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Susan H. Shakin-eshleman - One of the best experts on this subject based on the ideXlab platform.

  • The Amino Acid Following an Asn-X-Ser/Thr Sequon Is an Important Determinant of N-Linked Core Glycosylation Efficiency
    Biochemistry, 1998
    Co-Authors: J. L. Mellquist, Lakshmi Kasturi, Steven L. Spitalnik, Susan H. Shakin-eshleman
    Abstract:

    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.

  • 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, 1997
    Co-Authors: Lakshmi Kasturi, Hegang Chen, Susan H. Shakin-eshleman
    Abstract:

    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.

  • 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, 1995
    Co-Authors: Lakshmi Kasturi, James R. Eshleman, William H. Wunner, Susan H. Shakin-eshleman
    Abstract:

    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.

  • The Amino Acid Following an Asn-X-Ser/Thr Sequon Is an Important Determinant of N-Linked Core Glycosylation Efficiency
    Biochemistry, 1998
    Co-Authors: J. L. Mellquist, Lakshmi Kasturi, Steven L. Spitalnik, Susan H. Shakin-eshleman
    Abstract:

    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.

  • the amino acid following an asn x ser thr sequon is an important determinant of n linked core glycosylation efficiency
    Biochemistry, 1998
    Co-Authors: J. L. Mellquist, Steven L. Spitalnik, Lakshmi Kasturi, Susan H Shakineshleman
    Abstract:

    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.

  • 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, 1997
    Co-Authors: Lakshmi Kasturi, Hegang Chen, Susan H. Shakin-eshleman
    Abstract:

    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.

  • 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, 1995
    Co-Authors: Lakshmi Kasturi, James R. Eshleman, William H. Wunner, Susan H. Shakin-eshleman
    Abstract:

    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.

  • 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
    Journal of Biological Chemistry, 1995
    Co-Authors: Lakshmi Kasturi, James R. Eshleman, William H. Wunner, Susan H Shakineshleman
    Abstract:

    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.

Susan H Shakineshleman - One of the best experts on this subject based on the ideXlab platform.

  • the amino acid following an asn x ser thr sequon is an important determinant of n linked core glycosylation efficiency
    Biochemistry, 1998
    Co-Authors: J. L. Mellquist, Steven L. Spitalnik, Lakshmi Kasturi, Susan H Shakineshleman
    Abstract:

    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.

  • 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
    Journal of Biological Chemistry, 1995
    Co-Authors: Lakshmi Kasturi, James R. Eshleman, William H. Wunner, Susan H Shakineshleman
    Abstract:

    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.

J. L. Mellquist - One of the best experts on this subject based on the ideXlab platform.

  • The Amino Acid Following an Asn-X-Ser/Thr Sequon Is an Important Determinant of N-Linked Core Glycosylation Efficiency
    Biochemistry, 1998
    Co-Authors: J. L. Mellquist, Lakshmi Kasturi, Steven L. Spitalnik, Susan H. Shakin-eshleman
    Abstract:

    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.

  • the amino acid following an asn x ser thr sequon is an important determinant of n linked core glycosylation efficiency
    Biochemistry, 1998
    Co-Authors: J. L. Mellquist, Steven L. Spitalnik, Lakshmi Kasturi, Susan H Shakineshleman
    Abstract:

    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.

Kuo-ping Huang - One of the best experts on this subject based on the ideXlab platform.

  • Hypoxia/ischemia induces dephosphorylation of rat brain neuromodulin/GAP-43 in vivo.
    Journal of neurochemistry, 2008
    Co-Authors: Kuo-ping Huang, Freesia L. Huang, Hao‐chia Chen
    Abstract:

    The in vivo state of phosphorylation and the modification of two Cys residues of neuromodulin/ GAP-43 (Nm) were analyzed by electrospray ionization-mass spectrometry (ES-MS). The protein was purified from rat brain with homogenization buffer containing 1% Nonidet P-40, protease inhibitors, protein phosphatase inhibitors, and sulfhydryl reagent, 4-vinylpyridine. Nm was purified by HPLC and ion-exchange chromatography, and the various fractions were identified by ES-MS as unphosphorylated and mono-, di-, tri-, and tetraphosphorylated species. All of these Nm species contained 2 mol of added 4-vinylpyridine per mol of Nm, suggesting that the two Cys residues are in the reduced form in the brain. In vivo, the majority of Nm is in the phosphorylated form (∼80%), of which the levels of the mono- and diphospho forms are higher than those of the tri- and tetraphospho species. Four in vivo phosphorylation sites, Ser 41 , Thr 95 , Ser 142 , and Thr 172 , were identified by amino acid sequencing and tandem ES-MS of the peptides derived from Lys-C endoproteinase digestion. Among these sites, only Ser 41 is a known target of PKC, whereas the kinases responsible for the phosphorylation of the other three novel sites are unknown. Hypoxia/ischemia caused a preferential dephosphorylation of Ser 41 and Thr 172 , whereas Thr 95 is the least susceptible to dephosphorylation.

  • hypoxia ischemia induces dephosphorylation of rat brain neuromodulin gap 43 in vivo
    Journal of Neurochemistry, 2008
    Co-Authors: Kuo-ping Huang, Freesia L. Huang, Hao-chia Chen
    Abstract:

    The in vivo state of phosphorylation and the modification of two Cys residues of neuromodulin/ GAP-43 (Nm) were analyzed by electrospray ionization-mass spectrometry (ES-MS). The protein was purified from rat brain with homogenization buffer containing 1% Nonidet P-40, protease inhibitors, protein phosphatase inhibitors, and sulfhydryl reagent, 4-vinylpyridine. Nm was purified by HPLC and ion-exchange chromatography, and the various fractions were identified by ES-MS as unphosphorylated and mono-, di-, tri-, and tetraphosphorylated species. All of these Nm species contained 2 mol of added 4-vinylpyridine per mol of Nm, suggesting that the two Cys residues are in the reduced form in the brain. In vivo, the majority of Nm is in the phosphorylated form (∼80%), of which the levels of the mono- and diphospho forms are higher than those of the tri- and tetraphospho species. Four in vivo phosphorylation sites, Ser 41 , Thr 95 , Ser 142 , and Thr 172 , were identified by amino acid sequencing and tandem ES-MS of the peptides derived from Lys-C endoproteinase digestion. Among these sites, only Ser 41 is a known target of PKC, whereas the kinases responsible for the phosphorylation of the other three novel sites are unknown. Hypoxia/ischemia caused a preferential dephosphorylation of Ser 41 and Thr 172 , whereas Thr 95 is the least susceptible to dephosphorylation.