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

doi:10.1016/0006-291X(77)90512-5

Biochemical and Biophysical Research Communications

Volume 78, Issue 3, 10 October 1977, Pages 935-944

https://doi.org/10.1016/0006-291X(77)90512-5 Get rights and content

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.

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Glycans in breast milk are abundant and found as either free oligosaccharides or conjugated to proteins and lipids. Free human milk oligosaccharides (HMOs) function as prebiotics by stimulating the growth of beneficial bacteria while preventing the binding of harmful bacteria to intestinal epithelial cells. Bacteria have adapted to the glycan-rich environment of the gut by developing enzymes that catabolize glycans. The decrease in HMOs and the increase in glycan digestion products give indications of the active enzymes in the microbial population. In this study, we quantitated the disappearance of intact HMOs and characterized the glycan digestion products in the gut that are produced by the action of microbial enzymes on HMOs and glycoconjugates from breast milk. Oligosaccharides from fecal samples of exclusively breast-fed infants were extracted and profiled using nanoLC-MS. Intact HMOs were found in the fecal samples, additionally, other oligosaccharides were found corresponding to degraded HMOs and non-HMO based compounds. The latter compounds were fragments of N-glycans released through the cleavage of the linkage to the asparagine residue and through cleavage of the chitobiose core of the N-glycan. Marker gene sequencing of the fecal samples revealed bifidobacteria as the dominant inhabitants of the infant gastrointestinal tracts. A glycosidase from Bifidobacterium longum subsp. longum was then expressed to digest HMOs in vitro, which showed that the digested oligosaccharides in feces corresponded to the action of glycosidases on HMOs. Similar expression of endoglycosidases also showed that N-glycans were released by bacterial enzymes. Although bifidobacteria may dominate the gut, it is possible that specific minority species are also responsible for the major products observed in feces. Nonetheless, the enzymatic activity correlated well with the known glycosidases in the respective bacteria, suggesting a direct relationship between microbial abundances and catabolic activity.
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The enzyme did not recognize O-linked glycans or HMO (data not shown). In general the specificity of most known endoglycosidases is limited to high mannose glycans (for example EndoH (23)). EndoS from S. pyogenes acts solely on IgG (25), and the affinities of EndoEα from E. faecalis for proteins other than RNaseB have not been further studied (24).
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Alkaline treatment has contrasting effects on the structure of deglycosylated and glycosylated forms of glucose oxidase
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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.
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Single-strand-specific nucleases are multifunctional enzymes and widespread in distribution. Their ability to act selectively on single-stranded nucleic acids and single-stranded regions in double-stranded nucleic acids has led to their extensive application as probes for the structural determination of nucleic acids. Intracellularly, they have been implicated in recombination, repair and replication, whereas extracellular enzymes have a role in nutrition. Although more than 30 single-strand-specific nucleases from various sources have been isolated till now, only a few enzymes (S1 nuclease from Aspergillus oryzae, P1 nuclease from Penicillium citrinum and nucleases from Alteromonas espejiana, Neurospora crassa, Ustilago maydis and mung bean) have been characterized to a significant extent. Recently, some of these enzymes have been cloned, their crystal structures solved and their interactions with different substrates have been established. The detection, purification, characteristics, structure–function correlations, biological role and applications of single-strand-specific nucleases are reviewed.
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Biochemical and Biophysical Research Communications

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

Abstract

J. Biol. Chem

J. Biol. Chem

Arch. Biochem. Biophys

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Anal. Biochem

J. Biol. Chem

Identification of Oligosaccharides in Feces of Breast-fed Infants and Their Correlation with the Gut Microbial Community

Carboxypeptidase C including Carboxypeptidase Y

Endo-β-N-acetylglucosaminidases from infant gut-associated bifidobacteria release complex N-glycans from human milk glycoproteins

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

Single-strand-specific nucleases

The major exoglucanase secreted by Saccharomyces cerevisiae as a model to study protein glycosylation