Complete dissociation of gonadotropin receptor binding and signal transduction in mouse Leydig tumour cells. Obligatory role of glycosylation in hormone action.

Utilizing a clonal cell line of mouse testicular Leydig cells (MA-10 cells) the complete steroidogenic and other hormonal properties of chemically deglycosylated ovine lutropin (DG-LH) and human choriogonadotropin (DG-hCG) were evaluated. In these cells, with the LH receptor-steroidogenic mechanism tightly coupled and in which there are few, if any, spare receptors, both DG-LH and DG-hCG failed to elicit progesterone production, unlike fully glycosylated native LH and hCG. The receptor-binding activity of DG-LH and DG-hCG was 2-3 times that of LH and hCG in competition experiments with radiolabelled hormones. The typical phenomenon of rounding of MA-10 cells induced by LH and hCG was absent when cells were incubated with DG-LH or DG-hCG. This could be directly attributable to their failure to produce cyclic AMP as second messenger. DG-LH and DG-hCG inhibited cell shape changes and steroidogenesis caused by LH and hCG. The deglycosylated hormones were potent antagonists of the action of glycosylated hormones. Delaying DG-hCG (antagonist) addition for up to 1 h after initiation of hCG action was also very effective in preventing further activation of steroidogenesis. Similar effects were produced by addition of affinity-purified anti-hCG antibodies. In affinity cross-linking experiments, both hCG and DG-hCG bound to the same 90 kDa receptor. Studies with MA-10 cells thus provide unequivocal evidence that the presence of antennary sugars in LH and hCG (and perhaps in other similar hormones such as follicle-stimulating hormone and thyroid-stimulating hormone), is essential for signal transduction. Differences observed in the literature in other cellular systems may be attributed to differences in hormone-receptor-effector coupling.     

Antibody binding to the beta-subunit of deglycosylated chorionic gonadotropin converts the antagonist to an agonist

The murine Leydig tumor cell line, MLTC-1, has a gonadotropin-responsive adenylate cyclase system. Binding of human chorionic gonadotropin (hCG) stimulates the accumulation of cyclic AMP in these cells. Chemically deglycosylated hCG (DG-hCG) is an antagonist that binds with high affinity to the gonadotropin receptor, but fails to stimulate adenylate cyclase. This antagonism can be reversed if the binding of DG-hCG is followed by treatment of the DG-hCG-receptor complex with antibodies against hCG. Polyclonal antibodies against DG-hCG were raised in rabbits. These antibodies were strongly cross-reactive with hCG, bound to both the alpha- and beta-subunits of hCG and DG-hCG, and reversed the antagonism of DG-hCG. The antiserum was divided into two fractions by affinity chromatography on hCG-Sepharose. The fraction that was not retained reacted only with DG-hCG (DG-hCG antibodies) and, on Western blots, bound to both the alpha- and beta-subunits of DG-hCG. DG-hCG antibodies did not reverse the antagonism of DG-hCG. However, using 125I-protein A, we were able to detect binding of these antibodies to the cell surface DG-hCG-receptor complex. The fraction of antibodies retained by the affinity column reacted with both DG-hCG and hCG (DG-hCG/hCG antibodies). On Western blots, DG-hCG/hCG antibodies bound to the beta-subunit, but only weakly to the alpha-subunit of both hCG and DG-hCG. These antibodies also bound to the cell surface DG-hCG-receptor complex. In addition, DG-hCG/hCG antibodies were able reverse the antagonism of DG-hCG. Reversal of DG-hCG antagonism by the whole antiserum was blocked by the beta- but not the alpha-subunit of hCG. Polyclonal antiserum against the beta- but not the alpha-subunit of hCG reversed the antagonism of DG-hCG. From these results, we conclude that antibody binding to specific determinants common to both native and deglycosylated beta-subunit reverses the antagonism of DG-hCG. In addition, antibodies directed against unique determinants on the deglycosylated beta-subunit are not capable of reversing the antagonism of DG-hCG.     

Influence of carbohydrates on the antigenic structure of gonadotropins: distinction of agonists and antagonists.

The biological properties of glycosylated (native) and deglycosylated gonadotropins are different. The immunological characteristics of antibodies prepared against deglycosylated lutropin and human chorionic gonadotropin were investigated. Distinct antibodies of rabbit polyclonal antisera against deglycosylated lutropin and deglycosylated chorionic gonadotropin were separated by affinity chromatography on divinylsulfonyl-Sepharose-immobilized hormone or antagonist columns, respectively, in successive runs. Antibodies that could discriminate between agonist and antagonistic forms of the hormones could thus be obtained. In radioimmunoassays using 125I-labeled antagonists and respective antagonist antibodies, only the deglycosylated hormones or their deglycosylated alpha-subunits showed preferential reaction. Based on recombinations using different deglycosylated subunits, it was concluded that the loss of antennary sugars in the alpha-subunits was mainly responsible for the changes that led to the formation of antagonist-specific antibodies. Only the agonist-specific antibody could neutralize hormone action. Thus, the type and extent of glycosylation appears to influence the antigenic structure of these secreted glycoproteins.     

Deglycosylated human chorionic gonadotropin. An antagonist to desensitization and down-regulation of the gonadotropin receptor-adenylate cyclase system

Human chorionic gonadotropin (hCG) was deglycosylated with anhydrous HF and compared with native hCG for binding and biological activity. The deglycosylated hormone (DG-hCG) had the same affinity as hCG for gonadotropin receptors in murine Leydig tumor cells (MLTC-1) but was less than 1% as potent as hCG in stimulating cyclic AMP production in these cells. Exposure of MLTC-1 cells for 30 min to hCG caused a desensitization of hCG-stimulated adenylate cyclase activity, whereas DG-hCG did not induce desensitization even after 4 h. hCG induced down-regulation of hCG receptors; by 4 h, 40% of the receptors had disappeared, whereas there was no receptor loss in cells exposed to DG-hCG for the same time. By 6 h, receptor down-regulation began to occur in the DG-hCG-treated cells and could be mimicked by exposing the cells to dibutyryl cyclic AMP or cholera toxin. Thus, the small increase in cyclic AMP generated by DG-hCG appears to result in some loss of receptors. Cells were incubated with iodinated hCG or DG-hCG for 30 min, washed, and incubated in fresh medium. Both bound ligands were degraded as measured by disappearance of cell-associated radioactivity and appearance of trichloroacetic acid-soluble label in the medium. The half-lives were 3 and 6 h for hCG and DG-hCG, respectively. Our results indicate that DG-hCG in contrast to hCG does not cause either rapid desensitization of hCG-stimulated adenylated cyclase or rapid down-regulation of hCG receptors. Therefore, receptor occupancy alone is insufficient to induce these phenomena.     

Is deglycosylated human chorionic gonadotropin an antagonist to human chorionic gonadotropin? Characterization of deglycosylated human chorionic gonadotropin action in two testicular interstitial cell fractions

In order to determine the significance of carbohydrate residues of human chorionic gonadotropin (hCG) in receptor interaction and signal transduction leading to steroidogenesis, the effect of deglycosylated hCG (DG-hCG) was studied in vitro with two different hCG-responsive purified testicular interstitial cell fractions. Fraction I light cells, previously found to bind 125I-labeled hCG with high affinity without producing testosterone, also bound 125I-labeled DG-hCG with high affinity (Kd 7.2.10(-10) M) without stimulating testosterone production. Fraction IV heavier cells, which produced testosterone in response to hCG without detectable high-affinity hCG-binding sites, neither bound DG-hCG nor sufficiently produced cAMP and testosterone in response. With the addition of intact hCG, DG-hCG inhibited cAMP levels, although not sufficiently to inhibit testosterone production. This observation was contrary to previous studies in which DG-hCG was shown to be an antagonist to hCG action. We conclude that: (a) DG-hCG retains its binding activity in light cells and this high-affinity binding is unrelated to steroidogenesis; (b) DG-hCG does not bind to heavier cells with high affinity and loses its biological activity as result of deglycosylation; (c) DG-hCG actions in this study strengthen the concept of two different hCG-responsive cells in the rat interstitium which, if not separated, will yield misleading data supporting the coexistence of hCG high-affinity binding and biological response in the same cell; and (d) DG-hCG partially antagonizes the activation of adenylate cyclase but does not block testosterone production, thus questioning the usefulness of this analogue in antagonizing the action of native hCG in rat testis.     

The impact of glycosylation on monoclonal antibody conformation and stability

Antibody glycosylation is a common post-translational modification and has a critical role in antibody effector function. The use of glycoengineering to produce antibodies with specific glycoforms may be required to achieve the desired therapeutic efficacy. However, the modified molecule could have unusual behavior during development due to the alteration of its intrinsic properties and stability. In this study, we focused on the differences between glycosylated and deglycosylated antibodies, as aglycosyl antibodies are often chosen when effector function is not desired or unimportant. We selected three human IgG1 antibodies and used PNGase F to remove their oligosaccharide chains. Although there were no detected secondary or tertiary structural changes after deglycosylation, other intrinsic properties of the antibody were altered with the removal of oligosaccharide chains in the Fc region. The apparent molecular hydrodynamic radius increased after deglycosylation based on size-exclusion chromatography analysis. Deglycosylated antibodies exhibited less thermal stability for the CH2 domain and less resistance to GdnHCl induced unfolding. Susceptibility to proteolytic cleavage demonstrated that the deglycosylated version was more susceptible to papain. An accelerated stability study revealed that deglycosylated antibodies had higher aggregation rates. These changes may impact the development of aglycosyl antibody biotherapeutics.     

The impact of glycosylation on monoclonal antibody conformation and stability

Antibody glycosylation is a common post-translational modification and has a critical role in antibody effector function. The use of glycoengineering to produce antibodies with specific glycoforms may be required to achieve the desired therapeutic efficacy. However, the modified molecule could have unusual behavior during development due to the alteration of its intrinsic properties and stability. In this study, we focused on the differences between glycosylated and deglycosylated antibodies, as aglycosyl antibodies are often chosen when effector function is not desired or unimportant. We selected three human IgG1 antibodies and used PNGase F to remove their oligosaccharide chains. Although there were no detected secondary or tertiary structural changes after deglycosylation, other intrinsic properties of the antibody were altered with the removal of oligosaccharide chains in the Fc region. The apparent molecular hydrodynamic radius increased after deglycosylation based on size-exclusion chromatography analysis. Deglycosylated antibodies exhibited less thermal stability for the CH₂ domain and less resistance to GdnHCl induced unfolding. Susceptibility to proteolytic cleavage demonstrated that the deglycosylated version was more susceptible to papain. An accelerated stability study revealed that deglycosylated antibodies had higher aggregation rates. These changes may impact the development of aglycosyl antibody biotherapeutics.Key words: monoclonal antibody, glycosylation, stability, liquid chromatography-mass spectroscopy, Fourier transform infrared, fluorescence spectroscopy, size-exclusion chromatography, differential scanning calorimetry     

The role of carbohydrate in thyrotropin action assessed by a novel approach using enzymatic deglycosylation

Deglycosylation of thyrotropin (TSH) and gonadotropins by chemical methods virtually abolishes their biological activity without impairing receptor binding activity. Recent reports have suggested that enzymatic deglycosylation, using endoglycosidases caused a much smaller decrease, if any, in the potency of the glycoprotein hormones without altering the Vmax. However, in these studies complete removal of the carbohydrate chains from the hormones was not unequivocally documented. We have prepared completely deglycosylated bovine TSH by endoglycosidase F digestion of its subunits, which were more readily deglycosylated than the intact hormone. The deglycosylated subunits were separated from any incompletely digested subunits by concanavalin A affinity chromatography. Carbohydrate compositional analysis, using a highly sensitive pulsed amperometric detection method coupled to ion-exchange high performance liquid chromatography, was performed to ascertain the complete removal of the glycan moieties from the subunits. The deglycosylated subunits thus prepared were recombined to obtain deglycosylated TSH dimer. Receptor binding activity of bTSH was minimally affected by the carbohydrate removal. In an in vitro bioassay using stimulation of cyclic AMP production in FRTL-5 cells, deglycosylated bTSH showed reduced activity with a potency 5-10-fold lower than that of control, although the Vmax remained unaltered. In contrast, the deglycosylated bTSH showed a reduction in Vmax, when assayed for its adenylyl cyclase stimulating activity in bovine thyroid membranes. Previous reports using chemical methods have apparently overestimated the effects of deglycosylation, probably because of altered protein conformation, while those using endoglycosidases have apparently underestimated these effects, probably because of incomplete deglycosylation.     

Identification of the mucin core protein by cell-free translation of messenger RNA from bovine submaxillary glands

Bovine submaxillary mucin was purified and subjected to chemical deglycosylation by treatment at 20 degrees C with either anhydrous hydrogen fluoride or trifluoromethane sulfonic acid. Virtually all of the sialic acid, galactose, fucose, and over 90% of the N-acetylhexosamines were removed by these treatments. The amino acid compositions of the deglycosylated and native mucins were similar indicating that chemical deglycosylation did not cause significant degradation of the protein. Antiserum specific for the deglycosylated bovine submaxillary mucin was produced by immunization of rabbits with the deglycosylated mucin. RNA was isolated from bovine submaxillary glands by extraction with guanidine hydrochloride and further fractionated by chromatography on oligo(dT)-cellulose to yield poly(A)+ mRNA. The poly(A)+ mRNA was translated in a rabbit reticulocyte cell-free translation system using [35S]methionine, [3H]leucine, [3H]threonine, [3H]proline, or [3H]serine as radiolabel and the translation products were analyzed by gel electrophoresis and fluorography before and after immunoprecipitation with the antiserum. A labeled product of molecular weight 60,000 was present in the immunoprecipitates obtained in the absence but not in the presence of the unlabeled competitor deglycosylated mucin. It is concluded that the primary translation product of the bovine submaxillary gland gene is a 60,000-dalton protein and that the monomer subunit of the mucin is about 170,000. Thus, in the native state the mucin consists of several self-associating subunits.     

Enhanced thermal stability of chemically deglycosylated human choriogonadotropin

Exposure of aqueous solutions of native human choriogonadotropin (hCG), asialo-hCG (A-hCG), and chemically deglycosylated hCG (DG-hCG) to heat treatment revealed significant differences in their stability. Solutions of hCG and A-hCG were rapidly inactivated above 50 degrees C. On the other hand, solutions of DG-hCG were comparatively more stable under similar conditions as shown by the retention of significant receptor binding, immunological, and hormonal antagonistic activities. Heated solutions (100 degrees C) of hCG and A-hCG quickly lost their ability to enhance the fluorescence of the probe 1-anilino-8-naphthalenesulfonate (1,8-ANS) indicating dissociation into subunits. DG-hCG solutions were more stable in this respect suggesting significant preservation of conformational features required for the interaction with 1,8-ANS. Solutions of hCG and A-hCG which had been thermally denatured (100 degrees C, 10 min) required almost 48 h at 37 degrees C to regain complete ANS binding ability as well as receptor binding activity. Under the same conditions, heated solutions of DG-hCG completely regained these abilities in less than 2 h. A similar pattern was observed with acid (pH 2.0)-dissociated hCG, A-hCG, and DG-hCG. While heated solutions of hCG had no effect on the action of native hCG in vitro, heated DG-hCG solutions still retained their ability to antagonize the cyclic AMP accumulation or steroidogenesis induced by native hCG in rat interstitial cells. Thus, removal of carbohydrate residues (approximately 75% loss) from hCG renders the hormone more resistant to thermal denaturation.     

Enzymatic deglycosylation of the subunits of chorionic gonadotropin. Effects on formation of tertiary structure and biological activity

Both the O- and N-linked oligosaccharide moieties of the subunits of the placental glycoprotein hormone, human choriogonadotropin (hCG), are removed by treatment with a mixture of glycosidases produced by Streptococcus (Diplococcus) pneumoniae. The resulting deglycosylated subunits recombine with their native counterparts in good yield, and the reassociated hormones bind to gonadotropin receptors equally as well as the untreated hormone. Stimulation of steroidogenesis by the deglycosylated alpha-native beta recombinant, however, was markedly less than the stimulation by unmodified hCG both in terms of relative potency (0.10-0.15) and the maximal amount of steroid (40-50%) produced. The native alpha-deglycosylated beta recombinant produced a maximum level of steroid production of 80-90% that of control hCG although its relative potency had decreased approximately 4-fold. The data are in accord with results by others in which either hCG or lutropin was partially deglycosylated by treatment with anhydrous hydrofluoric acid. In addition, the effects of deglycosylation on the ability of each subunit to refold after reduction of their disulfide bonds was studied. Of particular interest is that, after deglycosylation, the beta subunit can correctly refold to a significant degree, in contrast to several unsuccessful attempts to demonstrate correct refolding of the unmodified beta subunit of either lutropin or hCG. Alpha subunit, as measured by a conformation sensitive radioimmunoassay, refolds with equal facility both before and after deglycosylation.     

Antibodies against human chorionic gonadotropin convert the deglycosylated hormone from an antagonist to an agonist

Chemical deglycosylation of human chorionic gonadotropin (hCG) produced an antagonist (DG-hCG) that specifically bound to hCG receptors but was no longer able to stimulate adenylate cyclase in the murine Leydig tumor cell line, MLTC-1. DG-hCG was restored to an agonist by incubating cells or membranes having the bound analogue with antibodies against hCG (anti-hCG). In the presence of anti-hCG, cyclic AMP accumulation and adenylate cyclase activity were stimulated over DG-hCG alone. There was no accumulation of cyclic AMP when the cells were exposed to anti-hCG alone or DG-hCG and normal serum or anti-hCG first then DG-hCG. Several different batches of anti-hCG were effective but their activity did not correlate with their affinity for DG-hCG or hCG. The effect of anti-hCG on DG-hCG activity was dose- and time-dependent. Maximal stimulation of cyclic AMP was achieved with antisera dilutions of 1:200 or less. When DG-hCG-treated cells were exposed to anti-hCG at 37 degrees C, there was a 10-min lag. The lag was eliminated when the cells were exposed to the antibodies at 4 degrees C for 3 h and then warmed to 37 degrees C. Adenylate cyclase was also activated when Fab fragments prepared by papain digestion of anti-hCG were used, whereas Fc fragments were not effective. Thus, the divalency of the anti-hCG is not the critical factor in the mechanism of antibody action. Our results suggest that anti-hCG converts DG-hCG from an antagonist to an agonist possibly by altering the conformation of the modified hormone.     

Development of a method for the efficient release of N-glycans from glycoproteins generating native deglycosylated proteins

In many cases, it is desirable to maintain the native status of the target glycoproteins when they are deglycosylated. However, most conventional deglycosylation process often causes the irreversible denaturation of the target glycoproteins. In the present study, we developed a deglycosylation method that could obtain the native deglycosylated proteins employing Png1p-ΔH1, which was confirmed to tolerate high concentration of dithiothreitol (DTT). To prove this process, ribonuclease B (RNase B) and Yeast carboxypeptidase (CPY) were employed as the targeting glycoproteins. Our results confirmed that both of them could be completely deglycosylated in the presence of high concentration DTT and could be refolded when DTT was removed. The circular dichroism spectroscopy (CD) measurement of refolded CPY and RNase B indicated that the structure of deglycosylated proteins had recovered their native status. This method offers the possibility of efficiently releasing N-linked glycans from glycoproteins and obtaining the native target proteins.     

Characterization and kinetic studies of deglycosylated dopamine beta-hydroxylase

Dopamine beta-hydroxylase (D beta H) (EC 1.14.17.1) from adrenal medulla is a glycoprotein with approximately 5% carbohydrate by weight. The oligosaccharide chains of this enzyme were enzymatically removed with various glycosidic enzymes (endoglycosidases D, F, and H; glycopeptidase F; alpha-mannosidase; neuraminidase; and beta-galactosidase). The time course of deglycosylation was monitored by polyacrylamide gel electrophoresis, and evidence for sugar removal was shown by a modification of the Western blot technique utilizing 125I-labeled concanavalin A and by amino acid analysis. Protein was detected in the gel by using specific antibodies and 125I-labeled protein A. Steady-state kinetic data of deglycosylated D beta H show minor differences between the native and the deglycosylated protein. The Km values for tyramine were 2.17 and 1.66 mM whereas the Km values for oxygen were 0.18 and 0.14 mM for the native and the deglycosylated protein, respectively. The Vmax values (pH 5.0) for the two forms of the enzyme were comparable, with the deglycosylated D beta H being 15% lower. These data indicate that the oligosaccharide moieties present on D beta H do not play a role in catalysis.     

Effects of deglycosylation of sodium channels on their structure and function

Voltage-gated sodium channels are important membrane proteins underlying electrical signaling in the nervous and muscular systems. They undergo rapid conformational changes between closed resting, activated, and inactivated states. Approximately 30% of the mass of the sodium channel is carbohydrate, present as glycoconjugate chains, mostly composed of N-acetylhexosamines and sialic acid. In this study, the effects of removing the carbohydrate on the functional and structural properties of highly purified sodium channels from Electrophorus electricus were investigated. After enzymatic deglycosylation, channels were reconstituted into planar lipid bilayers. In the presence of batrachotoxin, substates became evident and the single-channel conductance of the deglycosylated channels was slightly reduced relative to that of native channels, consistent with electrostatic effects due to the reduction in negative charge at the extracellular vestibule of the channel. The previously reported state-dependent changes in the circular dichroism spectra that are associated with the binding of the anticonvulsant drug Lamotrigine and batrachotoxin are also seen in the modified channels. Synchrotron radiation circular dichroism (SRCD) spectroscopy on the type of sugars found in the sodium channel showed that unlike most carbohydrates, these sugars produce a significant dichroic signal in the far-ultraviolet region. This can account for all of the measured SRCD-detected spectral differences between the native and deglycosylated channels, thereby indicating that no net change in protein secondary structure results from the deglycosylation procedure. Furthermore, thermal denaturation studies detected no significant differences in stability between native and deglycosylated channels. In summary, while the sugars of the voltage-gated sodium channels from electroplax are not essential for functional or structural integrity, they do appear to have a modulating effect on the conductance properties of these channels.     

Immunodetection of Botrytis-specific Invertase in Infected Grapes

Invertase (β-fructofuranosidase, EC 3.2.1.26) activity was shown to be stimulated in grape berries after infection with Botrytis cinerea . By using organism-specific extraction methods, evidence was found proving that both partners contribute to the increase in activity. Qualitative analysis of the extracts by SDS–PAGE showed a new invertase species in the botrytised material, with a molecular weight similar to that of Botrytis invertase (BIT). A method allowing the preparative isolation of homogeneous invertase from liquid cultures of B. cinerea in only three steps (ethanol precipitation, DEAE anion exchange and hydroxyapatite chromatography) with 50% recovery was developed. BIT appeared to be strongly glycosylated; interestingly, the amount of glycan seemed to vary and had a remarkable influence on the chromatographic behaviour of the enzyme. After chemical deglycosylation with TFMS, the BIT peptide was used for the production of polyclonal antibodies in chickens (anti-BIT-IgY). The antibodies recognised glycosylated as well as deglycosylated BIT, a partial denaturation of the protein being necessary for reaction. This indicates that the deglycosylation procedure had been successful and that the antibodies were in fact directed to the peptide moiety. However, after a short incubation at 70°C, native BIT was detectable by anti-BIT-IgY, while still active. Western blotting with extracts of diseased berries confirmed the fungal origin of the new invertase form. The anti-BIT-IgY proved highly specific, although some cross-reaction with a protein in Monilia laxa extracts occurred. The importance of careful immunogen preparation in the production of specific antibodies, and the potential of BIT as a target molecule for the immunological detection of B. cinerea are discussed.     

Partial deglycosylation of an anionic isoperoxidase from peach seeds – effect on enzyme activity, stability and antigenicity

An anionic isoperoxidase (EC 1.11.1.7) purified from peach seeds ( Prunus persica L. Batsch cv. Merry) was partially deglycosylated by glycopeptidase F (EC 3.2.2.18) treatment. A 40% deglycosylation resulted in an activity loss of 50% when assayed with o -dianisidine. 60% with guaiacol and 78% with 2,2'-azino-bis(3-ethyl)benzethiozoline-6-sulfonic acid (ABTS) as substrate. The indole-3-acetic acid oxidase activity loss was close to 55%. The partially deglycosylated isoperoxidase also showed a higher K_m value for H₂O₂ and higher values for Arrhenius activation energy and enthalpy of activation. There was a decrease in enzyme stability at 4°C after deglycosylation. Native and partially deglycosylated isoperoxidase reacted equally well in an enzyme-linked immunosorbent assay (ELISA) with rabbit polyclonal antibodies raised against the native enzyme. The carbohydrate moiety of this peach seed isoperoxidase appears to be important for enzyme activity and stability.     

Effects of carbohydrate removal on the structure and activity of bovine lutropin

Bovine lutropin was successfully deglycosylated by treatment with anhydrous HF at 0°C for 60 min. The overall loss of carbohydrate residues was about 70%. Fucose, mannose and galactosamine residues were removed completely. About six residues of N-acetylglucosamine were left in the deglycosylated hormone. This degree of deglycosylation did not reduce receptor binding ability but in vitro biological activity was completely lost. Consistent with these properties, the deglycosylated lutropin inhibited the in vitro biological action of the native lutropin. The results demonstrate that the full complement of carbohydrate residues including the sulfated hexosamines are not required for receptor binding but they are necessary to impart appropriate conformational features necessary for activation of the target cells.     

Role of carbohydrates in glycoprotein hormone signal transduction

The structure of the polypeptide chains and oligosaccharide moieties of the alpha and beta subunits of pituitary and placental glycoprotein hormones are known. The dimeric polypeptide structure (but not the carbohydrate) is important for binding of the hormone to specific receptors. The N-linked but not O-linked carbohydrates, on the other hand, are required in some manner to activate the effector system. Hormones with depleted carbohydrate content (deglycosylated hormones) interact with receptor but are unable to activate intracellular events. Because of such discordant properties, these forms act as competitive inhibitors of hormone action. Through a combination of chemical deglycosylation procedures and site-directed mutagenesis, the first site of N-glycosylation from the NH2 terminus of the common alpha subunit has been identified to be more critical for glycoprotein hormone signal transduction. Control of glycosylation by the endocrine milieu could contribute to regulation of hormone function by secreting variable forms of agonist/antagonist.     

Glycosylation of purified buffalo heart galectin-1 plays crucial role in maintaining its structural and functional integrity

A buffalo heart galectin-1 purified by gel filtration chromatography revealed the presence of 3.55% carbohydrate content, thus it is the first mammalian heart galectin found to be glycosylated in nature and emphasizes the need to perform deglycosylation studies. Physicochemical comparative analysis between the properties of the native and deglycosylated proteins was carried out to understand the significance of glycosylation. The deglycosylated protein exhibited lesser thermal and pH stability compared to the native galectin. When exposed to thiol blocking reagents, denaturants, and detergents, remarkable differences were observed in the properties of the native and deglycosylated protein. Compared to the native glycosylated protein, the deglycosylated galectin showed enhanced fluorescence quenching when exposed to various agents. CD and FTIR analysis showed that deglycosylation of the purified galectin and its exposure to different chemicals resulted in significant deviations from regular secondary structure of the protein, thus emphasizing the significance of glycosylation for maintaining the active conformation of the protein. The remarkable differences observed in the properties of the native and deglycosylated galectin add an important dimension to the significance of protein glycosylation and its associated biological and clinical relevance.