Human Reproduction, Vol. 16, No. 5, 825-830,
May 2001
© 2001 European Society of Human Reproduction and Embryology
Debates Continued |
Is there any physiological role for gonadotrophin oligosaccharide heterogeneity in humans?
II. A biochemical point of view
Wadsworth Center, New York State Department of Health, David Axelrod Institute for Public Health, 120 New Scotland Avenue, Albany, NY 1220, USA
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Abstract |
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 Top Abstract IntroductionStructure and function of...Functional aspects of...Partially agonistic glycoformsCarbohydrate as the bane...Oligosaccharide effects on...Protein stabilityConclusionsAcknowledgementsReferences |
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Heterogeneity of gonadotrophin oligosaccharides caused either by pharmacological intervention or which occurs naturally during normal physiological changes is well documented. Recent advances in structure determination of oligosaccharides has to some extent led to a better appreciation of how oligosaccharide heterogeneity may affect protein folding, stability, measurement and modulation of receptor binding. Here it is discussed how carbohydrate structure can impact upon gonadotrophin structure and function. It is well documented that oligosaccharides can serve as a cognate site for protein binding. One functional aspect of gonadotrophin glycosylation heterogeneity is the modulation of receptor binding affinity, yielding partially agonistic glycoforms. Carbohydrate heterogeneity is problematic for a clinical chemist if immunochemical assays are sensitive to heterogeneity. However, even measurements made without such interference may not accurately reflect the biological activity that is a collective result of all isoforms in the circulation, and perhaps of the genotype of each individual. Moreover, oligosaccharide heterogeneity may affect heterodimer stability, therefore, biological activity and immunochemical activity, not to mention clearance. It seems reasonable to conclude that from a biochemical point of view, oligosaccharide heterogeneity is of considerable importance. However, accurate measurement of isoforms in blood, and appropriate in-vitro bioassays that are insensitive to matrix effects are needed to define the physiological significance of each glycoform, and thereby better define target therapeutics and interpret diagnostic results.
Key words: glycoforms/gonadotrophins/hormone physiology/oligo-saccharides
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Introduction |
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TopAbstractIntroductionStructure and function of...Functional aspects of...Partially agonistic glycoformsCarbohydrate as the bane...Oligosaccharide effects on...Protein stabilityConclusionsAcknowledgementsReferences |
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The significance of the heterogeneity of structure of gonadotrophin oligosaccharides and its impact on hormone function differs for the systemic, cellular, and molecular universes. A systemic physiologist may consider the impact of structure and function of oligosaccharide glycoforms most significant for clearance, tissue compatibility, and quantification of gonadotrophins. For a cell biologist, the significance of oligosaccharide heterogeneity is in its impact upon protein trafficking through the endoplasmic reticulum and Golgi apparatus, for glycoprotein recognition, or in how its associated glycan structures simply serve as the molecular fingerprint of the repertoire of glycosyl-transferases within a particular genetic background. In this debate, the molecular point of view, from a biochemical endocrinologist, is given. The interest is not only in the structures of the gonadotrophin isoforms, but how they affect protein folding and stability, their measurement, and the nature of oligosaccharide unit interaction with either the gonadotrophin or its cognate receptor and whether these interactions affect the consummation of the signal transduction event at the level of the cell membrane.
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Structure and function of oligosaccharide heterogeneity |
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TopAbstractIntroductionStructure and function of...Functional aspects of...Partially agonistic glycoformsCarbohydrate as the bane...Oligosaccharide effects on...Protein stabilityConclusionsAcknowledgementsReferences |
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The dimensions of heterodimeric HCG are 75Åx35Åx30Å, thus a typical N-linked oligosaccharide measuring 30Å is expected to play a significant role in the functional properties of the gonadotrophins to which they are attached (Rudd et al., 1999
). Recent studies demonstrate that despite high local flexibility, solvent reorganization is a key factor that can stabilize distinct glycosidic linkage conformations (Woods et al., 1998). However, studying those conformations has been difficult. Despite the long residence time of these conformers as measured by nuclear magnetic resonance spectroscopy (NMR), unambiguous electron density for sugar ring carbons can usually only be assigned to the more rigid glycan cores in most X-ray crystal and NMR solution structures of glycoproteins. This difficulty has hindered a better understanding of glycoprotein hormone oligosaccharide structure and function (Rudd et al., 1999). For the three crystal structures of HCG that are available (GlcNAc – GlcNAcß1) (Lapthorn et al., 1994; Wu et al., 1994; Tegoni et al., 1999), and NMR structures available for the 
-subunit (Wu et al., 1994; Erbel et al., 2000) unambiguous electron density could only be assigned to certain of the asparagine-proximal glycan core sugar residues. However, recent breakthroughs in labelling strategies in mammalian cells and development of `carbohydrate filtration' NMR methods has led to greater resolution of the glycan structures on the 
-subunit, both in isolation, and in complex with the ß-subunit (Weller et al., 1996) This latter study provided two important structural discoveries: the glycan moieties of the -subunit beyond GlcNAc2 act without conformational restraint due to interactions with the -or ß-subunit, and the enzymatic capping of galactose residues with the addition of sialic acid did not alter the conformation of the glycan moieties. |
Functional aspects of gonadotrophin glycosylation |
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TopAbstractIntroductionStructure and function of...Functional aspects of...Partially agonistic glycoformsCarbohydrate as the bane...Oligosaccharide effects on...Protein stabilityConclusionsAcknowledgementsReferences |
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Discussion of the biochemical significance of the oligosaccharide heterogeneity of glycoprotein hormones is best appreciated after reviewing the chemical nature of the carbohydrate on the glycoprotein hormones (Baenziger and Green, 1988
). In addition, a full appreciation of the heterogeneity of oligosaccharides at each N-linked site can be obtained from NMR studies (Weisshaar et al., 1991a, b; Hiyama et al., 1992). Moreover, any opinion rendered about the biochemical significance of isoforms of the glycoprotein hormones would be best placed in a context of hormone action. Thus, a particular isoform must be at concentrations in blood that would allow for interaction with its cognate receptor. In the case of the follicle stimulating hormone (FSH) receptor, this would require nanomolar concentrations of each isoform of interest. Of interest was a report that circulating concentrations of FSH isoform bioactivity did not correlate with immunoactivity following treatment with gonadotrophin releasing hormone antagonist (Dahl et al., 1988). Concentrations of isoforms in blood have been determined at various stages of the menstrual cycle (Ulloa-Aguirre et al., 1995). Since circulating concentrations of isoforms with varied biological activity do vary with pharmacological intervention, or phase of the menstrual cycle, and concentrations are nanomolar, it is appropriate to investigate whether a variety of mechanisms underlie various isoform activities.
It is well recognized that one of the major functions of oligosaccharides in biology is to serve as a recognition epitope for protein binding. Oligosaccharide molecular interactions may include hydrophobic interactions, salt bridge formation, and hydrogen bonding. The glycoprotein hormone receptors are also glycosylated (Libert et al., 1989; Mcfarland et al., 1989a; Sprengel et al., 1990). If the T-cell receptors can serve as a paradigm, then glycosylation of the receptor may help prevent non-specific aggregation and provide a molecular scaffold that orients the binding face and leads to increased affinity (Rudd et al., 1999). This role of carbohydrate on the receptor seems offset by carbohydrate on the hormone that has less of a role in protein recognition, and more of a role in preventing high affinity protein-receptor binding (dampener). The suggestion that the glycoprotein hormone receptors may have a lectin-like activity (Mcfarland et al., 1989b) and that perhaps the hormone-attached carbohydrate moieties interact with the transmembrane helices of the receptor (Sprengel et al., 1990) has not been substantiated experimentally. Although the search for lectin-like sequences in the receptor has not proven useful, there is no reason to assume that the receptor does not interact with the hormone's associated carbohydrate. Oligosaccharide moieties bind to cellular proteins with high specificity, and N-linked oligosaccharides in particular modulate the homo- and hetero-dimerization of glycoproteins (Qasba, 2000). The preferred conformation of each asparagine linked oligosaccharide chain about the 1
3 and 16 linkages determines the overall shape of the glycan moiety and of the whole molecule (Qasba et al., 1997). Conformational variance of the Man1–3Manß linkage is not affected by substitutions on that antenna (Homans et al., 1987). Hormone activity will be impacted by isoform-specific oligosaccharides derived primarily from sugar residues distal to the glycosylation sequon (GalNAc-4-SO4 [LH and TSH] and sialic acid [FSH]). The gonadotrophin literature is replete with observations showing that removal of oligosaccharide from the N52 site increases human follicle stimulating hormone (hFSH) binding to cognate receptor with a commensurate decrease in signal transduction (Keene et al., 1994). It is important to point out that although the importance of N52 oligosaccharide in HCG is established (Matzuk et al., 1989), this is not the case with human thryroid stimulating hormone (hTSH). In this instance, deletion of either of the -subunit glycosylation sites does not decrease signal transduction, whereas deletion of both sites simultaneously greatly impairs signal transduction (Fares et al., 1996). The severity of the effect is directly related to the size of the Man1–6, Man linkage at N52 (Butnev et al., 1998). This linkage, of all other glycosidic linkages studied, appears to be less restrained, and at least three distinct conformers could be identified (Petrescu et al., 1999). Orientation of the 16 arm is affected by rotation about both the 
and 
angles (Qasba et al., 1997) which is influenced by certain key residues (Homans et al., 1986). Thus, in addition to enhancing stability of heterodimeric HCG, (Heikoop et al., 1998), the size of the Man1–6, Man linkage at N52 appears key to affecting intermolecular interactions between hormone and receptor. It remains to be shown if such glycosylation variants occur in nature.
Simply desialylating gonadotrophin and thereby removing the negatively charged sialic acid that caps the terminal galactose residues produces an increase in affinity with no effect on signal transduction. We have observed this to be true when comparing highly purified pituitary hFSH and equally pure hFSH expressed in insect cells. Both were prepared in this laboratory; the content of protein in each preparation was determined by amino acid analysis and the receptor binding activity of each preparation was compared using hFSH receptor expressed in Chinese hamster ovary cells (Kelton et al., 1992) (data not shown). High mannose forms of hFSH, produced in insect cells bound receptor with higher affinity than pituitary hFSH. These high mannose forms exhibited normal signal transduction properties compared to pituitary hFSH, (Ulloa-Aguirre et al., 1999) in direct contrast to reports to the contrary (Arey et al., 1997). Using anti-hFSH monoclonal antibody 46.3H6.B7 to capture hFSH and a polyclonal anti--subunit- antibody to detect hFSH captured, little difference was observed between the immunoreactivity of each preparation (data not shown). Isoforms of hFSH that arise from heterogeneity beyond the Man3GlcNAc2 core are therefore not expected to demonstrate more than a 10-fold difference in binding affinity. Such isoforms are not likely to be recognized differently by monoclonal antibodies, although as discussed below, this is an issue of some controversy. Following this line of logic, isoforms that are at concentrations at least as high as hFSH, and even in some cases, 10-fold lower, could reasonably give rise to a significant biological effect in vivo.
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Partially agonistic glycoforms |
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TopAbstractIntroductionStructure and function of...Functional aspects of...Partially agonistic glycoformsCarbohydrate as the bane...Oligosaccharide effects on...Protein stabilityConclusionsAcknowledgementsReferences |
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As stated above, the biochemical significance of glycoforms of the carbohydrate atN52 appears to be related to the length of its Man(
1–6)Man upper arm. Since forms of hormone with reduced size or no carbohydrate at N52 bind receptor with high affinity but with reduced signal transduction, they may be referred to as partial agonists that have only moderate intrinsic activity (Keene et al., 1994). As a result, the maximal effect that a glycoprotein hormone partial agonist can produce is lower than that of a full agonist. Unless partial agonist des N52 glycoforms can be shown to exist in nature, they are not likely to be physiologically significant. An early report suggested that isoforms of FSH exist, and could function as naturally occurring antagonists of FSH action (Dahl et al., 1988). Recently, it has been shown that a basic isoform of pituitary hFSH acts as a partial agonist, which can stimulate adenylate cyclase activity but blocks downstream effects such as steroidogenesis and secretion of tissue plasminogen activator (Timossi et al., 1998). Similar effects have been observed with basic isoforms of hTSH (Medri et al., 1994). It will be of great interest to determine the structure of the oligosaccharides associated with this particular glycoform, and whether the glycoform is present in the circulation. The structural nature may suggest previously unappreciated mechanisms of signal transduction that are modulated by oligosaccharide heterogeneity. Since this isoform does not block adenylate cyclase activation, it is unlikely to couple preferentially to the inhibitory subunit Gi. Its ability to block Bt2 cyclic AMP (cAMP) effects may provide further evidence that alternate signalling pathways for FSH exist (Maizels et al., 1998), and that cAMP enables or acts in concert with, but is not sufficient for the activation of, these pathways.
In any case, it appears that glycoforms can have variable signal transduction capacities. A consequence of glycoform heterogeneity that presently cannot be determined is its effect on: (i) final conformation of the hormone receptor complex; (ii) whether the hormone interacts with a lectin; (iii) if a particular glycoform predisposes the glycoform/receptor complex to interact with an alternate signalling pathway other than an Gs/adenylate cyclase pathway. This may be due to a specific carbohydrate-interaction with protein, or may arise from steric hindrance. Thus, less steric hindrance by smaller side chains may allow for tighter than normal binding that might lead to less than normal transduction.
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Carbohydrate as the bane of a clinical chemist |
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TopAbstractIntroductionStructure and function of...Functional aspects of...Partially agonistic glycoformsCarbohydrate as the bane...Oligosaccharide effects on...Protein stabilityConclusionsAcknowledgementsReferences |
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As mentioned above, glycoform variation can affect the immunoreactivity of each glycoprotein hormone, making measurement of glycoprotein hormones a challenge. The consequence of this is that estimates of circulatory concentrations of the hormones may be greatly varied because of the plethora of antibodies used for these determinations. Whether the major effect of oligosaccharide variation on immune reactivity is seen when the oligosaccharide is completely removed or is equally affected by, for example, variation in the degree of sialylation, is an open question and one of some controversy (Storring, 1992
; Jeffcoate, 1993). Differences are most likely to be detected when the antibodies used are made against heavily glycosylated pituitary preparations of the glycoprotein hormones. A key appears to be the judicious selection of antibodies, either monoclonal, or perhaps antipeptide antibodies in pairs, which are directed to parts of the molecule that are well away from oligosaccharide attachment, but are not subject to proteolysis or conformational changes. With judicious use of antibodies, it seems unlikely that hormone isoforms will vary significantly in antibody immunoreactivity unless their stability is compromised, leading to subunit dissociation. For example, antibodies can be generated that recognize carbohydrate moieties in addition to the peptide backbone. A good example of this is the crystal structure of two Fv (variable) fragments specific for the and ß subunits complexed to HCG (Tegoni et al., 1999). In this example, Ser26 from the anti-ß Fv interacts with the first GlcNAc residue on Asn52 of the -subunit. In this instance the structure anticipates the possibility that deglycosylation at Asn52 might affect that particular antibody reactivity with HCG. In an extensive examination of the differences between recombinant and pituitary hTSH, it was demonstrated that certain peptide regions of recombinant hTSH were not detectable by antipeptide antibodies, when compared to pituitary hTSH (Canonne et al., 1995). It was found that the degree of sialylation markedly affected measurement of hTSH. Thus removal of sialic acid did not alter the recognition of recombinant hTSH in various immunoassays, it did abolish binding of pituitary hTSH to anti-TSH monoclonal antibodies (Zerfaoui and Ronin, 1996). This speaks directly to the issue of circulating isoform heterogeneity impact on measurement of blood concentrations of glycoprotein hormones and the need to ascertain that one's assay is not confounded by sensitivity to isoform variation. |
Oligosaccharide effects on specificity |
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TopAbstractIntroductionStructure and function of...Functional aspects of...Partially agonistic glycoformsCarbohydrate as the bane...Oligosaccharide effects on...Protein stabilityConclusionsAcknowledgementsReferences |
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When addressing issues about the physiological significance of oligosaccharide heterogeneity on gonadotrophin function, the specificity of HCG for the TSH receptor is a case in point. The activity of HCG at the TSH receptor increases markedly with desialylation (Reddy et al., 1996
; Kraiem et al., 1997). However, full deglycosylation decreases HCG ability to stimulate TSH receptor-mediated adenyl cyclase activity. Basic isoforms of HCG were found to be more potent than native HCG at stimulating TSH receptor (Hoermann et al., 1995). One might predict that during pregnancy, changes in the isoform composition of HCG may impact upon thyroid function. |
Protein stability |
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TopAbstractIntroductionStructure and function of...Functional aspects of...Partially agonistic glycoformsCarbohydrate as the bane...Oligosaccharide effects on...Protein stabilityConclusionsAcknowledgementsReferences |
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Whether glycosylation affects intramolecular rearrangements during folding has been an open question. A comparison of deglycosylated HCG structures with fully glycosylated HCG revealed very similar overall backbone conformations (Tegoni et al., 1999
). However, in that study, it is not clear to what extent the Fv molecules stabilized a particular conformer of the glycosylated HCG structure. Whether greater differences would have been observed if free fully glycosylated HCG had been studied is an open question. It was also of interest that in the crystal of the fully glycosylated HCG-Fv(2), two HCG molecules formed a two-fold related dimer, which was not reported in the two independent deglycosylated HCG structures. The surface contacts involved -45–48 and ß104–106. Neither of these sequences has been implicated in receptor binding activity of the hormone. It seems likely that the most general effect of protein glycosylation is not on the overall protein fold, but may be an alteration in the dynamics or the range of conformers possible of the peptide backbone structure.
The impact of individual oligosaccharides on hormone activity may occur through interaction with the protein backbone or side chains derived primarily from the influence of the proximal sugar residues (Manß1,4GlcNAcß1,4GlcNAc core). Glycoforms can have variable conformational stability as shown by NMR studies of glycosylation variants of HCG (van Zuylen et al., 1997). For example, GlcNAcß1,2 are the primary determinants for HCG within-subunit interactions. GlcNAcAsn78 of the -subunit makes significant contacts with the amino acids in the free -subunit. In the heterodimer, the carbohydrate lies against the hydrophobic core formed between the ß-loops. It has been claimed that deglycosylated HCG is less active than fully glycosylated HCG because it is less stable at 37°C (Heikoop et al., 1998). However, chemically deglycosylated sheep lutrophin has been reported to have greater thermal stability than fully glycosylated sheep lutrophin (Sairam and Manjunath, 1982). Of course, there are many major differences between these two studies, including the species, the gonadotrophin, the extent of deglycosylation, as well as the fact that the sheep lutrophin is already properly folded when it is deglycosylated, whereas the HCG folded in the cell without the benefit of oligosaccharide.
The presence or absence of oligosaccharide on the -subunit may affect conformation of a hydrophobic patch in the -subunit (Lapthorn et al., 1994). We have previously shown that when residues in the long loop of ß-FSH that interact with this hydrophobic patch of the -subunit are substituted, the stability of the heterodimer is decreased (Roth and Dias, 1996). Therefore it seems reasonable to suggest that stability may be compromised if lack of oligosaccharide at N78 destabilises the conformation of the -subunit hydrophobic patch.
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Conclusions |
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TopAbstractIntroductionStructure and function of...Functional aspects of...Partially agonistic glycoformsCarbohydrate as the bane...Oligosaccharide effects on...Protein stabilityConclusionsAcknowledgementsReferences |
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A debate should stimulate further research on the most difficult and contentious of problems, such as the need to measure biological activity of isoforms in human blood and development of appropriate in-vitro bioassays that are insensitive to matrix effects of blood. That endeavour will not be embarked upon without adequate compelling rationale, derived from reductionist, mechanistic studies. The fact is that heterogeneity of oligosaccharides exist, changes in various physiological or pharmacological manipulations, and how this impacts upon diagnostics and protein structure are tangible pragmatic aspects of a modern debate. How oligosaccharide heterogeneity, even of currently available recombinant forms of gonadotrophins, impacts upon therapy can only be guessed at. In this regard, the physiology of each patient and how they might respond to particular isoforms is key. Availability of recombinant forms of gonadotrophins does not obviate the question posed. In fact, the glycoform distribution of recombinant FSH suggests that further refinements in processing are likely and are of practical interest (Lambert et al., 1995
). From a practical point of view, defining the target product will depend largely upon understanding the physiological significance of each glycoform. The fact is, that from a biochemical point of view, a major benefit of recombinant gonadotrophins to the patient aside from decreased production cost is lack of contamination with other proteins and gonadotrophins and not a more uniform glycoform preparation (Hayden et al., 1999). Indeed, the next level of therapy might include blended mixtures where FSH decreases with each dose and LH is held constant, as it has been shown that LH can continue follicle maturation in the presence of diminishing FSH concentrations (Sullivan et al., 1999). Thus the development of shorter acting forms of gonadotrophins with more rapid clearance and hence finer control of treatment, or even inhibitory forms of gonadotrophins, which can be used to ameliorate ovarian hyperstimulation will largely depend upon basic knowledge obtained from what might be considered academic pursuits. Rational design of gonadotrophin therapy (now based largely on oestradiol production as a response parameter), and accurate diagnosis of the mechanisms of infertility will require a better understanding of whether there is a physiological role of gonadotrophin oligosaccharide heterogeneity. In this regard, generation of conditional knockout animals, deficient in glycosylation pathways may provide some insights. In this way, animals could be reared to maturity, then induced to shut down oligosaccharide processing to study effects on reproduction. As pointed out above, development of immunoassays that can measure gonadotrophins without effect of glycosylation is trivial. One only needs the appropriate antibody pairs. A practical consequence of the foregoing debate however, is that one recognizes now, that just as one measures the `good' and `bad' cholesterol, the future of infertility diagnosis may well find one measuring the `good' and `bad' gonadotrophins. The idea is predicated on the fact that gonadotrophin immunoreactivity, does not and in its current form of assay never will translate into gonadotrophin bioactivity.
Will it ever be possible to measure gonadotrophin activity from an individual, and determine if it will behave similarly to another patient? There are problems here as well, particularly because these in-vitro bioassays are carried out in `defined' even `serum free' media, they are not frequently done with cells of human origin, and the concentrations of receptor expressed are so high that they do not replicate the in-vivo situation. Clearly, current research efforts are under the spotlight. This debate should stimulate the development of bioassays that will be conducted in serum or even whole blood, with cells of human gonadal origin plated upon gonadal derived matrices. One has to abandon the expectation of using a system conditioned to studying receptor binding and hormone structure and function, and expecting an outcome of understanding gonadotrophin isoform bioactivity. Instead, one needs to recondition the assay for different expectations, even if it means reinventing the assay and redefining the appropriate response parameters. Through the development of such assays and a better understanding of the consequences of nuances of oligosaccharide structure on gonadotrophin function a practical outcome of better diagnosis and treatment should be forthcoming.
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Acknowledgements |
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TopAbstractIntroductionStructure and function of...Functional aspects of...Partially agonistic glycoformsCarbohydrate as the bane...Oligosaccharide effects on...Protein stabilityConclusionsAcknowledgementsReferences |
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The author acknowledges the support of this work by HD18407 and the Wadsworth Center amino acid analysis/protein sequencing, protein biochemistry, and molecular genetics core facilities.
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Notes |
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1 To whom correspondence should be addressed. E-mail: James.Dias{at}wadsworth.org
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TopAbstractIntroductionStructure and function of...Functional aspects of...Partially agonistic glycoformsCarbohydrate as the bane...Oligosaccharide effects on...Protein stabilityConclusionsAcknowledgementsReferences |
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