Requirements of cleavage of high mannose oligosaccharides in glycoproteins by peptide N-glycosidase F.

Chu, Frederick K.

doi:10.1016/s0021-9258(17)42448-3

Cited by 123 publications

(10 citation statements)

References 25 publications

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“… 32 , 35 , 48 A possible reason for these discrepancies could be that the use of PNGase F in these studies, used to remove N-linked glycans, would not remove residual HexNAc(1) remaining from N-linked glycan degradation. 49 , 50 Unambiguous assignment of an O-linked HexNAc(1) in proximity to N-glycan sequons requires a parallel search for N-linked modifications and good fragmentation of the N sequon; otherwise, software pipelines will assign false O-linked glycan sites ( Figure 3 ).…”

Section: Results and Discussionmentioning

confidence: 99%

Suppression of O-Linked Glycosylation of the SARS-CoV-2 Spike by Quaternary Structural Restraints

et al. 2021

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Understanding the glycosylation of the envelope spike (S) protein of SARS-CoV-2 is important in defining the antigenic surface of this key viral target. However, the underlying protein architecture may significantly influence glycan occupancy and processing. There is, therefore, potential for different recombinant fragments of S protein to display divergent glycosylation. Here, we show that the receptor binding domain (RBD), when expressed as a monomer, exhibits O-linked glycosylation, which is not recapitulated in the native-like soluble trimeric protein. We unambiguously assign O-linked glycosylation by homogenizing N-linked glycosylation using the enzymatic inhibitor, kifunensine, and then analyzing the resulting structures by electron-transfer higher-energy collision dissociation (EThcD) in an Orbitrap Eclipse Tribrid instrument. In the native-like trimer, we observe a single unambiguous O-linked glycan at T323, which displays very low occupancy. In contrast, several sites of O-linked glycosylation can be identified when RBD is expressed as a monomer, with T323 being almost completely occupied. We ascribe this effect to the relaxation of steric restraints arising from quaternary protein architecture. Our analytical approach has also highlighted that fragmentation ions arising from trace levels of truncated N-linked glycans can be misassigned as proximal putative O-linked glycan structures, particularly where a paucity of diagnostic fragments were obtained. Overall, we show that in matched expression systems the quaternary protein architecture limits O-linked glycosylation of the spike protein.

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Section: Results and Discussionmentioning

confidence: 99%

Suppression of O-Linked Glycosylation of the SARS-CoV-2 Spike by Quaternary Structural Restraints

et al. 2021

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“…Purified gp55 was digested by N-GLYase, an endoglycosidase that catalyzes the hydrolysis of N-linked oligosaccharides (high-mannose, complex, and hybrid) at the β-aspartylglycosylamine bond between the innermost GlcNAc and the Asn residue (converting Asn to Asp; Tarentino et al, 1985;Chu, 1986). Removal of N-linked oligosaccharides from gp55 by N-GLYase reduced its average M r to ∼21 000 and increased the pI of both major isoforms to ∼5.3 (Figure 4), but did not significantly affect its ability to inhibit binding of sperm to eggs or to induce the acrosome reaction (Figure 5).…”

Section: Electrophoretic Analysis and Bioactivity Assays Of Gp55 Trea...mentioning

confidence: 99%

Characterization of a Mouse ZP3-Derived Glycopeptide, gp55, That Exhibits Sperm Receptor and Acrosome Reaction-Inducing Activityin Vitro

Litscher

Wassarman

1996

Biochemistry

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During fertilization, free-swimming mouse sperm bind to mZP3 (approximately 83 000 Mr), one of three zona pellucida glycoproteins, and once bound undergo the acrosome reaction, a type of cellular exocytosis [Wassarman, P. M., & Litscher, E. S. (1995) Curr. Top. Dev. Biol. 30, 1-19]. Sperm recognize and bind to specific serine/threonine-linked oligosaccharides located at the mZP3 combining site for sperm. Here, we examined certain characteristics of gp55, a approximately 55 000 Mr glycopeptide derived from the carboxy-terminal half of mZP3 polypeptide to which sperm bind [Rosiere, T. K., & Wassarman, P. M. (1992) Dev. Biol. 154, 309-317]. gp55 is heterogeneous with respect to Mr (approximately 47 000-62 000 Mr) and has a relatively low pI (approximately 4.3-4.5) compared to the polypeptide portion of the glycopeptide (pI approximately 6.5). gp55 inhibits binding of sperm to eggs (i.e., exhibits sperm receptor activity) and induces sperm to undergo the acrosome reaction in vitro at about the same concentrations required for intact mZP3 (approximately 50-200 nM). Each of three different size-fractions of gp55, separated by SDS-PAGE, also exhibits bioactivity in vitro. Removal of asparagine-linked (N-linked) oligosaccharides from gp55, by extensive digestion with N-glycanase, reduces its Mr to approximately 21 000 and increases it pI to approximately 5.3, but does not significantly affect its ability to inhibit binding of sperm to eggs or to induce sperm to undergo the acrosome reaction. Similarly, digestion of gp55 with either endo-beta-galactosidase or neuraminidase alters its Mr and/or pI, but does not significantly affect either of its bioactivities. These observations are consistent with the proposal that neither N-linked oligosaccharides nor sialic acid is an essential element of the mZP3 combining site for sperm. They also indicate that a relatively small mZP3 glycopeptide is able to induce sperm to undergo the acrosome reaction (i.e., cellular exocytosis) in vitro.

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“…And thirdly, the exogalactosylated monosaccharide bound to Na pump -subunit was insensitive to ^-elimination but it was released by PNGase. This enzyme specifically cleaves oligosaccharides at the asparaginyloligosaccharide linkage: Asn-GlcNAc bond (Plummer & Tarantino, 1981;Tarantino et al, 1985;Chu, 1986). Therefore, the specificity of this enzyme also points to a GlcNAc molecule as the monosaccharide bound to the -subunit.…”

Section: Discussionmentioning

confidence: 99%

A monosaccharide is bound to the sodium-pump .alpha.-subunit

Pedemonte

Kaplan²

1992

Biochemistry

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We have recently reported that the Na pump alpha-subunit has cytosolic-oriented oligosaccharides which were sensitive to cleavage by an enzyme specific for hydrolysis of N-linked glycans [Pedemonte et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9789-9793]. We now describe experiments that characterize the saccharides and further substantiate our previous findings. Bovine milk galactosyltransferase has been used in conjunction with radiolabeled UDP-galactose to label N-acetylglucosamine residues on the protein. The Na pump alpha-subunit contains some O-linked carbohydrates; however, the bulk (> 80%) of the radioactivity was found in oligosaccharides sensitive to peptide:N-glycosidase F degradation but not to alkaline hydrolysis. Alkaline hydrolysis produced degradation of the protein, and the [3H]Gal radiolabeled carbohydrates remained bound to peptides and were released by subsequent peptide N-glycosidase F treatment. The exogenously galactosylated sugars cleaved by the glycosidase were analyzed by liquid chromatography and had elution volumes identical to a galactose-N-acetylglucosamine disaccharide standard. Since the galactose was exogenously added, we propose that the N-linked glycans on the alpha-subunit of the Na pump are composed of a single sugar residue, which is probably N-acetylglucosamine.

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Requirements of cleavage of high mannose oligosaccharides in glycoproteins by peptide N-glycosidase F.

Cited by 123 publications

References 25 publications

Suppression of O-Linked Glycosylation of the SARS-CoV-2 Spike by Quaternary Structural Restraints

Suppression of O-Linked Glycosylation of the SARS-CoV-2 Spike by Quaternary Structural Restraints

Characterization of a Mouse ZP3-Derived Glycopeptide, gp55, That Exhibits Sperm Receptor and Acrosome Reaction-Inducing Activityin Vitro

A monosaccharide is bound to the sodium-pump .alpha.-subunit

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