Effects of enzymatic deglycosylation of human goiter thyroglobulin on its immunochemical properties

Thyroglobulin (Tg), isolated from soluble iodoproteins by ammonium sulphate fractionation, was enzymatically deglycosylated in vitro and analyzed by polyacrylamide gel electrophoresis, double immunodiffusion and non-commercial RIA. Carbohydrate and iodine content was chemically determined. By PAAGE deglycosylated Tg (dTg) showed the appearance of a major band in the 12S region and three slower migrating bands corresponding to higher aggregates than 19S Tg. In immunodiffusion by testing native and deglycosylated Tg against anti-native Tg antiserum it was shown the appearance of a spur of native on deglycosylated Tg. By RIA of native and deglycosylated Tg against anti-deglycosylated Tg antiserum it was shown a minor binding capacity of the anti-deglycosylated antibody against native Tg at high dilutions. The results demonstrate that the enzymatic deglycosylation release almost all the carbohydrates of goiter Tg and that the removal of the carbohydrates of Tg produces a loss of antigenic determinants of the molecule.     

Effect of deglycosylation of yeast invertase on its uptake and digestion in rat yolk sacs.

The uptake by rat yolk sacs of native invertase and invertase which was deglycosylated by treatment with endo-beta-N-acetylglucosaminidase was compared. The initial rate of uptake of the deglycosylated enzyme was severalfold greater and its accumulation leveled off much earlier than that of the native enzyme. Uptake rates of the deglycosylated and native forms of the enzyme were proportional to their concentration in the medium in the range employed and were inhibited about 85% by 10(-6) M glucagon in both cases. After preloading of yolk sacs with native invertase, the tissue level of activity remained relatively constant over a subsequent 6-h time period, while with the deglycosylated form, activity declined substantially. Since this difference appears not to be attributable to differences in thermal stability, it is suggested that the deglycosylated form of the protein is more susceptible to intracellular proteolytic digestion. In vitro studies on the digestion of these two forms of invertase by trypsin are consistent with this suggestion.     

Function of Mannan Chains of Yeast Repressible Acid Phosphatase on Its Enzymatic Properties

To find the function of the mannan chains covalently attached to yeast repressible acid phosphatase, the N-glycosidic carbohydrate chains were removed by endo-β-N-acetyl-glucosaminidase H under native conditions. Almost all of the N-glycosidic mannan chains were cleaved off by the glycosidase. The deglycosylated enzyme was shown to be a dimer structure as is the native enzyme. The deglycosylated enzyme retained enzyme activity, the same Km, and the same circular dichroism spectra as the native enzyme. These results indicate that the carbohydrate chains are not essential for maintaining the active enzyme structure, but the deglycosylated enzyme was shown to be more sensitive to acidic pH and high temperature.     

Role of the carbohydrate part of yeast acid phosphatase

Acid phosphatase, purified from the yeast Saccharomyces cerevisiae, was completely deglycosylated by endo-beta-N-acetylglucosaminidase H or by HF treatment. Three protein bands were obtained on sodium dodecyl sulfate (SDS)-electrophoresis, with molecular weights of 73,000, 71,000 and 61,500. The released carbohydrate chains varied in size from 12 to 142 mannose units. To study the role of carbohydrate chains in the structure and function of acid phosphatase, a comparison of the properties of the partially deglycosylated enzyme with the native one was performed. The 60% deglycosylated enzyme retained the original activity, and CD and fluorescence spectra showed that the native conformation of the enzyme was preserved. The 90% deglycosylated enzyme showed a pronounced loss of enzyme activity, accompanied by the disruption of the three-dimensional structure. The partially deglycosylated enzyme was less soluble and more susceptible to denaturing effects of heat, pH, urea, and guanidine hydrochloride. Under conditions of electrophoresis, the partially deglycosylated enzyme dissociated, indicating a possible role of carbohydrate chains in maintaining the dimeric structure of the enzyme. Susceptibility of acid phosphatase toward proteolysis was drastically increased by deglycosylation.     

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.     

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.     

Immunological similarity of the cationic and the anionic peanut peroxidase isozymes

Antibodies against both the native and the deglycosylated cationic peanut peroxidase (C.PRX) were used to probe the structural relationship of this isozyme with its anionic counterpart. Not only the native but also the deglycosylated forms of the cationic and the anionic peroxidases reacted with both antibodies. The activity of the cationic isozymes was inhibited by anti-native C.PRX. Similar but nevertheless distinct immunodetection patterns resulted from reaction of the partially digested cationic and anionic peroxidase peptides with antibodies directed to the deglycosylated as well as to the native C.PRX, suggesting a similarity in their polypeptide structures.     

Function of carbohydrate moiety in acid phosphatase of Rhodotorula glutinis

The properties of native and partially deglycosylated forms of acid phosphatase from Rhodotorula glutinis were compared. The removal of carbohydrate moiety resulted in higher thermostability and resistance to proteolysis whereas specific activity, pH optimum and Km value with p-nitrophenyl phosphate remained unchanged. The role of carbohydrate moiety in stabilization of the enzyme structure and protection against proteolysis is suggested.     

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.     

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.     

Chemical deglycosylation of hen ovomucoid: protective effect of carbohydrate moiety on tryptic hydrolysis and heat denaturation

Hen ovomucoid was chemically deglycosylated by treatment with trifluoromethanesulfonic acid at 0 degrees C for 60 min. About 75 mol% of the carbohydrate moiety was removed from the glycoprotein without changing its amino acid composition, and its trypsin inhibitory activity and immunoreactivity with specific antibodies remained unchanged. The deglycosylated ovomucoid was inactivated and degraded easily by an excess amount of trypsin, whereas the native glycoprotein was not. Furthermore, the biological and immunological activities of the deglycosylated ovomucoid were lowered by heat treatment more easily than those of the native ovomucoid. These results suggest that the carbohydrate moiety of ovomucoid contributes to the stability of the ovomucoid molecule against tryptic hydrolysis and heat denaturation.     

Purification and characterization of hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was purified 12 000-fold to homogeneity from yeast by a three-step procedure including acid precipitation, anion-exchange chromatography, and guanosine 5' -monophosphate affinity chromatography. The enzyme is a dimer consisting of two, probably identical, subunits of Mr 29 500. The enzyme recognized hypoxanthine and guanine, but not adenine or xanthine, as substrates. An antiserum against both native and denatured enzyme has been raised and shown to be specific for the enzyme. The antiserum has no affinity for Chinese hamster or human HPRT but does recognize subunits of yeast HPRT as well as some cyanogen bromide fragments of the enzyme.     

N-linked oligosaccharides of murine major histocompatibility complex class II molecule. Role in antigenic peptide binding, T cell recognition, and clonal nonresponsiveness.

To evaluate the functional role of the N-linked oligosaccharides of major histocompatibility complex (MHC) class II molecules, affinity-purified murine IAs class II molecules were deglycosylated in the presence of asparagine amidase enzyme. The deglycosylated IAs molecules were characterized by 12% SDS-polyacrylamide gel analysis under reduced and native conditions and the complete enzymatic removal of all three N-linked sugar components from the alpha/beta heterodimer was confirmed by lectin-link Western blot analysis. Like the native IAs molecules, the deglycosylated IAs molecules were fully capable of binding an antigenic peptide from myelin basic protein MBP(89-101). The kinetics of dissociation of preformed complexes of IAs.MBP(89-101) and deglycosylated IAs.MBP(89-101) were compared at 4 and at 37 degrees C. Both complexes were equally stable at 4 degrees C; however, at 37 degrees C the deglycosylated IAs.MBP(89-101) complexes showed an increased rate of dissociation as compared with the native IAs.MBP(89-101) complexes. When tested for their ability to recognize the T cell receptor on T cells, both complexes bound to cloned HS-1 T cells that recognize and respond to IAs.MBP(89-101). Finally, the complexes of deglycosylated IAs.MBP(89-101) were tested for the induction of in vitro nonresponsiveness and compared with native IAs.MBP(89-101) complexes. Both complexes were capable of inducing 95-100% nonresponsiveness in a proliferation assay. These results suggest that the N-linked oligosaccharide of MHC class II molecules may not be essential for either antigenic peptide binding or T cell recognition. In addition results obtained here provide evidence that the carbohydrate moities of MHC class II molecules may not be involved in induction of T cell clonal anergy.     

Extracellular phytase (E.C. 3.1.3.8) from Aspergillus ficuum NRRL 3135: purification and characterization

Extracellular phytase from Aspergillus ficuum, a glycoprotein, was purified to homogeneity in 3 column chromatographic steps using ion exchange and chromatofocusing. Results of gel filtration chromatography and SDS-polyacrylamide gel electrophoresis indicated the approximate molecular weight of the native protein to be 85-100-KDa. On the basis of a molecular weight of 85-KDa, the molar extinction coefficient of the enzyme at 280 nm was estimated to be 1.2 X 10(4) M-1 cm-1. The isoelectric point of the enzyme, as deduced by chromatofocusing, was about 4.5. The purified enzyme is remarkably stable at 0 degree C. Thermal inactivation studies have shown that the enzyme retained 40% of its activity after being subjected to 68 degrees C for 10 minutes, and the enzyme exhibited a broad temperature optimum with maximum catalytic activity at 58 degrees C. The Km of the enzyme for phytate and p-nitrophenylphosphate is about 40 uM and 265 uM, respectively, with an estimated turnover number of the enzyme for phytate of 220 per sec. Enzymatic deglycosylation of phytase by Endoglycosidase H lowered the molecular weight of native enzyme from 85-100-KDa to about 76-KDa; the digested phytase still retained some carbohydrate as judged by positive periodic acid-Schiff reagent staining of the electrophoresed protein. Immunoblotting of the phytase with monoclonal antibody 7H10 raised against purified native enzyme recognized not only native but also partially deglycosylated protein.     

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.     

Characterization of the kinetic, regulatory, and structural properties of ADP-glucose pyrophosphorylase from Chlamydomonas reinhardtii.

ADP-glucose pyrophosphorylase (ADP-Glc PPase) from Chlamydomonas reinhardtii cells was purified over 2000-fold to a specific activity of 81 units/mg protein, and its kinetic and regulatory properties were characterized. Inorganic orthophosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. Rabbit antiserum raised against the spinach leaf ADP-Glc PPase (but not the one raised against the enzyme from Escherichia coli) inhibited the activity of the purified algal enzyme, which migrated as a single protein band in native polyacrylamide gel electrophoresis. Two-dimensional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that the enzyme from C. reinhardtii is composed of two subunits with molecular masses of 50 and 53 kD, respectively. The molecular mass of the native enzyme is estimated to be 210 kD. Antisera raised against the spinach leaf holoenzyme and against the 51-kD spinach subunit cross-reacted with both subunits of the algal ADP-Glc PPase in immunoblot hybridization, but the cross-reaction was stronger for the 50-kD algal subunit than for the 53-kD subunit. No cross-reaction was observed when antiserum raised against the spinach leaf pyrophosphorylase 54-kD subunit was used. These results suggest that the ADP-Glc PPase from C. reinhardtii is a heterotetrameric protein, since the enzyme from higher plants and its two subunits are structurally more related to the small subunit of the spinach leaf enzyme than to its large subunit. This information is discussed in the context of the possible evolutionary changes leading from the bacterial ADP-Glc PPase to the cyanobacterial and higher plant enzymes.     

Deglycosylation of a native, protease-sensitive glycoprotein by peptide N-glycosidase F without protease inhibitors

The glycoprotein fibrinogen was deglycosylated in its native state and in the absence of protease inhibitors by peptide N-glycosidase F following removal of protease contaminants from the enzyme by HPLC. Fibrinogen is sensitive both to proteolysis by contaminants which may constitute as little as 0.2% of the enzyme protein and to denaturation by 1,10-o-phenanthroline, the only substance known to inhibit the proteolysis. Thus removal of protease contaminants from the enzyme is a prerequisite for the deglycosylation of native fibrinogen. The starting material for the present method is the final material obtained from the purification described by A. L. Tarentino, C. M. Gomez, and T. H. Plummer (1985, Biochemistry 24, 4565). Three sequential passages over a PolyCAT A (20 X .46 cm) cation-exchange column and elutions with a linear gradient of NaCl from 0 to 0.4 M were necessary to completely overcome the tenacious but noncovalent association of peptide N-glycosidase F with contaminants that proteolyze fibrinogen. All three chromatographic runs could be completed in 1 day. Using this "protease-free" enzyme at up to a 1:20 molar ratio, fibrinogen that is completely deglycosylated and native has been generated in order to determine the role of the carbohydrate moieties in its function.     

The Purification and Characterization of Soluble Acid Invertase from Coleoptiles of Wheat (Triticum aestivum L. cv. Avalon)

Soluble acid invertase from wheat coleoptiles was purified toelectrophoretic homogeneity. A comparison of molecular weightby SDS-PAGE and gel filtration suggested that the enzyme wasa monomer of M_r50 000. The enzyme was a glycoprotein and, afterchemical deglycosylation, possessed a M_rof 48000. A polyclonalantiserum was raised against the deglycosylated protein. Thiscross-reacted specifically with acid invertase. A putative precursorof invertase synthesized in a cell-free translation system wasdetected by SDS-PAGE and fluorography of the immunoprecipitatedpolypeptides. The distribution of acid invertase in wheat seedlingshoots was investigated both by visualizing invertase activityafter starch gel electrophoresis and by immunoblotting. Bothtechniques identified two forms of invertase in extracts ofthe primary leaf and only one form in extracts of coleoptiles.The low pH optimum and the glycoprotein nature of wheat coleoptileinvertase are consistent with a vacuolar location. Fructoseinhibited its activity, suggesting that enzyme activity couldbe modulated by end-product inhibition. Key words: Acid invertase, purification, antiserum, glycoprotein, Triticum aestivum, wheat, coleoptiles     

Alkaline treatment has contrasting effects on the structure of deglycosylated and glycosylated forms of glucose oxidase

X-ray crystallographic studies on glucose oxidase showed a strong interaction between carbohydrate and protein moieties of the glycoprotein. However, experimental studies under physiological conditions reported no influence of carbohydrate moiety on the structural and functional properties of glucose oxidase. In order to demonstrate the role of carbohydrate moiety on the structure and stability, we carried out a detailed comparative study on the pH-induced structural changes in the native and deglycosylated forms of glucose oxidase. Our studies demonstrate that at physiological pH both forms of enzyme have very similar structural and stability properties. Acid denaturation also showed similar structural changes in both forms of the enzyme. However, on alkaline treatment contrasting effects on the structure and stability of the two forms of enzyme were observed. The glycosylated enzyme undergoes partial unfolding with decreased stability at alkaline pH; however, a compaction of native conformation and enhanced stability of enzyme was observed for the deglycosylated enzyme under similar conditions. This is the first experimental demonstration of the influence of carbohydrate moiety on structure and stability of glucose oxidase. The studies also indicate the importance of pH studies in evaluating the effect of carbohydrate moiety on the structural and stability properties of glycoprotein.     

Subunit structure and immunological properties of wheat carboxypeptidase

Wheat carboxypeptidases I, II, III and IV from wheat seeds with isoelectric points of 4.8, 5.6, 6.0 and 6.5, respectively, were found to be homogeneous by the Ouchterlony double immunodiffusion technique using an antiserum of the enzyme III. In a previous paper [1], the native enzyme III (MW = 118 k) was separated into two 58 k subunits (MW = 58 k) and further divided into the 35 k and 25 k fragments (MW = 35 k and 25 k, respectively). The native enzyme III and the 58 k subunit produced a single precipitin line against the antiserum. The 35 k and 25 k fragments did not cross-react with the antiserum. The amino acid compositions of the 35 k and 25 k fragments were similar to each other. Amino-terminal amino acids of the 35 k and 25 k fragments were both glutamic acid. Carboxy-terminal groups of the 35k and 25k fragments were determined to be -(Gly, Ser)-Glu-OH and -Thr-Pro-Glu-OH, respectively. The Ouchterlony double immunodiffusion technique revealed the presence of a common antigen in a carboxypeptidase from wheat seeds and one from germinated wheat. Comparison of both enzymes is discussed.