Study of the carbohydrate moiety of human serum sex hormone-binding globulin

Sex hormone-binding globulin from human blood serum contains two biantennary N-linked oligosaccharide chains of the N-acetyllactosamine type and one O-linked oligosaccharide per one molecule of the glycoprotein. These conclusions have been based on the results of methylation analysis of the whole glycoprotein and investigation of the structures of its glycopeptides prepared using pronase digestion.     

Demonstration of heavy and light protomers of human testosterone-estradiol-binding globulin

Human testosterone-estradiol-binding globulin (hTeBG) was purified from pregnancy serum by sequential ammonium sulfate precipitation, affinity chromatography, and hydroxylapatite chromatography. An overall purification of 2800-fold was achieved with a 27% total yield. Apparent homogeneity of the final product was shown by polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate (SDS). The equilibrium dissociation constant (Kd) at 4 degrees C for 5 alpha-dihydrotestosterone (DHT) was estimated to be 1.94 +/- 0.95 X 10(-9) M. Analysis of the purified protein revealed microheterogeneity with regard to size on polyacrylamide gel in the presence of SDS and to charge on isoelectric focusing gels. The apparent molecular weight of native hTeBG determined by gradient gel electrophoresis was 115,000. SDS-polyacrylamide gel electrophoresis indicated that hTeBG is comprised of two molecular weight components of 53,000 and 46,000, which are designated as heavy (hTeBGH) and light (hTeBGL) protomers, respectively. Photolysis of purified hTeBG with [1,2-3H]17 beta-hydroxy-4,6-androstadien-3-one [( 3H]delta 6-testosterone) resulted in specific labeling of its binding sites. Analysis of photolabeled products by SDS-polyacrylamide gel electrophoresis revealed two radioactive products with electrophoretic mobilities identical to those of the hTeBGH and hTeBGL. The ratio of hTeBGH to hTeBGL was about 10:1. The H and the L protomers were separated and examined by peptide mapping using protease V8 and chymotrypsin. Comparison of the fragmentation patterns produced by these proteases revealed that hTeBGH and hTeBGL components were nearly identical. Removal of sialic acid or carbohydrate residues from hTeBG did not affect the presence of two molecular components. Isoelectric focusing of native hTeBG demonstrated three isoelectric variants with pIs at 4.75, 4.85 and 4.90. After treatment with neuraminidase and other glycosidases, only two isoelectric species were observed with more alkaline pIs. Although purified hTeBG appeared heterogeneous with regard to size and charge, it was remarkably homogeneous in its ability to absorb to Concanavalin A-Sepharose. We conclude that hTeBg, like the androgen binding proteins of the rabbit and rat, is a dimer whose monomer exhibits two protomeric forms.     

The role of the carbohydrate moiety in the biologic properties of fibrinogen

The carbohydrate moiety of some glycoproteins influences their secretion and functional properties. We have examined the importance of the oligosaccharide chains of fibrinogen in this regard. Fibrinogen was labeled de novo by the addition to rabbit hepatocyte monolayer cultures of either 3H-amino-acids or [2-3H] mannose, in the presence or absence of tunicamycin, a potent inhibitor of glycosylation. Inhibition of glycosylation, which ranged from 75 to 80%, was determined by incorporation of [2-3H]mannose as quantitated by gel filtration. Synthesis and secretion of fibrinogen were quantitated by 3H-amino-acid incorporation, using anti-fibrinogen immunoaffinity column chromatography of medium and cell homogenates. Tunicamycin did not appreciably inhibit fibrinogen synthesis, as compared to a 30-40% inhibition of overall protein synthesis, determined by incorporation of 3H-amino-acids into trichloroacetic acid-precipitable material. There was no evidence that secretion of fibrinogen was impaired. Fibrinogen from medium was copurified by adding cold plasma fibrinogen as carrier. Nonglycosylated fibrinogen was found to be functional as demonstrated by incorporation of radioactivity into clots of the copurified material at a rate identical to that of glycosylated fibrinogen. When clotted in the presence of Ca2+ and Factor XIII, cross-linking of glycosylated and nonglycosylated fibrin was demonstrable on fluorography of sodium dodecyl sulfate-polyacrylamide gels, showing disappearance of gamma-chain and appearance of gamma-gamma-dimers.     

The role of the carbohydrate moiety in the structure of the collagen fibril

Collagen isolated from different tendons of the steer was found to contain a firmly bound polysaccharide in amounts ranging from 0.29 to 0.85% depending on the location of the tendon. Viscosities of dilute acetic acid dispersions of the collagen varied in an inverse manner with the amount of bound polysaccharide. Collagen with a polysaccharide content of over 1% was no longer dispersable.Treatment of the collagen dispersions with dilute ficin extracts resulted in an increase in the viscosity followed by a decrease in the viscosity of the dispersion.These results are discussed with regard to the structure of the collagen fibril.     

The function of carbohydrate moiety and alteration of carbohydrate composition in human alkaline phosphatase isoenzymes

The relationship between the structure and function of alkaline phosphatase (orthoposphoric monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) isoenzymes is under investigation in a number of laboratories. The present study deals with the effects of glycosidase digestion on the alkaline phosphatase isoenzymes. Changes in physicochemcial properties, activity, affinity for various lectins and blood group antisera, carbohydrate composition and biological half-life were investigated. The desialylated hepatic enzyme was shown to be more heat labile and more sensitive to protease digestion in the presence of 0.5% sodium dodecyl sulfate than native hepatic enzyme. Helix contents of the native and desialated hepatic enzyme were calculated to be 39.0 and 30.8%, respectively, and apparent molecular weights 175,000 and 167,000, respectively. Intestinal enzyme preparations treated with alpha-mannosidase, exo-N-acetyl-Dglucosaminidase and endo-N-acetyl-D-glucosaminidase-D displayed a decrease in enzyme activity. Among these, the alpha-mannosidase-treated enzyme activity was the most clearly reduction. The maximum activity of the alpha-mannosidase-treated intestinal enzyme was observed to change from 40 mM Mg2+ to 5--10 mM Mg2+.     

Heterogeneity of horse transferrin: the role of carbohydrate moiety

Homozygous horse transferrin (Tf O) is highly heterogeneous. In starch gel electro-phoresis it gives at least 9 zones. Two main components (2a and 4b) were purified by rivanol and ammonium sulphate precipitation, DEAE-Sephadex chromatogra-phy and SP-Sephadex chromatography. Molecular weights of 75 200 and 80 500 for components 2a and 4b, respectively, were determined by sedimentation equilibrium ultracentrifugation. Amino acid compositions of the two components were similar, and there were no differences in the N -terminus (glutamic acid followed by glu-tamine) and the C -terminus (valine). Differences were found in carbohydrate composition between components 2a and 4b. Component 2a contained 10 moles of sugar components per mole of protein (4 hexoses, 4 hexosamines and 2 sialic acids), while component 4b contained twice the number of both total carbohydrates and individual sugar components. Carbohydrates were identified as mannose and galac-tose (ratio mannose:galactose = 1.5:l), N -acetylglucosamine and N -acetylneu-raminic acid. At present it is not clear whether the difference between the two components resides solely in the difference of carbohydrate contents. It is proposed that component 2a has one diantennary glycan, while component 4b has two.     

Heterogeneity of horse transferrin: the role of carbohydrate moiety

Homozygous horse transferrin (Tf O) is highly heterogeneous. In starch gel electrophoresis it gives at least 9 zones. Two main components (2a and 4b) were purified by rivanol and ammonium sulphate precipitation, DEAE-Sephadex chromatography and SP-Sephadex chromatography. Molecular weights of 75 200 and 80 500 for components 2a and 4b, respectively, were determined by sedimentation equilibrium ultracentrifugation. Amino acid compositions of the two components were similar, and there were no differences in the N-terminus (glutamic acid followed by glutamine) and the C-terminus (valine). Differences were found in carbohydrate composition between components 2a and 4b. Component 2a contained 10 moles of sugar components per mole of protein (4 hexoses, 4 hexosamines and 2 sialic acids), while component 4b contained twice the number of both total carbohydrates and individual sugar components. Carbohydrates were identified as mannose and galactose (ratio mannose: galactose approximately equal to 1.5:1), N-acetylglucosamine and N-acetylneuraminic acid. At present it is not clear whether the difference between the two components resides solely in the difference of carbohydrate contents. It is proposed that component 2a has one diantennary glycan, while component 4b has two.     

The carbohydrate moiety of human glycophorin in CDG syndrome.

Human glycophorin, the major sialoglycoprotein of erythrocyte membranes, was isolated from erythrocytes of healthy individuals and four patients with CDG syndrome. Sugar analysis revealed lower carbohydrate content in three out of four CDG-glycophorin samples. In order to characterize closer the glycosylation differences between glycophorin samples in health and disease, reaction with four biotinylated lectins was performed, using ELISA procedure on polystyrene microplates. Results obtained so far strongly suggest that both N- and O-glycans of glycophorin are affected in CDG syndrome.     

Structural study on the carbohydrate moiety of human placental alkaline phosphatase

Alkaline phosphatase purified from human placenta contains a single asparagine-linked sugar chain in one molecule. The sugar chain was quantitatively liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction, and separated by paper electrophoresis into one neutral and two acidic fractions. By a combination of sequential exoglycosidase digestion and methylation analysis, the structures of oligosaccharides in the neutral fraction were confirmed to be as follows: Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc. The acidic oligosaccharide fractions were mixtures of mono- and disialyl derivatives of the neutral fraction. All the sialic acid residues of the sugar chains occur as the NeuAc alpha 2----3Gal group. In the case of monosialyl derivatives, the N-acetylneuraminic acid was exclusively linked to the Man alpha 1----3 arm.     

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.     

Structural studies of the carbohydrate moiety of human antithrombin III

Human antithrombin III contains four asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively released as radioactive oligosaccharides from the polypeptide portion by hydrazinolysis followed by N-acetylation and NaB³H₄ reduction. All of the oligosaccharides, thus obtained, contain N-acetylneuraminic acid. A same neutral nonaitol was released from all acidic oligosaccharides by sialidase treatment. By combination of the sequential exoglycosidase digestion and methylation analysis, their structures were elucidated as NeuAcα2 → 6Galβ1 → 4GlcNAcβ1 → 2Manα1 → 6-(NeuAcα2 → 6Galβ1 → 4GlcNAcβ1 → 2Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc, Galβ1 → 4GlcNAcβ1 → 2Manα1 → 6(NeuAcα2 → 6Galβ1 → 4GlcNAcβ1 → 2Manαl → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc, and NeuAcα2 → 6Galβ1 → 4GlcNAcβ1 → 2Manα1 → 6(Galβ1 → 4GlcNAcβ1 → 2Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc.     

Isolation and characterization of the testosterone-estradiol-binding globulin from human plasma. Use of a novel affinity column.

This report concerns the purification and characterization of the testosterone-estradiol-binding globulin (TeBG) from human plasma. Cohn fraction IV was submitted sequentially to ammonium sulfate preciptation, affinity chromatography, gel filtration, and isoelectric focusing. The final product was homogeneous in polyacrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. Its activity was demonstrated by the finding of slightly more than one binding site/mole for dihydrotestosterone. Association constants (M-1) at 4 and 37degreesC were ascertained for three steroids: dihydrotestosterone; 2.4 x 10(9) and 0.99 x 10(9); testosterone, 1.1 x 10(9) and 0.35 x 10(9); estradiol, 0.60 x 10(9) and 0.22 x 10(9). TeBG is a glycoprotein having a molecular weight of 94000 and both the amino acid and carbohydrate content are presented along with other physical properties.     

Microheterogeneity of the carbohydrate moiety of the human erythrocyte glucose transporter

The carbohydrate moiety of the human erythrocyte glucose transporter was isolated using two independent methods: hydrazinolysis andN-glycanase treatment. The major structure observed was constituted of complex-type carbohydrate chains carrying repetitive units ofN-acetyllactosamine. This structure exhibited microheterogeneity: a broad variability in the number of repetitive units, presence of branched structures and substitution by fucosyl residues. Moreover, significant amounts of bi-antennary and hybrid structures were present.     

The carbohydrate moiety of band-3 glycoprotein of human erythrocyte membranes.

Band-3 glycoprotein was purified from human blood-group-A erythrocyte membranes by selective solubilization and gel chromatography on Sepharose 6B in the presence of sodium dodecyl sulphate. The purified glycoprotein was subjected to hydrazinolysis in order to release the carbohydrate moiety. The released oligosaccharides were N-acetylated and applied to a column of DEAE-cellulose. Most of the band-3 oligosaccharides obtained were found to be free of sialic acids. When this neutral fraction was subjected to gel chromatography on a column of Sephadex G-50, two broad peaks were observed indicating that the band-3 glycoprotein was heterogeneous in the size of the oligosaccharide moieties. All fractions from gel chromatography were found to contain galactose, mannose, N-acetylglucosamine and fucose. The higher-molecular-weight (mol.wt. 3000-8000) peak consisted of fucose, mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine in a molar proportion of 1.6:3.0:8.4:10.5:0.2. Most of these oligosaccharides were digested with a mixture of beta-galactosidase and beta-N-acetylhexosaminidase after alpha-L-fucosidase treatment to give a small oligosaccharide with the structure alpha Man2-beta Man-beta GlcNAc-GlcNAc. Methylation studies and limited degradation by nitrous acid deamination showed that the oligosaccharides contained the repeating disaccharide Gal beta 1----4GlcNAc beta 1----3, with branching points at C-6 of some of the galactose residues. These results indicate that a major portion of the band-3 oligosaccharide has a common core structure, with heterogeneity in the numbers of the repeating disaccharides, and contains fucose residues both in the peripheral portion and in the core portion. Haemagglutination tests were also carried out to determine the blood-group specificities of the glycoprotein and the results demonstrated the presence of both blood-group-H and I antigenic activities.     

Sunflower seed protein: size and charge heterogeneity in subunits of the globulin fraction

The subunit heterogeneity of the globulin fraction of sunflower seeds was investigated by two dimensional electrophoresis, using isoelectric focusing in the first dimension and sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension. Under non reducing conditions, intermediary subunits B, C and D (molecular weight 54 000, 48 000 and 40 000, respectively) were focused within a pI range 5.4-6.0 but intermediary subunits A (molecular weight 60 000) focused within a pI range 6.3-6.8. Under reducing conditions the electrophoretic patterns show that intermediary subunits consist in large "acidic" and small "basic" subunits linked by disulphide bonds. The large subunits of B species are more acidic and less heterogeneous than the corresponding subunits of the A species. These results confirm that helianthinin had a "legumin-type" structure.     

The carbohydrate moiety of the acid carboxypeptidase fromAspergillus saitoi

Acid carboxypeptidase fromAspergillus saitoi is a glycoprotein that contains both N-and O-linked sugar chains. The N-glycanase released high-mannose type oligosaccharides that were separated into eight components on HPLC. One, which had a unique structure of Man₁₁GlcNAc₂, was characterized. Mild alkali treatment of the carboxypeptidase, under conditions that effect -elimination, yieldedd-mannose. Deglycosylation of the carboxypeptidase with endo--N-acetylglucosaminidase and -mannosidase effected the reduction of the molecular mass from 72 kDa to 60 kDa. Partial changes of CD spectra of the native and the deglycosylated enzymes indicate that some conformational changes on the peptide of the enzyme occurred after deglycosylation. Other enzymatic properties, such as catalytic activity, pH, and thermal stability and resistivity to protease digestion, did not appear to change. Tunicamycin halted secretion of the carboxypeptidase extracellularly.     

Biological function of the carbohydrate component of human sex hormone-binding globulin

The role of the carbohydrate component of sex steroid-binding globulin (SBP) from human blood in the glycoprotein interaction with the recognition system for SBP-estrogen complexes in human decidual endometrium plasma membrane was studied. It was shown that the removal of N-acetylneuraminic acid residues from the oligosaccharide chains of SBP did not affect the steroid-binding or immunochemical properties of the glycoprotein. At the same time, the above modification of the glycoprotein resulted in a loss by SBP of its ability to specifically interact with the membrane recognition system. It is concluded that the oligosaccharide chains of SBP are involved in the formation of determinants needed for recognition of the SBP-estrogen complexes by endometrium cell plasma membranes.