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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Key words: glycoforms/gonadotrophins/hormone physiology/oligo-saccharides
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Introduction |
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Structure and function of oligosaccharide heterogeneity |
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Functional aspects of gonadotrophin glycosylation |
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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
1
6 linkages determines the overall shape of the glycan moiety and of the whole molecule (Qasba et al., 1997
). Conformational variance of the Man
13Manß 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 Man
16, 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
1
6 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 Man
16, 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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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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Oligosaccharide effects on specificity |
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Protein stability |
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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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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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Notes |
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