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four O-glycans on Ser121, Ser127, Ser131 and Ser138).
25
Complex bian-
tennary N-glycans represented in Fig. 2 show a wide coverage of the protein
surface in the axis of the molecule. However, the C-terminal peptide pre-
sent on the b-subunit (often designed as CTP) containing four attached
O-linked glycans is absent of hCG crystal structure because the 34
C-terminal amino acids have been deleted to crystallize the protein. Re-
cently, Laurence A. Cole raised the problem that biologically active hCG is
a very acidic glycoprotein (pI 3.5) while the three-dimensional structural
model stands for a biologically inactive, C-terminal peptide-less, oligo-
saccharide-less basic molecule of pI
B
8.5 missing approximately 50% of its
molecular weight.
26
As a result, the overall three-dimensional structure of
the native dimer remains largely unknown. Based on two independent
approaches (epitope analysis and homology-based modeling approach),
CTP however was hypothesized to overlap the amino acid residues a7-14
and a56-60
27
as shown in cyan in Fig. 2B.
3.2 Glycan structure of natural gonadotropins
All N-linked glycans are based on a common core pentasaccharide
Man
3
GlcNAc
2
. They differ in branching and distribute in three main
classes defined as high-mannose, hybrid and complex chains. Complex
glycans are widely represented in circulating glycoproteins and are often
referred as serum type oligosaccharides. Despite a highly similar amino
acid sequences, the glycosylation pattern of gonadotropins is exquisitely
specific for each hormone. LH is the only gonadotropin to contain
GlcNAc-Gal-GalNAc-sulfate branch in place of the regular GlcNAc-Gal-
Neu5Ac.
3.2.1 Natural FSH. Pituitary FSH was found to be highly sialylated
and 71% of glycoforms of the International Standard 83/575 are acidic
forms (pI
o
3.5).
28
Variations in the sialic acid content as well as differ-
ences in glycan heterogeneity determine a wide panel of FSH glycosyla-
tion variants.
29
Pituitary FSH is mainly composed of bi-and triantennary
structures covered with sialic acid (88%) compared to GalNAc-sulfate
(7%).
30
At least 20 to 30 isoforms circulate in blood during the menstrual
cycle.
31
During the early phase of a new ovulatory cycle, stimulation of the
ovary is mediated by long-lived acidic isoforms of FSH. As follicles begin
to develop at midcycle, the pituitary secretes short-lived less acidic FSH
isoforms so that only the largest follicles continue to grow. In the luteal
phase more acidic glycoforms are again secreted (Table 1).
31-33
FSH
stimulates the secretion of inhibins from the follicular cells, and in
Table 1 Distribution of acidic and complex glycoforms of FSH during the ovarian cycle.
Acidic glycoforms are determined as variants with pIr4.3 and complex glycoforms as
non-retained material from Concanavalin A chromatography. Adapted from Anobile et al.,
1998.
33
Early to mid-follicular
Late-follicular
Midcycle
Luteal
Menopause
Acidic
64%
63%
50%
71%
83%
Complex
90%
90%
63%
93%
34%
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