Robert B. Trimble

Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-β-N-acetylglucosaminidase H

The ability of endo-beta-acetylglucosaminidase H (Endo H) from Streptomyces plicatus to hydrolyze high-mannose oligosaccharides from glycoproteins is influenced by numerous factors, including the tertiary structure of the substrate glycoproteins, the amount of Endo H used, the time of incubation, and the presence or absence of reagents that affect protein configuration. Endo H levels below 10 to 20 milliunits/ml may incompletely hydrolyze oligosaccharides, regardless of the incubation time, because even though the enzyme remains active, it becomes trapped or sequestered and is unavailable. Endo H activity can be potentiated by first denaturing substrate glycoproteins in a 1.2-fold weight excess of sodium dodecyl sulfate prior to hydrolysis. However, low levels of Endo H are sensitive to inactivation by sodium dodecyl sulfate, with considerable activity being lost over 4 h when the unbound detergent concentration exceeds protein by 0.02% (0.2 mg/ml). Other denaturants such as the Tritons, the zwittergents, the Brij series, or octylglucoside do not enhance or inhibit Endo H removal of oligosaccharides, but the chaotropic salt sodium thiocyanate at 0.5 M enhances Endo H action on some glycoproteins, particularly bovine thyroglobulin. Under denaturing conditions, proteolytic contaminants are a potential problem. Addition of 1 mM phenylmethylsulfonyl fluoride to Endo H incubations completely inhibits the residual Endo H-associated protease(s). Furthermore, Endo H is unaffected by a wide range of proteolytic inhibitors that may be used to protect substrate glycoproteins.

The use of endo-β-N-acetylglucosaminidase H in characterizing the structure and function of glycoproteins

Abstract Endo-β-N-acetylglucosaminidase H from Streptomyces plicatus can be useful in determining both the molecular weight of the protein moiety of glycoproteins and their inherent number of oligosaccharide chains. In the case of carboxypeptidase Y the molecular mass of the carbohydrate free protein was confirmed as 51,000 daltons. The native enzyme was shown to contain 4 oligosaccharide chains each averaging about 14 mannose residues. On treatment of mung bean nuclease I with the endoglycosidase, the molecular mass decreased from 39,000 to 31,000 daltons. The peptides produced on reduction of this enzyme with thiol were 18,700 and 12,500 daltons, indicating that carbohydrate had been present on both. Penicillium nuclease P1 was decreased in size from 40,000 to 30,000 daltons by the endoglycosidase. Although most of the carbohydrate was removed from each of the native enzymes by the endoglycosidase, denaturation of the glycoproteins was necessary to effect complete removal. Enzyme activitywas not affected by carbohydrate depletion of these glycoproteins, a result consistent with similar studies on other oligosaccharide-containing enzymes.

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.

The Accumulation of Man6GlcNAc2-PP-dolichol in theSaccharomyces cerevisiae Δalg9 Mutant Reveals a Regulatory Role for the Alg3p α1,3-Man Middle-arm Addition in Downstream Oligosaccharide-lipid and Glycoprotein Glycan Processing

N-Glycans in nearly all eukaryotes are derived by transfer of a precursor Glc3Man9GlcNAc2 from dolichol (Dol) to consensus Asn residues in nascent proteins in the endoplasmic reticulum. The Saccharomyces cerevisiae alg(asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide-lipid properly, and thealg9 mutant, accumulates Man6GlcNAc2-PP-Dol. High-field 1H NMR and methylation analyses of Man6GlcNAc2released with peptide-N-glycosidase F from invertase secreted by Δalg9 yeast showed its structure to be Manα1,2Manα1,2Manα1,3(Manα1,3Manα1,6)-Manβ1,4GlcNAcβ1,4GlcNAcα/β, confirming the addition of the α1,3-linked Man to Man5GlcNAc2-PP-Dol prior to the addition of the final upper-arm α1,6-linked Man. This Man6GlcNAc2 is the endoglycosidase H-sensitive product of the Alg3p step. The Δalg9Hex7–10GlcNAc2 elongation intermediates were released from invertase and similarly analyzed. When compared withalg3 sec18 and wild-type core mannans, Δalg9 N-glycans reveal a regulatory role for the Alg3p-dependent α1,3-linked Man in subsequent oligosaccharide-lipid and glycoprotein glycan maturation. The presence of this Man appears to provide structural information potentiating the downstream action of the endoplasmic reticulum glucosyltransferases Alg6p, Alg8p and Alg10p, glucosidases Gls1p and Gls2p, and the Golgi Och1p outerchain α1,6-Man branch-initiating mannosyltransferase.

Separation of high-mannose isomers from yeast and mammalian sources using high-pH anion-exchange chromatography

The biosynthesis of oligosaccharides N-linked at AsnXxxThr(Ser) sequons of glycoproteins is initiated by the co-translational transfer of a Glc,Man,GlcNAc, from dolichyl pyrophosphate’. The three glucose residues are rapidly removed by two glucosidases, one a( I+ 2)and the other a( 1 +3)-specific’. These first steps are identical in yeast and in man, and likely to be the same in plants’? however, the subsequent removal and addition of the peripheral mannoses are very different. The array of oligo-mannosyl structures which eventually occupy each glycosylation site are a function, in part, of the concerted action of mannosidases and mannosyl transferases on the archetypal Man,GlcNAc, structure’. These enzymes often display specificity for not only the type of residue and anomeric linkage, but also for its branch location. For example, a yeast mannosidase removes a single (l-+2)-x-linked mannose from a specific branch of the Man,GlcNAcz oligosaccharide4,‘. A yeast (1+6)-a-mannosyl transferase places a single mannose on one specific branch which is essential for elongation reaction?. Trimming by at least three mammalian mannosidases accounts for the heterogeneity of high-mannose structures at individual glycosylation sites (discussed in ref. 7). Thus, chromatographic methods which can separate not only by size and ring substitutions (linkage), but also according to branch isomerism, are required to understand the structural glycobiology of iv-linked oligosaccharide biosynthesis. High-pH anion-exchange chromatography (h.p.a.e.c.) has been shown to separate many oligosaccharide isomers (both neutral and charged) which differ only in a single linkagex~‘4~‘7). H owever, certain isomers were difficult to separate using

Characterization of cellular oligosaccharides from normal and cystic fibrotic fibroblasts using sequential endoglycosidase digestions

A method was developed for obtaining detailed oligosaccharide profiles from [2-3H]mannose- or [6-3H]fucose-labeled cellular glycoproteins. The oligosaccharides were segregated first according to class, using endo-beta-N-acetylglucosaminidase H (Endo H) to release the high mannose species, and then with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F), which provided a complete array of complex oligosaccharide chains. The high mannose and complex oligosaccharides were fractionated subsequently according to net negative charge on QAE-Sephadex. High resolution gel filtration on TSK HW-40(S) resolved the neutral high mannose population into species of the type Man9-5 N-acetylglucosamine. Desialylation of the complex chains with neuraminidase allowed resolution of these oligosaccharides into their corresponding asialo bi-, tri-, and tetraantennary species. Fibroblasts from normal and cystic fibrosis cells were analyzed for differences in their glycosylation patterns using these techniques. Over 95% of the [2-3H]mannose-labeled glycoproteins were susceptible to the combined glycosidase digestions, but no difference in either the high mannose or complex oligosaccharides were observed. Nonetheless, the methodology developed in this study provides an important new approach for investigating oligosaccharides of different cell types and variants of the same type. Metabolic changes induced in cellular glycoproteins, as illustrated by use of the processing inhibitor swainsonine, demonstrated the versatility of this procedure for investigating questions relating to glycoprotein structure and enzyme specificity. Thus, by employing a variation of this method, it was possible to confirm the location of fucose in the core of PNGase F-released hybrid oligosaccharides by the subsequent release with Endo H of the disaccharide, fucosyl-N-acetylglucosamine.

[62] Endo-β-N-acetylglucosaminidase H from Streptomyces plicatus

Publisher Summary Endo-β- N -acetylglucosaminidase H (endo H) offers great utility in studies on the biosynthesis of glycoproteins and the relationship of oligosaccharides to function of specific glycoproteins. Demand for this endoglycosidase has escalated markedly. This chapter describes the materials and methods used for the cloning procedure of the gene for Endo H adjacent to the λP L promoter of the plasmid vector pKC30 3 . The construction of plasmid has been described, which was used in the purification process. There is a detailed, step-by-step procedure of purification of endo H.

In vitro synthesis of deoxynucleotide kinase, dihydrofolate reductase and deoxycytidylate hydroxymethylase from RNA transcripts of T2 phage DNA

Abstract RNA transcripts of specified lengths were isolated from an in vitro system containing T2 phage DNA and E. coli RNA polymerase. The informational content of these RNA segments was monitored for deoxynucleotide kinase, dihydrofolate reductase and deoxycytidylate hydroxymethylase with a cell-free protein synthesizing system from E. coli . All three enzymes were translated from RNA chains of 2,300 nucleotides, but deoxycytidylate hydroxymethylase appeared not to be translated from chains of 1,500 nucleotides. Only deoxynucleotide kinase synthesis was directed by transcripts 700–900 nucleotides long. Based on the recently reported subunit molecular weight of this enzyme (1), the gene for deoxynucleotide kinase would appear to be located adjacent to its promoter.

[56] Endo-β-N-acetylglucosaminidase L from Streptomyces plicatus

Publisher Summary This chapter presents the procedure for purification and assay of endo-β-N acetylglucosaminidase L from Streptomyces plicatus. For purification, Streptomyces plicatus is grown at 30 ° on particulate chitin in l-liter batches as described previously for the purification of Endo-H. Eighteen liters of culture media are filtered to yield 15 liters of clear yellow fluid, containing about 1.2 mg of protein and 11 milliunits of Endo-L/ml. The steps involve in the purification are following: zinc acetate and ammonium sulfate precipitations; pH4.2 precipitation; agarose Chromatography; sulfopropyl-Sephadex C-25 chromatography; phenyl-Sepharose CL-4B chromatography; and sulfopropyl-Sephadex C-25 chromatography. Endo-L is obtained in a 40% yield with an overall purification of 100-fold. On electrophoresis in nondenaturing Tris-glycine acrylamide gel or SDS slab gel electrophoresis, a single band is evident at step, however, when the combined protein at this step is subjected to thin-layer isolectric focusing, two species of enzyme are resolved, one with an isolectric point at 4.20, and the other at 4.25. The assay is based on the release of [ 3 H]dansyl-AsnGlcNAc from the substrate [ 3 H]dansyl-Asn(GlcNAc)2Man. The substrate and products are separated by paper chromatography in 1-butanol, ethanol, water and located by their fluorescence under UV light. [ 3 H]Dansyl- AsnGlcNAc is eluted and quantitated by liquid scintillation spectrometry.

The influence of potassium and rifampicin on the expression of bacteriophage T2 prereplicative genes in vitro and in vivo

Abstract The transcription of specific mRNA from T2 phage DNA by purified Escherichia coli RNA polymerase was markedly affected by the concentration of KCl. Thus the genes for dihydrofolate reductase and deoxynucleotide kinase were transcribed at 50 m m KCl, but at 200 m m KCl the synthesis of the kinase mRNA was nearly eliminated and that of the reductase was increased slightly. Rifampicin-resistant initiation complexes formed at 200 m m KCl failed to produce appreciable kinase mRNA despite reduction of the KCl concentration to 50 m m following initiation of RNA chain elongation. This result suggests that the RNA polymerase holoenzyme does not bind to deoxynucleotide kinase promoters in the presence of 200 m m KCl in a manner capable of initiating synthesis of this message. Initiation complex formation at 50 m m KCl, however, permitted the synthesis of deoxynucleotide kinase mRNA at 200 m m KCl indicating that once formed the initiation complex is capable of producing message even at high salt. The KCl effect on gene expression was reproduced in a coupled E. coli cell-free system directed by T2 DNA. However, in this case the inhibition of protein synthesis at high concentrations of KCl was due primarily to the chloride ions and could be alleviated by substituting acetate for chloride. The synthesis of mRNA in the coupled system was similar with either anion. Analysis of proteins synthesized in the coupled system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, like deoxynucleotide kinase, the synthesis of several additional gene products was influenced by the concentration of KCl. The genes coding for these products, including deoxynucleotide kinase, belong to a class whose transcription is reduced or eliminated in vivo on addition of rifampicin at 1 min postinfection and are referred to as “quasilate.” Evidence is presented to indicate that the mRNAs for deoxynucleotide kinase, dihydrofolate reductase, and deoxycytidylate deaminase are produced in excess of the levels needed for optimal synthesis of these enzymes during normal infection.