From the Unit of Glycobiology, Developmental
Endocrinology Branch, NICHD, National Institutes of Health, Bethesda,
Maryland 20892 and the
Department of Microbiology and
Immunology/Mass Spectrometry Resource, Boston University Medical
Center, Boston, Massachusetts 02118
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ABSTRACT |
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Glycoprotein hormone subunit, in its free
form (free
), is a major placental product. Its glycosylation was
found to change dramatically during the advancement of pregnancy. In
this study, we have analyzed these glycosylation changes in five normal
pregnancies. Binding to Lens culinaris lectin increased
dramatically in all subjects between weeks 14 and 17 from the last
menstrual period, indicating more core fucosylation as well as possible
changes in branching of glycans. Studies using Datura
stramonium agglutinin confirmed that the type of triantennary
branching changed in this period of pregnancy. The precise structural
nature of these changes was determined by high-pH anion-exchange
chromatography and electrospray ionization mass spectrometry. Amounts
of core fucosylation and of triantennary glycans increased
substantially from early to late second trimester, and a shift was
observed from 1
4/1
3- toward predominantly 1
6/1
6-branched
triantennary structures. The glycosylation changes occurred in all five
individuals at the same time period in gestation, suggesting
developmental regulation of
N-acetylglucosaminyltransferases IV and V and
6-fucosyltransferase during normal pregnancy. These enzymatic
activities also appear to be affected in malignant transformation of
the trophoblast. Our findings have important implications for the
proposed use of specific forms of glycosylation as markers for cancer,
as the relative amounts of these glycans in normal pregnancy will be determined by gestational age.
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INTRODUCTION |
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Glycoprotein hormone subunit is common to the heterodimeric
hormones chorionic gonadotropin, luteinizing hormone,
follicle-stimulating hormone, and thyroid-stimulating hormone. However,
in its free form (free
subunit), it is an important placental (1,
2) and pituitary (3) product, and it has been shown to have functions that are independent of the dimeric hormones (4-7). Glycosylation of
free
differs from glycosylation of the combined form (8, 9). The
combination of
and
for heterodimer formation takes place in the
endoplasmic reticulum prior to processing of the immature glycans. In
subunits that have not combined with a
subunit, enzymes from
the post-translational glycosylation machinery have access to substrate
sites that are normally protected by the
subunit of the
heterodimer. As a result, the free form of
subunit generally
contains more elaborate oligosaccharide branching as well as higher
amounts of core fucosylation than
subunit obtained from dissociated
hormone (8, 9). These characteristic glycosylation patterns prevent
secreted free
subunits from combining with
subunits that might
be encountered extracellularly, thus ensuring a population of free
molecules (9, 10).
The structural diversity of complex-type N-linked glycans is
initiated by GlcNAc branching of the trimannosyl core and continues with the action of different glycosyltransferases that further extend
these antennae (11). Specifically, the activity of
N-acetylglucosaminyltransferase IV initiates the
14/1
3-branch of complex glycans, whereas the action of
N-acetylglucosaminyltransferase V initiates the
1
6/1
6-branch. In most human epithelial tissues, expression of
1
6/1
6-branching is low, whereas in malignancy, expression of this
branch is increased, and the resulting oligosaccharides are considered
to be significant markers of carcinoma (12, 13). However, a contrasting
pattern seems to exist in normal and malignant pregnancies. A
literature survey of pregnancy-related glycoproteins and
oligosaccharides (Table I) (14-25)
showed that in transformed placental tissues as well as in glycans
isolated during the early part of pregnancy, a large amount of
1
4/1
3-branching is expressed, whereas 1
6/1
6-branching seems
to be typical for glycoproteins obtained from the final stages of
pregnancy.
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It is our hypothesis that the expression of 14/1
3-branched
glycans in early pregnancy reflects the implantation and placentation process during the invasion of trophoblast tissue. Consequently, glycosylation patterns of pregnancy-related glycoproteins should change
concurrently with the decline of the invasiveness of trophoblast tissue
during the early second trimester of normal pregnancy. This theory is
supported by the different glycosylation patterns found on
hCG1 (8, 15, 26-28) and on
free
subunit from normal pregnancy (8, 26), choriocarcinoma
(22-24), and non-trophoblastic neoplasms (25, 29). In addition, less
highly charged isoforms of hCG were found in late pregnancy (30), and
further studies of its electrophoretic mobility suggested that its
glycosylation patterns change during the early second trimester of
pregnancy (31). Previously, we have presented lectin data that implied
increased branching and higher incorporation of fucose into
carbohydrate moieties in late pregnancy (32). In the present study, we
have analyzed the glycan structures of free
from five individuals throughout their normal pregnancies to determine the exact nature of
the glycosylation changes and to define the time in pregnancy at which
they occur. Structural analysis of these glycans suggests which
glycosyltransferases are involved and contributes to the understanding
of normal and pathologic glycobiology in pregnancy.
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EXPERIMENTAL PROCEDURES |
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Materials--
Reference preparations of hCG (CR 125), hCG
(CR 119), and hCG
(CR 119) were provided by Drs. S. Birken and R. Canfield through the Center for Population Research. Oligosaccharide
standards and
-fucosidase were obtained from Oxford GlycoSystems
Ltd. (Abingdon, United Kingdom).
-Fucosidase was used to remove core
fucose from glycans to create non-core-fucosylated standards.
Tri(6/6)-F oligosaccharide was kindly donated by Dr. Harald S. Conradt
(Gesellschaft für Biotechnologische Forschung, Braunschweig,
Germany). Neuraminidase (Vibrio cholerae) was obtained from
Calbiochem. BSA (Pentex fraction V) was purchased from Miles Inc.
(Kankakee, IL). Sephadex G-100 (superfine) was obtained from Amersham
Pharmacia Biotech. LcH-agarose and DSA gel were purchased from E-Y
Laboratories, Inc. (San Mateo, CA). Centricon-10 and Microcon-10
concentration devices were obtained from Amicon, Inc. (Beverly,
MA).
Immunoassays--
Intact hCG was assayed by RIA using a
monoclonal antibody, A03C9 (Monoclonal Antibodies Inc., Sunnyvale, CA),
with cross-reactivity for free and
subunits estimated at 1.3 and 0.3%, respectively. Free
subunit was assayed by RIA using
polyclonal antiserum SB6, with <0.6% cross-reactivity for hCG (33).
Free
subunit was assayed by RIA using an
-specific monoclonal
antibody (BioMerica, Newport Beach, CA). The cross-reactivity of hCG
with the free
monoclonal antibody was <0.1% (34, 35).
Isolation of Free --
Five healthy pregnant volunteers
provided 24-h urine collections throughout their full-term
uncomplicated pregnancies. A sample was removed from each 24-h urine
collection and was assayed for hCG, free
, and creatinine. Free
was isolated as described previously (32). Essentially, each urine
specimen was precipitated with 2 volumes of acetone at pH 5.5 and
4 °C, followed by centrifugation. The precipitates were resuspended
in distilled water and dialyzed against 50 mM ammonium
acetate for 72 h at 4 °C, followed by centrifugation. The
supernatants from each sample were lyophilized and redissolved in
water. If not all material dissolved, then the pellets were redissolved, dialyzed, and centrifuged as described above. The second
set of supernatants of each sample was combined with the first. Free
was isolated from each sample by gel filtration on a Sephadex G-100
superfine column (1.6 × 100 cm) run in 0.2 M ammonium
acetate (pH 7.4) at 4 °C at a flow rate of 5 ml/h. Fractions of 2 ml
were collected into tubes containing BSA (2 mg/tube) and assayed by RIA
for free
subunit, free
subunit, and intact hCG, respectively.
Fractions containing free
subunit were pooled and lyophilized.
Lectin Affinity Chromatography--
Samples from different time
points throughout the second trimester of each individual pregnancy
were subjected to affinity chromatography on LcH and DSA lectin
columns. To ascertain that the columns were not initially overloaded
with glycoproteins, materials from the unbound fractions were reapplied
to fresh columns. Additional tests showed that no matrix effects from
the eluent interfered with assays of free in the various
fractions.
Liberation of Desialylated N-Linked Glycans--
Immunopurified
free samples were desialylated with neuraminidase, desalted, and
concentrated using Microcon-10 concentrators. Subsequently, the samples
were denatured by heating at 100 °C for 3-4 min in 0.2 M sodium phosphate (pH 8.0) containing 1% SDS and 0.1 M
-mercaptoethanol, followed by addition of EDTA (final concentration of 10 mM), Nonidet P-40 (final concentration
of 5%), and recombinant glycerol-free
peptide-N4-(N-acetyl-
-D-glucosaminyl)asparagine
amidase F (25-100 units/mg of glycoprotein; Genzyme, Cambridge, MA).
The mixture was incubated at 37 °C for 18 h; a second aliquot
of enzyme was added, and the incubation was continued for another
20 h. The reaction was stopped by addition of an equal volume of
10% trichloroacetic acid, and the precipitate was eliminated by
centrifugation. The pellets were washed with methanol (containing 5%
water) to remove residual trichloroacetic acid and detergent and
examined for complete release of the carbohydrate chains by acid
hydrolysis with trifluoroacetic acid, followed by monosaccharide
analysis by HPAEC-PAD (36). The liberated glycans in the supernatant
were purified on a column (0.7 × 28 cm) of Bio-Gel P-4 (mesh
200-400) run in water at room temperature at a flow rate of 5 ml/h.
Fractions of 0.5 ml were collected, and the released glycans were
pooled and lyophilized. Elution positions of the glycans were
established with oligosaccharide standards, detected by hexose assay
using phenol-sulfuric acid reagents (37).
HPAEC-PAD of Released Glycans-- The glycans were resolved by HPAEC-PAD on a CarboPac PA-100 column (0.4 × 25 cm; Dionex Corp., Sunnyvale, CA) and detected with an electrochemical detector (ED40) set in the "carbohydrate" waveform, controlled by a personal computer using PeakNet chromatography software (38). The column was eluted with 250 mM NaOH with a 10% gradient of 0.5 M sodium acetate at a flow rate of 1 ml/min.
Periodate Oxidation and Reduction-- Periodate oxidation of the glycans (39, 40) was performed by incubation in 9 mM NaIO4, buffered with 0.1 M sodium acetate at pH 5.5, for 3 days at 4 °C in the dark. The reaction was quenched with 3 µl of ethylene glycol and incubated overnight under the same conditions. The product was neutralized with 0.1 M NaOH, reduced by the direct addition of 5 mg of solid NaBD4, and kept at room temperature for an additional 16 h. Excess reducing agent was destroyed by the addition of 5 µl of acetic acid, and the solutions were dried in a vacuum centrifuge. Borate was removed by repeated addition and drying with methanol. The samples were vacuum-desiccated overnight prior to methylation.
Methylation--
Each preparation (1-2 µg) was dissolved in
200 µl of a NaOH/Me2SO suspension (41). After 1 h at
room temperature, 50 µl of methyl iodide was added, and the
suspensions were left for 1 h at room temperature with occasional
vortexing (39). The methylated product was extracted by adding 1 ml of
chloroform, and the suspensions were backwashed four times with 2-3 ml
of 30% acetic acid. The chloroform layer was dried down and stored at
20 °C.
Electrospray Ionization Mass Spectrometry--
ESI-MS was
performed on a TSQ 700 triple quadrupole mass spectrometer (Finnigan
MAT, San Jose, CA) equipped with an electrospray ion source (Analytica
Inc., Branford, CT). Samples were dissolved in methanol/water solutions
(6:4, v/v) containing 0.25 mM NaOH and analyzed by syringe
pump flow injection directly into the electrospray chamber through a
stainless steel hypodermic needle at a flow rate of 0.85 µl/min. The
voltage difference between the needle tip and the source electrode was
3.5 kV.
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RESULTS |
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Lectin Affinity Chromatography--
Free subunit was purified
from 24-h urine collections throughout the pregnancies of five healthy
individuals. In all individuals, the total amount of free
production increased as pregnancy progressed. Typical recovery achieved
was 95% of the initial free
immunoreactivity. In all procedures,
loss of material was minimized by adding small amounts of BSA to the
eluent. BSA also prevented loss of highly purified free
on
membranes of the devices used for desalting.
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HPAEC-PAD of Released Glycans--
Glycans were liberated with
peptide-N4-(N-acetyl--D-glucosaminyl)asparagine
amidase F from immunopurified desialylated free
from volunteer A
during the early (weeks 13-15 from LMP) and late (week 26 from LMP)
second trimester of pregnancy (Fig. 3A). Using HPAEC-PAD,
the glycans were resolved on a CarboPac PA-100 column (Fig.
4). Elution conditions were established
by which base-line separation of the oligosaccharide standards was
obtained (Fig. 4C). Analysis of early pregnancy free
glycans (Fig. 4A) showed the presence of Di-F (22%) and Di
(31%), with retention times of 11.8 and 13.1 min, respectively, and of
triantennary (13%, 14.5-15.5 min) and tetraantennary (12%, 17.5 min)
glycans (Fig. 5). In addition, minor
amounts of hybrid-type glycans (5%, 9.0 min) were detected. Late
second trimester free
glycans (Fig. 4B) contained 9%
less Di (22%), about equal amounts of Di-F (22%), and 7% more
triantennary structures (20%) as compared with the early free
sample. Oligosaccharide standards suggested that the twin peaks at 14.7 and 15.2 min represent Tri(6/6) and Tri(4/3), respectively (data not
shown), implying increased relative amounts of Tri(6/6) in the late
free
sample. The relative amounts of tetraantennary (10%) and
hybrid-type (3%) glycans were both ~2% lower in late free
. In
addition, an unidentified peak eluting at 16.0 min was more prominent
(+3%) in the late pregnancy free
sample. Taken together, these
data show increased branching, specifically by generation of Tri(6/6)
in the late free
sample. However, definitive conclusions could not
be made due to coelution of several specific glycans. Particularly,
some fucosylated triantennary glycans were found to coelute with Di-F
at 11.8 min. Therefore, further structural analysis of the glycans was
performed.
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Electrospray Ionization Mass Spectrometry--
Enzymatically
released and methylated glycans from immunopurified desialylated free
samples from volunteers A and B and from a hCG
reference
preparation were analyzed by ESI-MS (Table II). Profiles from early (weeks 13-15
from LMP) and late (week 26 from LMP) second trimester free
glycans
from volunteer A are shown in Fig. 6. A
unique feature of electrospray ionization is the generation of multiply
charged ions (z) from a single molecular species. These
provide, together with the detected ion mass to charge ratio
(m/z), a direct indication of the relative
molecular mass through the following relationship: relative molecular
mass = z(m/z
23), where
23 is the mass of the adherent sodium cation. The percentages shown in
Table II represent molar ratios of the individual glycans as a
summation of all of their detected charge states (z).
Branching of the glycans increased from early to late pregnancy, as is
evidenced by a decrease in the relative amounts of Mono
m/z 822 (2+) and Di m/z
1047 (2+)/705 (3+) glycans and a concurrent increase in the relative
amounts of Tri m/z 1272 (2+)/855 (3+) and Tri-F
m/z 1359 (2+)/913 (3+) glycans in the late free
samples (Fig. 6 and Table II). Furthermore, complete disappearance
of all hybrid-type and monoantennary glycans was observed in late
pregnancy free
from both subjects (Table II). Additionally, core
fucosylation increased as pregnancy progressed, as evidenced by
increased relative amounts of core-fucosylated glycans (Di-F,
m/z 1134 (2+)/763 (3+) and Tri-F,
m/z 1359 (2+)/ 913 (3+)), concurring with lower
amounts of non-fucosylated glycans (e.g. Di,
m/z 1047 (2+)/705 (3+)). The total increase in
core-fucosylated glycans from early to late pregnancy was 57.6% in
volunteer A and 25.2% in volunteer B. Nearly 100% of the glycans were
core-fucosylated in both subjects at the end of pregnancy (Table II).
The hCG
reference preparation contained mainly hybrid-type (31.9%),
monoantennary (42.5%), and some diantennary (24.5%) glycans (Table
II); only a small amount of core fucosylation was detected (0.6%), and
no tri- or tetraantennary glycans were found.
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DISCUSSION |
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Previously, we found evidence that glycosylation of free from
the third trimester of pregnancy is different from that from the first
trimester (32). In this study, we have analyzed these changes in five
individual pregnancies throughout the second trimester to determine the
exact nature of the glycosylation changes and to identify when they
occur. Using LcH and DSA lectin analysis, we observed that the binding
properties of free
in all five individuals underwent marked changes
beginning at around week 14 of pregnancy (Fig. 3).
Binding to LcH requires glycans with a trimannosyl core and
fucosylation at the innermost GlcNAc residue (42). We observed increased binding to LcH (mean difference of 35.6 ± 6.0%) as
pregnancy progressed, which was initially interpreted as reflecting an
increase in core fucosylation. Interestingly, compositional analysis
revealed that free subunits from early pregnancy contained enough
fucose to account for at least one core-fucosylated glycan per subunit, yet many of those molecules were unable to bind to LcH. Therefore, it
was proposed that 1
4/1
3-branching, which prevents binding to LcH,
might be more abundant in early pregnancy.
DSA interacts more strongly with 16/1
6-branched tri- and
tetraantennary glycans than with 1
4/1
3-branched structures; thus, it is particularly useful in discerning differently branched
triantennary glycans (43, 44). Dissociated hCG
subunit passed
through the DSA column without any interaction, confirming that this
subunit, formerly combined with hCG
, does not contain any tri- or
tetraantennary glycans (8-10, 45). In contrast, some free
interacted with DSA, and the amount increased from a mean of 17.4 ± 4.5% in the early part to 51.3 ± 2.2% in the late part of
the second trimester of pregnancy (mean difference of 33.9 ± 5.0%). The major changes in free
interaction with DSA took place
during the same period in which the changes in LcH binding were
observed (Fig. 3). Taken together, the lectin data suggest that during
weeks 14-17, an increase in core fucosylation occurs, and there is a
shift in the type of branching of the glycans of free
, from the
presence of 1
4/1
3-branched structures to higher amounts of
1
6/1
6-branched structures.
HPAEC-PAD analysis of released glycans from free of volunteer A
during the early and late second trimester of pregnancy indicated a
decrease in diantennary and an increase in triantennary structures
(Fig. 4). Standards indicated that this was essentially due to
generation of more 1
6/1
6-branched glycans in the late free
sample. The ESI-MS data provided accurate molar ratios of individual
N-linked glycans (Table II) and structurally confirmed the
conclusions from the lectin affinity and HPAEC-PAD experiments. Nearly
100% of the glycans isolated from free
from the third trimester
were core-fucosylated. In addition, hybrid and monoantennary glycans
disappeared in late pregnancy. ESI-MS analysis of ODM glycans from
early and late second trimester free
samples demonstrated that
virtually all of the increase in branched structures was due to
increased 1
6/1
6-branched triantennary glycans (Figs. 7 and 8 and
Table II).
The ESI-MS analysis of volunteers A and B (Table II) correlated well
with the DSA binding properties observed for these two individuals
(Fig. 3, A and B). There was considerably more
heterogeneity between individuals in early pregnancy as evidenced by
both DSA binding and structural analysis. This may reflect normal
variations between individual pregnancies or lack of precision in
dating gestational age based on LMP. As the shift in glycosylation
nears completion at the end of the second trimester, there is greater uniformity among pregnancies on the basis of both DSA binding and
structural analysis by ESI-MS. It is important to note that when
glycans are released from free and examined individually, observed
differences are likely to be smaller than those from experiments
involving the intact glycoprotein. Changes in structure of only one of
the two N-linked glycans can lead to altered affinity of the
entire free
molecule for a specific lectin.
Previous compositional analysis has shown that urinary free ,
pooled during the second and third trimesters of pregnancy, is fully
sialylated and that both glycosylation sites are occupied with intact
glycans (8). Furthermore, 97.3% of the Gal residues of early second
trimester free
were found to be sialylated. These data indicate
that the glycans on free
isolated from pregnancy urine have not
been partially degraded. In addition, sialic acid is expected to be
predominantly present in
2
3-linkage on the N-acetyllactosamine antennae of free
since human
placenta contains almost exclusively the Gal
1
4GlcNAc-R
2
3-sialyltransferase variant of the possible sialyltransferases
that specifically elongate these branches (46).
The observation that the glycosylation changes occur in all five
pregnancies within a narrow window of gestational time suggests that
the activities of the enzymes involved are developmentally regulated.
Specifically, the activities of
N-acetylglucosaminyltransferases IV and V and of
6-fucosyltransferase seem to be affected during weeks 14-17 of
pregnancy. Our data, together with previously published structural
analyses of pregnancy-related glycoproteins (Table I), imply that
during this period in time, there is a large increase in
N-acetylglucosaminyltransferase V activity and perhaps a
corresponding decrease in N-acetylglucosaminyltransferase IV
activity. The activity of
6-fucosyltransferase appears to increase
during the same period and stays at a high level throughout the early
third trimester of pregnancy, as illustrated by the almost complete
core fucosylation of free
glycans in late pregnancy (Table II).
Subsequently,
6-fucosyltransferase activity may decline, as aging
trophoblast tissue is associated with a decrease in core fucosylation
of complex glycans (47).
During pregnancy, free subunits are secreted by cytotrophoblasts,
in which little or no
subunit is expressed, and by
syncytiotrophoblasts, in which free
and free
subunits as well
as hCG are produced (48). In early pregnancy, part of the
cytotrophoblast population becomes invasive, penetrating into the
endometrium and eventually into the superficial layers of the
myometrium and uterine blood vessels (49). These invasive cells behave
much like tumor cells and likely give rise to pathologic conditions
such as choriocarcinoma when appropriate regulatory factors are not
recognized. The type of triantennary branching observed on early
pregnancy free
was similar to glycan structures found on hCG
associated with invasive mole and choriocarcinoma, reflecting increased
N-acetylglucosaminyltransferase IV activity (Table I) (50).
Furthermore, the changes that we observed in the glycosylation of free
occur during the time frame that coincides with the decline in
cytotrophoblast invasiveness as normal pregnancy progresses. Thus,
changes in the state of trophoblast differentiation appear to be
associated with alterations in glycosyltransferase activity.
Changes in glycosylation may have functional significance for free receptor binding, signal transduction, or circulatory clearance, as has
been observed for the heterodimeric glycoprotein hormones (51).
Alternatively, the changes in glycan structures on free
may not be
directly involved in
function, but rather, could be coincidental to
its synthesis within cells in which the general glycosylation machinery
has differentiated, reflecting altered functional status of the cell.
Increased branching and core fucosylation as well as changes in the
relative amounts of triantennary isomers may reflect the change from an
invasive state to a more nurturing role for the placenta during the
second trimester of pregnancy.
In conclusion, glycosylation of free changes dramatically during
the early part of the second trimester of pregnancy. Similar changes
occurred in all five pregnancies examined, suggesting that there is
developmental regulation of the placental glycosylation machinery
during normal pregnancy. Our findings have important implications for
the proposed use of specific forms of glycosylation as markers for
cancer in pregnancy (50). Since activities of the same enzymes appear
to be altered in certain stages of gestational development and in
malignant transformation of the trophoblast, the relative amounts of
these glycan markers in normal pregnancy will be determined by
gestational age.
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ACKNOWLEDGEMENTS |
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We thank Dr. Harald S. Conradt for the kind
gift of oligosaccharide standards and Paulette O'Connell for excellent
assistance with the purification of free .
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FOOTNOTES |
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* The mass spectral studies carried out at the Boston University Mass Spectrometry Resource were supported by National Institutes of Health Grants NCRR 5P41RR10888 (to C. E. Costello, Principal Investigator) and RO1 GM54045 (to V. N. R., Principal Investigator).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Pharma Bio-Research Laboratories B. V., Westerbrink 3, 9405BJ Assen, The Netherlands.
¶ Present address: Human Genome Sciences, Inc., Rockville, MD 20850.
** To whom correspondence should be addressed: Contraception and Reproductive Health Branch, NICHD, National Institutes of Health, Bldg. 61E, Rm. 8B13, Bethesda, MD 20892. Tel.: 301-496-1661; Fax: 301-480-1972; E-mail: BlitheD{at}exchange.nih.gov.
1 The abbreviations used are: hCG, human chorionic gonadotropin; BSA, bovine serum albumin; LcH, Lens culinaris lectin; DSA, Datura stramonium agglutinin; RIA, radioimmunoassay; HPAEC-PAD, high-pH anion-exchange chromatography with pulsed amperometric detection; ESI-MS, electrospray ionization mass spectrometry; LMP, last menstrual period; ODM, oxidation-deuterioreduction and methylation; Fuc, L-fucose. All sugars were of the D-configuration unless noted otherwise.
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REFERENCES |
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