Laboratoire de Glycobiologie Structurale et Fonctionnelle, Unité Mixte de Recherche du CNRS N°8576, Université des Sciences et Technologies de Lille, F-59655 Villeneuve dAscq, France and 2Laboratoire Français du Fractionnement et des Biotechnologies, Département Recherche, 59, rue de Trévise, F-59011 Lille, France
Received on September 17, 1999; revised on November 18, 1999; accepted on December 1, 1999.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: biological activity/electrospray ionization mass spectrometry/glycosylation/recombinant glycoprotein/sialylation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human Epo is a 166 amino acid glycoprotein containing three complex type N-glycans located on Asn residues at position 24, 38, and 83, respectively, and one mucin type O-glycan located on Ser-126. Exhaustive structural studies of N-glycans isolated from urinary human Epo (uHuEpo) (Takeuchi et al., 1988; Tsuda et al., 1988
; Rahbek-Nielsen et al., 1997
) and from recombinant human Epo (rHuEpo) produced either by CHO cell line (Epo-CHO) (Sasaki et al., 1987
, 1988; Takeuchi et al., 1988
; Rice et al., 1992
; Rush et al., 1993
, 1995; Linsley et al., 1994
; Watson et al., 1994
; Hokke et al., 1995
) or by BHK cell line (Epo-BHK) (Tsuda et al., 1988
; Nimtz et al., 1993
; Morimoto et al., 1996
) have been achieved. These later differ from those of uHuEpo by (1) the presence of NeuGc (less than 3% of total sialic acid) which exhibits a strong immunogenicity in adult human, (2) the kind of sialyl linkages (only
23 sialyl linkage), and (3) a number of LacNAc repeat slightly increased (up to three per molecule for some glycans).
The N-glycan moieties of Epo have been shown to be implicated in various physical and biological processes such as the secretion of the molecule, the control of the lifetime, the homing to bone marrow and the maintaining of an active conformation (Dordal et al., 1985; Tsuda et al., 1990
; Takeuchi and Kobata, 1991
). For the first point, it has been evidenced that glycans of Asn-38 and Asn-83 plays an important role since the prevention of one of these two glycosylation sites by site directed mutagenesis abolishes the secretion of Epo (Dubè et al., 1988
; Delorme et al., 1992
). For the second point, several studies have shown that the capping of external Gal residues by sialic acid residues prevented the recognition of Epo by the hepatic asialo glycoprotein binding lectin (Fukuda et al., 1989
; Spivak and Hogans, 1989
; Imai et al., 1990
; Higuchi et al., 1992
). The number of antennae seems also be important since Takeuchi et al. (1988)
revealed a positive correlation between this number and the in vivo activity. They suggested that branching part of N-glycans could act either as an anti-filtration system (e.g., by the kidney) or as a targeting signal to bone marrow. Finally, enzymatic trimmings of N-glycans to the pentasaccharide structure increased in vitro activities whereas the subsequent removing of the two Man residues of the core leads to a decreased activity (Takeuchi et al., 1990
), suggesting that the inner core could correspond to the minimal glycan structure necessary to maintain the active conformation of Epo.
In an attempt to produce a rHuEpo exhibiting a glycosylation pattern as close as possible to the natural Epo (i.e., 23 and
26 sialylation or absence of NeuGc), human Epo gene has been expressed in a human lymphoblastoid cell line named RPMI 1788. A similar strategy was followed by Yanagi et al. (1989)
by using an other lymphoblastoid cell line, the Namalwa cells. However, this rHuEpo was not fully characterized.
In this paper, we report, on one hand, the structural characterization of both glycan moieties and protein backbone and on the other hand, the in vivo and in vitro biological studies of this rHuEpo termed Epo-RPMI. The results show that despite unusual structural features observed in N-glycans, Epo-RPMI displays biological activities similar to both uHuEpo and Epo-CHO.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Monosaccharide analysis
The monosaccharide content of Epo-RPMI was determined according to Zanetta et al. (1999).
On the basis of three Man residues, the molar ratios of Fuc, Gal, GalNAc, NeuAc, and GlcNAc residues are, respectively, 1.4, 6.0, 0.2, 3.1, and 9.3 (it has been taken into account that the Asn-linked GlcNAc is poorly detected after methanolysis and derivatization). As described for the uHuEpo, these results indicate that Epo-RPMI glycans are highly sialylated and are mainly represented by N-glycans. O-Glycans appear weakly represented in regard to the very low quantity of GalNAc residues.
The high content of Fuc residues could indicate the presence of difucosylated N-glycans whereas the high level of Gal and GlcNAc residues could reflect the presence of numerous LacNAc repeats. Thus, from the monosaccharide composition established above, we have hypothesized that in N-glycans, the six Gal residues were linked to six GlcNAc residues in a tetraantennae structure. Taking into account the two core GlcNAc residues, we are left with one GlcNAc residue that could correspond to a bisecting GlcNAc, thus explaining a total of nine GlcNAc residues.
Sialylation pattern study of N-glycans.
The sialylation level and the type of sialyl linkage were carried out according to the Cointe et al. (1998) strategy. Following the trideuteromethylation step, it was found that 81.5% of N-glycans are sialylated. This result is in agreement with the NeuAc content obtained from the monosaccharide analysis. The analysis of partially ethylated methylgalactoside residues (Figure 1a) revealed the presence of 6-Et-Gal and 3-Et-Gal derivatives (63% and 37%, respectively) indicating that sialic acids are bound to Gal either via an
26 or an
23 sialyl linkage in a gross ratio of 2 to 1.
|
Glycopeptide fractionation and identification.
To isolate the four glycosylation sites, the pyridyl-ethylated Epo-RPMI was digested by endoproteinase Glu-C. Following RP-HPLC fractionation, peptides and glycopeptides were identified by Edman degradation. As shown in Figure 2, Asn-24 and Asn-38 glycopeptides were eluted as three peaks (3,4,5, and 12,13,14, respectively) whereas Asn-83 and Ser-126 glycopeptides were eluted in the two broad peaks 18 and 19, respectively. These results reveal the microheterogeneity of glycan chains associated to each glycosylation site.
|
ESI-MS analysis of glycopeptides and methylation analysis
The ESI-MS analysis of sialylated glycopeptides led to extremely complex spectra due to numerous peaks. To make the interpretation easier, each glycopeptide was chemically desialylated prior to the ESI-MS analysis. Structure of each glycan was deduced from its molecular mass obtained by the difference between the measured molecular mass of the glycopeptide species and the molecular mass of the corresponding peptide backbone calculated from the Edman degradation data. Since it is not possible to distinguish a LacNAc repeat unit containing-triantennary N-glycan and a tetraantennary N-glycan by simple molecular mass measurement, the higher branch form possible of a glycan was chosen. In the same manner, it was postulated that an extra mass of 203 corresponded to a bisecting GlcNAc residue.
Both molecular masses observed on each ESI-MS spectra, the monosaccharide content as well as a putative structure of each glycan deduced from these masses and the corresponding theoretical molecular masses are listed in Table I. Examination of this table leads to several observations. First, N-glycans are nearly all fucosylated. Second, the Asn-24 glycosylation site appears the most heterogeneous site with 26 different glycans whereas only 10 and 12 are observed on the Asn-38 and Asn-83 glycosylation sites, respectively. Third, biantennary and triantennary glycans are mainly located on Asn-24 (34% and 21%, respectively) and Asn-38 (6% and 8%, respectively), whereas tetraantennary glycans are mainly present on Asn-38 (32%) and Asn-83 (100%).
|
Third, 10%, 74%, and 74% of glycans present respectively on Asn-24, Asn-38, and Asn-83, revealed a high level of LacNAc repeat. To confirm this point, the Cointe et al. (1998) strategy was used to distinguish Gal residues substituted on their C-3 hydroxyl group either by a sialic acid residue or a GlcNAc residue of a LacNAc repeat and thus quantify the level of LacNAc repeats. For this purpose, the peak area ratio of methyl-3-O-acetyl-2,4,6-tri-O-methyl-D-galactopyrannoside derivatives (2,4,6-Me-Gal) which could correspond to a Gal residue substituted by a GlcNAc residue to the sum of the different methyl galactopyrannoside derivatives (methyl-2,3,4,6-tetra-O-methyl-D-galactopyrannoside, 2,4,6-Me-Gal, 3-Et-Gal and 6-Et-Gal) was calculated. The value of 30% is in agreement with that deduced from the ESI-MS data (20%, Table I) and with the molar composition of monosaccharide giving six Gal residues for three Man residues. It is the first time that LacNAc repeats are reported for N-glycans isolated from Asn-24 glycosylation site of an Epo molecule. In the same manner, the presence of five LacNAc repeats in Asn-83 glycans of Epo has neither been reported so far.
At last, it must be mentioned that 20% of glycan chains linked to Asn-24 (and 4% linked to Asn-38) appear truncated since one or two Gal residues but also GlcNAc residues are missing (Table I).
Determination of sialyl-Lewis x motif
From the analysis of methyl glycoside derivatives corresponding to GlcNAc residues, it was speculated that sialyl Lewisx (sLex) motifs could be present on some glycans. To verify this hypothesis, Epo-RPMI was allowed to react by ELISA with the anti-sLex monoclonal IgM. As observed in Figure 3, a strong specific response was observed. After desialylation with sialidase, Epo-RPMI was not recognized by these antibodies but strongly reacted with anti-human CD 15 monoclonal antibodies which are specific of Lex motifs. These two experiments support the presence of sLex motif in the Epo-RPMI.
|
|
|
|
|
A second experiment was performed with collection of blood every hour from 15 h to 24 h after SC injection. To compare the half-life of the two Epo molecules, 15 h was chosen as reference time for the maximal Epo concentration (Y max). As shown in Table II, the mean values were 22.5 h and 22.1 h for Epo-RPMI and Epo-CHO, respectively. According to these results, Epo-RPMI and Epo-CHO display the same behavior in the blood of rabbits.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In contrast to these results, N-glycan chains of the Epo-RPMI molecule displayed several unusual structural characteristics. The first one concerns the presence in a high level of bisecting GlcNAc residue (~80%, as quantified by gas-phase chromatography analysis). The transfer of this residue is catalyzed by the UDP-N-acetyl glucosamine:ß-D-mannoside ß1,4-N-acetylglucosaminyltransferase III (GlcNAc-TIII) which is known to prevent the action of subsequent glycosyltransferases such as GlcNAc-TII, GlcNAc-TIV, GlcNAc-TV responsible of the antennae biosynthesis and core 16 fucosyltransferase (Schachter et al., 1983
). Because all tetraantennary N-glycans are fucosylated on their Asn-linked GlcNAc residue and contain a bisecting GlcNAc residue, it must be postulated that the action of GlcNAc-TIII is a late event in the biosynthetic process of these glycans. To explain the high GlcNAc-TIII activity observed in the RPMI cells, it must be kept in mind that they originate from EBV-transformed peripheral blood circulating B lymphocytes in which a such strong activity has been reported (Narasimhan et al., 1988
).
The precise role of bisecting GlcNAc is not well understood. It has been observed that its presence in glycans modify their in vitro recognition by either E- and L-PHA lectins (Cummings and Kornfeld, 1982) or by Gal-binding lectins as reported for a CHO-Lec 10 mutant (Campbell and Stanley, 1984
; Stanley et al., 1991
). Thus, as the hepatic clearance of Epo involves the recognition of terminal Gal residues, it can be thought that the clearance of desialylated Epo-RPMI could be decreased. However, our data indicate no change in its clearance since it disappears from the blood stream in less than 15 min following injection. Moreover, the presence of the bisecting GlcNAc residue does not modify the in vivo message transduction as revealed by the reticulocyte production which was very similar to Epo-CHO.
The second unusual structural feature of Epo-RPMI concerns the high level of LacNAc repeats present in some N-glycan chains. Thus, 6% of tetraantennary N-glycans contain from four to five LacNAc repeats. Such a number had never been described for an Epo molecule, even if Hokke et al. (1990) and Sasaki et al. (1987)
reported for the Epo-CHO a tendency to contain more LacNAc repeat (until three LacNAc repeats for some N-glycans) than the uHuEpo (Takeuchi et al., 1988
). The relationship between LacNAc repeats in glycans and biological functions are not clearly elucidated. It is thought that highly polymerized glycan chains could decrease the filtration rate of the kidney leading to an increase of the half-life of these molecules. However, as demonstrated by Fukuda et al. (1989)
, glycoproteins containing more than three LacNAc repeats were efficiently captured by hepatic lectin, with or without sialic acid at its nonreducing termini. The presence of 6% of such structures in Epo-RPMI does not significantly modify its half-life since it was found comparable to that of Epo-CHO.
As often observed, poly-LacNAc repeats can provide the backbone structure for additional modifications as for example the fucosylation by the 1,3/4 fucosyltransferase(s) to generate either Lewis a, Lewis x, sialyl Lewis a, or sialyl Lewis x motifs (Lowe et al., 1990
). As revealed by ELISA, Epo-RPMI displays sLex motifs and the fact that the desialylation increases strongly the anti-Lex response means that the majority of the
3 linked Fuc residues are located to the terminal GlcNAc residue. Thus, it is the first time that a sLex containing Epo molecule is reported. During inflammatory processes, sLex motif at the surface of leukocytes allows their recognition by E- and P-selectins expressed by endothelial cells and, as reported by Mulligan et al. (1993)
, these cellcell interactions can be inhibited by soluble molecules bearing this epitope. Thus, it can be envisaged that the Epo-RPMI could play a similar competitive role in chronic inflammation suffering patients, leading to a decreased amount of Epo available for the stimulation of erythroid cells.
In the field of the recombinant glycoprotein production, it is well known that the final structure of glycoprotein sugar chains is determined by the tuning of putative signals in polypeptide backbone, of operating enzyme depending on the cell line and of the culture conditions. In this study, our results have confirmed the influence plays by the protein since Asn-83 glycosylation site of Epo is exclusively occupied by tetraantennary N-glycans whereas Asn-24 is occupied by bi-, tri-, and tetraantennary glycans in an equal ratio. Such a distribution of glycan chains in the Epo is observed whatever the mammalian cell line used for its production. As already discussed (Takeuchi and Kobata, 1991), there are probably signals on the protein chain which direct the glycan biosynthesis. Unlike rHuEpo produced either by CHO- or BHK- cell lines, the Epo-RPMI has revealed the presence of sLex motif. This structural feature must be considered as dependent of the cell line since the sLex is also detected in a recombinant IgG1 synthesized by the RPMI 1788 cells and in the IgM naturally produced by these cells (unpublished observations). On the same way, the presence of bisecting GlcNAc residue in 80% of glycans synthesized in a cell line exhibiting a strong GlcNAc-TIII activity, constitute an other example of the influence played by the enzymatic equipment of the host cell.
In conclusion, the unusual glycan structures of Epo-RPMI do not modify the in vitro and in vivo biological activities measured in animals. However, its therapeutic use will require more biological studies since the influence of these features on the behavior of this molecule in human remains questionable.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Microsep concentrators (cutoff 10 kDa) were from Filtron. HPLC analysis were carried out on a Spectra Physics apparatus equipped with a C18 column (Vydac 250 x 4.6 mm; 5 µm particle size). Amino acid sequence and PTH analysis were performed with an Applied Biosystems 473A and 120A, respectively, according to the manufacturer recommendations.
The ESI-MS mass experiments were carried out on a Quattro II triple-quadripole mass spectrometer (Micromass) with an API ion source in positive ion electrospray mode.
Production and purification of the Epo-RPMI
Epo-RPMI was produced by LFB from the culture supernatant of genetically engineered human B lymphoblastoid RPMI 1788 cells (ATCC number CCL 156) as described in the patent PCT91/00636. Large-scale batch production of supernatant was achieved by cell culture in a 200 l bioreactor in a FCS containing medium. After harvesting, Epo-RPMI was purified successively by anion exchange chromatography on a Q-Sepharose, immunoaffinity on anti-Epo monoclonal antibodies bound to Sepharose beads, weak anion exchange chromatography on a DEAE Sepharose gel, and a size exclusion chromatography by using a S200 sephacryl support. The purified product was prepared in PBS for analytical study and in a 20 mM citrate buffer, pH 6.9 supplemented with 0.25% human albumin in order to reproduce Epo-CHO formulation for biological activity measurement. The specific activity of Epo-RPMI (137,000 IU/mg) was found to be comparable to that Epo-CHO (129,000 IU/mg) as reported by Browne et al. (1986).
Monosaccharide content analysis and sialylation pattern study
Monosaccharide analysis was carried out by dissolving Epo-RPMI (50 µg) in a 5 µg ml1 lysine solution (1 ml) used as internal standard and lyophilized. After methanolysis with a methanol/HCl 0.5 M solution (500 µl) for 16 h at 80°C and drying, monosaccharides were derivatized and quantified by gas chromatography according to the method described by Zanetta et al. (1999).
The sialylation level and the 23 to
26 sialyl linkage ratio was determined by treating 50 µg of Epo-RPMI using the Cointe et al. (1998)
procedure. Partially deuteromethylated methyl glycosides were analyzed by GC-MS to calculate the sialylation level whereas partially ethylated methyl glycosides were analyzed by GC to determine the
23 to
26 sialyl linkage ratio. Concerning the identification of sialic acids, they were released from the Epo-RPMI by treatment with a 2 M acetic acid solution for 3 h at 80°C. Prior their analysis by RP-HPLC, they were derivatized with DMB according to Hara et al. (1987)
.
Enzymatic cleavage of Epo-RPMI and identification of peptide and glycopeptide
Epo-RPMI (2 mg) was dissolved in a 6 M guanidine-HCl solution (100 µl) containing 0.1 M NH4HCO3, pH 8.0 and reduced 1 h at 37°C by DTT (0.1 mg). Free thiol groups were then alkylated with 1 µl of 4-vinylpyridine (1 h at 37°C). After desalting by ultrafiltration using a Microsep concentrator and freeze drying, the pyridyl-ethylated Epo-RPMI was dissolved in 1 ml of 50 mM NaHCO3 pH 7.8 and submitted to digestion by Endoproteinase Glu-C for 8 h at 37°C (final enzyme to substrate mass ratio of 1/100). After fractionation by RP-HPLC on a C18 column (Vydac 250 x 4.6mm; 5 µm particle size) eluted with a linear gradient of acetonitrile solution containing 0.05% of TFA (0.5% per min) and monitored at 206 nm, peptides and glycopeptides were identified by automated Edman degradation.
Electrospray ionization mass spectrometry
Samples were dissolved in water/methanol/acetic acid 49/49/2 at a concentration of approximately 14 pmol µl1 and the solution was infused into the electrospray ion source by a Harvard syringe pump at a flow rate of 5 µl min1. Typically, best conditions were defined for a cone voltage of 65 V. Spectra were recorded in MCA mode from 1500 to 2300 m/z at a scan speed of 10 s and smoothed three times.
ELISA technique
For the measurement of Epo concentration, microtiter plates were coated with an affinity purified polyclonal rabbit anti-Epo. After blocking of excess reactive, standard Epo (range concentration 8500 mIU ml1) or samples were incubated overnight at 4°C. After washing with a 0.05% Tween-20 in 0.9 % NaCl solution (used for each washing step), a biotinylated mouse anti-Epo monoclonal antibody was added together with a streptavidinperoxidase solution for 1.5 h at room temperature before developing the reaction and reading at 405 nm. This assay was validated against BioMerieux EIA.
For sLex and Lex detection, microtiter plates were coated with Epo, asialo-Epo, or with BSA (used as negative control) by incubation overnight at 4°C and washed. Excess reactive sites were then blocked, and monoclonal IgM anti-sLex or monoclonal anti-human CD15 biotin conjugate (diluted 1/2000) were added and incubated at room temperature for 2 h. After washing, anti-murin IgM biotin conjugate (1/4000; 2 h at room temperature) and next streptavidinperoxidase (1/2000; 30 min at room temperature) or directly streptavidinperoxidase were added to detect sLex or anti CD15, respectively. After the addition of OPD, the reaction was stopped with a 1M HCl solution and the absorbance was measured at 492 nm.
In vitro assay for Epo activity
The DAE7 and UT-7 cell lines used to evaluate the Epo activity (Nicolis et al., 1993, Sakaguchi et al., 1987
) were maintained in DMEM (from Sigma) supplemented with 10% FCS and 2 IU/ml (UT-7) or 10 IU/ml (DAE7) of Epo. After washing of cells with IMDM culture medium (from Sigma), increasing amounts of Epo from 0.003 to 0.2 IU ml1 (or 0.05 to 1 for UT-7) in 100 µl of IMDM with 10% fetal calf serum were added to 100 µl of medium containing about 20,000 cells in a 96-well culture plate. After 3 days at 37°C and 7% CO2, 20 µl of a MTT solution (Page et al., 1988
) were brought in each well of the plate and an additional incubation of 2 h at 37°C was performed. One hundred microliters was removed, and 100 µl of an isopropanol-HCl solution were added and mixed thoroughly to dissolve the dark blue crystals before reading at 570 nm.
In vivo bioassay in mice
The 6- to 10-week-old female mice were SC injected once a day for three consecutive days with the Epo solution (one mouse for one dose, one for the negative control, i.e., 20 mM citrate buffer pH 6.9 containing 0.25% human albumin). On the fourth day, blood was collected in EDTA-Na containing tube and diluted to 1/2000 before counting the red blood cells on the Counter Coulter. Reticulocytes were numbered by counting eight fields of 200 cells according to the technique described by Hayakawa et al. (1992) using the Nile blue sulfate and the brilliant cresyl blue.
Pharmacokinetic evaluation
The rate of disappearance of Epo molecules in the blood of rabbits was determined after a slow IV (90 IU kg1 as reference to human dose) or SC (360 IU kg1) injection of Epo (0.3 ml kg1). Blood samples (3 ml) were collected at different times and the serum Epo concentration were determined by ELISA as described above. For the measurement of half-life, the rate of disappearance of EPO was semilogarithmically plotted versus time. Calculation of the regression line derived by the least-squares method allowed estimation of the Epo serum half-life given by the formula y = a LOG (x) + b where y represents the half of the maximal concentration (in mIU/ml) and x-value, the half-life time (in hours). The calculation of ([Epo] T15 [Epo] T1)/2 given the y value for the distribution phase whereas the calculation of [Epo] T1/2 given the y value for the elimination phase (N.B. T15 and T1 express the 15 min and 1 h time points).
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Campbell,C. and Stanley,P. (1984) A dominant mutation to ricin resistance in Chinese hamster ovary cells induces UDP-GlcNAc:glycopeptide ß-4-N-acetylglucosaminyltransferase III activity. J. Biol. Chem., 259, 1337013378.
Cointe,D., Leroy,Y. and Chirat,F. (1998) Determination of the sialylation level and of the ratio -(2
3)/
-(2
6) sialyl linkages of N-glycans by methylation and GC/MS analysis. Carbohydr. Res., 311, 5159.[ISI][Medline]
Cummings,R.D. and Kornfeld,S. (1982) Characterization of the structural determinants required for the high affinity interaction of asparagine-linked oligosaccharides with immobilized Phaseolus vulgaris leukoagglutinating and erythroagglutinating lectins. J. Biol. Chem., 257, 1123011234.
Delorme,E., Lorenzini,T., Giffin,J., Martin,F., Jacobsen,F., Boone,T. and Elliott,S. (1992) Role of glycosylation on the secretion and biological activity of erythropoietin. Biochemistry, 31, 98719876.[ISI][Medline]
Dordal,M.S., Wang,F.F. and Goldwasser,E. (1985) The role of carbohydrate in erythropoietin action. Endocrinology, 116, 22932299.[Abstract]
Dubè,S., Fisher,J.W. and Powell,J.S. (1988) Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion and biological function. J. Biol. Chem., 263, 1751617521.
Fukuda,M.N., Sasaki,H., Lopez,L. and Fukuda,M. (1989) Survival of recombinant erythropoietin in the circulation: the role of carbohydrates. Blood, 73, 8489.[Abstract]
Hara,S., Takemori,Y., Yamaguchi,M., Nakamura,M. and Ohkura,Y. (1987) Fluorometric High-Performance Liquid Chromatography of N-acetyl- and N-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins and glycolipids. Anal. Biochem., 164, 138145.[ISI][Medline]
Hayakawa,T., Wada,M., Mizuno,K., Abe,S., Miyashita,M. and Ueda,M. (1992) Simple in vivo bioassay without radioisotopes for recombinant human erythropoietins. Biologicals, 20, 243251.[ISI][Medline]
Higuchi,M., Oh-eda,M., Kuboniwa,H., Tomonoh,K., Shimonaka,Y. and Ochi,N. (1992) Role of sugar chains in the expression of the biological activity of human erythropoeitin. J. Biol. Chem., 267, 77037709.
Hokke,C.H., Bergwerff,A.A., van Dedem,G.W.K., van Oostrum,J., Kamerling,J.P. and Vliegenthart,J.F.G. (1990) Sialylated carbohydrate chains of recombinant human glycoproteins expressed in Chinese hamster ovary cells contain traces of N-glycolylneuraminic acid. FEBS Lett., 275, 914.[ISI][Medline]
Hokke,C.H., Bergwerff,A.A., van Dedem,G.W.K., Kamerling,J.P. and Vliegenthart,J.F.G. (1995) Structural analysis of the sialylated N- and O-linked carbohydrate chains of recombinant human erythropoietin expressed in Chinese Hamster Ovary cells. Sialylation patterns and branch of N-acetyllactosamine units. Eur. J. Biochem., 228, 9811008.[Abstract]
Imai,N., Higuchi,M., Kawamura,A., Tomonoh,K., Oh-eda,M., Fujiwara,M., Shimonaka,Y. and Ochi,N. (1990) Physicochemical and biological characterization of asialoerythropoietin. Suppressive effects of sialic acid in the expression of biological activity of human erythropoietin in vitro. Eur. J. Biochem., 194, 457462.[Abstract]
Inoue,N., Takeuchi,M., Ohashi,H. and Suzuki,T. (1995) The production of recombinant human erythropoietin. Biotechnol. Annu. Rev., 1, 297313.[Medline]
Jelkmann,W. (1992) Erythropoietin: structure, control of production and function. Physiol. Rev., 72, 449489.
Klein,A., Diaz,S., Ferreira,I., Lamblin,G., Roussel,P. and Manzi,A.E. (1997) New sialic acids from biological sources identified by a comprehensive and sensitive approach: liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) of SIA quinoxalinones. Glycobiology, 7, 421432.[Abstract]
Lai,P.-H., Everett,R., Wang,F.-F., Arakawa,T. and Goldwasser,E. (1986) Structural characterization of human erythropoietin. J. Biol. Chem., 261, 31163121.
Linsley,K.B., Chan,S.-Y., Chan,S., Reinhold,B.B., Lisi,P.J. and Reinhold,V.N. (1994) Applications of electrospray mass spectrometry to erythropoietin N- and O-linked glycans. Anal. Biochem., 219, 207217.[ISI][Medline]
Lowe,J.B., Stoolman,L.M., Nair,R.P., Larsen,R.D., Berhend,T.L. and Marks,R.M. (1990) ELAM-1-dependent cell adhesion to vascular endothelium determined by a transfected human fucosyltransferase cDNA. Cell, 63, 475484.[ISI][Medline]
Morimoto,K., Tsuda,E., Said Abdu,A., Uchida,E., Hatakeyama,S., Ueda,M. and Hayakawa,T. (1996) Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialyltransferase. Glycogonjugate J., 13, 10131020.
Mulligan,M.S., Paulson,J.C., De Frees,S., Zheng,Z.L., Lowe,J.B. and Ward,P.A. (1993) Protective effects of oligosaccharides in P-selectin-dependent lung injury. Nature, 364, 149151.[ISI][Medline]
Narasimhan,S., Lee,J.W., Cheung,R.K., Gelfand,E.W. and Schachter,H. (1988) ß-1,4-Mannosyl-glycoprotein ß-1,4-N-acetylglucosaminyltransferase III activity in human B and T lymphocyte lines and in tonsillar B and T lymphocytes. Biochem. Cell Biol., 66, 889900.[ISI][Medline]
Nicolis,S., Ottolenghi,S., Papayannopoulos,T., Baiocchi,M., Migliaccio,G., Adamson,J. and Migliaccio,A.R. (1993) Dependance for the proliferative response to erythropoietin on an established erythroid differentiation program in a human hematopoietic cell line, UT-7. Exp. Hematol., 21, 665670.[ISI][Medline]
Nimtz,M., Martin,W., Wray,V., Klöppel,K.D., Augustin,J. and Conradt,H.S. (1993) Structures of sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK-21 cells. Eur. J. Biochem., 213, 3956.[Abstract]
Noguchi,A., Mukuria,C.J., Suzuki,E. and Naiki,M. (1995) Immunogenicity of N-glycolylneuraminic acid-containing carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. J. Biochem., 117, 5962.[Abstract]
Page,M., Bejaoui,N., Cinq-Mars,B. and Lemieux,P. (1988) Optimization of the tetrazolium-based colorimetric assay for the measurement of cell number and cytotoxicity. Int. J. Immunopharmacol., 10, 785793.[ISI][Medline]
Rahbek-Nielsen,H., Roepstorff,P., Reischl,H., Wozny,M., Koll,H. and Haselbeck,A. (1997) Glycopeptide profiling of human urinary erythropoietin by matrix-assisted laser desorption/ionization mass spectrometry. J. Mass Spectrom., 32, 948958.[ISI][Medline]
Rice,K.G., Takahashi,N., Namiki,Y., Tran,A.D., Lisi,P.J. and Lee,Y.C. (1992) Quantitative mapping of the N-linked sialyl oligosaccharides of recombinant erythropoietin: combination of direct high-performance anion-exchange chromatography and 2-aminopyridine derivatization. Anal. Biochem., 206, 278287.[ISI][Medline]
Rush,R.S., Derby,P.L., Strickland,T.W. and Rohde,M.F. (1993) Peptide mapping and evaluation of glycopeptide microheterogeneity derived from endoproteinase digestion of erythropoietin by affinity high-performance capillary electrophoresis. Anal. Chem., 65, 18341842.[ISI][Medline]
Rush,R.S., Derby,P.L., Smith,D.M., Merry,C., Rogers,G., Rohde,M.F. and Katta,V. (1995) Microheterogeneity of erythropoietin carbohydrate structure. Anal. Biochem., 67, 14421452.
Sakaguchi,M., Koishihara,Y., Tsuda,H., Fujimoto,K., Shibuya,K., Kawakita,M. and Takatsuki,K. (1987) The expression of functional erythropoietin receptors on an interleukin-3 dependent cell line. Biochem. Biophys. Res. Commun., 146, 712.[ISI][Medline]
Sasaki,H., Bothner,B., Dell,A. and Fukuda,M. (1987) Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA. J. Biol. Chem., 262, 1205912076.
Sasaki,H., Ochi,N., Dell,A. and Fukuda,M. (1988) Site specific glycosylation of human recombinant erythropoietin: analysis of glycopeptides or peptides at each glycosylation site by fast atom bombardment mass spectrometry. Biochemistry, 27, 86188626.[Medline]
Schachter,H., Narasimhan,S., Gleeson,P. and Vella,G. (1983) Control of branching during the biosynthesis of asparagine-linked oligosaccharides. Can. J. Biochem. Cell Biol., 61, 10491066.
Spivak,J.L. and Hogans,B. (1989) The in vitro metabolism of recombinant human erythropoietin in the rat. Blood, 73, 9099.[Abstract]
Stanley,P., Sundaram,S. and Sallustio,S. (1991) A subclass of cell surface carbohydrates revealed by a CHO mutant with two glycosylation mutations. Glycobiology, 1, 307314.[Abstract]
Takeuchi,M. and Kobata,A. (1991) Structures and functional roles of the sugar chains of human erythropoietins. Glycobiology, 1, 337346.[Abstract]
Takeuchi,M., Takasaki,S., Miyazaki,H., Kaato,T., Hoshit,S., Kochibe,N. and Kobata,A. (1988) Comparative study of the asparagine-linked sugar chains of human erythropoietins purified from urine and the culture medium of recombinant Chinese Hamster Ovary cells. J. Biol. Chem., 263, 36573663.
Takeuchi,M., Takasaki,S., Shimada,M. and Kobata,A. (1990) Role of sugar chains in the in vitro biological activity of human erythropoietin produced in recombinant Chinese hamster ovary cells. J. Biol. Chem., 265, 1212712130.
Tsuda,E., Goto,M., Murakami,A., Akai,K., Ueda,M., Kawanishi,G., Takahashi,N., Sasaki,R., Chiba,H., Ishihara,H., Mori,M., Tejima,S., Endo,S. and Arata,Y. (1988) Comparative structural study of N-linked oligosaccharides of urinary and recombinant erythropoietins. Biochemistry, 27, 56465654.[ISI][Medline]
Tsuda,E., Kawanishi,G., Ueda,M., Masuda,S. and Sasaki,R. (1990) The role of carbohydrate in recombinant human erythropoietin. Eur. J. Biochem., 188, 405411.[Abstract]
Watson,E., Bhide,A. and van Halbeek,H. (1994) Structure determination of the intact major sialylated oligosaccharide chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. Glycobiology, 4, 227237.[Abstract]
Yanagi,H., Yoshima,T., Ogawa,I. and Okamoto,M. (1989) Recombinant human erythropoietin produced by Namalwa cells. DNA, 8, 419427.[ISI][Medline]
Zanetta,J.-P., Timmerman,P. and Leroy,Y. (1999) Gas-liquid chromatography of the heptafluorobutyrate derivatives of the O-methyl-glycosides on capillary columns: a method for the quantitative determination of the monosaccharide composition of glycoproteins and glycolipids. Glycobiology, 9, 255266.