The use of mannan-Sepharose 4B affinity chromatography for the purification of endo-beta-N-acetylglucosaminidase from Bacillus alvei

In order to facilitate the isolation of endo-beta-N-acetylglucosaminidase for the structural analysis of glycoconjugates, we have isolated a strain of Bacillus alvei which produces a high level of endo-beta-N-acetylglucosaminidase. We have also devised a simple procedure for the purification of endo-beta-N-acetylglucosaminidase from B. alvei using mannan-Sepharose affinity chromatography. By using this method, endo-beta-N-acetylglucosaminidase was purified 3300-fold with 85% yield from the crude enzyme obtained by ammonium sulfate precipitation of the culture medium. The molecular weight of this enzyme was estimated to be about 66 000 by gel filtration. When using (Man)6(GlcNAc)2-Asn-Dns as substrate, the optimal activity occurs at pH 6.5 with Km of 1.9 mM. The action of endo-beta-N-acetylglucosaminidase toward several glycopeptides was also studied.     

Purification and properties of an iminopeptidase from culture media of Streptomyces plicatus.

The degradation of the prosequence of the secreted enzyme endo-beta-N-acetylglucosaminidase H from Streptomyces plicatus is not elucidated. Both the primary structure of this segment and the finding that the secreted species contain ragged aminoterminal ends of specific structure suggested that a dipeptidylaminopeptidase might mature this enzyme. Therefore, we tested the culture medium of Streptomyces plicatus for prolin-specific peptidases. Proline iminopeptidase was purified about 800-fold to homogeneity from the culture medium. Dipeptidylaminopeptidase, the enzyme that seemed most likely to process the prosequence of endo-beta-N-acetylglucosaminidase H, could not be detected.     

Complete amino acid sequence of endo-beta-N-acetylglucosaminidase from Flavobacterium sp

The complete amino acid sequence of endo-beta-N-acetylglucosaminidase from Flavobacterium sp. has been determined by analysis of peptides after cleavage with lysyl endopeptidase, pepsin and chymotrypsin. The protein consists of a single polypeptide chain consisting of 267 amino acid residues and a molecular mass of 27972 Da. The sequence of Flavobacterium endo-beta-N-acetylglucosaminidase is very close to that of the Streptomyces enzyme (endo-H), having 60% similarity and very similar hydropathy profiles. Similarities were also found between Flavobacterium endo-beta-N-acetylglucosaminidase and chitinases from Bacillus circulans, Serratia marcescens and Phaseolus vulgaris.     

Identification of an endo-beta-N-acetylglucosaminidase gene in Caenorhabditis elegans and its expression in Escherichia coli

We report the identification, molecular cloning, and characterization of an endo-beta-N-acetylglucosaminidase from the nematode Caenorhabditis elegans. A search of the C. elegans genome database revealed the existence of a gene exhibiting 34% identity to Mucor hiemalis (a fungus) endo-beta-N-acetylglucosaminidase (Endo-M). Actually, the C. elegans extract contained endo-beta-N-acetylglucosaminidase activity. The putative cDNA for the C. elegans endo-beta-N-acetylglucosaminidase (Endo-CE) was amplified by polymerase chain reaction from the Uni-ZAP XR library, cloned, and sequenced. The recombinant Endo-CE expressed in Escherichia coli exhibited substrate specificity mainly for high-mannose type oligosaccharides. Man(8)GlcNAc(2) was the best substrate for Endo-CE, and Man(3)GlcNAc(2) was also hydrolyzed. Biantennary complex type oligosaccharides were poor substrates, and triantennary complex substrates were not hydrolyzed. Its substrate specificity was similar to those of Endo-M and endo-beta-N-acetylglucosaminidase from hen oviduct. Endo-CE was confirmed to exhibit transglycosylation activity, as seen for some microbial endo-beta-N-acetylglucosaminidases. This is the first report of the molecular cloning of an endo-beta-N-acetylglucosaminidase gene from a multicellular organism, which shows the possibility of using this well-characterized nematode as a model system for elucidating the role of this enzyme.     

Induction and Purification of Endo-beta-N-Acetylglucosaminidase from Arthrobacter protophormiae Grown in Ovalbumin

Arthrobacter protophormiae produced a high level of extracellular endo-beta-N-acetylglucosaminidase when cells were grown in a medium containing ovalbumin. The enzyme was induced by the glycopeptide fraction of ovalbumin prepared by pronase digestion. Production of the enzyme was also induced by glycoproteins such as yeast invertase and bovine ribonuclease B but not by monosaccharides such as mannose, N-acetylglucosamine, and galactose. The enzyme was purified to homogeneity as demonstrated by polyacrylamide gel electrophoresis and has an apparent molecular weight of about 80,000. The enzyme showed a broad optimum pH in the range of pH 5.0 to 11.0. The enzyme hydrolyzed all heterogeneous ovalbumin glycopeptides, although the hydrolysis rates for hybrid type glycopeptides were very low. The substrate specificity of A. protophormiae endo-beta-N-acetylglucosaminidase was very similar to that of Endo-C(II) from Clostridium perfringens. Therefore, the enzyme induction by A. protophormiae seems to have a close relation to the substrate specificity of the enzyme.     

Characterization of endo-beta-N-acetylglucosaminidase from alkaliphilic Bacillus halodurans C-125

The genome sequencing project on alkaliphilic Bacillus halodurans C-125 revealed a putative endo-beta-N-acetylglucosaminidase (Endo-BH), which consists of a signal peptide of 24 amino acids, a catalytic region of 634 amino acids exhibiting 50.1% identity with the endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A), and a C-terminal tail of 220 amino acids. Transformed Escherichia coli cells carrying the Endo-BH gene exhibited endo-beta-N-acetylglucosaminidase activity. Recombinant Endo-BH hydrolyzed high-mannose type oligosaccharides and hybrid type oligosaccharides, and showed transglycosylation activity. On deletion of 219 C-terminal amino acid residues of Endo-BH, the wild type level of activity was retained, whereas with deletions of the Endo-A homolog domain, the proteins were expressed as inclusion bodies and these activities were reduced. These results suggest that the enzymatic properties of Endo-BH are similar to those of Endo-A, and that the C-terminal tail does not affect the enzyme activity. Although the C-terminal tail region is not essential for enzyme activity, the sequence is also conserved among endo-beta-N-acetylglucosaminidases of various origins.     

Arylsulfatase A from human tissues contains an endo-beta-N-acetylglucosaminidase F-resistant oligosaccharide

Homogeneous arylsulfatase A from human placenta, liver and urine contains two nonidentical subunits of 59 and 54 kDa. The two subunits are immunologically identical. The relative amount of low molecular weight subunits is only 20-30% of the total enzyme protein. Treatment of the enzyme under various conditions with endo-beta-N-acetylglucosaminidase F results in a decrease in the apparent molecular weight of both subunits by 1-2 kDa. a value that corresponds to the loss of a single N-linked oligosaccharide. However, as judged by carbohydrate staining, endo-beta-N-acetylglucosaminidase F does not remove all carbohydrate from the subunits or from glycopeptides of arylsulfatase A. In contrast, human prostatic acid phosphatase, a glycoprotein with a high content of mannose, hybrid and complex oligosaccharides is completely deglycosylated under identical experimental conditions. Several attempts to deglycosylate arylsulfatase A by chemical methods were unsuccessful due to poor recovery of the protein. From the present studies we conclude that arylsulfatase A contains an endo-beta-N-acetylglucosaminidase F resistant, perhaps O-linked carbohydrate.     

Characterization of the intracellular processing and secretion of hepatic lipase in FU5AH rat hepatoma cells

The processing and secretion of newly synthesized hepatic lipase was characterized in FU5AH rat hepatoma cells. Pulse-chase experiments revealed two immunoreactive species with apparent molecular weights of 55,400 and 57,600. The 55.4 kDa species was detectable only in cell extracts, whereas the 57.6 kDa species was present in both cell extracts and media. Following a 5 min pulse with L-[35S]methionine and a 10 min chase, these two species represented only 0.003% of the total labelled protein. Quantitation of the 55.4 kDa and 57.6 kDa species in a chase time course taken together with their respective sensitivity and resistance to digestion with endo-beta-N-acetylglucosaminidase H indicates that the 55.4 kDa species is a high mannose precursor to the mature 57.6 kDa enzyme which contains only complex N-linked oligosaccharides. From a time course of endo-beta-N-acetylglucosaminidase H digestion, it was determined that hepatic lipase contains a minimum of two N-linked oligosaccharides. Treatment of the 55.4 kDa species with endo-beta-N-acetylglucosaminidase H yields a protein with a kDa value similar to that observed after treatment of the mature secreted enzyme with endo-beta-N-acetylglucosaminidase F or trifluoromethanesulfonic acid. Therefore, processing of N-linked oligosaccharides is probably the only post-translational modification responsible for the observed change in the apparent molecular weight of hepatic lipase. The half-residence times of hepatic lipase in the endoplasmic reticulum-cis Golgi region and in the cell were estimated at 34 min and 57 min, respectively. Newly synthesized hepatic lipase in Fu5AH cells is secreted constitutively and is not stored in an intracellular pool. Finally, little of the newly synthesized enzyme is degraded during the course of a 1 h chase.     

Determination of glycosylation sites using a protein sequencer and deglycosylation of native yeast invertase by endo-beta-N-acetylglucosaminidase.

Endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae was tested for its capacity to release N-linked sugar chains from native yeast invertase. The enzyme liberated about 80% of the sugar chains from the native invertase. Deglycosylated invertase was digested by chymotrypsin or pepsin, and twelve N-acetylglucosamine-containing glycopeptides were isolated. The amino acid sequences of these glycopeptides were analyzed by a protein sequencer, and the elution position of 4-L-aspartylglycosylamine was directly identified by conventional sequencing. The endo-beta-N-acetylglucosaminidase was found to remove mainly nine sugar chains from native invertase.     

Biosynthesis of intestinal microvillar proteins. The effect of swainsonine on post-translational processing of aminopeptidase N.

The post-translational processing of pig small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in organ-cultured mucosal explants. Exposure of the explants to swainsonine, an inhibitor of Golgi mannosidase II, resulted in the formation of a Mr-160000 polypeptide, still sensitive to endo-beta-N-acetylglucosaminidase H. Swainsonine caused only a moderate inhibition of transport of the enzyme through the Golgi complex and the subsequent expression in the microvillar membrane. This may imply that the trimming of the high-mannose core and complex glycosylation of N-linked oligosaccharides is not essential for the transport of aminopeptidase N to its final destination. A different type of processing was observed to take place in the presence of swainsonine, resulting in a considerable increase in apparent Mr (from 140000 to 160000). This processing could not be ascribed to N-linked glycosylation, since treatment of the Mr-160000 polypeptide with endo-beta-N-acetylglucosaminidase H only decreased its apparent Mr by 15000. The susceptibility of the mature Mr-166000 polypeptide, but not the Mr-140000 polypeptide, to mild alkaline hydrolysis suggests that aminopeptidase N becomes glycosylated with O-linked oligosaccharides during its passage through the Golgi complex. Aminopeptidase N was not labelled by [3H]palmitic acid, indicating that the processing of the enzyme does not include acylation.     

Identification of amino acid residues essential for the substrate specificity of Flavobacterium sp. endo-beta-N-acetylglucosaminidase

The gene encoding the endo-beta-N-acetylglucosaminidase from Flavobacterium sp. (Endo-Fsp) was sequenced. The Endo-Fsp gene was overexpressed in Escherichia coli cells, and was purified from inclusion bodies after denaturation by 8 M urea. The renatured Endo-Fsp had the same optimum pH and substrate specificity as the native enzyme. Endo-Fsp had 60% sequence identity with the endo-beta-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H), and the putative catalytic residues were conserved. Site-directed mutagenesis was done at conserved residues based on the three-dimensional structure and mutagenesis of Endo-H. The mutant of Glu-128, corresponding to Glu-132 in Endo-H and identified as an active site residue, was inactivated. Mutagenesis around the predicted active site of Endo-Fsp reduced the enzymatic activity. Moreover, the hydrolytic activity toward hybrid-type oligosaccharides was decreased compared to that toward high-mannose type oligosaccharides by mutagenesis of Asp-126 and Asp-127. Therefore, site-directed mutagenesis of some of these conserved residues indicates that the predicted active sites are essential to the enzymatic activity of Endo-Fsp, and may have similar roles in catalysis as their counterparts in Endo-H.     

Purification of a 51 kDa endo-β-N-acetylglucosaminidase from Staphylococcus aureus

A bacteriolytic enzyme obtained from the culture fluid of Staphylococcus aureus FDA 209P was purified to homogeneity utilizing dye-ligand affinity column chromatography, hydrophobic interaction high pressure liquid chromatography (HPLC) and hydroxyapatite HPLC. Subsequent characterizations indicated that the purified enzyme acted as endo-beta-N-acetylglucosaminidase. The molecular weight determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was 51,000 and the isoelectric point was higher than 10. The optimum pH for the enzyme activity on whole cells of Micrococcus luteus as a substrate was 8.0. Some heavy metal cations (Cu2+ and Zn2+) inhibited the enzyme activity at a concentration of 0.1 mM and others (Ba2+, Mg2+ and Co2+) showed a stimulating effect at a concentration of 1 mM.     

Purification of S1 nuclease to homogeneity and its chemical, physical and catalytic properties

An S1 nuclease preparation was used to purify the enzyme to homogeneity. The enzyme had an isoelectric point of 4.2, and a high content of hydrophobic amino acids, especially tyrosine. It exhibited low 3'-ribonucleotidase activity. Circular dichroism analysis suggested that the contents of alpha-helix, beta-structure and random coil are 25%, 31% and 44%, respectively. The enzyme contained about 3 g atoms Zn/mol and the removal of Zn from the enzyme by addition of EDTA resulted in disruption of its secondary structure with resultant inactivation. From Con A-Sepharose chromatography, we suggest that the enzyme is a high-mannose glycoprotein. After treatment with endo-beta-N-acetylglucosaminidase H under moderate conditions, a small part of the enzyme was converted to a form lacking the sugar side chain. This form of the enzyme was as thermostable as the parent enzyme, suggesting that the sugar side chain may not be involved in thermostability of the enzyme.     

Structural and functional characterization of ovotransferrin produced by Pichia pastoris

Ovotransferrin is an egg white protein with complex disulfide and bilobal structures, which is derived from the same gene as chicken serum transferrin. We demonstrate here the structural and functional characteristics of bilobal ovotransferrin, produced at a high level using Pichia pastoris expression system. The recombinant protein was secreted into the medium, and the secretion signal peptide was processed correctly. The secretion level was almost 100 mg/l culture and the yield after purification by two-step anion exchange chromatography was 57 mg/l. The CD spectrum and fluorescence spectra indicate the correct folding of the recombinant protein. The analyses for the Fe3+ binding ability by urea-PAGE and visible absorption spectrum revealed that two Fe3+ sites exist in a recombinant ovotransferrin molecule as in the egg white protein. Endoglycosidases, such as endo-beta-N-acetylglucosaminidase H (Endo-H), peptide:N-glycosidase F (PNGaseF), and endo-beta-N-acetylglucosaminidase from Mucor hiemalis, showed differential activities for the native Fe3+-loaded, native Fe3+-free, and denatured forms of recombinant ovotransferrin; only the first enzyme displayed the cleavage ability for all the ovotransferrin forms. The results from the enzyme specificity and from the molecular weight difference for the intact and deglycosylated proteins were consistent with the view that recombinant ovotransferrin have one N-linked carbohydrate chain which mainly consists of two GlcNac and 10 mannoses.     

Purification and properties of endo-beta-N-acetylglucosaminidase L from Streptomyces plicatus.

An enzyme, previously described as endo-beta-N-acetylglucosaminidase L (Tarentino, A.L., and Maley, F. (1974) J. Biol. Chem. 249, 811-817) because of its apparent specificity for Man(GlcNAc)2Asn, has been purified to homogeneity. The enzyme has now been found to hydrolyze (GlcNAc)3 to (GlcNAc)2 plus GlcNAc, and (GlcNAc)4 to 2(GlcNAc)2, at twice the rate observed for Man(GlcNAc)2Asn. Removal of the asparagine from the latter compound reduces the rate of hydrolysis by about 30-fold. Reduction of (GlcNAc)3 to GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc-ol eliminates this compound as a substrate for endo-beta-N-acetylglucosaminidase L. However, the reduction of (GlcNAc)4 does not affect its rate of hydrolysis. Endo-beta-N-acetylglucosaminidase L consists of a single polypeptide chain with a molecular weight of 49,500 +/- 400, which on isoelectric focusing separates into two closely migrating bands; a major with a pI of 4.25 and a minor one with a pI 4.20. Both bands possess similar enzyme activities and amino acid compositions, but differ slightly in their tryptic peptide maps.     

Purification,characterization, and molecular cloning of a novel keratan sulfate hydrolase,endo-beta-N-acetylglucosaminidase,from Bacillus circulans

Keratan sulfate (KS) is degraded by various enzymes including endo-beta-galactosidase, keratanase, and keratanase II, which are used for the structural analysis of KS. We purified a novel KS hydrolase, endo-beta-N-acetylglucosaminidase, from the cell pellet and conditioned medium of Bacillus circulans, by sequential chromatography using DE52 and phenyl-Sepharose columns with approximately 63- and 180-fold purity and 58 and 12.5% recovery, respectively. Like keratanase II of Bacillus sp. Ks36, the enzyme, designated Bc keratanase II, hydrolyzed KS between the 4GlcNAcbeta1-3Gal1 structure (endo-beta-N-acetylglucosaminidase), but not hyaluronan, heparan sulfate, heparin, and chondroitin sulfate C, demonstrating a strict specificity to KS. The enzyme digested shark cartilage KS to disaccharides and tetrasaccharides and bovine cornea KS to hexasaccharide, indicating that it prefers highly sulfated KS. Distinct from keratanase II of strain Ks36, the enzyme digested shark cartilage KS at an optimal temperature of 55 degrees C. Based on partial peptide sequencing of the enzyme, we molecularly cloned the gene, which encodes a protein with a predicted molecular mass of approximately 200 kDa. From the deduced protein sequence, Bc keratanase II contained a domain at the C terminus, homologous to the S-layer-like domain of pullulanase from Thermoanaerobacterium thermosulfurigenes and endoxylanase from Thermoanaerobacterium saccharolyticum, and a carbohydrate-binding domain, which may serve to specifically recognize KS chains. A full-length recombinant enzyme showed keratanase II activity. These results may prove useful for the structural analysis of KS toward achieving an understanding of its function.     

Synthesis of neoglycoenzymes with homogeneous N-linked oligosaccharides using immobilized endo-beta-N-acetylglucosaminidase A

A procedure for the enzymatic synthesis of neoglycoenzymes is described. The gene encoding endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was overexpressed in Escherichia coli as a fusion protein linked to glutathione S-transferase (GST). GST-Endo-A fusion was extracted as a soluble protein. The fusion protein was purified to homogeneity with glutathione-Sepharose 4B and showed transglycosylation activity toward high-mannose-type glycopeptides without removing the GST moiety. The GST-Endo-A immobilized on glutathione-Sepharose 4B retained its transglycosylation activity. The immobilized enzyme could transfer (Man)(6)GlcNAc en bloc to partially deglycosylated ribonuclease B without damaging its enzyme activity. The immobilized GST-Endo-A should be very useful for synthesizing active neoglycoenzymes attached with homogeneous N-linked oligosaccharides.     

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.     

Arylsulfatase a from normal human lung and lung tumors showed different patterns of microheterogeneity

Arylsulfatase A was purified from human lung to apparent homogeneity as determined by electrophoresis in the presence of sodium dodecyl sulfate. The enzyme from normal lung as well as that from lung adenocarcinoma showed considerable microheterogeneity when examined by isoelectric focussing, with an isoelectric point (pI) ranging from 5.1 to 4.6. The tumor enzyme was more heterogeneous and contained more acidic components than the normal lung enzyme. The cause of the charge heterogeneity was examined by treatment with exogenous hydrolases. Upon treatment with sialidase, phosphatase or endo-beta-N-acetylglucosaminidase H (endoglycosidase H), the acidic enzyme forms shifted to an alkaline region on isoelectric focussing gels. Combined treatment of the arylsulfatase A with endoglycosidase H and sialidase resulted in complete loss of the most acidic components to give the less acidic components with pI 5.1, 5.0, and 4.9. These results strongly suggest that the charge heterogeneity of arylsulfatase A is due not only to sialylation but also to phosphorylation at the carbohydrate moiety of the enzyme, and the extent of substitution by acidic groups is markedly increased in the tumor enzyme.     

Further studies on endo-beta-N-acetylglucosaminidase D1.

The purification procedure for endo-beta-N-acetylglucosaminidase D was improved to yield an enzyme preparation which was homogeneous upon gel electrophoresis. The molecular weight of the enzyme as estimated by Sephadex G-200 column chromatography was 280,000, while SDS-gel electrophoresis after reduction with 2-mercaptoethanol gave a value of 150,000. The purified enzyme did not show any chitinase, hyaluronidase or lysozyme activity. In the presence of exoglycosidases removing peripheral sugars, the endoglycosidase acted on serum glycoproteins such as transferrin and fetuin. The enzyme also hydrolyzed an oligosaccharide, (Man)5(GlcNAc)2, indicating that the peptide portion of substrates does not have much effect on susceptibility to the enzyme.