The Experts below are selected from a list of 5760 Experts worldwide ranked by ideXlab platform
Takashi Tonozuka - One of the best experts on this subject based on the ideXlab platform.
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The side chain of a glycosylated Asparagine Residue is important for the stability of isopullulanase.
Journal of biochemistry, 2014Co-Authors: Takatsugu Miyazaki, Hiroyuki Yashiro, Atsushi Nishikawa, Takashi TonozukaAbstract:N-glycosylation has been shown to be important for the stability of some glycoproteins. Isopullulanase (IPU), a polysaccharide-hydrolyzing enzyme, is a highly N-glycosylated protein, and IPU deglycosylation results in a decrease in thermostability. To investigate the function of N-glycan in IPU, we focused on an N-glycosylated Residue located in the vicinity of the active site, Asn448. The thermostabilities of three IPU variants, Y440A, N448A and S450A, were 0.5-8.4°C lower than the wild-type enzyme. The crystal structure of endoglycosidase H (Endo H)-treated N448A variant was determined. There are four IPU molecules, Mol-A, B, C and D, in the asymmetric unit. The conformation of a loop composed of amino acid Residues 435-455 in Mol-C was identical to wild-type IPU, whereas the conformations of this loop in Mol-A, Mol-B and Mol-D were different from each other. These results suggest that the Asn448 side chain is primarily important for the stability of IPU. Our results indicate that mutation of only N-glycosylated Asn Residue may lead to incorrect conclusion for the evaluation of the function of N-glycan. Usually, the structures of N-glycosylation sites form an extended configuration in IPU; however, the Asn448 site had an atypical structure that lacked this configuration.
Boyle D - One of the best experts on this subject based on the ideXlab platform.
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Increased deamidation of Asparagine during human senile cataractogenesis.
Molecular Vision, 2000Co-Authors: Takemoto L, Boyle DAbstract:To identify and quantitate deamidation of a specific Asparagine Residue of gammaS-crystallin that preferentially undergoes deamidation during the process of human senile cataractogenesis. Reverse phase chromatography, together with synthetic peptide standards, was used to resolve the amidated and deamidated forms of Asparagine-143 in the gammaS-crystallin sequence 131-145 from total tryptic digests of the central, nuclear region of human cataractous and normal lenses. Identities of the resolved peptides co-eluting with synthetic peptide standards were confirmed by mass spectral analysis. The synthetic peptide standards were also used to quantitate the amount of deamidation occurring in individual cataractous and normal lenses. In all lenses analyzed, there was greater deamidation of Asparagine-143 in cataractous lenses, as compared with age-matched normal lenses. The results demonstrate, for the first time, that increased deamidation of a specific Asparagine Residue is present in proteins from the human cataractous lens.
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Increased deamidation of Asparagine during human senile cataractogenesis.
Molecular vision, 2000Co-Authors: Larry J. Takemoto, Boyle DAbstract:PURPOSE To identify and quantitate deamidation of a specific Asparagine Residue of gammaS-crystallin that preferentially undergoes deamidation during the process of human senile cataractogenesis. METHODS Reverse phase chromatography, together with synthetic peptide standards, was used to resolve the amidated and deamidated forms of Asparagine-143 in the gammaS-crystallin sequence 131-145 from total tryptic digests of the central, nuclear region of human cataractous and normal lenses. Identities of the resolved peptides co-eluting with synthetic peptide standards were confirmed by mass spectral analysis. The synthetic peptide standards were also used to quantitate the amount of deamidation occurring in individual cataractous and normal lenses. RESULTS In all lenses analyzed, there was greater deamidation of Asparagine-143 in cataractous lenses, as compared with age-matched normal lenses. CONCLUSIONS The results demonstrate, for the first time, that increased deamidation of a specific Asparagine Residue is present in proteins from the human cataractous lens.
Richard L. Schowen - One of the best experts on this subject based on the ideXlab platform.
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Racemization of an Asparagine Residue during Peptide Deamidation
Journal of the American Chemical Society, 2003Co-Authors: Bei Li, Ronald T. Borchardt, Elizabeth M. Topp, David Vandervelde, Richard L. SchowenAbstract:The Asn Residue in the pentapeptide Asn-Gln-Asn-Glu-Gly undergoes racemization at the Cα center in the course of deamidation of this Residue through a succinimide intermediate. The succinimide intermediate is known to racemize at the corresponding center, leading to racemized products of deamidation. Return of this intermediate to reactant Asn is very unlikely in dilute solution where attack of product ammonia on the succinimide is precluded, and Asn racemization has not been previously observed. We give evidence that the observed racemization occurs at the tetrahedral-intermediate stage preceding the succinimide intermediate. The observation is significant for protein stability in vivo and in vitro and has importance in medicine, food chemistry, and archaeological/palaeological dating.
Takatsugu Miyazaki - One of the best experts on this subject based on the ideXlab platform.
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The side chain of a glycosylated Asparagine Residue is important for the stability of isopullulanase.
Journal of biochemistry, 2014Co-Authors: Takatsugu Miyazaki, Hiroyuki Yashiro, Atsushi Nishikawa, Takashi TonozukaAbstract:N-glycosylation has been shown to be important for the stability of some glycoproteins. Isopullulanase (IPU), a polysaccharide-hydrolyzing enzyme, is a highly N-glycosylated protein, and IPU deglycosylation results in a decrease in thermostability. To investigate the function of N-glycan in IPU, we focused on an N-glycosylated Residue located in the vicinity of the active site, Asn448. The thermostabilities of three IPU variants, Y440A, N448A and S450A, were 0.5-8.4°C lower than the wild-type enzyme. The crystal structure of endoglycosidase H (Endo H)-treated N448A variant was determined. There are four IPU molecules, Mol-A, B, C and D, in the asymmetric unit. The conformation of a loop composed of amino acid Residues 435-455 in Mol-C was identical to wild-type IPU, whereas the conformations of this loop in Mol-A, Mol-B and Mol-D were different from each other. These results suggest that the Asn448 side chain is primarily important for the stability of IPU. Our results indicate that mutation of only N-glycosylated Asn Residue may lead to incorrect conclusion for the evaluation of the function of N-glycan. Usually, the structures of N-glycosylation sites form an extended configuration in IPU; however, the Asn448 site had an atypical structure that lacked this configuration.
Remy Loris - One of the best experts on this subject based on the ideXlab platform.
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interplay between metal binding and cis trans isomerization in legume lectins structural and thermodynamic study of p angolensis lectin
Journal of Molecular Biology, 2006Co-Authors: Abel Garciapino, L Buts, Lode Wyns, Remy LorisAbstract:The interplay between metal binding, carbohydrate binding activity, stability and structure of the lectin from Pterocarpus angolensis was investigated. Removal of the metals leads to a more flexible form of the protein with significantly less conformational stability. Crystal structures of this metal-free form show significant structural rearrangements, although some structural features that allow the binding of sugars are retained. We propose that substitution of an Asparagine Residue at the start of the C-terminal β-strand of the legume lectin monomer hinders the trans-isomerization of the cis-peptide bond upon demetallization and constitutes an intramolecular switch governing the isomer state of the non-proline bond and ultimately the lectin phenotype.
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Interplay between metal binding and cis/trans isomerization in legume lectins: structural and thermodynamic study of P. angolensis lectin.
Journal of Molecular Biology, 2006Co-Authors: Abel Garcia-pino, L Buts, Lode Wyns, Remy LorisAbstract:The interplay between metal binding, carbohydrate binding activity, stability and structure of the lectin from Pterocarpus angolensis was investigated. Removal of the metals leads to a more flexible form of the protein with significantly less conformational stability. Crystal structures of this metal-free form show significant structural rearrangements, although some structural features that allow the binding of sugars are retained. We propose that substitution of an Asparagine Residue at the start of the C-terminal β-strand of the legume lectin monomer hinders the trans-isomerization of the cis-peptide bond upon demetallization and constitutes an intramolecular switch governing the isomer state of the non-proline bond and ultimately the lectin phenotype.
