Anthony L. Tarentino

Enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum.

Publisher Summary This chapter discusses purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum . It describes the purification and properties of two distinct hydrolases with specificity directed at the invariant pentasaccharide core region of asparagine-linked glycans. One of these enzymes,peptide- N- ( N- acetyl- β -glucosaminyl)asparagine amidase (PNGase), is actually an amidase (amidohydrolase) that hydrolyzes the glycosylamine linkage of a wide variety of glycoprotein/glycopeptide substrates, generating an aspartic acid residue at the site of hydrolysis, and liberating 1-amino oligosaccharide. The latter slowly hydrolyzes nonenzymatically to ammonia and an oligosaccharide with a di- N -acetyl chitobiose unit on the reducing end. All oligosaccharide-cleaving enzymes can be assayed, using the fluorescent dansyl (Dns) derivatives of their preferred substrates. Qualitative assays can be followed by simple paper chromatography. Quantitative determination is more readily accomplished by chromatographic separation of deglycosylated product from original substrate in a high-performance liquid chromatographic (HPLC) assay.

Asparagine-linked glycosylation of the scrapie and cellular prion proteins☆

Post-translational modification of the scrapie prion protein (PrP) is thought to account for the unusual features of this protein. Molecular cloning of a PrP cDNA identified two potential Asn-linked glycosylation sites. Both the scrapie (PrPSc) and cellular (PrPC) isoforms were susceptible to digestion by peptide N-glycosidase F (PNGase F) but resistant to endoglycosidase H as measured by migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PNGase F digestion of PrPC yielded two proteins of Mr26K and 28K; however, the 26-k species was only a minor component. In contrast, PNGase F digestion of PrPSc yielded equimolar amounts of two proteins of Mr26K and 28K. The significance of this altered stoichiometry between the 26- and 28-kDa deglycosylated forms of PrP during scrapie infection remains to be established. Both isoforms as well as PrP 27-30, which is produced by limited proteolysis of PrPSc, exhibited a reduced number of charge isomers after PNGase F digestion. The molecular weight of PrP 27-30 was reduced from 27K-30K by PNGase F digestion to 20K-22K while anhydrous hydrogen fluoride or trifluoromethanesulfonic acid treatment reduced the molecular weight to 19K-21K and 20K-22K, respectively. Denatured PrP 27-30 was radioiodinated and then assessed for its binding to lectin columns. PrP 27-30 was bound to wheat germ agglutinin (WGA) or lentil lectins and eluted with N-acetylglucosamine or alpha-methyl-mannoside, respectively. Digestion of PrP 27-30 with sialidase prevented its binding to WGA but enhanced its binding to Ricinus communis lectin. These findings argue that PrP 27-30 probably possesses Asn-linked, complex oligosaccharides with terminal sialic acids, penultimate galactoses, and fucose residues attached to the innermost N-acetyl-glucosamine. Whether differences in Asn-linked oligosaccharide structure between PrPC and PrPSc exist and are responsible for the distinct properties displayed by these two isoforms remain to be established.

A comparison of the substrate specificities of endo-β-N-acetylglucosaminidases from Streptomyces griseus and Diplococcus pneumoniae

Abstract The substrate specificities of the endo-β-N-acetylglucosaminidases from Diplococcus pneumoniae and Streptomyces griseus were compared and found to differ considerably. The enzyme from D. pneumoniae released Asn-GlcNAc-Fuc-containing glycopeptides from exoglycosidase-treated acidic IgM glycopeptides but was limited in its capacity to hydrolyze ovalbumin glycopeptides larger than Asn(GlcNAc)2(Man)5. In contrast, the enzyme from S. griseus hydrolyzed this and larger neutral oligosaccharides but could not hydrolyze the above fucose-containing IgM glycopeptides. Removal of the fucose residue, however, converted the latter to an active substrate for the S. griseus enzyme, thus broadening its substrate range to encompass most of those substrates hydrolyzed by the D. pneumoniae endoglycosidase.

Chapter 5 Enzymatic Approaches for Studying the Structure, Synthesis, and Processing of Glycoproteins

Publisher Summary This chapter highlights the enzymatic approaches for studying the structure, synthesis, and processing of glycoproteins. Enzymes that hydrolyze specifically at the inner-core di- N -acetylchitobiose moiety comprise a broad group of glycosidic enzymes known as “endo- β -N-acetylglucosaminidases,” or more commonly, “endoglycosidases.” Among these, the bacterial enzymes Endo H and Endo F are widely used because of their different substrate specificities and their commercial availability. Protein conformational effects can influence greatly the course and extent of deglycosylation by rendering the susceptible oligosaccharides at certain glycosylation sites partially or completely “inaccessible” to endoglycosidase action. Various denaturants have been employed to expose otherwise inaccessible oligosaccharide cores, including ionic, nonionic, and zwitterionic detergents as well as chaotropic salts and thiols. For the oligosaccharide cleaving enzymes to be effective, they must be used in conjunction with highly sensitive analytical techniques for characterizing the carbohydrate or protein moieties. The simplest method of determining whether deglycosylation has occurred is by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of control and enzyme-treated material. A significant decrease in molecular weight (i.e., increase in electrophoretic mobility) is a good indication that Asn-linked oligosaccharides have been enzymatically released.

Structural characterization of intact, branched oligosaccharides by high performance liquid chromatography and liquid secondary ion mass spectrometry

We report results of a mass-spectrometric-based strategy for determining the detailed structural features of N-linked oligosaccharides from glycoproteins. The method was used to characterize a series of intact, high mannose oligosaccharides isolated from human immunoglobulin M (IgM). The IgM was purified from a patient with Waldenstroms macroglobulinemia. The strategy included releasing the oligosaccharides by digestion of the purified glycoprotein with endoglycosidase H, separating the released oligosaccharides by high resolution gel filtration, and derivatizing the resulting reducing termini with the uv-absorbing moiety, ethyl p-aminobenzoate. This particular derivative facilitates HPLC detection and provides centers for protonation and deprotonation enhancing liquid secondary ion mass spectra. Positive and negative ion spectra contained molecular species of similar abundance. However, fragment ion peaks yielding sequence information were significantly more prominent in the negative ion mass spectra. Furthermore, it was obvious that the fragmentation patterns differed substantially for linear and branched oligomers. For linear oligosaccharides, a smooth envelope of fragment ions was observed; from low to high mass there was an ordered decrease in ion abundance from both the reducing and nonreducing termini. This pattern of fragment ions was not observed for branched oligosaccharides since in these cases fragments at certain masses could not arise by single bond cleavages. Therefore, these fragments were either significantly reduced in abundance or absent as compared with identical fragments formed from linear molecules. Importantly, 200 pmol of an oligosaccharide could be derivatized, separated, and detected by mass spectrometry, allowing identification of previously unreported minor components of the IgM oligosaccharides. Therefore, this experimental strategy is particularly useful for the purification and detailed structural characterization of low abundance oligosaccharides isolated from heterogeneous biological samples.

The purification and properties of a β-aspartyl N-acetylglucosylamine amidohydrolase from hen oviduct

Abstract An enzyme capable of hydrolyzing 1- l -β-aspartamido[-2-acetamido]-1,2-dideoxy β- d -glucose (Asn-GlcNAc) and related glycopeptides, such as those from ovalbumin, ribonuclease B, and transferrin, was purified about 1500-fold from hen oviduct. Substitution of the amino or carboxyl groups of the aspartate moiety prevented the enzyme from hydrolyzing the substrate. The products of the reaction were aspartate, ammonia, and N -acetylglucosamine, with 1-amino- N -acetylglucosamine an apparent intermediate. The molecular weight of the protein, determined by sucrose density gradient centrifugation and gel filtration, fell in the range of 101,000–110,000. The enzyme was irreversibly inhibited by 5-diazo-4-oxo- sl -norvaline (DONV), an asparaginase inhibitor, with a K i of 9.5 × 10 −6 m .

On the nature of α-mannosidase-resistant linkages in glycoproteins

The mannose residue in Asn-(GlcNAc)2(Man)1, a glycosyl-asparagine sequence common to RNase B, ovalbumin, and many other glycoproteins, is resistant to hydrolysis by α-mannosidases from jack bean meal and hen oviduct. This residue is hydrolyzed, however, by a partially purified β-mannosidase fraction from hen oviduct. Similar results were obtained with partially purified α- and β-mannosidases from rat epididymis.

The use of iodinated lectins for determining the degree of deglycosylation of high-mannose glycoproteins by endo-β-N-acetylglucosaminidase H

Abstract A method which demonstrates that the removal of polymannosyl chains from glycoproteins by endo-β- N -acetylglucosaminidase H can be monitored reliably using only submicrogram quantities of glycoprotein is described. Glycoproteins and their endoglycosidase-treated forms are subjected to electrophoresis on SDS-polyacrylamide gels, which are then overlaid with [ 125 I]concanavalin A or [ 125 I]wheat germ agglutinin. The degree to which these lectins bind is measured by autoradiography. The complete loss of [ 125 I]concanavalin A binding by glycoproteins such as deoxyribonuclease I, ovalbumin, carboxypeptidase Y, and invertase is associated with the removal of their oligosaccharide chains. Invertase, unlike the above mannose-containing glycoproteins, acquires the capacity to bind [ 125 I]wheat germ agglutinin only upon partial or complete deglycosylation, a finding substantiated by wheat germ agglutinin-Sepharose column chromatography. In addition to providing a procedure for monitoring the enzymatic deglycosylation of mannose-containing glycoproteins, the lectin-gel binding technique is shown to provide an estimate of the mannose content of neutral glycoproteins at levels which cannot be detected by conventional methods. In some cases, this method may be useful in distinguishing between N - and O -glycosidic linkages where the oligosaccharide is predominantly mannosyl.

Multiple forms of a highly purified β-N-acetylhexosaminidase from hen oviduct

Abstract A β- N -acetylhexosaminidase was purified from hen oviduct to near homogeneity. At least two forms of this enzyme, a major (I) and a minor (II), were shown to exist by polyacrylamide gel electrophoresis and isoelectric focusing. Their molecular weights, as determined by gel electrophoresis, were approximately 118,000 and 158,000. On isoelectric focusing, two peaks of activity were obtained, one at pH 6.45, the other at 6.86. β- N -acetylglucosaminidase could not be separated from β- N -acetylgalactosaminidase activity by any of the procedures employed. Of a number of compounds tested as substrates, there was no difference in the ratio of hydrolysis rates of N -acetylhexosaminidase I–II, except with tri- N -acetylchitotriose and tetra- N -acetylchitotetrose. At a concentration of 6 μ m Hg ++ , the enzyme was completely and selectively inhibited. The inhibition was reversed entirely by thiols. Of about 20 other cations tested, only Ag + inhibited the enzyme, but amounts 200 times greater were required to effect the same degree of inhibition as Hg ++ .

Structure of the oligosaccharide portion of human hepatitis B surface antigen.

The hepatitis B surface antigen, which constitutes the currently available vaccine, is the empty envelope of the hepatitis B virus. We investigated the carbohydrate structures of the envelope glycoproteins. The intact oligosaccharides were enzymatically released from the coat glycoproteins using peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F and isolated by gel permeation chromatography. Cesium ion liquid secondary ion mass spectra of the intact, underivatized oligosaccharides showed molecular weights of 1932, 2078, and 2223. The mixture included partially and totally sialylated structures, a fraction (approximately 8%) of which were substituted with a single terminal fucose residue; no desialylated oligosaccharides were detected. The reducing termini of the oligomers were derivatized by reduction of the Schiff base formed using p-aminobenzoic acid ethyl ester, and fragmentation patterns identical to those produced from standard biantennary complex oligosaccharides were obtained. Methylation linkage analysis of the oligosaccharides showed that the carbohydrate composition and the mannose branching patterns also resembled those of a biantennary oligosaccharide. The results of this study indicate that glycosylation of the hepatitis B surface antigen, which takes place in the liver, is typical of other serum glycoproteins made in the liver; and this analytical strategy, including cesium ion liquid secondary ion mass spectrometry, is an effective approach for the structural analysis of complex carbohydrates available in only the 1-10 micrograms sample size range.