
Sigma. Pcptide.N’-(N-acetyl-~~-~lucosnrTlinyl)ns]~ara~i~~c (PNGascaF) from Nuvobacfcriun~ nleningouepliclrnl
alnidasc I: wns purchased
from Bochringcr Mannheim, 2.2. Liberation of N4~tkcd carbollydmtc ctlmins The N-linkedcarbohydrate chains of kztuPA and EPO were relensed by treatment with non-immobilized PNGase.F cssentialiy as described [9,8]. In case of EPO a complete liberation of oliposaccharides was obtained without the addition of Non-idet P-40, as indicated by SDS-PAGE. Released carbohydrate chains were collected by gel-permeation chromatography on Bio-Gel P-100 (BioRad) using 50 mM (k2tuPA) or 25 mM (EPO) NHdHCO,, pH 9,0, as cluent. In each case, the pooled carbohydrate fraction was scparatcd according to charge by FPLC on Mono Q (Pharmacia), followed by HPLC of the subfractions on Lichrosorb-NH2 (Chrompack). 2.3. Analysis of sialic acids The analysis of sialic acids was carried out essentially as described 191. Briefly, aliquots of sialoglycoproteins (0.25-I mg) in 0.2 ml of 2 M acetic acid were heated for 3 h at 80°C, and released sialic acids wereconverted with 1,2-diamino-4,5-methylene-dioxybenzene (DMB) into fluorescent derivatives. WAcetylneuraminic acid (NeuSAc) and N-glycolylneuraminic acid (NeuSGc) were used as standards. HPLC analysis was carried out on a Kratos Spectroflow 400 HPLC system equipped with a Chromspher Cls (Chrompack) reversed-phase CO]t!mn (25 x 0.46 cm) and Kratos 9lsO fluorescence detector operating at an excitation wavelength of 393 nm, detection emission at wavelengths > 448 nm using a cut-off filter. Elutions were performed
Division)
0 1990 Federation of European Biochemical Societies
9
NCUSAC iruscc
NcuSAc
3.2. Identjficarion of NeuSGc-containing chains in kztuPA md EPO
NeuSGc
-0
_A
IO (ml)
r
0
,
IO (ml)
0
IO (1111)
Fig. 1. HPLC Profile of DMB derivatives of sialic acids, released from different glycoproteins, on a Chromspher CI~ column (25 x 0.46 cm). The elution was carried out with acetonitrile : methanol : water (9 : I : 84, v/v/v) at a flow rate of 1 ml/min. (A) human serotransferrin; (B) bovine fibrinogen; (C) recombinant human FSPI, expressed in CM0 cells,
10
1C the HPLC profile of the DMEi sialic acids derived from recombinant human FSH is presented, showing peaks at clution positions corresponding witfa NeuSCc and NeuSAc in a ratio of 3:97. Similar patterns were found for recombinant kztuPA, EPO, and PA. These findings strongly suggest that the occurrence of NcuSGc as a constituent of carbohydrate chains in rl;lcombinanr glycoprotcins expressed in not:nal CHO cells, is a general feature. N-linked
The pool ot N-linked carbohydrate chains of kztuPA, released by PNGase-F, was separated on Mcrlo Q, yielding 4 carbohydrate-positive peaks (K l-N4) having the same retention volumes as mono-, di-, tri-, and tetra-sialo N-type antennary structures, respectively [8], Each FPLC fraction was iurther separated by HPLC on Lichrosorb-NHz. Tn the context of this paper, the WPLC subfractions N2.2 and N2.3 of FPLC fraction N2 (Fig. 2) will be discussed in more detail. The major fraction N2.2 represents the following conventional disialo diantennary compound:
The ‘I-I-NMK
spccrrum shows the characteristic structural reporters for the cclnstituting Gal., GlcNAc, Man and Fuc residues, being identical to those of 1~2.2 (see also [7]). However, as compared to N2.2, thcaintensity of the NAc signal of NcuSAc at 6 = 2.031 ppm is halvcd, whcrcas a new singlet at 6 = 4.121 ppm is observed, diagnostic for the N-glycolyl group of NcuSGc [8]. In addition, two sialic acid H-3e double doublets arc observed at d = 2.958 pprn (NeuSAc) and 6 = 2.775 ppm (NeuSGc), whereas in the H-3a chemical-shift region 4 triplets occur at 6 = 1.797 ppm (Neu5Ac, Manaul-3 branch), 6 = 1.801 ppm (NcuSAc, Manor l-6 branch), f = 1.813 ppm (NcuSGc, Manoll-3 branch), and 6 = 1.820 :?pm (NeuSGc, Mancxl-6 branch), The assignments of the H-3 signals arc based 0;1comparison with the related signals in N2,2, and on shift increments going from NeuSAc to NeuSGc in ~~2-6linked sialicacid-containing diantennary str\ictures (As = + O.OlV
0.019 ppm) [8] and in CI-!inkcd ,~iecv2-3Gnl~~l-3GalNAcc\l (H-3c, AS = i-O.013 ppm; I-l-3& A& = -i-O.017 ppm) [13]. The rclarivc rctcntion volume of N2,3 with rcspcct to N2.2 is in accordance with the rcplaccmcnt of only one Ncu§Ac rcsiduc by NcuSGc (compnrc with the disialo(a2-6) diantcnnacy carbohydrate chains from bovine fibrinogen [8]), The pool of N-linked carbohydrate chains of EPO released by PNGasc-F VW fractionated on Mono Q, yielding 4 carbohydrate-positive peaks (Al-A4) at the same retention positions as mentioned for k2tuPA (see above). Each FPLC fraction was further subfracrionatcd by HYLC on Lichrosorb-NHz. In the context of this report, the HPLC subfractions A4.4 and A4.5 of FPLC fraction A4 (Fig. 3) will be discussed in more der.all. ‘H-NMR ana!gsis of subfraction 144.4 demonstrated the presence of the fo!l;;wing tetrasialo tetraantennary compound:
NeuS Accr%-3Gal/3 l-4GlcNAcP l-6 NeuS AcaZ-3Galp l-4GlcNAcP I -&anol l-6 , ,
FllCCXll-6
Manp l-4GlcNAcp I-4&cNAc
[A&4]
Neu5Acc&3Gal/31-4GlcNAc~l-~Mancy1-3 MeuSAcrxZ-3Galpl-4GlcNAc@l-4
11
FEBS LETTERS
Volume 275, number 1,2
,’ A.4.4
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v,
60
0
120 (ml)
Fig. 3. HPLC Fractionation pattern at 205 nm of the EPO FPLC fraction A4 on a Lichrosorb-NI-iz 10 pm column (25 X 0.46 cm). The elution was carried out isocraticaliy wirh 30 mM K~HPO+/KHZPO~, PI-I7.0 : acetonitrile (37.5 : 62.5, v/v) at a flow rate of 2 ml/min at ambient temperature. Fractions were collected as indicated.
The NMR data of A4.4 are in agreemknt with those reported for the tetrasialo(Neu5Ac(u2-3) tetraantennary
November 1990 .
oligosaccharide from recombinant human FSH [14]. The four ~~2-3linked NeuSAc residues give rise to the characteristic set of structural reporters:at 6 = 2.031 ppm (4x ‘NAc), 6 = 1.804 ppm (4 x H-3a), and 6 = 2.756 ppm. (4 x H-3e). Comparison of the ‘H-YMR spectra of the subfractions A4.4 and A4.5 shows that the niajor component in subfraction A4.5 has the same characteristic structural reporters for the constituting Gal, GlcNAd, Man and Fuc residues as those found for A4.4 (see, also [14]). Howeyer, in case of A4.$ the 6region covering the H-2 signal for al-6 linked Man, the H-3 signal of Gal (4 x), and the H-5 signal of Fuc, contains an additional singlet at 6 = 4.121 ppm,‘typical for the NGc signal of ‘NeuSGc [g]. As corhpared 90 the relative intensity of the NAc singlet of NeuSAc in the NAc region of the NMR spectrum of A4.4, the intensity of the NAc singlet in the spectrum of A4;S is reduced to about 75%. Furthermore, additional signals are piesent in the sialic acid H-3 regions, Double doublets for H-3e are observed at 6 = 2.757 ppm (major, Neu5Ac) and 6 = 2.794 ppm (minor, Neu5G@, whereas’ triplets .are detected for H-3a at S = I.804 ppm (major, Neu§Ac) and d = 1.821 ppm (minor, NeuSGc). Based on the : NMR, data and the relative retention volume of A45 as compared to 84.4 (&.s = 1.3 x 1p~d.4)[a], it can be concluded that overall one of the NeuSAc residues has been replaced by NeuSGc in the tetrasialo tetraantennary structure. Whether or not a specific antenna is involved needs to be clarified.
Gal0 14GlcNAcBl-ZMancul -6 [A4.5]
(NeuSAc&3)sNeuSGcc&3 GalP II-4GlcNAcfi l -2F
l-3
@alp14GlcNAcBl-4
4. DISCUSSION In view of the literature data on the recombinant variants of human interferon-y [15], hCG f6], human FSH [14], human interferon-@1 [16-183, bovine lutropin [ 191, human EPO [%O-221, human #A [23-2>]; human transforming growth factor-@1 precursor [26], human immunodeficiency virus envelope glycoprotein gp120 [%7,28], human CD4 [29,30], and human interleukia-5 [311pit can be concluded that CM0 cells can synthesize glycoproteins with (phosphorylated) oligomannose, hybrid, as well as IV-acetyllactosamine type of structures, depending on the- protein involved. 12
The last type compris& mono-, di-; tri-, tri’-, and tetraantennary glycans. Also pslj+V-acetyllactosamin@ sequences can occur. The terminal NeuSAc residue is always exclusively linked in (~2-3 .position to Galpl4GlcNAc, indicating the absence of O-Gal a%-6-sialyltransferase activity. It has been shown that the terminal, sialylation of N-linked glycans in CHQ cells can be altered by expresBion df this transferase [32]. Structures with bisecting GlcMAc 1141, the antennary element GalNAc~l-4GlcNAc [ 191, peripheral Fuccul-3GlcNAc [33] and the antennary element Galm1-3Gal@l-4 [4] are not produced. Recently, also the transfection of a human al-3 fucosyltransferase gene
Volume 275, number 1,2
PEBS LETTERS
into CHO cells has been reported [33]. Sialylated Olinked oligosaccharides as NeuTAca2-3Galpl-3GalNAc and NeuSAccr2-3Galp1-3[NeuSAccr2-6)GalNAc are synthesized in a normal.vir&y, .includitig’ a2-3 as well as ~2-6 linked NeuSAc, as found for the recombinant variants of hCq [6], human EPO 1203, human interleukin-2 [34], and human granulocyte-colonystimulating factor [35]. This means that the CHO cell ’ has normal: a-GalNAc ,rr2-&sialyltransfeiase activity. Cbmparison of the carbohydrate chains in those cases in which the native and recombinant glycoproteins have both been analyzed, has made it clear that from a carbohydrate’point of view, the CHO cell line is highly attractive. In this report we have furnished data that recombinant glycoproteins expressed in CHQ cells contain carbohydrate chains with a small percentage (-3%) of 1~2-3linked Neu5Gc. The occurrence of Neu5Gc in norma1 human tissue and soluble glycoproteins has not been established conclusively 136,371. Preliminary investigations ‘have shown that glycoconjugate-bound NeuSGc is present regularly in a&mounts lower than 0.01% of the total sialic acid fraction in human tissues and in blood plasma glycoproteins [38]. However, there are indicatibns from studies with mice that low amounts of Neu§Gc in glycosonjtigates might be of nutritional origin [39]. So far, the occurrence of?+acetglneuramirrate mono-oxygenase (EC 1.14.99.18) activity, responsible for the conversion of CMP-NeuSAc into CMPNeuSGc [39,40]; has not been demonstrated to come to expression ifi normal ad.ult human tissue [+Il]. Extensive studies Erave shown that when normal adult humans are exposed to sera of animal species, irnmunogenic responses may occur. The so-called Hanganutziu-D&her (serum-sickness) antibodies are directed, towards glycoconjugates containing terminal NeuSGc, c&3 linked to @-Gal [$2,44]. Wanganutziu-Deicher antigens have alsd been detected in sera and tissues of patients suffering from various diseases including cancer, having never received sera from animals [43,45]. Literature data have indicated the occurrence of Neu5Gc in fetal human tissue 1463, in certain tumors [47,48], and in various human tumor cell lines 149,501. lin case of human colon cancer, NeuSGccontaining gangliosides were demonstrated to occur in amounts < i %I l&j. The demonstration of NeuSGc,as an oricofetal antigen in humans suggests that postnatally the expression of Neu:jGc is completely suppressed, but that re-expression of NeuSGc can take place in certain disease states.
This investigation was supported by the A cknowledgemetils: Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Orgallization for Scientific Research (NWO), and the Netherlands Program for Innovation Oriented Carbohydrate Research (IOP-kj with financial aid from the Ministry of Esoriomis Affairs and the Ministry of Agriculttir~,
Novembei
1990
REFERENCES [l] Kobata, A. (1984) in: The Carbohydrates of Glycoproteins,‘v~l. 2 (Ginsbbrg, V. and Robbins, P.\Y. cds) pp. k7-161, Wiley and Sons, New York. [2] Rademacher, T.W., Parekh, R.B, and Dwek, R.A. (L989) A;;nu. Rev. Biochem. 57, 785-838. [3] Paulson, J.C. (1989) Trends Biochem. Sci. 14, 272-276. [4] Parekh, R.B., Dwek, R.A., Edge, C.J. and Rademachw, ‘F.W. (1989) Trends Bioteshnoi. 7, 117-622. [S] Knight, P. (1989) Biotechnology 7, X5-40. [6] Kamerling, J.P.. H&d, K. and ‘Vliegentha.rr, J.F.G. ;lPPo) Biotherapy, in press. [71 Dam~m, J.&L., Kemerling, J.P., Van Dedcm, G.W.E, aad :‘iiegenthart, J.F.G. (1987) Glysoconjugate J. 4, 229-144, [S] Damm, J.B.L,, Bergwerff, AA., HBrd, K.: Kamerling, J.P. and Vliegenthart, J..F.G. (1989) Resl. Trav. shim. Pays-Bas 108, 351-.359. [9] Hera, S., Yamagushi, M., Takemori, Y ., Furuhata, I!., Ogura, I-K.and Nakamura, M. (1989), Anal, Biochcm. 179, 16%166, [IO) Vliegenthart, J.F.G., Dorland, L. and Van Halbeek, H, (19h3) hdv. Carbohydr. Chem. Biochem. 41, 209-374. [i 11 Kamerling, J.P. and Vliegendlaut, J.F.G. .(1989) is: Clinical tiioshemistry - Principles; Methods, &@ications (Curt&, N.Ch. ani! Roth, M,‘eds), vol. 1? Mass Spectrometry (Lawson, A.M. ed), pp. 175-263, F?!altsr de c’lruyter, Berlin. /12] Spik, G., Bayard, R., Fournet, R., Strecker, O., BouquClet, S. and Montreuil, J. (1975) FE&S Lett. 50, 296-29X [13] Savage, A.V.,, iCoppen, F.L., Schiphorst, W33.C.M., Trippelvite, E.iO.‘w.,Van Halbeek, H., yliegenthart, J.F.G. aud Van den Bijnden, D.H. (1986) Eur. J. Biochem. 160, 123-129. f14,]Hhrd, K., Mekking, A.,, Qamm, J.U.L., Kamerling, J.P., De Boer, W., Wijnands, R.A. and Vliegenthart,J.P.0. (1990) Eur. J. Biostiem., in press. [15] Mutsaers, J.H.G.M., Kamerling, J.P., Dcvos, R., Guisez, Y., ‘Fiers, ‘W. and Vliegenthart, J.F.G. (19861 Imon, J ,, ih~.a.
i.~'q
I~}I C'arr, ,~,A,, llclldil)~, .~01,1~.,l,'.l~ll:l.Wa,~cCm:Uh (i., S~,.¢cl.
{,:ol Mu~.til1'~Ofc,I!,A., Mile~ki, M,, Varkh A, ;~,;
