The Carboxyl-Terminal Region Is a Determinant for the Intracellular Behavior of the Chorionic Gonadotropin ß Subunit: Effects on the Processing of the Asn-Linked Oligosaccharides
Mesut Muyan1 and
Irving Boime
Department of Molecular Biology & Pharmacology Washington
University School of Medicine St. Louis, Missouri 63110
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ABSTRACT
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The placental hormone human CG (hCG) consists of
two noncovalently linked
- and ß-subunits similar to the other
glycoprotein hormones LH, FSH, and TSH. These heterodimers share a
common
subunit but differ in their structurally distinct ß
subunits. The CGß subunit is distinguished among the ß subunits by
the presence of a C-terminal extension with four serine-linked
oligosaccharides (carboxyl terminal peptide or CTP). In previous
studies we observed that deleting this sequence decreased assembly of
the truncated CGß subunit (CGß114) with the
-subunit and
increased the heterogeneity of the secreted forms of the uncombined
subunit synthesized in transfected Chinese hamster ovary (CHO) cells.
The latter result was attributed to alterations in the processing of
the two N-linked oligosaccharides. To examine at what step this
heterogeneity occurs, the CGß and CGß114 genes were transfected
into wild-type and mutant CHO cell lines that are defective in the late
steps of the N-linked carbohydrate-processing pathway. We show here
that removal of the CTP alters the processing of the core mannosyl unit
of the subunit to complex forms at both glycosylation sites and that
the oligosaccharides contain polylactosamine. Although it has been
presumed that there is little intramolecular interaction between the
CTP and the proximal domains of the subunit, our data suggest that the
CTP sequence participates in the folding of the newly synthesized
subunit, which is manifest by the posttranslational changes observed
here.
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INTRODUCTION
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Human CG (hCG), is a member of the glycoprotein hormone family,
which includes the pituitary hormones LH, FSH, and TSH (1, 2, 3, 4, 5). They are
heterodimers containing two nonidentical subunits,
and ß (1, 2, 3, 4, 5).
Within an animal species, the amino acid sequences of the
subunits
are identical, and, although the ß subunit determines the biological
specificity of the hormones, there is significant amino acid sequence
similarity among them (1, 2, 3, 4, 5). This is readily apparent for the LHß
and CGß subunits as they share >80% sequence identity, which is
presumably responsible for the similar biological activity of the
corresponding dimers (1, 2, 3, 4, 5). CGß is distinguished among the ß
subunits by the presence of a C-terminal extension with four O-linked
oligosaccharides (5) designated CTP. Sequence analysis showed that a
1-bp deletion and 2-bp insertion in the CGß gene relative to the
LHß coding sequence accounts for the extended open reading frame of
CGß (5). Several studies have suggested that this extension plays no
significant role in the overall conformation of the subunit (6, 7, 8), but
it is associated with maintaining the prolonged half-life of hCG
compared with the other hormones (9). However, our previous studies
demonstrated that the intracellular behavior of the glycoprotein
hormone ß subunits involves an interaction between regions in the
amino and carboxyl ends of the subunit (10), suggesting the CTP
contributes to the maturation of the newly synthesized subunit. This
hypothesis was supported by the observation that truncation of CGß at
residue 114 (CGß114) decreased its ability to form heterodimers and
that CGß114 exhibited marked heterogeneity compared with the
wild-type (WT) subunit when secreted from transfected Chinese hamster
ovary cells (CHO) cells (11). The latter was attributed to
modifications of the Asn-linked oligosaccharides reflecting a change in
the structure of the subunit devoid of the CTP (11). This observation
may, in part, explain the basis for the synthesis of hormone-specific
oligosaccharides; since the amino acid sequence of the common
subunit is identical among the glycoprotein hormones, the specificity
of N-linked oligosaccharide processing is linked to the ß subunit
(2, 3, 4).
To characterize the posttranslational modification affected by the
truncation, the CGß and the CGß114 genes were transfected into WT
and mutant CHO cell lines, which are defective in the late steps of the
N-linked carbohydrate processing pathway, and the products were probed
with ß subunit-specific antiserum. Because defined glycoprotein
intermediates accumulate, these mutant cell lines provide a model for
elucidating at what step the changes in heterogeneity occurs in the
truncated subunit. We show that removal of the carboxyl-terminal region
of the CGß subunit alters terminal processing of the core glycosyl
units of the subunit, and the major modification is the formation of
polylactosamine-containing structures. The data also suggest that the
carboxyl-terminal region is involved in the folding of the CGß
subunit, reflected by changes in the processing of the N-linked
carbohydrates.
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RESULTS
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Removal of the 31-amino acid carboxy-terminal region
(carboxy-terminal peptide or CTP) from CGß resulted in a variant
(CGß114) secreted from transfected CHO cells that was much more
heterogeneous than the WT subunit. We attributed this to altered
processing of the N-linked oligosaccharides (11). The initial steps in
the processing of N-linked carbohydrates is the trimming of the
high-mannose oligosaccharide unit to
Man5GlcNAc2 (12, 13). Further modification of
this intermediate is catalyzed by the Golgi enzyme N-acetylglucosaminyl
transferase I, which is a pivotal step in the synthesis of fully
processed N-linked oligosaccharides (12, 13). Because the heterogeneity
of the glycoproteins primarily occurs at the processing of the core
glycan unit to complex forms (2, 4, 14, 15), we examined whether the
changes in the N-linked oligosaccharides of the secreted CGß114 occur
at the Man5GlcNAc2 step. The WT CGß and the
mutant CGß114 genes were transfected into the CHO mutant cell line
15B, which lacks the Golgi enzyme N-acetylglucosaminyl transferase I
and, thus, synthesizes glycoproteins bearing only the
Man5GlcNAc2 core (16). If the heterogeneity of
CGß114 is related to altered processing beyond this point, CGß114
secreted from 15B cells should exhibit little or no heterogeneity
compared with the form secreted from WT-CHO cells. CGß synthesized in
WT-CHO cells displays two forms reflecting the presence of one (N1) or
two (N2) N-linked oligosaccharides (Ref. 17 and Fig. 1
, lane 1) and CGß114 exhibits the
heterogeneity reported previously (lane 3). The migration of CGß114
differs from CGß because of the absence of the CTP and its associated
O-linked oligosaccharides (11). Compared with WT-CHO cells (lane 3),
CGß114 secreted from 15B cells is more homogeneous; i.e.
the ladder of bands associated with the N2 form of the wild type is
absent in CGß114 (lane 4). As expected, the CGß-secreted subunit
from 15B cells was likewise homogeneous (lane 2). That the ß subunits
secreted from 15B cells bear only noncomplex oligosaccharides was shown
by their quantitative digestion with endoglycosidase H (data not
shown). This enzyme hydrolyzes high mannose and hybrid N-linked, but
not complex, oligosaccharides (18). These results show that removal of
the carboxy terminus of CGß alters the processing of the variant
beyond the Man5GlcNAc2 step.

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Figure 1. Analysis of the Secreted Forms of the CGß and
CGß114 Subunits from the WT-CHO and the Glycosylation Mutant 15B
Cells
Cells were labeled overnight with 25 µCi/ml 35S-labeled
Pro-Mix, and the media were immunoprecipitated with the ß antiserum,
subjected to SDS-PAGE, and autoradiographed. The migration of the CGß
species bearing two (N2) and one (N1) asparagine-linked
oligosaccharides in the electropherograms is indicated. The numbers 34
and 31 correspond to the molecular weights of the N2 (34,000) and N1
(31,000) forms of the CGß subunit; the 24 refers to 24,000, the mol
wt of the dimer form of the subunit (see Ref. 39).
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Because heterogeneity of glycoproteins is often due to variations in
sialic acid content, we treated CGß or CGß114 secreted from WT-CHO
cells with neuraminidase (Fig. 2A
) (19).
If the heterogeneity of CGß114 is due to differences in sialic acid
content, removal by neuraminidase should lead to a more homogeneous
form compared with the untreated subunit. Although CGß and CGß114
are neuraminidase sensitive, as evident by the faster migrating
electrophoretic forms compared with the untreated samples, the
heterogeneity of CGß114 is unchanged (lane 4). These results suggest
that the modified oligosaccharides in CGß114 are not due to
alterations in the sialic acid content. Further support for this
conclusion is provided by studies with the mutant CHO cell line, 1021
(20). These cells are deficient in the Golgi transport of CMP-sialic
acid and, consequently, synthesize glycoproteins devoid of terminal
sialic acid residues (20). Similar to that seen for
neuraminidase-treated CGß114, the electrophoretic pattern of the
variant is still observed (panel B, lane 8). As expected, the subunits
recovered from 1021 cells were not sensitive to neuraminidase (data not
shown). It is curious that CGß114 secreted from 1021 cells is much
more heterogeneous than the form secreted from WT-CHO cells (compare
lanes 6 and 8); even WT CGß subunit synthesized in 1021 cells is more
heterogeneous than that normally observed for the subunit recovered
from WT cells (lanes 5 and 7). It should be noted that the experimental
approaches using neuraminidase and 1021 cells to delineate sialic acid
contribution to the heterogeneity of the subunits are biologically
distinct: neuraminidase removes sialic acid from already synthesized
protein whereas protein secreted from 1021 cells reflects the
consequence of inhibiting intracellular sialic acid addition. Thus, in
the case of CGß and CGß114 synthesized in 1021 cells, the presence
or absence of sialic acid may affect the extent of the modification
(see Discussion).

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Figure 2. Analysis of Sialic Acid in CGß114
Panel A shows the effects of neuraminidase (neura) treatment of the
CGß and CGß114 subunits secreted from WT-CHO cells. Precipitates
were incubated without (-) (lanes 1 and 3) or with (+) (lanes 2 and 4)
enzyme for 16 h at 37 C. Panel B displays the secretion of CGß
and CGß114 subunits from WT-CHO (lanes 5 and 6) and the mutant 1021
cells (lanes 7 and 8). The labeling and immunoprecipitation are
described in the legend of Fig. 1 .
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A modification that results in the heterogeneity of Asn-linked
oligosaccharides in several glycoproteins is the addition of
poly-N-acetyllactosamine (PL). These structures are composed
of repeating disaccharide units [Galß (1, 2, 3, 4)
GlcNAc (1, 2, 3)]n
attached to the Man3GlcNAc2 core (21, 22). To
assess whether the oligosaccharides on CGß114 contain this
modification, immunoprecipitates of secreted CGß or CGß114 from
CHO-WT cells were treated with endo-ß-D-galactosidase
(endo-ß-gal) (21, 22), which hydrolyzes ß-galactosyl linkages in
the N-acetyllactosamine moiety (23) (Fig. 3
). The WT CGß subunit was resistant
(lane 2), but CGß114 was digested by the enzyme as indicated by the
disappearance of the protein-ladder compared with the untreated subunit
(lane 4). Both subunits secreted from 1021 cells were sensitive to the
enzyme (lanes 6 and 8) and, as expected, the enzyme had no effect on
the subunits derived from 15B cells (lanes 10 and 12). The secreted
forms of glycosylation variants of CGß114, in which either of the two
N-linked glycosylation sites at Asn-13 and -30 were mutated, also
exhibited the characteristic polylactosamine modification (data not
shown). These data imply that alterations of the CGß114
oligosaccharides are due, to poly-N-acetyllactosamine
addition to the mannose core of the immature N-linked carbohydrates at
both glycosylation sites.

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Figure 3. Endo-ß-Galactosidase (endo-ß-gal) Treatment of
the CGß and CGß114 Subunits Secreted from the WT-CHO and the Mutant
1021 and 15B Cells
WT-CHO (lanes 14), 1021 (lanes 58), and 15B (lanes 912) cells
expressing the CGß and CGß114 subunits were labeled, and medium
samples were immunoprecipitated as described in legend of Fig. 1 .
Precipitates were incubated overnight at 37 C in the absence (-)
(lanes 1, 3, 5, 7, 9, and 11) or presence (+) (lanes 2, 4, 6, 8, 10,
and 12) of endo-ß-gal and subjected to SDS-PAGE.
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Assembly of CGß114 with
and the Heterogeneity of Asn-Linked
Oligosaccharides
The quaternary structure, i.e. the heterodimeric
configuration, of multisubunit glycoproteins influences the processing
pattern of their N-linked oligosaccharides (24, 25, 26, 27). To examine whether
the electrophoretic heterogeneity of CGß114 is altered when combined
with the
subunit, WT-CHO cells coexpressing the
and CGß114
subunits were labeled overnight, and equal aliquots of media were
precipitated with the
and ß antisera (Fig. 4
). The mature N2 form of CGß114
comigrates with the
subunit (11), and thus electrophoretic analysis
of the heterodimeric forms is difficult (lanes 1 and 2). To examine
only the combined forms of the subunits, samples were subjected to
multiple immunoprecipitation and electrophoresis. The first step was to
capture the dimer with
antiserum since only the dimer form of ß
will be precipitated. The proteins are then resolved by SDS-PAGE to
separate the labeled subunits from the antiserum; the electrophoresis
was performed under nonreducing conditions to minimize a loss in
immunoreactivity of the subunits to the subsequent precipitation. After
eluting the dimer/uncombined subunits (arrow), the mixture
was boiled to ensure complete dissociation of the heterodimer. The ß
antiserum was then used in the next step. This approach permits
analysis of only the dimer form of ß subunit. These precipitates were
then treated with the endo-ß-gal and electrophoresed under reducing
conditions. The data show that the heterodimeric forms of CGß114 are
relatively homogeneous (lane 3) and resistant to endo-ß-gal digestion
(lane 4). Based on the earlier experiments, we predicted that
uncombined CGß114 would be heterogeneous. To test this point the
supernate obtained after precipitation with
antiserum described in
lane 1 of panel A was precipitated again with ß antiserum (ß
-super). As expected the uncombined CGß114 displayed the
characteristic heterogeneity (lane 5) and was sensitive to the enzyme
(lane 6). These results show that assembly modifies the processing of
the PL-modified N-linked carbohydrates of the CGß114 subunit.
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DISCUSSION
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Although they arise from the processing of a common preassembled
glycan unit, the mature Asn-linked oligosaccharides of secretory
glycoproteins exhibit differences in the number and location of
peripheral branches and terminal modifications (for review, see Refs.
2, 3, and 4). A major determinant for this specificity is encoded in
the primary amino acid sequence (for review, see Refs. 28 and 29).
However, these determinants are not well understood. Smith and
Baenziger (30, 31) have shown that a sequence in the glycoprotein
hormone subunits is associated with the recognition by a GalNAc
transferase in the biosynthesis of sulfated Asn-linked
oligosaccharides. From the results presented here it is evident that
presence or absence of the CTP influences the processing of the
Asn-linked oligosaccharides, although it is not clear how such
sequences cause these posttranslational modifications. They may contain
regions specifically recognized by the enzymes in the oligosaccharide
pathway, or they could hinder accessibility of sites in the 114 region.
Deleting the CTP might change the conformation of the truncated protein
such that recognition by one or more glycosyl transferases is
modified.
A critical question arises as to whether or not these alterations
reflect that seen in vivo. The carbohydrate modifications
observed could reflect one or more epitopes in the ß subunit that
contribute to the specificity of the oligosaccharide processing
in vivo. As shown here, the heterodimeric form of CGß114
does not exhibit the posttranslational changes observed for the
noncombined subunit. Therefore, due to differences in the configuration
of the combined subunit, assembly prevents the heterogeneity. This
result is consistent with earlier investigations showing that the
conformation of the heterodimer induced by the unique ß subunit is an
important element in gonadotropin-specific oligosaccharide processing
(2, 4, 27).
The ß subunits exhibit distinct intracellular fates (10, 25, 32, 33)
and have different carboxyl ends: LHß and TSHß have stretches of
hydrophobic residues at their carboxyl termini and bear biantennary
oligosaccharides ending with GalNAc-linked sulfate, whereas the FSHß
subunit lacking such an extension contains triantennary sugars
terminating with Gal and sialic acid (14, 15). Because CHO cells lack
the pituitary-specific GalNAc- and sulfo-transferases it is difficult
to recapitulate the processing conditions in the gonadotrope. The use
of mutant and/or chimeric subunits expressed in a pituitary cell line
could provide a strong approach to define the intracellular role of the
carboxyl terminus on assembly and hormone-specific processing of the
N-linked oligosaccharides.
It is intriguing that the N-linked oligosaccharides of CGß114
synthesized in 1021 cells bear apparently more PL compared with the
subunit secreted from WT-CHO cells. This suggests that the absence or
presence of sialic acid affects the synthesis and elongation of PL.
Thus, competition for the terminal Gal residue by the PL-specific
glucosaminyl (21, 22, 34) and sialyl transferases could be responsible
for the observed heterogeneity of CGß subunits. The CGß114 mutant
may be a poor substrate for sialyl transferase and thus lack of the
sialic acid cap would favor PL addition. This could also explain why
the WT subunit synthesized in 1021 cells contains PL, since in the
absence of sialic acid a fraction of the subunit is recognized by the
glucosaminyl transferase. These data imply that altering the rate of
sialic acid addition could be a determinant in the specificity of
Asn-linked carbohydrate processing. It could also explain the
appearance of numerous charged species that contribute to the
heterogeneity observed in glycoprotein hormones purified from pituitary
and placenta (2, 35).
It has been widely accepted that the CTP sequence does not contribute
significantly to the overall secondary/tertiary structure of the CGß
subunit. This was based on the following observations: 1) The native
hCGß subunit or the synthetic CTP peptide competed at the same
molarity with radiolabeled hCG for monoclonal antibodies to the CTP (6, 7); 2) the immunoreactivity of the denatured or native CGß subunit to
these monoclonal antibodies is comparable (6, 7); 3) heterodimers
bearing the CGß114 have the same receptor-binding affinity as the WT
CG dimer (9); 4) the CTP sequence could be fused to the FSHß subunit
without significantly affecting signal transduction of the chimera (8);
and 5) x-ray diffraction failed to resolve the carboxy-terminal region
(36, 37). There is sufficient evidence, however, to support the
hypothesis that the CTP sequence interacts with other domains within
the subunit and ultimately participating in the overall folding of the
subunit. Studies using the LHß and CGß subunit chimeras showed a
cooperativity between the amino and carboxyl ends of the subunits (10).
Because the carboxyl domains in those chimeras included residues
87145, it was not possible to restrict conclusions to the CTP
sequence. Our current data that deleting the CTP sequence alters
processing of the oligosaccharides, and reduces heterodimer formation
compared with CGß (11), support the hypothesis that the CTP with its
attached O-linked oligosaccharides is an interactive domain within the
native subunit.
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MATERIALS AND METHODS
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Materials
Cell culture media and reagents were prepared by the Washington
University Center for Basic Research. Dialyzed calf serum, the neomycin
analog G418, and Immunoprecipitin were purchased from GIBCO-BRL (Grand
Island, NY). Endoglycosidase H and neuraminidase were purchased from
Boehringer-Mannheim (Indianapolis, IN).
Peptide-N-glycosidase F was purchased from New England
BioLabs (Beverly, MA). Endo-ß-gal was obtained from V-Labs
(Covington, LA). Rabbit polyclonal antiserum against the
or the
CGß subunit was prepared in this laboratory.
[35S]Pro-Mix and [35S]cysteine were
purchased from Amersham (Arlington Heights, IL) and ICN (Costa Mesa,
CA), respectively.
Plasmid Construction, Transfection, and Selection of Stable
Clones
The construction of mammalian expression plasmids
pM2CGß and pM2CGß114 (truncated at amino
acid 114) were described previously (10, 17). The mutation was within a
BglII and BamHI fragment, which was inserted into
the unique BamHI site of the expression vector,
pM2 (9). Transfection of the WT-CHO cells and the mutant
cell lines 15B (16) and 1021 (20) with CGß subunit, with or without
the
subunit gene, and the isolation of stable clones were described
(10, 11, 38).
Cell Labeling and Immunoprecipitation
CHO clones were grown in 12-well plates to near confluency
before labeling. Cells were washed once with PBS and with Hams F-12
labeling medium [minus methionine and/or cysteine] and labeled
overnight (16 h) in this medium containing 25 µCi/ml of
35S-labeled Pro-Mix or [35S] cysteine.
Samples were precleared with normal rabbit serum. Samples were
precipitated with CGß antiserum, resolved on SDS-PAGE, and
autoradiographed. Treatment of samples with endoglycosidase H,
peptide-N-glycosidase F, and neuraminidase was according to
Keene et al. (38). Samples were treated with endo-ß-gal
according to the suppliers instruction. Briefly, immunoprecipatates
were dissolved in 50 µl of 50 mM sodium acetate buffer
(pH 5.5) containing 1% ß-mercaptoethanol, boiled for 3 min, and
centrifuged. The supernates were incubated at 37 overnight (18 h) with
2.5 mU of endo-ß-gal.
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ACKNOWLEDGMENTS
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We are grateful to Drs. Alison Jackson, Jacques Baenziger, Diana
Blithe, David Ben-Menahem, and Vicenta Garcia-Compayo for their
critical reading of the manuscript and to Dr. Susan Daniels-McQueen for
her technical advice. We thank Mary Wingate for her invaluable
assistance in preparing the manuscript.
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FOOTNOTES
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Address requests for reprints to: Dr. Irving Boime, Department of Molecular Biology & Pharmacology, 660 South Euclid Avenue, Campus Box 8103, St. Louis, Missouri 63110.
1 Present address: The University of Rochester Medical Center,
Department of Biochemistry, 601 Elmwood Avenue, Rochester, New York
14642. 
Received for publication December 1, 1997.
Revision received February 5, 1998.
Accepted for publication February 10, 1998.
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REFERENCES
|
---|
-
Pierce JG, Parsons TF 1981 Glycoprotein hormones:
structure and function. Annu Rev Biochem 50:465495[CrossRef][Medline]
-
Baenziger JU, Green ED 1988 Pituitary glycoprotein hormone
oligosaccharides: structure, synthesis and function of the
asparagine-linked oligosaccharides on lutropin, follitropin and
thyrotropin. Biochim Biophys Acta 947:287306[Medline]
-
Baenziger JU 1996 Glycosylation: to what end for the
glycoprotein hormones? Endocrinology 137:15201522[Medline]
-
Bielinska M, Boime I 1995 The glycoprotein hormone family:
structure and function of the carbohydrate chains. In: Montreuil J,
Vliegenthart JFG, Schachter H (eds) Glycoproteins. Elsevier Science
B.V., The Netherlands, pp 565616
-
Talmadge K, Vamvakopoulos NC, Fiddes JC 1984 Evolution of the
genes for the ß subunits of human chorionic gonadotropin and
luteinizing hormone. Nature 307:3740[Medline]
-
Birken S, Agosto G, Amr S, Nisula B, Cole L, Lewis J,
Canfield R 1988 Characterization of antisera distinguishing
carbohydrate structures in the ß-carboxyl terminal region of human
chorionic gonadotropin. Endocrinology 122:20542063[Abstract]
-
Krichevsky A, Birken S, OConnor J, Acevedo H, Bikel K,
Lustbader J, Hartree A, Canfield R 1994 Development, characterization,
and application of monoclonal antibodies to the native and synthetic
ß COOH-terminal portion of human chorionic gonadotropin (hCG) that
distinguish between the native and desialylated forms of hCG.
Endocrinology 134:11391145[Abstract]
-
Fares F, Suganuma N, Nishimori K, LaPolt P, Hsueh AJW, Boime
I 1992 Design of a long-acting follitropin agonist by fusing the
carboxy terminal sequence of the chorionic gonadotropin ß subunit to
the follitropin ß subunit. Proc Natl Acad Sci USA 89:43044308[Abstract]
-
Matzuk M, Hsueh A, LaPolt P, Tsafriri A, Keene J, Boime I 1990 The biological role of the carboxy-terminal extension of human
chorionic gonadotropin ß subunit. Endocrinology 126:376383[Abstract]
-
Matzuk M, Spangler M, Camel M, Suganuma N, Boime I 1989 Mutagenesis and chimeric genes define determinants in the ß subunits
of human chorionic gonadotropin and lutropin for secretion and
assembly. J Cell Biol 109:14291438[Abstract]
-
Muyan M, Furuhashi M, Sugahara T, Boime I 1996 The
carboxyl-terminal region of the ß-subunits of LH and CG
differentially influence secretion and assembly of the heterodimers.
Mol Endocrinol 10:16781687[Abstract]
-
Kornfeld R, Kornfeld S 1985 Assembly of asparagine-linked
oligosaccharides. Annu Rev Biochem 54:631664[CrossRef][Medline]
-
Verbert A 1995 Biosynthesis 2b. From
Glc3Man9GlcNAc2-protein to
Man5GlcNAc2-protein: transfer en bloc and
processing. In: Montreuil J, Vliegenthart JFG, Schachter H (eds)
Glycoproteins. Elsevier, Amsterdam, pp 145152
-
Green ED, Baenziger JU 1988 Asparagine-linked oligosaccharides
on lutropin, follitropin and thyrotropin. I. Structural elucidation of
the sulfated and sialylated oligosaccharides on bovine, ovine and human
pituitary glycoprotein hormones. J Biol Chem 263:2535[Abstract/Free Full Text]
-
Green ED, Baenziger JU 1988 Asparagine-linked oligosaccharides
on lutropin, follitropin and thyrotropin. II. Distribution of sulfated
and sialylated oligosaccharides on bovine, ovine and human pituitary
glycoprotein hormones. J Biol Chem 263:3644[Abstract/Free Full Text]
-
Gottlieb C, Baenziger JU, Kornfeld S 1975 Deficient uridine
diphosphate-N-acetylglucosamine: glycoprotein
N-acetyl-glucosaminyltransferase activity in a clone of Chinese
hamster ovary cells with altered surface glycoproteins. J Biol
Chem 250:33033309[Abstract]
-
Matzuk MM, Boime I 1988 Site-specific mutagenesis defines the
intracellular role of the asparagine-linked oligosaccharides of
chorionic gonadotropin ß subunit. J Biol Chem 263:1710617111[Abstract/Free Full Text]
-
Tarentino AL, Maley F 1974 Purification and properties of an
endo-ß-N-acetylglucosaminidase from Streptomyces griseus.
J Biol Chem 249:811817[Abstract/Free Full Text]
-
Hoyer LL, Roggentin P, Schauer RM, Vimr ER 1991 Purification and properties of cloned Salmonella
typhimurium LT2 sialidase with virus-typical kinetic
preference for sialyl alpha 23 linkages. J Biochem 110:462467[Abstract]
-
Deutscher SL, Nuwayhid N, Stanley P, Briles EI, Hirschberg CB 1984 Translocation across Golgi vesicle membranes: a CHO glycosylation
mutant deficient in CMP-sialic acid transport. Cell 39:295299[Medline]
-
Feizi T 1981 Carbohydrate differentiation antigens. Trends
Biochem Sci 6:333335[CrossRef]
-
Fukuda M 1985 Cell surface glycoconjugates as
onco-differentiation markers in hematopoietic cells. Biochim Biophys
Acta 780:119150[Medline]
-
Nakagawa H, Yamada TM, Chien J-L, Gardas A, Kitamikado M, Li
S-C, Li Y-T (1980) Isolation and characterization of an
endo-ß-galactosidase from a new strain of Escherichia
freundii. J Biol Chem 255:59555959
-
Dahms NM, Hart GW 1986 Influence of quaternary structure on
glycosylation. Differential subunit association affects the
site-specific glycosylation of the common beta chain from Mac-1 and
LFA-1. J Biol Chem 261:1318613196[Abstract/Free Full Text]
-
Corless C, Matzuk M, Ramabhadran T, Krichevsky A, Boime I 1987 Gonadotropin beta subunits determine the rate of assembly and the
oligosaccharide processing of hormone dimer in transfected cells.
J Cell Biol 104:11731181[Abstract]
-
Corless C, Bielinska M, Ramabhadran T, Daniels-McQueen S,
Otani T, Reitz B, Tiemeier D, Boime I 1987 Gonadotropin alpha subunit:
determinants of the oligosaccharide processing of the combined and free
forms. J Biol Chem 262:1419714203[Abstract/Free Full Text]
-
Blithe DL 1994 Glycoprotein Hormones. In: Lustbader JW, Puett
D, Ruddon RW (eds) Serono Symposia, USA. Springer-Verlag, New York, pp
156166
-
Manzella SM, Hooper LV, Baenziger JU 1996 Oligosaccharides
containing beta 1,4-linked N-acetylgalactosamine, a paradigm for
protein specific glycosylation. J Biol Chem 271:1211712120[Free Full Text]
-
Camphausen RT, Yu H-A, Cumming DA 1995 Biosynthesis 6. The
role of polypeptide in the biosynthesis of protein-linked
oligosaccharides. In: Montreuil J, Vliegenthart JFG, Schachter H (eds)
Glycoproteins. Elsevier, Amsterdam, pp 391414
-
Smith PL, Baenziger JU 1988 A pituitary N-acetylgalactosamine
transferase that specifically recognizes glycoprotein hormones. Science 242:930933[Medline]
-
Smith PL, Baenziger JU 1992 Molecular basis of recognition by
the glycoprotein hormone-specific N-acetylgalactosamine-transferase.
Proc Natl Acad Sci USA 89:329333[Abstract]
-
Matzuk M, Kornmeier T, Whitfield K, Kourides I, Boime I 1989 The glycoprotein
subunit is critical for secretion and stability of
the human thyrotropin ß-subunit. Mol Endocrinol 2:95100[Abstract]
-
Keene J, Matzuk M, Boime I 1989 Expression of recombinant
human choriogonadotropin in Chinese hamster ovary glycosylation
mutants. Mol Endocrinol 3:20112017[Abstract]
-
Do K-Y, Fregien N, Pierce M, Cummings RD 1994 Modification of
glycoproteins by N-acetylglucosamyltransferase V is greatly influenced
by accesibility of the enzyme to oligosaccharide acceptors. J Biol
Chem 269:2345623464[Abstract/Free Full Text]
-
Grotjan Jr HE 1989 Oligosaccharide structures of the anterior
pituitary and placental glycoprotein hormones. In: Keel BA, Grotjan Jr
HE (eds) Microheterogeneity of Glycoprotein Hormones. CRC Press, Boca
Raton, FL, pp 2352
-
Lapthorn AJ, Harris DC, Littlejohn A, Lustbader JW, Canfield
RE, Machin KJ, Morgan FJ, Isaacs NW 1994 Crystal structure of human
chorionic gonadotropin. Nature 369:455461[CrossRef][Medline]
-
Wu H, Lustbader J, Liu Y, Canfield R, Hendrickson W 1994 Structure of human chorionic gonadotropin at 2.6 A resolution from MAD
analysis of the selenomethionyl protein. Structure 2:545558[Medline]
-
Keene J, Matzuk M, Otani T, Fauser B, Galway A, Hsueh A, Boime
I 1989 Expression of biologically active human follitropin in Chinese
hamster ovary cells. J Biol Chem 264:47694775[Abstract/Free Full Text]
-
Matzuk M, Krieger M, Corless C, Boime I 1987 Effects of
preventing O-glycosylation on the secretion of human chorionic
gonadotropin in Chinese hamster ovary cells. Proc Natl Acad Sci USA 84:63546358[Abstract]