Blood, 1 February 2003, Vol. 101, No. 3, pp. 1204-1204
CORRESPONDENCE
To the editor:
Differences between the N-glycans of human serum
erythropoietin and recombinant human erythropoietin
The differences between the N-glycans of human
erythropoietin (hEPO), extracted from serum, and recombinant human
erythropoietin (rhEPO) reported by Skibeli et al1 have
received attention in the editorials of 2 other journals. One suggested
that these differences might be involved in the etiology of the
autoimmune pure red cell aplasia found in some patients treated with
rhEPO.2 The other suggested that these differences might
form the basis for a test to detect the doping of athletes with
rhEPO.3 A review of the data of Skibeli et al, however,
indicates that the differences they have reported between the
N-glycans of hEPO and rhEPO are not consistent with the
differences between the physicochemical properties of these EPOs found
in other laboratories. This discrepancy may be a consequence of the
effects on the N-glycans of hEPO of the conditions used by
Skibeli et al to extract hEPO from serum in order to compare it with
unextracted rhEPO.
Skibeli et al reported that hEPO lacked the tetra-acidic
(tetra-sialylated) N-glycans found in the rhEPOs, and was
also lower in its content of tri-acidic
N-glycans.1 These findings are summarized in
their Table1,1 which also permits comparison of the
N-glycan charges of different EPOs, as calculated, for example, by the
method of Hermentin et al.4 This suggests that the total
negative charge of the N-glycans of hEPO is < 70%
that of the rhEPOs, and implies that hEPO is less acidic than the
rhEPOs. Thus the sialic acid residues of the N-glycans of
EPO represent up to 12 of a possible total of 14 sialic acid residues
in EPO, and make a major contribution to the net negative charge of
EPO, as indicated by the fact that the pI of intact EPO is in the range of about 2.5 to 4.0,5 and the pI of desialylated EPO is
about 8.5.6 However, other studies have consistently shown
hEPO to be more acidic than rhEPO,7-9 and, indeed, this is
the basis for an established test for doping.9
The discrepancy between the findings of Skibeli et al and those of
others is probably due to the conditions used to extract the hEPO from
serum to compare it with unextracted rhEPOs. Although the data in
Figure 8 of Skibeli et al is said to demonstrate "the similarity of
sugar profiles from rhEPO with or without a bead-extraction step,"
comparisons of the areas under the 2 elution profiles indicates that
the extraction procedure has reduced the recovery of all oligosaccharides with elution times of 60 minutes or more, representing tri- and tetrasialylated N-glycans, and has increased the
relative recovery of most of the neutral, mono- and di-sialylated
N-glycans.1 This effect of the extraction
procedure probably represents some desialylation of the
N-glycans of EPO, since the 20min-treatment with 10mmol/l
HCl, used to dissociate the antibody-bound EPO, is also commonly used,
albeit at 80°C rather than ambient temperature, for the quantitative
desialylation of N-glycans.10
Patrick L. Storring and C-T. Yuen
Correspondence: Patrick L. Storring, National Institute
for Biological Standards and Control, Blanche Lane, South Mimms,
Potters Bar, Herts EN6 3QG, United Kingdom; e-mail:
pstorring{at}nibsc.ac.uk
References
1.
Skibeli V, Nissen-Lie G, Torjesen P.
Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin.
Blood.
2001;98:3626-3634[Abstract/Free Full Text].
2.
Bunn HF.
Drug-induced autoimmune red-cell aplasia.
N Engl J Med.
2002;346:522-523[Free Full Text].
3.
Jenkins P.
Doping in sport.
Lancet.
2002;360:99-100[CrossRef][Medline]
[Order article via Infotrieve].
4.
Hermentin P, Witzel R, Kanzy EJ, et al.
The hypothetical N-glycan charge: a number that characterizes protein glycosylation.
Glycobiology.
1996;6:217-230[Abstract/Free Full Text].
5.
Tam RC, Coleman SL, Tiplady RJ, Storring PL, Cotes PM.
Comparisons of human, rat and mouse erythropoietins by isoelectric focusing: differences between serum and urinary erythropoietins.
Br J Haematol.
1991;79:504-511[Medline]
[Order article via Infotrieve].
6.
Imai N, Higuchi M, Kawamura A, et al.
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.
1990;194:457-462[Medline]
[Order article via Infotrieve].
7.
Storring PL, Gaines Das RE.
The International Standard for Recombinant DNA-derived Erythropoietin: collaborative study of four recombinant DNAderived erythropoietins and two highly purified human urinary erythropoietins.
J Endocrinol.
1992;134:459-484[Abstract].
8.
Wide L, Bengtsson C, Berglund B, Ekblom B.
Detection in blood and urine of recombinant erythropoietin administered to healthy men.
Med Sci Sports Exerc.
1995;27:1569-1576.
9.
Lasne F, De Ceaurriz J.
Recombinant erythropoietin in urine.
Nature.
2000;405:635[Medline]
[Order article via Infotrieve].
10.
Sonnenburg JL, van Halbeek H, Varki A.
Characterization of the acid stability of glycosidically linked neuraminic acid: use in detecting de-N-acetyl-gangliosides in human melanoma.
J Biol Chem.
2002;277:17502-17510[Abstract/Free Full Text].
Response:
Inherent charge properties of the isolated sugar parts of human
serum erythropoietin
Drs Storring and Yuen claim our
data1 to be inconsistent with other reports describing the
molecular differences between endogenous human erythropoietin (hEPO)
and recombinant human EPO (rhEPO) due to desialylation of human serum
EPO caused by the extraction conditions used in our study. But they do
not take into consideration that we analyzed EPO from human serum,
while the reports they refer to2-6 all investigated EPO
from human urine, a completely different matrix.
This is important as charge profiles of glycoproteins undergo changes
during renal secretion.7,8 In fact, 2 of the
papers Storring and Yuen refer to2,5 described human serum
EPO as more basic than human urinary EPO when
analyzing the intact glycoprotein by isoelectric focusing. Furthermore,
one of the other papers referred to as contradictory to our
study3 showed a more basic charge pattern of human urinary
EPO than the one obtained for rhEPO. In addition, Storring has used
this urinary EPO preparation in his own work when comparing the
isoelectric pattern of different batches of rhEPO with human urinary
EPOs,4 reproducing the findings of Imai et
al.3 Storring and Yuen also mentioned the shift in
isoelectric point to 8.5 caused by the desialylation of
hEPO,3 as an illustration of the contribution of sialic acids to the net charge of hEPO without commenting that Imai et al used
recombinant hEPO and not endogenous hEPO.
Furthermore, during discussion of our results, Storring and Yuen did
not take into consideration the fact that our findings of a reduced
sialylation of the glycans of human serum EPO referred to the isolated
sugar part, which cannot be directly compared to studies of the charge
pattern of the intact glycoprotein. This point has previously been
elaborated by Tsuda et al,9 who reported that the glycans
from rhEPO contained more sialic acids than glycans from human urinary
EPO, indicating that sugar from human urinary EPO is more basic than
sugar from rhEPO.
In addition, we have presented results obtained from the analyses of
serum EPO from anemic patients that must be taken into consideration
when interpreting our results. In our paper all relevant reports,
including the papers mentioned by Drs Storring and Yuen, were
thoroughly referred to and discussed.
Venke Skibeli, Gro Nissen-Lie, and Peter Torjesen
Correspondence: Venke Skibeli, Section for Doping
Analysis, Hormone Laboratory, Aker University Hospital, 0514 Oslo,
Norway; e-mail: venke.skibeli{at}ioks.uio.no
References
1.
Skibeli V, Nissen-Lie G, Torjesen P.
Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin.
Blood.
2001;98:3626-3634[Abstract/Free Full Text].
2.
Tam RC, Coleman SL, Tiplady RJ, Storring PL, Cotes PM.
Comparisons of human, rat and mouse erythropoietins by isoelectric focusing: differences between serum and urinary erythropoietins.
Br J Haematol.
1991;79:504-511[Medline]
[Order article via Infotrieve].
3.
Imai N, Higuchi M, Kawamura A, et al.
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.
1990;194:457-462[Medline]
[Order article via Infotrieve].
4.
Storring PL, Gaines Das RE.
The international standard for recombinant DNA-derived erythropoietin: collaborative study of four recombinant DNA-derived erythropoietins and two highly purified human urinary erythropoietins.
J Endocrinol.
1992;134:459-484[Abstract].
5.
Wide L, Bengtsson C, Berglund B, Ekblom B.
Detection in blood and urine of recombinant erythropoietin administered to healthy men.
Med Sci Sports Exerc.
1995;27:1569-1576.
6.
Lasne F, De Ceaurriz J.
Recombinant erythropoietin in urine.
Nature.
2000;405:635[Medline]
[Order article via Infotrieve].
7.
Abraham PA, Katz SA, Opsahl JA, Miller RP, Stanchfield WR Jr, Andersen RC.
Renal secretion and hepatic clearance of human multiple renin forms.
Hypertens.
1990;16:669-676[Abstract/Free Full Text].
8.
Opsahl JA, Abraham PA, Katz SA.
Acute stimulation of renin secretion changes the multiple form profile of active plasma renin.
Am J Hypertens.
1991;4:126-130[Medline]
[Order article via Infotrieve].
9.
Tsuda E, Goto M, Murakami A, et al.
Comparative structural study of N-linked oligosaccharides of urinary and recombinant erythropoietins.
Biochem.
1988;26:5646-5654.
