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Susan H. Shakin-eshleman - One of the best experts on this subject based on the ideXlab platform.
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The Amino Acid Following an Asn-X-Ser/Thr Sequon Is an Important Determinant of N-Linked Core Glycosylation Efficiency
Biochemistry, 1998Co-Authors: J. L. Mellquist, Lakshmi Kasturi, Steven L. Spitalnik, Susan H. Shakin-eshlemanAbstract:Many eukaryotic proteins are modified by Asn-linked (N-linked) glycosylation. The number and position of oligosaccharides added to a protein by the enzyme oligosaccharyltransferase can influence its expression and function. N-Linked glycosylation usually occurs at Asn residues in Asn-X-Ser/Thr Sequons where X not equal Pro. However, many Asn-X-Ser/Thr Sequons are not glycosylated or are glycosylated inefficiently. Inefficient glycosylation at one or more Asn-X-Ser/Thr Sequons in a protein results in the production of heterogeneous glycoprotein products. These glycoforms may differ from one another in their level of expression, stability, antigenicity, or function. The signals which control the efficiency of N-linked glycosylation at individual Asn residues have not been fully defined. In this report, we use a site-directed mutagenesis approach to investigate the influence of the amino acid at the position following a Sequon (the Y position, Asn-X-Ser/Thr-Y). Variants of rabies virus glycoprotein containing a single Asn-X-Ser/Thr Sequon at Asn37 were generated. Variants were designed with each of the twenty common amino acids at the Y position, with either Ser or Thr at the hydroxy (Ser/Thr) position. The core glycosylation efficiency of each variant was quantified using a cell-free translation/glycosylation system. These studies reveal that the amino acid at the Y position is an important determinant of core glycosylation efficiency.
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The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein
Glycobiology, 1998Co-Authors: Boguslaw S. Wojczyk, Susan H. Shakin-eshleman, William H. Wunner, Magda Stwora-wojczyk, Steven L. SpitalnikAbstract:Rabies virus glycoprotein is important in the biology and pa-thogenesis of neurotropic rabies virus infection. This trans-membrane glycoprotein is the only viral protein on thesurface of virus particles, is the viral attachment protein thatfacilitates virus uptake by the infected cell, and is the targetof the host humoral immune response to infection. The extra-cellular domain of this glycoprotein has N-glycosylationSequons at Asn37, Asn247, and Asn319. Appropriate glyco-sylation of these Sequons is important in the expression of theglycoprotein. Soluble forms of rabies virus glycoprotein wereconstructed by insertion of a stop codon just external to thetransmembrane domain. Using site-directed mutagenesisand expression in transfected eukaryotic cells, it was possibleto compare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and sol-uble forms, respectively, of the same glycoprotein. Thesestudies yielded the surprising finding that although any of thethree Sequons permitted cell surface expression of full-lengthrabies virus glycoprotein, only the N-glycan at Asn319 per-mitted secretion of soluble rabies virus glycoprotein. Despiteits biological and medical importance, it has not yet beenpossible to determine the crystal structure of the full-lengthtransmembrane form of rabies virus glycoprotein which con-tains heterogeneous oligosaccharides. The current studiesdemonstrate that a soluble form of rabies virus glycoproteincontaining only one Sequon at Asn319 is efficiently secreted inthe presence of the N-glycan processing inhibitor 1-deoxy-mannojirimycin. Thus, it is possible to purify a conformation-ally relevant form of rabies virus glycoprotein that containsonly one N-glycan with a substantial reduction in its micro-heterogeneity. This form of the glycoprotein may be particu-larly useful for future studies aimed at elucidating thethree-dimensional structure of this important glycoprotein.Key words: glycoproteins/N-linked glycosylation/rabiesvirus/SequonIntroductionRabies virus is a medically important neurotropic virus (forreview, see Fishbein and Robinson, 1993). It has a negative-strand RNA genome that encodes five proteins, including oneglycoprotein. Rabies virus glycoprotein (RGP) of the ERA strainis a 505 amino acid type I membrane glycoprotein thatoligomerizes into trimers (Whitt et al. , 1991; Gaudin et al. , 1992)and that contains three Asn-X-Ser/Thr potential N-glycosylationsites (Sequons) in the extracellular domain (Anilionis et al. , 1981).RGP is important in the biology and pathogenesis of rabies virusinfection. It is the only viral protein on the surface of virusparticles anchored in the viral envelope, it is the viral attachmentprotein that facilitates virus uptake via the host cell receptor, andit is the target of the host humoral immune response (for review,see Coll, 1995). In addition, RGP itself is efficacious as a vaccine(Kieny et al., 1984; Prehaud et al., 1989; Xiang et al., 1994).Appropriate glycosylation of RGP is important for its properexpression and function. For example, nonglycosylated RGP isnot expressed at the cell surface (Burger et al., 1991; Shakin-Eshleman et al. , 1992) and is not effective as a vaccine (Yelvertonet al., 1983; Lathe et al., 1984). In addition, it is critical that oneof its three Sequons is glycosylated for RGP to be expressed at thecell surface (Shakin-Eshleman et al. , 1992). Finally, for monogly-cosylated forms of RGP, the level of cell surface expressioncorrelates with the efficiency of glycosylation at the correspon-ding Sequon (Shakin-Eshleman et al. , 1992; Katsuri et al. , 1995).We previously constructed a soluble, truncated form of RGP(denoted RGP(WT)T434) by inserting a stop codon amino-ter-minal to the transmembrane domain (Shakin-Eshleman et al.,1993). This soluble form of RGP was appropriately glycosylated,assembled, and secreted by transfected cells (Wojczyk et al.,1995). It was also appropriately antigenic and protective as avaccine (Wojczyk et al., 1995; Xiang et al., 1995). These resultsled to the current studies which compare the effects of site-spe-cific glycosylation on the cell-surface expression and secretion oftransmembrane and soluble forms, respectively, of the sameglycoprotein. This investigation yielded the surprising findingthat although any of the three Sequons permitted cell surfaceexpression of full-length RGP, only the N-glycan at Asn319permitted secretion of soluble RGP.In addition, despite its biological and medical importance, ithas not yet been possible to structurally characterize RGP indetail. Crystallization studies have been hampered by the need fordetergents to solubilize the full-length transmembrane glycopro-tein and by the microheterogeneity of the oligosaccharides onmature RGP. The current studies use site-directed mutagenesisfollowed by eukaryotic expression in the presence of N-glycanprocessing inhibitors to demonstrate the possibility of purifyinga simplified yet conformationally relevant form of RGP, that is,a soluble form of recombinant RGP that contains only oneN-glycan with a substantial reduction in its microheterogeneity.This form of RGP may be particularly useful for future studies
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Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr Sequons that are poor oligosaccharide acceptors.
Biochemical Journal, 1997Co-Authors: Lakshmi Kasturi, Hegang Chen, Susan H. Shakin-eshlemanAbstract:N-linked glycosylation can profoundly affect protein expression and function. N-linked glycosylation usually occurs at the Sequon Asn-Xaa-Ser/Thr, where Xaa is any amino acid residue except Pro. However, many Asn-Xaa-Ser/Thr Sequons are glycosylated inefficiently or not at all for reasons that are poorly understood. We have used a site-directed mutagenesis approach to examine how the Xaa and hydroxy (Ser/Thr) amino acid residues in Sequons influence core-glycosylation efficiency. We recently demonstrated that certain Xaa amino acids inhibit core glycosylation of the Sequon, Asn37-Xaa-Ser, in rabies virus glycoprotein (RGP). Here we examine the impact of different Xaa residues on core-glycosylation efficiency when the Ser residue in this Sequon is replaced with Thr. The core-glycosylation efficiencies of RGP variants with different Asn37-Xaa-Ser/Thr Sequons were compared by using a cell-free translation/glycosylation system. Using this approach we confirm that four Asn-Xaa-Ser Sequons are poor oligosaccharide acceptors: Asn-Trp-Ser, Asn-Asp-Ser, Asn-Glu-Ser and Asn-Leu-Ser. In contrast, Asn-Xaa-Thr Sequons are efficiently glycosylated, even when Xaa=Trp, Asp, Glu or Leu. A comparison of the glycosylation status of Asn-Xaa-Ser and Asn-Xaa-Thr Sequons in other glycoproteins confirms that Sequons with Xaa=Trp, Asp, Glu or Leu are rarely glycosylated when Ser is the hydroxy amino acid residue, and that these Sequons are unlikely to serve as glycosylation sites when introduced into proteins by site-directed mutagenesis.
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The Amino Acid at the X Position of an Asn-X-Ser Sequon Is an Important Determinant of N-Linked Core-glycosylation Efficiency
The Journal of biological chemistry, 1996Co-Authors: Susan H. Shakin-eshleman, Steven L. Spitalnik, Lakshmi KasturiAbstract:N-Linked glycosylation is a common form of protein processing that can profoundly affect protein expression, structure, and function. N-Linked glycosylation generally occurs at the Sequon Asn-X-Ser/Thr, where X is any amino acid except Pro. To assess the impact of the X amino acid on core glycosylation, rabies virus glycoprotein variants were generated by site-directed mutagenesis with each of the 20 common amino acids substituted at the X position of an Asn-X-Ser Sequon. The efficiency of core glycosylation at the Sequon in each variant was quantified in a rabbit reticulocyte lysate cell-free translation system supplemented with canine pancreas microsomes. The presence of Pro at the X position completely blocked core glycosylation, whereas Trp, Asp, Chi, and Leu were associated with inefficient core glycosylation. The other variants were more efficiently glycosylated, and several were fully glycosylated. These findings demonstrate that the X amino acid is an important determinant of N-linked core-glycosylation efficiency.
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The Hydroxy Amino Acid in an Asn-X-Ser/Thr Sequon Can Influence N-Linked Core Glycosylation Efficiency and the Level of Expression of a Cell Surface Glycoprotein
The Journal of biological chemistry, 1995Co-Authors: Lakshmi Kasturi, James R. Eshleman, William H. Wunner, Susan H. Shakin-eshlemanAbstract:N-Linked glycosylation usually occurs at the Sequon, Asn-X-Ser/Thr. In this Sequon, the side chain of the hydroxy amino acid (Ser or Thr) may play a direct catalytic role in the enzymatic transfer of core oligosaccharides to the Asn residue. Using recombinant variants of rabies virus glycoprotein (RGP), we examined the influence of the hydroxy amino acid on core glycosylation efficiency. A variant of RGP containing a single Asn-X-Ser Sequon at Asn37 was modified by site-directed mutagenesis to change the Sequon to either Asn-X-Cys or Asn-X-Thr. The impact of these changes on core glycosylation efficiency was assessed by expressing the variants in a cell-free transcription/translation/glycosylation system and in transfected tissue culture cells. Substitution of Cys at position 39 blocks glycosylation, whereas substitution of Thr dramatically increases core glycosylation efficiency of Asn37 in both membrane-anchored and secreted forms of RGP. The substitution of Thr for Ser also dramatically enhances the level of expression and cell surface delivery of RGP when the Sequon at Asn37 is the only Sequon in the protein. Novel forms of membrane-anchored and secreted RGP which are fully glycosylated at all three Sequons were also generated by substitution of Thr at position 39.
Lakshmi Kasturi - One of the best experts on this subject based on the ideXlab platform.
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The Amino Acid Following an Asn-X-Ser/Thr Sequon Is an Important Determinant of N-Linked Core Glycosylation Efficiency
Biochemistry, 1998Co-Authors: J. L. Mellquist, Lakshmi Kasturi, Steven L. Spitalnik, Susan H. Shakin-eshlemanAbstract:Many eukaryotic proteins are modified by Asn-linked (N-linked) glycosylation. The number and position of oligosaccharides added to a protein by the enzyme oligosaccharyltransferase can influence its expression and function. N-Linked glycosylation usually occurs at Asn residues in Asn-X-Ser/Thr Sequons where X not equal Pro. However, many Asn-X-Ser/Thr Sequons are not glycosylated or are glycosylated inefficiently. Inefficient glycosylation at one or more Asn-X-Ser/Thr Sequons in a protein results in the production of heterogeneous glycoprotein products. These glycoforms may differ from one another in their level of expression, stability, antigenicity, or function. The signals which control the efficiency of N-linked glycosylation at individual Asn residues have not been fully defined. In this report, we use a site-directed mutagenesis approach to investigate the influence of the amino acid at the position following a Sequon (the Y position, Asn-X-Ser/Thr-Y). Variants of rabies virus glycoprotein containing a single Asn-X-Ser/Thr Sequon at Asn37 were generated. Variants were designed with each of the twenty common amino acids at the Y position, with either Ser or Thr at the hydroxy (Ser/Thr) position. The core glycosylation efficiency of each variant was quantified using a cell-free translation/glycosylation system. These studies reveal that the amino acid at the Y position is an important determinant of core glycosylation efficiency.
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the amino acid following an asn x ser thr Sequon is an important determinant of n linked core glycosylation efficiency
Biochemistry, 1998Co-Authors: J. L. Mellquist, Steven L. Spitalnik, Lakshmi Kasturi, Susan H ShakineshlemanAbstract:Many eukaryotic proteins are modified by Asn-linked (N-linked) glycosylation. The number and position of oligosaccharides added to a protein by the enzyme oligosaccharyltransferase can influence its expression and function. N-Linked glycosylation usually occurs at Asn residues in Asn-X-Ser/Thr Sequons where X not equal Pro. However, many Asn-X-Ser/Thr Sequons are not glycosylated or are glycosylated inefficiently. Inefficient glycosylation at one or more Asn-X-Ser/Thr Sequons in a protein results in the production of heterogeneous glycoprotein products. These glycoforms may differ from one another in their level of expression, stability, antigenicity, or function. The signals which control the efficiency of N-linked glycosylation at individual Asn residues have not been fully defined. In this report, we use a site-directed mutagenesis approach to investigate the influence of the amino acid at the position following a Sequon (the Y position, Asn-X-Ser/Thr-Y). Variants of rabies virus glycoprotein containing a single Asn-X-Ser/Thr Sequon at Asn37 were generated. Variants were designed with each of the twenty common amino acids at the Y position, with either Ser or Thr at the hydroxy (Ser/Thr) position. The core glycosylation efficiency of each variant was quantified using a cell-free translation/glycosylation system. These studies reveal that the amino acid at the Y position is an important determinant of core glycosylation efficiency.
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Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr Sequons that are poor oligosaccharide acceptors.
Biochemical Journal, 1997Co-Authors: Lakshmi Kasturi, Hegang Chen, Susan H. Shakin-eshlemanAbstract:N-linked glycosylation can profoundly affect protein expression and function. N-linked glycosylation usually occurs at the Sequon Asn-Xaa-Ser/Thr, where Xaa is any amino acid residue except Pro. However, many Asn-Xaa-Ser/Thr Sequons are glycosylated inefficiently or not at all for reasons that are poorly understood. We have used a site-directed mutagenesis approach to examine how the Xaa and hydroxy (Ser/Thr) amino acid residues in Sequons influence core-glycosylation efficiency. We recently demonstrated that certain Xaa amino acids inhibit core glycosylation of the Sequon, Asn37-Xaa-Ser, in rabies virus glycoprotein (RGP). Here we examine the impact of different Xaa residues on core-glycosylation efficiency when the Ser residue in this Sequon is replaced with Thr. The core-glycosylation efficiencies of RGP variants with different Asn37-Xaa-Ser/Thr Sequons were compared by using a cell-free translation/glycosylation system. Using this approach we confirm that four Asn-Xaa-Ser Sequons are poor oligosaccharide acceptors: Asn-Trp-Ser, Asn-Asp-Ser, Asn-Glu-Ser and Asn-Leu-Ser. In contrast, Asn-Xaa-Thr Sequons are efficiently glycosylated, even when Xaa=Trp, Asp, Glu or Leu. A comparison of the glycosylation status of Asn-Xaa-Ser and Asn-Xaa-Thr Sequons in other glycoproteins confirms that Sequons with Xaa=Trp, Asp, Glu or Leu are rarely glycosylated when Ser is the hydroxy amino acid residue, and that these Sequons are unlikely to serve as glycosylation sites when introduced into proteins by site-directed mutagenesis.
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The Amino Acid at the X Position of an Asn-X-Ser Sequon Is an Important Determinant of N-Linked Core-glycosylation Efficiency
The Journal of biological chemistry, 1996Co-Authors: Susan H. Shakin-eshleman, Steven L. Spitalnik, Lakshmi KasturiAbstract:N-Linked glycosylation is a common form of protein processing that can profoundly affect protein expression, structure, and function. N-Linked glycosylation generally occurs at the Sequon Asn-X-Ser/Thr, where X is any amino acid except Pro. To assess the impact of the X amino acid on core glycosylation, rabies virus glycoprotein variants were generated by site-directed mutagenesis with each of the 20 common amino acids substituted at the X position of an Asn-X-Ser Sequon. The efficiency of core glycosylation at the Sequon in each variant was quantified in a rabbit reticulocyte lysate cell-free translation system supplemented with canine pancreas microsomes. The presence of Pro at the X position completely blocked core glycosylation, whereas Trp, Asp, Chi, and Leu were associated with inefficient core glycosylation. The other variants were more efficiently glycosylated, and several were fully glycosylated. These findings demonstrate that the X amino acid is an important determinant of N-linked core-glycosylation efficiency.
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The Hydroxy Amino Acid in an Asn-X-Ser/Thr Sequon Can Influence N-Linked Core Glycosylation Efficiency and the Level of Expression of a Cell Surface Glycoprotein
The Journal of biological chemistry, 1995Co-Authors: Lakshmi Kasturi, James R. Eshleman, William H. Wunner, Susan H. Shakin-eshlemanAbstract:N-Linked glycosylation usually occurs at the Sequon, Asn-X-Ser/Thr. In this Sequon, the side chain of the hydroxy amino acid (Ser or Thr) may play a direct catalytic role in the enzymatic transfer of core oligosaccharides to the Asn residue. Using recombinant variants of rabies virus glycoprotein (RGP), we examined the influence of the hydroxy amino acid on core glycosylation efficiency. A variant of RGP containing a single Asn-X-Ser Sequon at Asn37 was modified by site-directed mutagenesis to change the Sequon to either Asn-X-Cys or Asn-X-Thr. The impact of these changes on core glycosylation efficiency was assessed by expressing the variants in a cell-free transcription/translation/glycosylation system and in transfected tissue culture cells. Substitution of Cys at position 39 blocks glycosylation, whereas substitution of Thr dramatically increases core glycosylation efficiency of Asn37 in both membrane-anchored and secreted forms of RGP. The substitution of Thr for Ser also dramatically enhances the level of expression and cell surface delivery of RGP when the Sequon at Asn37 is the only Sequon in the protein. Novel forms of membrane-anchored and secreted RGP which are fully glycosylated at all three Sequons were also generated by substitution of Thr at position 39.
Steven L. Spitalnik - One of the best experts on this subject based on the ideXlab platform.
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N-glycosylation at one rabies virus glycoprotein Sequon influences N-glycan processing at a distant Sequon on the same molecule.
Glycobiology, 2005Co-Authors: Boguslaw S. Wojczyk, Noriko Takahashi, Matthew T. Levy, David W. Andrews, William R. Abrams, William H. Wunner, Steven L. SpitalnikAbstract:Rabies glycoprotein (RGP(WT)) contains N-glycosylation Sequons at Asn(37), Asn(247), and Asn(319), although Asn(37) is not efficiently glycosylated. To examine N-glycan processing at Asn(247) and Asn(319), full-length glycosylation mutants, RGP(-2-) and RGP(--3), were expressed, and Endo H sensitivity was compared. When the Asn(247) Sequon is present alone in RGP(-2-), 90% of its N-glycans are high-mannose type, whereas only 35% of the N-glycans at Asn(319) in RGP(--3) are high-mannose. When both Sequons are present in RGP(-23), 87% of the N-glycans are of complex type. The differing patterns of Endo H sensitivity at Sequons present individually or together suggests that glycosylation of one Sequon affects glycosylation at another, distant Sequon. To explore this further, we constructed soluble forms of RGP: RGP(WT)T441His and RGP(--3)T441His. Tryptic glycopeptides from these purified secreted proteins were isolated by HPLC and characterized by a 3D oligosaccharide mapping technique. RGP(WT)T441His had fucosylated, bi- and triantennary complex type glycans at Asn(247) and Asn(319). However, Asn(247) had half as many neutral glycans, more monosialylated glycans, and fewer disialylated glycans when compared with Asn(319). Moreover, when comparing the N-glycans at Asn(319) on RGP(--3)T441His and RGP(WT)T441His, the former had 30% more neutral, 28% more monosialylated, and 33% fewer disialylated glycans. This suggests that the N-glycan at Asn(247) allows additional N-glycan processing to occur at Asn(319), yielding more heavily sialylated bi- and triantennary forms. The mechanism(s) by which glycosylation at one Sequon influences N-glycan processing at a distant Sequon on the same glycoprotein remains to be determined.
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The Amino Acid Following an Asn-X-Ser/Thr Sequon Is an Important Determinant of N-Linked Core Glycosylation Efficiency
Biochemistry, 1998Co-Authors: J. L. Mellquist, Lakshmi Kasturi, Steven L. Spitalnik, Susan H. Shakin-eshlemanAbstract:Many eukaryotic proteins are modified by Asn-linked (N-linked) glycosylation. The number and position of oligosaccharides added to a protein by the enzyme oligosaccharyltransferase can influence its expression and function. N-Linked glycosylation usually occurs at Asn residues in Asn-X-Ser/Thr Sequons where X not equal Pro. However, many Asn-X-Ser/Thr Sequons are not glycosylated or are glycosylated inefficiently. Inefficient glycosylation at one or more Asn-X-Ser/Thr Sequons in a protein results in the production of heterogeneous glycoprotein products. These glycoforms may differ from one another in their level of expression, stability, antigenicity, or function. The signals which control the efficiency of N-linked glycosylation at individual Asn residues have not been fully defined. In this report, we use a site-directed mutagenesis approach to investigate the influence of the amino acid at the position following a Sequon (the Y position, Asn-X-Ser/Thr-Y). Variants of rabies virus glycoprotein containing a single Asn-X-Ser/Thr Sequon at Asn37 were generated. Variants were designed with each of the twenty common amino acids at the Y position, with either Ser or Thr at the hydroxy (Ser/Thr) position. The core glycosylation efficiency of each variant was quantified using a cell-free translation/glycosylation system. These studies reveal that the amino acid at the Y position is an important determinant of core glycosylation efficiency.
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the amino acid following an asn x ser thr Sequon is an important determinant of n linked core glycosylation efficiency
Biochemistry, 1998Co-Authors: J. L. Mellquist, Steven L. Spitalnik, Lakshmi Kasturi, Susan H ShakineshlemanAbstract:Many eukaryotic proteins are modified by Asn-linked (N-linked) glycosylation. The number and position of oligosaccharides added to a protein by the enzyme oligosaccharyltransferase can influence its expression and function. N-Linked glycosylation usually occurs at Asn residues in Asn-X-Ser/Thr Sequons where X not equal Pro. However, many Asn-X-Ser/Thr Sequons are not glycosylated or are glycosylated inefficiently. Inefficient glycosylation at one or more Asn-X-Ser/Thr Sequons in a protein results in the production of heterogeneous glycoprotein products. These glycoforms may differ from one another in their level of expression, stability, antigenicity, or function. The signals which control the efficiency of N-linked glycosylation at individual Asn residues have not been fully defined. In this report, we use a site-directed mutagenesis approach to investigate the influence of the amino acid at the position following a Sequon (the Y position, Asn-X-Ser/Thr-Y). Variants of rabies virus glycoprotein containing a single Asn-X-Ser/Thr Sequon at Asn37 were generated. Variants were designed with each of the twenty common amino acids at the Y position, with either Ser or Thr at the hydroxy (Ser/Thr) position. The core glycosylation efficiency of each variant was quantified using a cell-free translation/glycosylation system. These studies reveal that the amino acid at the Y position is an important determinant of core glycosylation efficiency.
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The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein
Glycobiology, 1998Co-Authors: Boguslaw S. Wojczyk, Susan H. Shakin-eshleman, William H. Wunner, Magda Stwora-wojczyk, Steven L. SpitalnikAbstract:Rabies virus glycoprotein is important in the biology and pa-thogenesis of neurotropic rabies virus infection. This trans-membrane glycoprotein is the only viral protein on thesurface of virus particles, is the viral attachment protein thatfacilitates virus uptake by the infected cell, and is the targetof the host humoral immune response to infection. The extra-cellular domain of this glycoprotein has N-glycosylationSequons at Asn37, Asn247, and Asn319. Appropriate glyco-sylation of these Sequons is important in the expression of theglycoprotein. Soluble forms of rabies virus glycoprotein wereconstructed by insertion of a stop codon just external to thetransmembrane domain. Using site-directed mutagenesisand expression in transfected eukaryotic cells, it was possibleto compare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and sol-uble forms, respectively, of the same glycoprotein. Thesestudies yielded the surprising finding that although any of thethree Sequons permitted cell surface expression of full-lengthrabies virus glycoprotein, only the N-glycan at Asn319 per-mitted secretion of soluble rabies virus glycoprotein. Despiteits biological and medical importance, it has not yet beenpossible to determine the crystal structure of the full-lengthtransmembrane form of rabies virus glycoprotein which con-tains heterogeneous oligosaccharides. The current studiesdemonstrate that a soluble form of rabies virus glycoproteincontaining only one Sequon at Asn319 is efficiently secreted inthe presence of the N-glycan processing inhibitor 1-deoxy-mannojirimycin. Thus, it is possible to purify a conformation-ally relevant form of rabies virus glycoprotein that containsonly one N-glycan with a substantial reduction in its micro-heterogeneity. This form of the glycoprotein may be particu-larly useful for future studies aimed at elucidating thethree-dimensional structure of this important glycoprotein.Key words: glycoproteins/N-linked glycosylation/rabiesvirus/SequonIntroductionRabies virus is a medically important neurotropic virus (forreview, see Fishbein and Robinson, 1993). It has a negative-strand RNA genome that encodes five proteins, including oneglycoprotein. Rabies virus glycoprotein (RGP) of the ERA strainis a 505 amino acid type I membrane glycoprotein thatoligomerizes into trimers (Whitt et al. , 1991; Gaudin et al. , 1992)and that contains three Asn-X-Ser/Thr potential N-glycosylationsites (Sequons) in the extracellular domain (Anilionis et al. , 1981).RGP is important in the biology and pathogenesis of rabies virusinfection. It is the only viral protein on the surface of virusparticles anchored in the viral envelope, it is the viral attachmentprotein that facilitates virus uptake via the host cell receptor, andit is the target of the host humoral immune response (for review,see Coll, 1995). In addition, RGP itself is efficacious as a vaccine(Kieny et al., 1984; Prehaud et al., 1989; Xiang et al., 1994).Appropriate glycosylation of RGP is important for its properexpression and function. For example, nonglycosylated RGP isnot expressed at the cell surface (Burger et al., 1991; Shakin-Eshleman et al. , 1992) and is not effective as a vaccine (Yelvertonet al., 1983; Lathe et al., 1984). In addition, it is critical that oneof its three Sequons is glycosylated for RGP to be expressed at thecell surface (Shakin-Eshleman et al. , 1992). Finally, for monogly-cosylated forms of RGP, the level of cell surface expressioncorrelates with the efficiency of glycosylation at the correspon-ding Sequon (Shakin-Eshleman et al. , 1992; Katsuri et al. , 1995).We previously constructed a soluble, truncated form of RGP(denoted RGP(WT)T434) by inserting a stop codon amino-ter-minal to the transmembrane domain (Shakin-Eshleman et al.,1993). This soluble form of RGP was appropriately glycosylated,assembled, and secreted by transfected cells (Wojczyk et al.,1995). It was also appropriately antigenic and protective as avaccine (Wojczyk et al., 1995; Xiang et al., 1995). These resultsled to the current studies which compare the effects of site-spe-cific glycosylation on the cell-surface expression and secretion oftransmembrane and soluble forms, respectively, of the sameglycoprotein. This investigation yielded the surprising findingthat although any of the three Sequons permitted cell surfaceexpression of full-length RGP, only the N-glycan at Asn319permitted secretion of soluble RGP.In addition, despite its biological and medical importance, ithas not yet been possible to structurally characterize RGP indetail. Crystallization studies have been hampered by the need fordetergents to solubilize the full-length transmembrane glycopro-tein and by the microheterogeneity of the oligosaccharides onmature RGP. The current studies use site-directed mutagenesisfollowed by eukaryotic expression in the presence of N-glycanprocessing inhibitors to demonstrate the possibility of purifyinga simplified yet conformationally relevant form of RGP, that is,a soluble form of recombinant RGP that contains only oneN-glycan with a substantial reduction in its microheterogeneity.This form of RGP may be particularly useful for future studies
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The Amino Acid at the X Position of an Asn-X-Ser Sequon Is an Important Determinant of N-Linked Core-glycosylation Efficiency
The Journal of biological chemistry, 1996Co-Authors: Susan H. Shakin-eshleman, Steven L. Spitalnik, Lakshmi KasturiAbstract:N-Linked glycosylation is a common form of protein processing that can profoundly affect protein expression, structure, and function. N-Linked glycosylation generally occurs at the Sequon Asn-X-Ser/Thr, where X is any amino acid except Pro. To assess the impact of the X amino acid on core glycosylation, rabies virus glycoprotein variants were generated by site-directed mutagenesis with each of the 20 common amino acids substituted at the X position of an Asn-X-Ser Sequon. The efficiency of core glycosylation at the Sequon in each variant was quantified in a rabbit reticulocyte lysate cell-free translation system supplemented with canine pancreas microsomes. The presence of Pro at the X position completely blocked core glycosylation, whereas Trp, Asp, Chi, and Leu were associated with inefficient core glycosylation. The other variants were more efficiently glycosylated, and several were fully glycosylated. These findings demonstrate that the X amino acid is an important determinant of N-linked core-glycosylation efficiency.
Mihika Gangolli - One of the best experts on this subject based on the ideXlab platform.
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sequin multiscale imaging of mammalian central synapses reveals loss of synaptic connectivity resulting from diffuse traumatic brain injury
Neuron, 2020Co-Authors: Andrew D Sauerbeck, Mihika Gangolli, Sydney J Reitz, Maverick H Salyards, Christopher Hemingway, Tejaswi Makkapati, Martin Kerschensteiner, Maud Gratuze, David M HoltzmanAbstract:Summary The brain’s complex microconnectivity underlies its computational abilities and vulnerability to injury and disease. It has been challenging to illuminate the features of this synaptic network due to the small size and dense packing of its elements. Here, we describe a rapid, accessible super-resolution imaging and analysis workflow—SEQUIN—that quantifies central synapses in human tissue and animal models, characterizes their nanostructural and molecular features, and enables volumetric imaging of mesoscale synaptic networks without the production of large histological arrays. Using SEQUIN, we identify cortical synapse loss resulting from diffuse traumatic brain injury, a highly prevalent connectional disorder. Similar synapse loss is observed in three murine models of Alzheimer-related neurodegeneration, where SEQUIN mesoscale mapping identifies regional synaptic vulnerability. These results establish an easily implemented and robust nano-to-mesoscale synapse quantification and characterization method. They furthermore identify a shared mechanism—synaptopathy—between Alzheimer neurodegeneration and its best-established epigenetic risk factor, brain trauma.
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sequin multiscale imaging of mammalian central synapses reveals loss of synaptic microconnectivity resulting from diffuse traumatic brain injury
bioRxiv, 2019Co-Authors: Andrew D Sauerbeck, Mihika Gangolli, Sydney J Reitz, Maverick H Salyards, Christopher Hemingway, Tejaswi Makkapati, Martin Kerschensteiner, David L Brody, Terrance T KummerAbstract:The complex microconnectivity of the mammalian brain underlies its computational abilities, and its vulnerability to injury and disease. It has been challenging to illuminate the features of this synaptic network due in part to the small size and exceptionally dense packing of its elements. Here we describe a rapid and accessible super-resolution imaging and image analysis workflow--SEQUIN--that identifies, quantifies, and characterizes central synapses in animal models and in humans, enabling automated volumetric imaging of mesoscale synaptic networks without the laborious production of large histological arrays. Using SEQUIN, we identify delayed cortical synapse loss resulting from diffuse traumatic brain injury. Similar synapse loss is observed in an Alzheimer disease model, where SEQUIN mesoscale mapping of excitatory synapses across the hippocampus identifies region-specific synaptic vulnerability to neurodegeneration. These results establish a novel, easily implemented and robust nano-to-mesoscale synapse quantification and molecular characterization method. They furthermore identify a mechanistic link--synaptopathy--between Alzheimer neurodegeneration and its best-established epigenetic risk factor, brain trauma.
David M Holtzman - One of the best experts on this subject based on the ideXlab platform.
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sequin multiscale imaging of mammalian central synapses reveals loss of synaptic connectivity resulting from diffuse traumatic brain injury
Neuron, 2020Co-Authors: Andrew D Sauerbeck, Mihika Gangolli, Sydney J Reitz, Maverick H Salyards, Christopher Hemingway, Tejaswi Makkapati, Martin Kerschensteiner, Maud Gratuze, David M HoltzmanAbstract:Summary The brain’s complex microconnectivity underlies its computational abilities and vulnerability to injury and disease. It has been challenging to illuminate the features of this synaptic network due to the small size and dense packing of its elements. Here, we describe a rapid, accessible super-resolution imaging and analysis workflow—SEQUIN—that quantifies central synapses in human tissue and animal models, characterizes their nanostructural and molecular features, and enables volumetric imaging of mesoscale synaptic networks without the production of large histological arrays. Using SEQUIN, we identify cortical synapse loss resulting from diffuse traumatic brain injury, a highly prevalent connectional disorder. Similar synapse loss is observed in three murine models of Alzheimer-related neurodegeneration, where SEQUIN mesoscale mapping identifies regional synaptic vulnerability. These results establish an easily implemented and robust nano-to-mesoscale synapse quantification and characterization method. They furthermore identify a shared mechanism—synaptopathy—between Alzheimer neurodegeneration and its best-established epigenetic risk factor, brain trauma.
