Stabilization of cellobiase by covalent coupling to soluble polysaccharide.

Cellobiase from Aspergillus niger was glycosylated by covalent coupling to cyanogen bromide activated dextran. The conjugated enzyme retained 62% of the original specific activity exhibited by the native cellobiase. The optimum pH as well as the pH stability of the conjugated form remain almost the same as for the native enzyme. Compared to the native enzyme, the conjugated form exhibited a higher optimal reaction temperature and energy of activation, a higher K(m) (Michaelis constant) and lower Vmax (maximal reaction rate), and improved thermal stability. The thermal deactivation of the native and conjugated cellobiase obeyed the first-order kinetics. The calculated half-life values of heat inactivation at 60, 70 and 80 degrees C was 10.7, 6.25, and 4.05 h, respectively, whereas at these temperatures the native enzyme was less stable (half-life of 3.5, 1.69, and 0.83 h, respectively). The deactivation rate constant at 80 degrees C for the conjugated cellobiase is about 7.9 x 10(-2) h-1, which is lower than that of the native enzyme (36.0 x 10(-2) h-1). The activation energy for denaturation of the native enzyme is about 10.58 kcal/mol, which is 7.25 kcal/mol lower than that of the conjugated enzyme. The effect of different surfactants and some metal ions on the activity of the conjugated cellobiase has been investigated.     

Influence of heat treatment on rabbit liver ferritin

In order to enhance the stability of beta-galactosidase, we conjugated the enzyme with dextran T-10 (Mr approx. 10 000). The conjugate contained 9-10 mol dextran/mol protein (beta-galactosidase, Mr 68 000), and the specific activity retained after conjugation was 90 +/- 4% (n = 3) of the initial activity. Uptake and degradation of native and conjugated beta-galactosidase in isolated hepatocytes and nonparenchymal liver cells was studied. There was a marked increase in stability against degradation in both cell types when beta-galactosidase was conjugated with Dextran. The degradation of dextran-conjugated enzyme was reduced by 35% in hepatocytes and by 43% in nonparenchymal cells, after 80 and 40 min, respectively, as compared with the free enzyme. However, there was insignificant difference between the uptake of native and conjugated enzyme into the liver cells. Upon intravenous infusion into rats, native and conjugated enzyme were cleared from plasma with only a slight difference in the clearance rate. The observed stability of dextran-conjugated beta-galactosidase towards cellular degradation was in accordance with the in vitro experiments. The conjugate showed marked thermal stability at 50 degrees C and enhanced resistance towards proteolysis by the broad specific protease subtilopeptidase A. This demonstrates that dextran conjugation may be used as a means of stabilizing lysosomal enzymes for therapeutic purposes.     

Secretion of an active nonglycosylated form of the repressible acid phosphatase of Saccharomyces cerevisiae in the presence of tunicamycin at low temperatures

The role of mannan chains in the formation and secretion of active acid phosphatase of yeast (Saccharomyces cerevisiae), a repressible cell surface mannoprotein, was studied in yeast protoplast systems by using tunicamycin at various temperatures. At 30 degrees C, tunicamycin-treated protoplasts did not produce active acid phosphatase; however, at 25 or 20 degrees C they formed and secreted active enzyme. This form of acid phosphatase gave 59-, 57-, and 55-kDa bands on SDS-PAGE which neither bound to concanavalin A Sepharose, nor changed in molecular weight upon treatment with endoglycosidase H, indicating that the peptides are nonglycosylated. The nonglycosylated form, like its glycosylated counterpart, is a dimer on the basis of gel permeation chromatography. The Km for para-nitrophenyl-phosphate and Ki for inorganic phosphate of both glycosylated and nonglycosylated acid phosphatases were almost the same. These results suggested that 1) the conformation of the nonglycosylated acid phosphatase secreted at low temperatures is probably identical with that of the glycosylated one, and 2) the conformation of acid phosphatase is very important for its secretion. The rate of intracellular transport of nonglycosylated acid phosphatase is about one-fourth that of the glycosylated enzyme, indicating that glycosylation facilitates the transport of acid phosphatase proteins.     

Enhanced stability of β-galactosidase in parenchymal and nonparenchymal liver cells by conjugation with dextran

In order to enhance the stability of β-galactosidase, we conjugated the enzyme with dextran T-10 (M_r approx. 10 000). The conjugate contained 9–10 mol dextran/mol protein (β-galactosidase, M_r 68 000), and the specific activity retained after conjugation was 90 ± 4% (n = 3) of the initial activity. Uptake and degradation of native and conjugated β-galactosidase in isolated hepatocytes and nonparenchymal liver cells was studied. There was a marked increase in stability against degradation in both cell types when β-galactosidase was conjugated with Dextran. The degradation of dextran-conjugated enzyme was reduced by 35% in hepatocytes and by 43% in nonparenchymal cells, after 80 and 40 min, respectively, as compared with the free enzyme. However, there was insignificant difference between the uptake of native and conjugated enzyme into the liver cells. Upon intravenous infusion into rats, native and conjugated enzyme were cleared from plasma with only a slight difference in the clearance rate. The observed stability of dextran-conjugated β-galactosidase towards cellular degradation was in accordance with the in vitro experiments. The conjugate showed marked thermal stability at 50°C and enhanced resistance towards proteolysis by the broad specific protease subtilopeptidase A. This demonstrates that dextran conjugation may be used as a means of stabilizing lysosomal enzymes for therapeutic purposes.     

Site-selective glycosylation of subtilisin Bacillus lentus causes dramatic increases in esterase activity

Using site directed mutagenesis combined with chemical modification, we have developed a general and versatile method for the glycosylation of proteins which is virtually unlimited in the scope of proteins and glycans that may be conjugated and in which the site of glycosylation and the nature of the introduced glycan can be carefully controlled. We have demonstrated the applicability of this method through the synthesis of a library of 48 glycosylated forms of the serine protease subtilisin Bacillus lentus (SBL) as single, pure species. As part of our ongoing program to tailor the activity of SBL for use in peptide synthesis, we have screened these enzymes for activity against the esterase substrate succinyl-Ala-Ala-Pro-Phe-S-benzyl. Gratifyingly, 22 enzymes displayed greater than wild type (WT) activity. Glycosylation at positions 62, in the S2 pocket, resulted in five glycosylated forms of SBL that were 1.3- to 1.9-fold more active than WT. At position 217, in the S1' pocket, all glycosylations increased kcat/KM up to a remarkable 8.4-fold greater than WT for the glucosylated enzyme L217C-S-beta-Glc(Ac)3. Furthermore, the ratio of amidase to esterase activity, (kcat/KM)esterase/(kcat/KM)amidase (E/A), is increased relative to wild type for all 48 glycosylated forms of SBL. Again, the most dramatic changes are observed at positions 62 and 217 and L217C-S-beta-Glc(Ac)3 has an E/A that is 17.2-fold greater than WT. The tailored specificity and high activity of this glycoform can be rationalized by molecular modeling analysis, which suggests that the carbohydrate moiety occupies the S1' leaving group pocket and enhances the rate of deacylation of the acyl-enzyme intermediate. These glycosylated enzymes are ideal candidates for use as catalysts in peptide synthesis as they have greatly increased (kcat,KM)esterase and severely reduced (kcat/KM)amidase and will favor the formation of the amide bond over hydrolysis.     

Structure of human thymidylate synthase suggests advantages of chemotherapy with noncompetitive inhibitors

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms. The emergence of resistance to the treatment is often related to the increased levels of TS in cancer cells, which have been linked to the elimination of TS binding to its own mRNA upon drug binding, a feedback regulatory mechanism, and/or to the increased stability to intracellular degradation of TS.drug complexes (versus unliganded TS). The active site loop of human TS (hTS) has a unique conformation resulted from a rotation by 180 degrees relative to its orientation in bacterial TSs. In this conformation, the enzyme must be inactive, because the catalytic cysteine is no longer positioned in the ligand-binding pocket. The ordered solvent structure obtained from high resolution crystallographic data (2.0 A) suggests that the inactive loop conformation promotes mRNA binding and intracellular degradation of the enzyme. This hypothesis is supported by fluorescence studies, which indicate that in solution both active and inactive forms of hTS are present. The binding of phosphate ion shifts the equilibrium toward the inactive conformation; subsequent dUMP binding reverses the equilibrium toward the active form. Thus, TS inhibition via stabilization of the inactive conformation should lead to less resistance than is observed with presently used drugs, which are analogs of its substrates, dUMP and CH(2)H(4)folate, and bind in the active site, promoting the active conformation. The presence of an extension at the N terminus of native hTS has no significant effect on kinetic properties or crystal structure.     

Antioxidant and Enzyme Inhibitory Activities of Rhoifolin Flavonoid: In Vitro and in Silico Studies

Rhoifolin (apigenin-7-O-β-neohesperidoside) belongs to the class of flavonoids and was reported to exhibit anti-inflammatory, cytotoxic, antidiabetic, hepatoprotective, and cardioprotective activities. The current study presents the in-vitro evaluation of the antioxidative effects of rhoifolin by many assays, namely DPPH, CUPRAC, ABTS, phosphomolybdenum, and FRAP. Enzyme inhibitory potential was also evaluated for acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, amylase, and glucosidase enzymes. While results revealed weak antioxidant activities for rhoifolin, the compound demonstrated some promising enzyme inhibitory effects against BChE (4.03 mg GALAE/g) and tyrosinase (7.44 mg KAE/g) but was not active on AChE. Regarding anti-diabetic enzymes, the compound was active on amylase but did not show any inhibition effect on glucosidase. In-silico molecular docking study was performed for rhoifolin on the active site of NADPH oxidase, BChE, and amylase enzymes to verify the observed enzyme inhibitory effect. Good binding affinities were observed for rhoifolin on all the docked enzymes, revealing numerous hydrogen bonds, carbon-hydrogen, van der Waals interactions. This is the first study to evaluate the enzyme inhibition potential of rhoifolin. We concluded that the increase in the degree of glycosylation might decrease the antioxidant abilities of flavonoids and that rhoifolin had moderate enzyme inhibition abilities to be investigated in future studies.     

Chemical stabilization of glucoamylase from Aspergillus niger against thermal inactivation

The applicability of crosslinking an enzyme to an oxidized polysaccharide by reductive alkylation to enhance thermostability has been investigated for glucoamylase from Aspergillus niger. Direct covalent coupling of the enzyme to periodate-oxidized dextran in the presence of NaBH(3)CN results in a conjugate which has thermal properties similar to those of the native enzyme. Our working hypothesis postulates that enhancement of thermostability will result from rigidification of the protein's conformation subsequent to the formation of multiple covalent bonds between the protein and the support. On the basis of the known characteristics of glucoamylase from Aspergillus niger, it would seem necessary to introduce additional amino groups in the polypeptide chain of the protein. The incorporation of new amino groups was performed in two phases. First, the glycosidic part of glucoamylase was oxidized by periodate and the resulting aldehyde groups were reductively aminated by a diaminoalkane and NaBH(3)CIM. Secondly, additional amino groups were introduced on carboxyl functions into the previously aminated glucoamylase by a diaminoalkane and a water-soluble carbodiimide in the presence of maltose to protect the active site. The final derivative was then coupled to periodate-oxidized dextran T-70 in the presence of NaBH(3)CN. Starting with native glucoamylase, three successive operations give rise to a conjugate which retained 27% of the initial activity when measured with soluble starch and 39% when measured with maltopentaose. Using substrates of various sizes, it was observed that steric hindrance at the active site may result from covalent coupling to dextran T-70. It was demonstrated in heat inactivation experiments that the thermostability of the conjugate was in all cases superior to that of the native enzymes. Finally, it was observed that the operational stability of the conjugate was at least twice that of native glucoamylase at 70 degrees C on 18% maltodextrin. Additional experiments rule out the possibility that thermosta-bilization of the complex is due to other reasons than the increase in the amino content of the protein prior to crosslinking. Neither chemical modification, reticulation nor change in the net charge of the protein resulted in a derivative of glucoamylase which presented enhanced thermostability after conjugation. We conclude that for enzymes which have a low content of available amino groups, the thermostabilization method proposed previously by the present authors may still be applicable if additional amino groups are introduced into the protein prior to its crosslinking to an oxidized polysaccharide. This new example reinforces the generality of this method of stabilization.     

Intramolecular crosslinking of gamma-glutamyl transpeptidase

gamma-Glutamyl transpeptidase (rat kidney) is a heterodimeric glycoprotein (subunit molecular weights 52,000 and 25,000). In addition to its single-chain biosynthetic precursor (Mr 78,000), glycosylated high molecular weight forms (Mr 85,000-95,000) have been reported in various rat tissues as well as during in vitro translation of its mRNA. Studies reported here suggest that these might be attributed to the anomalous behavior of intramolecularly crosslinked species. Thus, chemical crosslinking of the purified enzyme (as well as enzyme on the renal brush border membranes) by bifunctional reagents such as dimethyl suberimidate and by an active site-directed reagent, diazotized p-amino-hippurate, produces stable heterodimers which exhibit molecular weights identical to that of the native enzyme when subjected to gel filtration. However, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the crosslinked species exhibit apparent Mr values of 85,000 to 110,000, depending upon the crosslinking agent used. Protein glycosylation alone does not account for such anomalous electrophoretic behavior; the extent and the regions of the enzyme involved in formation of crosslinks appear to exert considerable constraints upon their conformation even in denaturing media.     

N-glycosylation and residue 96 are involved in the functional properties of UDP-glucuronosyltransferase enzymes

The recent cloning of several human and monkey UDP-glucuronosyltransferase (UGT) 2B proteins has allowed the characterization of these steroid metabolic enzymes. However, relatively little is known about the structure-function relationship, and the potential post-translational modifications of these proteins. The mammalian UGT2B proteins contain at least one consensus asparagine-linked glycosylation site NX(S/T). Endoglycosidase H digestion of the human and monkey UGT2B proteins demonstrates that only UGT2B7, UGT2B15, UGT2B17, and UGT2B20 are glycosylated. Although UGT2B15 and UGT2B20 contain three and four potential glycosylation sites, respectively, site-directed mutagenesis revealed that both proteins are glycosylated at the same first site. In both proteins, abolishing glycosylation decreased glucuronidation activity; however, the K(m) values and the substrate specificities were not affected. Despite the similarities between UGT2B15 and UGT2B20, UGT2B20 is largely more labile than UGT2B15. Treating HK293 cells stably expressing UGT2B20 with cycloheximide for 2 h decreased the enzyme activity by more than 50%, whereas the activity of UGT2B15 remained unchanged after 24 h. The UGT2B20 protein is unique in having an isoleucine at position 96 instead of an arginine as found in all the other UGT2B enzymes. Changing the isoleucine in UGT2B20 to an arginine stabilized enzyme activity, while the reciprocal mutation in UGT2B15 R96I produced a more labile enzyme. Secondary structure predictions of UGT2B proteins revealed a putative alpha-helix in this region in all the human and monkey proteins. This alpha-helix is shortest in UGT2B20; however, the helix is lengthened in UGT2B20 I96R. Thus, it is apparent that the length of the putative alpha-helix between residues 84 and 100 is a determining factor in the stability of UGT2B enzyme activity. This study reveals the extent and importance of protein glycosylation on UGT2B enzyme activity and that the effect of residue 96 on UGT2B enzyme stability is correlated to the length of a putative alpha-helix.     

The effect of glycosylation on the folding kinetics of erythropoietin

Glycosylation is a common posttranslational modification that generally increases protein solubility and thermodynamic stability. Less is known about how this modification influences protein folding, particularly folding processes involving intermediate species. In the present report, folding comparisons of a nonglycosylated erythropoietin (EPO) mutant are made with the fully glycosylated EPO, which was recently shown to fold by a three-state on-pathway mechanism. The absence of glycosylation did not alter the folding mechanism of EPO but did greatly decrease the stability of the intermediate species, change the rate-limiting step of the folding reaction, and accelerate the folding kinetics to both the intermediate state and the native state. Surprisingly, glycosylation stabilized the intermediate species to a greater extent than it increased the EPO equilibrium stability. These results suggest that glycosylation impedes the latter EPO folding steps rather than accelerating them by biasing particular folding pathways, as previously proposed for folding reactions initiated from unfolded ensembles with minimal residual structure. Due to the specific biological processes modulated by EPO glycosylation, however, there may be little evolutionary pressure to fold on a faster, more direct pathway at the expense of biological function, particularly given the protective role glycosylation has at preventing EPO aggregation. Lastly, evidence that is consistent with glycosylation destabilizing the unfolded state to some degree and contributing to the greater equilibrium stability of the glycosylated EPO is presented.     

Effect of limited proteolysis in the 8th loop of the barrel and of antibodies on porcine pancreas amylase activity.

The porcine pancreatic alpha-amylase is a (beta/alpha)8-barrel protein, containing domains A and B (peptide sequence 1-403) and a distinct C-domain (peptide sequence 404-496). Separation of the terminal C-domain from the A and B domains has been attempted by limited proteolysis in the hinge region. Subtilisin was found to hydrolyse amylase between residues 369 and 370 situated in the loop between the eighth beta-strand and alpha-helix. The cleaved amylase was isolated by chromatofocusing and found to retain about 60% of the activity of the native enzyme, while the isolated fragments were inactive. Antigen binding fragments prepared from polyclonal antibodies to native amylase and the CNBr-fragment P1 (peptide sequence 395-496) respectively, were tested for influence on the enzyme activity. Antibodies directed against P1 had no effect whereas antibodies against the peptide sequence 1-394 and amylase respectively inhibited hydrolysis of substrates having four or more glucose residues but not of shorter oligomaltosides. Crystallographic analysis revealed that changes in the region of residue 369 might affect the conformation of the active site as well as of a second binding site. This site, located on the enzyme surface, is proposed to be required for the hydrolysis of larger substrates.     

Serine scanning: a tool to prove the consequences of N-glycosylation of proteins

N-Glycosylation of proteins is a common posttranslational modification in eukaryotes. Often this results in enhanced protein stability through protection by the attached sugar moieties. Due to its 13 potential N-glycosylation motifs (N-X-T/S), recombinant hydroxynitrile lyase isoenzyme 5 from almonds (PaHNL5) is secreted by the heterologous host Pichia pastoris in a massively glycosylated form, and it shows extraordinary stability at low pH. The importance of N-glycosylation in general, and individual glycosylation sites in particular for stability at low pH were investigated. To identify especially important glycosylation sites asparagine from all N-X-S/T-motifs was replaced by serine. Thus, critical sites, which contributed to overall enzyme activity and/or stability, were identified individually. One glycosylation site revealed to be essential for stability at low pH. After enzymatic deglycosylation, leaving only one acetylglucosamine attached to asparagines, PaHNL5 retained most of its stability at low pH. Protonation effects in the active site as well as higher-order aggregational events upon incubation in low pH were excluded. This study provides evidence for the interconnection of N-glycosylation and stability at low pH for PaHNL5. Moreover, serine scanning was proven to be applicable for quick identification of critical glycosylation sites.     

Effects of ligand binding and conformational switching on intracellular stability of human thymidylate synthase

Thymidylate synthase (TS) is the target in colon cancer therapeutic protocols utilizing such drugs as 5-fluorouracil and raltitrexed. The effectiveness of these treatments is hampered by emerging drug resistance, usually related to increased levels of TS. Human TS (hTS) is unique among thymidylate synthases from all species examined as its loop 181-197 can assume two main conformations related by rotation of 180 degrees. In one conformation, "active", the catalytic Cys-195 is positioned in the active site; in the other conformation, "inactive", it is at the subunit interface. Also, in the active conformation, region 107-128 has one well-defined conformation while in the inactive conformation this region assumes multiple conformations and is disordered in crystals. The native protein exists in apparent equilibrium between the two conformational states, while the enzyme liganded with TS inhibitors assumes the active conformation. The native protein has been reported to bind to several mRNAs, including its own mRNA, but upon ligation, RNA binding activity is lost. Ligation of TS by inhibitors also stabilizes it to turnover. Since currently used TS-directed drugs stabilize the active conformation and slow down the enzyme degradation, it is postulated that inhibitors of hTS stabilizing the inactive conformation of hTS should cause a down-regulation in enzyme levels as well as inactivate the enzyme.     

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.     

Human 11beta-hydroxysteroid dehydrogenase type 1 is enzymatically active in its nonglycosylated form

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) is a microsomal enzyme responsible for the reversible interconversion of active 11beta-hydroxyglucocorticoids into inactive 11-ketosteroids and by this mechanism regulates access of glucocorticoids to the glucocorticoid receptor. The enzyme has also been proven to participate in xenobiotic carbonyl compound detoxification. 11beta-HSD 1 is anchored within the membranes of the endoplasmic reticulum (ER) by its N-terminus, whereby its active site protrudes into the lumen of the ER. In the primary structure of 11beta-HSD 1 three Asn-X-Ser glycosylation motifs have been identified. However, the importance of N-linked glycosylation of 11beta-HSD 1 for catalytic activity has been controversely discussed. To clarify if glycosylation is essential for enzyme activity, we performed deglycosylation experiments of native 11beta-HSD 1 from human liver as well as site-directed mutagenesis to remove potential glycosylation sites upon overexpression in Pichia pastoris. The altered proteins were examined regarding their catalytic activity towards their physiological glucocorticoid substrates. The molecular size of the various 11beta-HSD 1 forms was analyzed by immunoblotting with a polyclonal antibody raised against 11beta-HSD 1 protein from human liver. By stepwise enzymatic deglycosylation of native 11beta-HSD 1 we could demonstrate that all potential glycosylation sites carry N-linked oligosaccharide residues under physiological conditions. Interestingly, complete deglycosylation did not affect enzyme activity, neither in the reductive (cortisone) nor in the oxidative (cortisol) direction. Upon overexpression in the yeast P. pastoris, 11beta-HSD 1 did not undergo glycosylation, but, in spite of this, yielded a fully active enzyme. Our results conclusively demonstrate that 11beta-HSD 1 does not need to be glycosylated to perform its physiological role as glucocorticoid oxidoreductase.     

A role of glycosyl moieties in the stabilization of bitter gourd (Momordica charantia) peroxidase

The possible role of carbohydrate moieties in the stabilization of proteins has been investigated by using bitter gourd peroxidase as a model system. A comparative study of glycosylated and non-glycosylated isoenzymes of bitter gourd peroxidase was performed at various temperatures, pH, water-miscible organic solvents, detergents and chaotropic agent like urea. The pH-optima and temperature-optima of both glycosylated and non-glycosylated isoforms of bitter gourd peroxidase remained unchanged. The probes employed were changes in the enzyme activity and fluorescence. The glycosylated form of peroxidase retained greater fraction of enzyme activity against the exposure caused by various physical and chemical denaturants. The unfolding of both forms of enzyme in the presence of high urea concentrations, studied by fluorescence, indicated greater perturbations in the conformation of non-glycosylated preparation. The different properties examined thus indicated that glycosylation plays an important role in the stabilization of native conformation of proteins against the inactivation caused by various types of denaturants.     

Expression of soluble bovine pancreatic ribonuclease A in Pichia pastoris and its purification and characterization

A Pichia pastoris expression system for bovine pancreatic RNase A was constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion. Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated. After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture. N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A. Glycosylated RNase A had a decreased kcat, 60-70% of the activity of wildtype RNase A, as in the case of RNase B. Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since Tm of the glycosylated RNase A was decreased by 6 degrees C. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc. The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.     

Mutational analysis ofN-linked glycosylation of esterase 6 inDrosophila melanogaster

The primary sequence of the esterase 6 (EST6) enzyme ofDrosophila melanogaster contains four potential N-linked glycosylation sites, at residues 21, 399, 435, and 485. Here we determine the extent to which EST6 is glycosylated and how the glycosylation affects the biochemistry and physiology of the enzyme. We have abolished each of the four potential glycosylation sites by replacing the required Asn residues with Gln byin vitro mutagenesis. Five mutant genes were made, four containing mutations of each site individually and the fifth site containing all four mutations. Germline transformation was used to introduce the mutant genes into a strain ofD. melanogaster null for EST6. Electrophoretic and Western blot comparisons of the mutant strains and wild-type controls showed that each of the four potential N-linked glycosylation sites in the wild-type protein is glycosylated. However, the fourth site is not utilized on all EST6 molecules, resulting in two molecular forms of the enzyme. Digestion with specific endoglycosidases showed that the glycan attached at the second site is of the high-mannose type, while the other three sites carry more complex oligosaccharides. The thermostability of the enzyme is not affected by abolition of the first, third, or fourth glycosylation sites but is reduced by abolition of the second site. Anomalously, abolition of all four sites together does not reduce thermostability. Quantitative comparisons of EST6 activities showed that abolition of glycosylation does not affect the secretion of the enzyme into the male sperm ejaculatory duct, its transfer to the female vagina during mating, or its subsequent translocation into her hemolymph. However, the activity of the mutant enzymes does not persist in the female's hemolymph for as long as wild-type esterase 6. The latter effect may compromise the role of the transferred enzyme in stimulating egg-laying and delaying receptivity to remating.     

Relative stability of recombinant versus native peroxidases from Phanerochaete chrysosporium.

Two types of glycosylated peroxidases are secreted by the white-rot fungus Phanerochaete chrysosporium, lignin peroxidase (LiP) and manganese peroxidase (MnP). The thermal stabilities of recombinant LiPH2, LiPH8, and MnPH4, which were expressed without glycosylation in Escherichia coli, were lower than those of corresponding native peroxidases isolated from P. chrysosporium. Recovery of thermally inactivated recombinant enzyme activities was higher than with that of the thermally inactivated native peroxidases. Removal of N-linked glycans from native LiPH8 and MnPH4 did not affect enzyme activities or thermal stabilities of the enzymes. Although LiPH2, LiPH8, and MnPH4 contained O-linked glycans, only the O-linked glycans from MnPH4 could be removed by O-glycosidase, and the glycan-depleted MnPH4 exhibited essentially the same activity as nondeglycosylated MnPH4, but thermal stability decreased. Periodate-treated MnPH4 exhibited even lower thermal stability than O-glycosidase treated MnPH4. The role of O-linked glycans in protein stability was also evidenced with LiPH2 and LiPH8. Based on these data, we propose that neither N- nor O-linked glycans are likely to have a direct role in enzyme activity of native LiPH2, LiPH8, and MnPH4 and that only O-linked glycans may play a crucial role in protein stability of native peroxidases.