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.     

Chick cartilage chondroitin sulfate proteoglycan core protein. I. Generation and characterization of peptides and specificity for glycosaminoglycan attachment

Peptides were derived from the large chondroitin sulfate proteoglycan from chick cartilage by clostripain digestion. Using differential chondroitinase ABC and keratanase treatment and direct carbohydrate analysis, three major peptides of 86, 75, and 27 kDa were shown to bear only chondroitin sulfate chains. Another major peptide of 65 kDa was shown to contain both chondroitin sulfate and keratan sulfate chains, allowing it to be separated from the peptides derived from the chondroitin sulfate domain by DEAE-cellulose chromatography. An additional new peptide (100 kDa) containing keratan sulfate chains was found only in clostripain digests of proteoglycan-hyaluronate-link protein aggregates. Unlike any of the other peptides derived from clostripain digestion of proteoglycan monomer or aggregate, this peptide had the properties of a functional hyaluronate binding region. All of these peptides were purified to apparent homogeneity by preparative electroelution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and deglycosylated with anhydrous hydrogen fluoride. Automated Edman degradation of the two largest chondroitin sulfate peptides revealed that they had unique N termini and several unrecognized residues, which were all subsequently revealed to be modified serine residues following deglycosylation. The keratan sulfate-bearing peptide also had a unique N terminus, which contained a single unrecognized residue, even after HF deglycosylation. Finally, the N terminus of the hyaluronate binding region was blocked. These studies allow estimates of core peptide masses in the absence of carbohydrate as well as provide primary amino acid sequence for O-xylosylated serine residues in the multiply substituted proteoglycans.     

Effect of long-chain fatty acids on the binding of thyroxine and triiodothyronine to human thyroxine-binding globulin

The effect of long-chain fatty acids on the binding of thyroxine to highly purified human thyroxine-binding globulin has been studied by equilibrium dialysis performed at pH 7.4 and 37 degrees C. At a fixed molar ratio of 2000:1 of fatty acid to thyroxine-binding globulin, the degree of binding inhibition based on the percent change in nK value relative to the control as determined from Scatchard plots was: palmitic, 0%; stearic, 0%; oleic, 76%; linoleic, 69%; and linolenic, 61%. At a 500:1 molar ratio of oleic acid to thyroxine-binding globulin, equivalent to 0.125 mM free fatty acid in serum, thyroxine binding was inhibited by 18%, increasing to 93% at a 4500:1 molar ratio. At molar ratios of oleic acid to thyroxine-binding globulin of 1000:1, 2000:1 and 4000:1, the degree of inhibition of triiodothyronine binding was 24%, 41% and 76%, respectively. The results indicate that the unsaturated long-chain fatty acids are potent inhibitors of thyroxine binding to thyroxine-binding globulin, whereas the saturated fatty acids have little or no effect on thyroxine binding.     

Reductive cleavage of Xaa-proline peptide bonds by mild alkaline borohydride treatment employed to release O-glycosidically linked carbohydrate units of glycoproteins

During the deglycosylation reaction of fish egg polysialoglycoproteins under the conditions of 1 M NaBH4 in 0.1 M NaOH at 37 degrees C for 48 h, a marked loss of the glycine content has been encountered, besides the serine and threonine residues to which the carbohydrate units are linked. The chemical basis behind this phenomenon has been elucidated by amino acid analysis first of the major glycopeptides (carbohydrate-(O)Thr-Gly-Pro-Ser) derived from desialylated polysialoglycoproteins and subsequently six proline-containing peptides before and after treatment under similar conditions. It has thus been established that -Xaa-Pro- sequences are remarkably susceptible to reductive cleavage under such mild aqueous conditions. In view of the finding that the reductive cleavage of insulin B-chain, which contains a single proline residue adjacent and C-terminal to a threonine residue, led to about 80% loss of the threonine residue, deglycosylation with alkaline borohydride reagents warrants a special comment. The decreased amounts of serine or threonine residues cannot be related simply to the degree of glycosylation of these residues. The above results are therefore discussed in the relation to other work.     

Reduced serum free thyroxine concentration in postmenopausal women receiving oestrogen treatment.

Thyroid hormone state was assessed in a group of postmenopausal women who had received long term treatment with oestrogen. Serum concentrations of total thyroxine, triiodothyronine, and thyroxine binding globulin were raised compared with those in a control group given placebo; serum concentrations of thyroid stimulating hormone did not differ between the groups. Oestrogen treatment resulted in a significant decrease in the serum free thyroxine concentration and in the ratio of thyroxine to thyroxine binding globulin, which supports the view that oestrogen is the causative factor of the physiological reduction in free thyroid hormone during pregnancy.     

Allosteric modulation of hormone release from thyroxine and corticosteroid-binding globulins

The release of hormones from thyroxine-binding globulin (TBG) and corticosteroid-binding globulin (CBG) is regulated by movement of the reactive center loop in and out of the β-sheet A of the molecule. To investigate how these changes are transmitted to the hormone-binding site, we developed a sensitive assay using a synthesized thyroxine fluorophore and solved the crystal structures of reactive loop cleaved TBG together with its complexes with thyroxine, the thyroxine fluorophores, furosemide, and mefenamic acid. Cleavage of the reactive loop results in its complete insertion into the β-sheet A and a substantial but incomplete decrease in binding affinity in both TBG and CBG. We show here that the direct interaction between residue Thr(342) of the reactive loop and Tyr(241) of the hormone binding site contributes to thyroxine binding and release following reactive loop insertion. However, a much larger effect occurs allosterically due to stretching of the connecting loop to the top of the D helix (hD), as confirmed in TBG with shortening of the loop by three residues, making it insensitive to the S-to-R transition. The transmission of the changes in the hD loop to the binding pocket is seen to involve coherent movements in the s2/3B loop linked to the hD loop by Lys(243), which is, in turn, linked to the s4/5B loop, flanking the thyroxine-binding site, by Arg(378). Overall, the coordinated movements of the reactive loop, hD, and the hormone binding site allow the allosteric regulation of hormone release, as with the modulation demonstrated here in response to changes in temperature.     

Deglycosylated human follitropin: characterization and effects on adenosine cyclic 3',5'-phosphate production in porcine granulosa cells

Chemical deglycosylation of gonadotropins with hydrogen fluoride (HF) has facilitated the investigation of the structure-function relationship of the individual peptide and oligosaccharide moieties in the mechanism of hormone action. These studies have dealt almost exclusively with lutropin or human choriogonadotropin. We report here the chemical characterization and biological properties of deglycosylated human follitropin (degly hFSH). Results indicate that deglycosylation of hFSH by HF removes 89% of the total carbohydrate without disruption of the peptide chain or significant loss of amino acid residues. However, a change in the conformation of the molecule was observed by measurement of the far-ultraviolet circular dichroic spectrum. The degly hFSH showed a 44% reduction in binding when tested in a FSH radioimmunoassay utilizing a polyclonal antibody. Binding of the degly hFSH to FSH-responsive tissues showed that the altered hormone bound with equal or better avidity than the intact hormone while the association constants were approximately the same for both preparations. The degly hFSH alone did not stimulate the FSH-stimulatable adenylyl cyclase (AC) activity of cellular homogenates of small follicle porcine granulosa cells. Furthermore, degly hFSH was a potent antagonist of hFSH-stimulatable AC activity when coincubated with intact hFSH. In intact granulosa cells, both the hFSH and the degly hFSH stimulated cAMP production and release by these cells. However, the degly hFSH was one-tenth as effective as the intact hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of bacterial glycogen: purification and characterization of ADPglucose pyrophosphorylase with modified regulatory properties from Escherichia coli B mutant CL1136-504

The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT₄). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT₄, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT₄ to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT₄ and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT₄/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively.     

The function of the human factor V carbohydrate moiety in blood coagulation

Human factor V was subjected to desialation and deglycosylation to investigate the function of the molecular carbohydrate moiety. Removal of 90% of the sialic acid residues resulted in a 1.5-2-fold increase in clotting activity, and up to 70% deglycosylation in a concurrent decrease in clotting activity. Desialation had no effect on thrombin-induced activation, whereas deglycosylated factor V activation was impaired. Lectin-blot experiments with sialic-acid-specific Limax flavus agglutinin (LFA), galactose-specific Ricinus communis agglutinin (RCA-II) and mannose-specific concanavalin A on thrombin-induced factor V fragments revealed the presence of carbohydrate residues in fragments B, C1, D and F1F2. Interestingly, sialic acid was present in C1 whilst galactose was not detectable. Fragment F1F2 contained terminal galactose residues. LFA and RCA-II inhibited the procoagulant activity of native factor V and of desialated factor V respectively. These investigations distinctly indicate the important role of the human factor V carbohydrate moiety in the process of blood coagulation.     

Human insulin-like growth factor binding protein-6 is O-glycosylated.

Insulin-like growth factor binding protein-6 is abundant in cerebrospinal fluid and has a marked preferential binding affinity for IGF-II over IGF-I. The present study demonstrates that IGFBP-6 is O-glycosylated but not N-glycosylated. Carbohydrate analysis revealed the presence of approximately 20-30 carbohydrate residues/molecule. Galactosamine, galactose and sialic acid were most abundant, with glucosamine and fucose present in lower concentrations. Mannose was not detected. Enzymatic deglycosylation did not alter the high affinity of IGF binding protein-6 for IGF-II (Ka 4.4 +/- 2.2 x 10(11) M-1) or its preference for IGF-II over IGF-I. Glycosylation of IGFBP-6 may affect its secretion, in vivo stability or localization, but does not affect its ligand binding properties.     

Neonatal screening for congenital hypothyroidism by measurement of plasma thyroxine and thyroid stimulating hormone concentrations.

Neonatal screening for congenital hypothyroidism was introduced in the City of Birmingham in 1980 by measuring concentrations of both thyroid stimulating hormone and thyroxine in plasma. Over two years 30 108 babies were tested. Thirty one babies were recalled because of thyroid stimulating hormone concentrations greater than 40 mU/l, of whom 12 were treated with replacement thyroxine. Six babies were found to have low thyroxine concentrations because of reduced thyroxine binding globulin and five raised thyroxine values because of increased thyroxine binding globulin. As a result of this study screening was continued with measurement of thyroid stimulating hormone only as the primary test for congenital hypothyroidism, the thyroxine value being measured only when the concentration of thyroid stimulating hormone exceeded 20 mU/l.     

Biochemical, biological, and immunological properties of chemically deglycosylated human choriogonadotropin

A chemical method of deglycosylation of human choriogonadotropin (hCG) was used to assess the role of carbohydrate moiety in the maintenance of quaternary structure and functional parameters such as receptor binding, immunological activity, and in vitro biological response. Treatment of purified hCG with anhydrous HF at 0 degrees C for 60 min was effective in removing more than 75% of the carbohydrate moiety. This extent of deglycosylation altered its chromatographic characteristics as revealed by retarded behavior on Sephadex G-100 and failure to be retained on concanavalin A-Sepharose. The electrophoretic heterogeneity present in native hCG was markedly reduced by deglycosylation. The deglycosylated hCG was stable in the lyophilized form and retained its quaternary structure as revealed by the fluorescence probe 8-anilino 1-naphthalene sulfonic acid, receptor binding, and immunological activities. Unlike receptor binding and immunological activities, which were fully retained, the ability of the hormone to stimulate cyclic AMP accumulation in vitro in rat interstitial cells was completely abolished.     

Comparison of the amino acid sequences of hen plasma-, yolk-, and white-riboflavin binding proteins

The amino acid sequence of hen egg yolk-riboflavin binding protein (yolk-RBP) was determined by conventional methods. The sequence was identical with that of hen egg white-riboflavin binding protein except that their carboxyltermini were different, that of yolk-RBP lacked 11 or 13 amino acid residues, while hen plasma-RBP had the same C-terminal sequence as white-RBP. This indicated that the C-terminal 11 or 13 amino acid residues in plasma-RBP might be cleaved off during the incorporation from the blood into the oocyte or in the yolk fluid. Yolk-RBP had the same characteristics as white-RBP, such as N-terminal pyroglutamic acid, polymorphism in the amino acid sequence (Lys/Asn) at the fourteenth residue from the N-terminal end, carbohydrate chains attached to both Asn(36) and Asn(147) residues, and phosphate groups bound to some serine residues in the sequence of Ser(185) to Ser(197) as a cluster. These results led us to the conclusion that yolk- and white-RBPs are bio-synthesized from the same gene in the different organs (liver and oviduct). The carbohydrate composition of yolk-RBP was identical to that of plasma-RBP but different from that of white-RBP showing that the processing of the carbohydrate chains in the liver was different from that in the oviduct.     

Enzymatic deglycosylation of human thyroglobulin: fluorescence studies

The interaction between the carbohydrate and the amino acid residues in human thyroglobulin has been studied. Previous reports showed that the removal of the two terminal carbohydrates of the complex chains leads to an increase in thyroglobulin binding to thyroid membranes. In our study, after enzymatic release with glycosidases of the sugar moieties from thyroglobulin, a time-dependent decrease in tryptophan fluorescence has been observed. This decrease was also associated with a shift in the emission peak from 335 to 340 nm. The strong quenching of tryptophan emission was also accompanied by a decrease in the exposure of tryptophan residues, as shown by a Stern-Volmer analysis with the neutral quencher acrylamide. These data, together with the increase in fluorescence of the dansylated deglycosylated thyroglobulin, strongly suggest that a significant conformational change of thyroglobulin follows the deglycosylation of the protein.     

Chemical deglycosylation of ovine pituitary lutropin. A study of the reaction conditions and effects on biochemical, biophysical and biological properties of the hormone

The oligomeric glycoprotein hormone, ovine lutropin was treated with anhydrous HF at 0 degrees C for 30, 60 and 180 min and at 23 degrees C for 60 and 180 min. The products, designated deglycosylated lutropin 1 (DGLH-1) to deglycosylated lutropin 5 (DGLH-5) respectively, were characterized by gel filtration, concanavalin A-Sepharose binding, disc electrophoresis, amino acid analysis, carbohydrate composition and spectral properties. The preparations were also evaluated for receptor binding activity and immunological activity and bioassayed in vitro in collagenase-dispersed rat interstitial cells. In DGLH-1, fucose and galactosamine were removed completely, and there was a 94% decrease in hexoses and 39% decrease in N-acetylglucosamine. Reaction with HF at 0 degrees C for 1 or 3h led to removal of all hexoses and additional loss of hexosamines. Reactions at 23 degrees C for either 1 or 3h were not of additional value in deglycosylation and none of the reaction conditions yielded the apohormone. All the five deglycosylated hormone preparations were not retained on immobilized-concanavalin A columns and on Sephadex G-100 they were eluted with an increased V(e)/V(0) ratio consistent with the loss of carbohydrate residues. Loss of all but the last of the N-acetylglucosamine residues decreased the abnormality of lutropin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but did not eliminate it. Receptor binding activities of DGLH-1 and DGLH-2 were not different from that of the native hormone, but that of DGLH-3 was slightly decreased and the products obtained at 23 degrees C (DGLH-4 and DGLH-5) had lower activity. Immunoreactivities followed a similar pattern. None of the derivatives had activity in the bioassay in vitro. All of the five derivatives inhibited the action of the native hormone in the bioassay in vitro. Their hormonal antagonistic activity was consistent with the receptor binding activity, with DGLH-5 being the least potent in this respect. The DGLH-4 and DGLH-5 preparations had undergone conformational changes as revealed by 8-anilinonaphthalene-1-sulphonate fluorescence, but this did not result in loss of quaternary structure.     

Deglycosylation of chondroitin sulfate proteoglycan by hydrogen fluoride in pyridine

The original deglycosylation procedure using HF/pyridine has been modified for maximal removal of carbohydrate from chondroitin sulfate proteoglycan, with minimal alteration of the core protein. Gas-liquid chromatography analysis after treatment for various times showed that 95% of xylose and mannose and 70-85% of other sugars were removed within 30 min, indicating that almost all chondroitin sulfate chains and about 80% of N- and O-linked oligosaccharides were removed. In contrast to the loss of carbohydrate, no change in amino acid composition or loss of immunoreactivity occurred. Longer treatment of up to 16 h resulted in little additional removal of carbohydrate, but did cause a significant decrease in solubility and recovery of the deglycosylated product. Optimal removal of xylose residues after about 1 h was also shown by maximal acceptor activity of the product in a xylosyltransferase assay. Rapid removal of the HF reagent by vacuum evacuation and ion-exchange chromatography, coupled with the reduced time of treatment allowed recovery of an intact, homogenous protein core that is amenable to structural and sequence studies.     

Enzymatic generation of triplet acetone by deglycosylated horseradish peroxidase

A study of the effects of deglycosylation of horseradish peroxidase on protein conformation, as well as on its catalytic activity of oxidation of isobutyraldehyde or its enol form to triplet acetone and formic acid, was performed. The loss of carbohydrates leads to structural modifications of this enzyme. This is confirmed by a change in the circular dichroism spectrum, an increase in tryptophan's environment polarity, and a loss of the chiral specificity toward D- and L-tryptophan. Deglycosylation does not destroy either the peptide backbone or the amino acid residues and does not affect the heme group content of the protein. The rates of oxygen uptake and light emission observed when horseradish peroxidase oxidizes isobutyraldehyde or the trimethylsilyl enol ether form of the latter are reduced when the enzyme is 70% deglycosylated. Concomitantly, the acting deglycosylated enzyme becomes inactivated during the course of the reaction. It appears that the carbohydrate moiety plays an important role in the protection of the peroxidase from damaging effects induced by triplet acetone and in the stabilization of the three-dimensional structure of this enzyme.     

Comparison of the modelled thyroxine binding site in TBG with the experimentally determined site in transthyretin.

The structure of cleaved thyroxine-binding globulin (TBG) has been modelled on the crystal structure of cleaved alpha 1-antitrypsin (a member of the serine proteinase inhibitor, serpin, superfamily) based on the high sequence homology exhibited by the two proteins. Particular attention was paid to the identification and modelled characteristics of the thyroxine binding site. The primary aim of the study was to compare the site qualitatively with the crystallographically determined binding site of transthyretin, the other major transporter of thyroxine, in an attempt to explain the higher binding affinity of the site compared with the known thyroxine binding site in transthyretin (10(10) versus 10(8) M-1). The proposed binding site shares some similar characteristics with the transthyretin binding site but also includes a cluster of aromatic residues which are entirely absent in transthyretin. It is proposed that this might account for the substantial difference in binding affinities.     

The amino acid sequences of two large glycopeptides derived from the carbohydrate-carrying region of α1-acid glycoprotein

Specific fragmentation with cyanogen bromide and subsequent reduction and carboxymethylation of α₁-acid glycoprotein, a normal human plasma globulin, permitted isolation of a large fragment which was shown to represent the amino-terminal half and to contain the total carbohydrate moiety of this protein. The amino acid sequences of two large glycopeptides derived from this fragment were established. One glycopeptide was composed of 22 amino acid residues and one carbohydrate unit, and the other consisted of 65 amino acid residues and carried four carbohydrate units.     

Specific labeling of the thyroxine binding site in thyroxine-binding globulin: determination of the amino acid composition of a labeled peptide fragment isolated from a proteolytic digest of the derivatized protein

[125I] Thyroxine has been covalently bound to the thyroxine binding site in thyroxine-binding globulin by reaction with the bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. An average of 0.47 mol of [125I] thyroxine was incorporated per mol protein; nonspecific binding amounted to 8%. A labeled peptide fragment was isolated from a proteolytic digest of the derivatized protein by HPLC and its amino acid composition was determined. Comparison with the amino acid sequence of thyroxine-binding globulin indicated partial correspondence of the labeled peptide with two possible regions in the protein. These regions also coincide with part of the barrel structure present in the closely homologous protein, alpha 1-antitrypsin.