The Experts below are selected from a list of 5841 Experts worldwide ranked by ideXlab platform
Kelley W. Moremen - One of the best experts on this subject based on the ideXlab platform.
-
substrate recognition and catalysis by gh47 α Mannosidases involved in asn linked glycan maturation in the mammalian secretory pathway
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Yong Xiang, Khanita Karaveg, Kelley W. MoremenAbstract:Maturation of Asn-linked oligosaccharides in the eukaryotic secretory pathway requires the trimming of nascent glycan chains to remove all glucose and several mannose residues before extension into complex-type structures on the cell surface and secreted glycoproteins. Multiple glycoside hydrolase family 47 (GH47) α-Mannosidases, including endoplasmic reticulum (ER) α-Mannosidase I (ERManI) and Golgi α-Mannosidase IA (GMIA), are responsible for cleavage of terminal α1,2-linked mannose residues to produce uniquely trimmed oligomannose isomers that are necessary for ER glycoprotein quality control and glycan maturation. ERManI and GMIA have similar catalytic domain structures, but each enzyme cleaves distinct residues from tribranched oligomannose glycan substrates. The structural basis for branch-specific cleavage by ERManI and GMIA was explored by replacing an essential enzyme-bound Ca2+ ion with a lanthanum (La3+) ion. This ion swap led to enzyme inactivation while retaining high-affinity substrate interactions. Cocrystallization of La3+-bound enzymes with Man9GlcNAc2 substrate analogs revealed enzyme–substrate complexes with distinct modes of glycan branch insertion into the respective enzyme active-site clefts. Both enzymes had glycan interactions that extended across the entire glycan structure, but each enzyme engaged a different glycan branch and used different sets of glycan interactions. Additional mutagenesis and time-course studies of glycan cleavage probed the structural basis of enzyme specificity. The results provide insights into the enzyme catalytic mechanisms and reveal structural snapshots of the sequential glycan cleavage events. The data also indicate that full steric access to glycan substrates determines the efficiency of mannose-trimming reactions that control the conversion to complex-type structures in mammalian cells.
-
substrate recognition and catalysis by gh47 α Mannosidases involved in asn linked glycan maturation in the mammalian secretory pathway
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Yong Xiang, Khanita Karaveg, Kelley W. MoremenAbstract:Maturation of Asn-linked oligosaccharides in the eukaryotic secretory pathway requires the trimming of nascent glycan chains to remove all glucose and several mannose residues before extension into complex-type structures on the cell surface and secreted glycoproteins. Multiple glycoside hydrolase family 47 (GH47) α-Mannosidases, including endoplasmic reticulum (ER) α-Mannosidase I (ERManI) and Golgi α-Mannosidase IA (GMIA), are responsible for cleavage of terminal α1,2-linked mannose residues to produce uniquely trimmed oligomannose isomers that are necessary for ER glycoprotein quality control and glycan maturation. ERManI and GMIA have similar catalytic domain structures, but each enzyme cleaves distinct residues from tribranched oligomannose glycan substrates. The structural basis for branch-specific cleavage by ERManI and GMIA was explored by replacing an essential enzyme-bound Ca2+ ion with a lanthanum (La3+) ion. This ion swap led to enzyme inactivation while retaining high-affinity substrate interactions. Cocrystallization of La3+-bound enzymes with Man9GlcNAc2 substrate analogs revealed enzyme–substrate complexes with distinct modes of glycan branch insertion into the respective enzyme active-site clefts. Both enzymes had glycan interactions that extended across the entire glycan structure, but each enzyme engaged a different glycan branch and used different sets of glycan interactions. Additional mutagenesis and time-course studies of glycan cleavage probed the structural basis of enzyme specificity. The results provide insights into the enzyme catalytic mechanisms and reveal structural snapshots of the sequential glycan cleavage events. The data also indicate that full steric access to glycan substrates determines the efficiency of mannose-trimming reactions that control the conversion to complex-type structures in mammalian cells.
-
family 47 α Mannosidases in n glycan processing
Methods in Enzymology, 2006Co-Authors: Steven W Mast, Kelley W. MoremenAbstract:Abstract α‐Mannosidases in eukaryotic cells are involved in both glycan biosynthetic reactions and glycan catabolism. Two broad families of enzymes have been identified that cleave terminal mannose linkages from Asn‐linked oligosaccharides (Moremen, 2000), including the Class 1 Mannosidases (CAZy GH family 47 (Henrissat and Bairoch, 1996)) of the early secretory pathway involved in the processing of N‐glycans and quality control and the Class 2 Mannosidases (CAZy family GH38 [Henrissat and Bairoch, 1996]) involved in glycoprotein biosynthesis or catabolism. Within the Class 1 family of α‐Mannosidases, three subfamilies of enzymes have been identified (Moremen, 2000). The endoplasmic reticulum (ER) α1,2‐Mannosidase I (ERManI) subfamily acts to cleave a single residue from Asn‐linked glycans in the ER. The Golgi α‐Mannosidase I (GolgiManI) subfamily has at least three members in mammalian systems (Herscovics et al., 1994; Lal et al., 1994; Tremblay and Herscovics, 2000) involved in glycan maturation in the Golgi complex to form the Man5GlcNAc2 processing intermediate. The third subfamily of GH47 proteins comprises the ER degradation, enhancing α‐Mannosidase‐like proteins (EDEM proteins) (Helenius and Aebi, 2004; Hirao et al., 2006; Mast et al., 2005). These proteins have been proposed to accelerate the degradation of misfolded proteins in the lumen of the ER by a lectin function that leads to retrotranslocation to the cytosol and proteasomal degradation. Recent studies have also indicated that ERManI acts as a timer for initiation of glycoprotein degradation via the ubiquitin‐proteasome pathway (Hosokawa et al., 2003; Wu et al., 2003). This article discusses methods for analysis of the GH47 α‐Mannosidases, including expression, purification, activity assays, generation of point mutants, and binding studies by surface plasmon resonance.
-
energetics of substrate binding and catalysis by class 1 glycosylhydrolase family 47 α Mannosidases involved inn glycan processing and endoplasmic reticulum quality control
Journal of Biological Chemistry, 2005Co-Authors: Khanita Karaveg, Kelley W. MoremenAbstract:Abstract Nascent glycoproteins are subject to quality control in the lumen of the endoplasmic reticulum (ER) where they can either be effectively folded with the aid of a collection of ER chaperones or they can be targeted for disposal in a process known as ER-associated degradation. Initiation of the ER disposal process involves selective trimming of N-glycans by ER α-Mannosidase I and subsequent recognition by the ER degradation-enhancing α-Mannosidase-like protein family of lectins, both members of glycosylhydrolase family 47. The kinetics and energetics of substrate binding and catalysis by members of this family were investigated here by the analysis of wild type and mutant forms of human ER α-Mannosidase I. The contributions of several amino acid residues and an enzyme-associated Ca2+ ion to substrate binding and catalysis were demonstrated by a combination of surface plasmon resonance and enzyme kinetic analyses. One mutant, E330Q, shown previously to alter general acid function within the catalytic site, resulted in an enzyme that possessed increased glycan binding affinity but compromised glycan hydrolysis. This mutant protein was used in a series of glycan binding studies with a library of mannose-containing ligands to examine the energetics of Man9GlcNAc2 substrate interactions. These studies provide a framework for understanding the nature of the unusual substrate interactions within the family 47 Mannosidases involved in glycan maturation and ER-associated glycoprotein degradation.
-
energetics of substrate binding and catalysis by class 1 glycosylhydrolase family 47 α Mannosidases involved inn glycan processing and endoplasmic reticulum quality control
Journal of Biological Chemistry, 2005Co-Authors: Khanita Karaveg, Kelley W. MoremenAbstract:Abstract Nascent glycoproteins are subject to quality control in the lumen of the endoplasmic reticulum (ER) where they can either be effectively folded with the aid of a collection of ER chaperones or they can be targeted for disposal in a process known as ER-associated degradation. Initiation of the ER disposal process involves selective trimming of N-glycans by ER α-Mannosidase I and subsequent recognition by the ER degradation-enhancing α-Mannosidase-like protein family of lectins, both members of glycosylhydrolase family 47. The kinetics and energetics of substrate binding and catalysis by members of this family were investigated here by the analysis of wild type and mutant forms of human ER α-Mannosidase I. The contributions of several amino acid residues and an enzyme-associated Ca2+ ion to substrate binding and catalysis were demonstrated by a combination of surface plasmon resonance and enzyme kinetic analyses. One mutant, E330Q, shown previously to alter general acid function within the catalytic site, resulted in an enzyme that possessed increased glycan binding affinity but compromised glycan hydrolysis. This mutant protein was used in a series of glycan binding studies with a library of mannose-containing ligands to examine the energetics of Man9GlcNAc2 substrate interactions. These studies provide a framework for understanding the nature of the unusual substrate interactions within the family 47 Mannosidases involved in glycan maturation and ER-associated glycoprotein degradation.
Annette Herscovics - One of the best experts on this subject based on the ideXlab platform.
-
stimulation of erad of misfolded null hong kong α1 antitrypsin by golgi α1 2 Mannosidases
Biochemical and Biophysical Research Communications, 2007Co-Authors: Nobuko Hosokawa, Linda O Tremblay, Kazuhiro Nagata, Annette HerscovicsAbstract:Abstract Terminally misfolded or unassembled proteins are degraded by the cytoplasmic ubiquitin-proteasome pathway in a process known as ERAD (endoplasmic reticulum-associated protein degradation). Overexpression of ER α1,2-Mannosidase I and EDEMs target misfolded glycoproteins for ERAD, most likely due to trimming of N -glycans. Here we demonstrate that overexpression of Golgi α1,2-Mannosidase IA, IB, and IC also accelerates ERAD of terminally misfolded human α1-antitrypsin variant null (Hong Kong) (NHK), and mannose trimming from the N -glycans on NHK in 293 cells. Although transfected NHK is primarily localized in the ER, some NHK also co-localizes with Golgi markers, suggesting that mannose trimming by Golgi α1,2-Mannosidases can also contribute to NHK degradation.
-
edem3 a soluble edem homolog enhances glycoprotein endoplasmic reticulum associated degradation and mannose trimming
Journal of Biological Chemistry, 2006Co-Authors: Kazuyoshi Hirao, Linda O Tremblay, Yuko Natsuka, Taku Tamura, Ikuo Wada, Daisuke Morito, Shunji Natsuka, Pedro Romero, Barry Sleno, Annette HerscovicsAbstract:Abstract Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing α-Mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I α-Mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded α1-antitrypsin variant (null (Hong Kong)) and of TCRα. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded α1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent α1,2-Mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of α1,2-Mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has α1,2-Mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.
-
structure of penicillium citrinum alpha 1 2 Mannosidase reveals the basis for differences in specificity of the endoplasmic reticulum and golgi class i enzymes
Journal of Biological Chemistry, 2002Co-Authors: Yuri D Lobsanov, Annette Herscovics, Francois Vallee, Anne Imberty, T Yoshida, Patrick Yip, Lynne P HowellAbstract:Class I alpha1,2-Mannosidases (glycosylhydrolase family 47) are key enzymes in the maturation of N-glycans. This protein family includes two distinct enzymatically active subgroups. Subgroup 1 includes the yeast and human endoplasmic reticulum (ER) alpha1,2-Mannosidases that primarily trim Man(9)GlcNAc(2) to Man(8)GlcNAc(2) isomer B whereas subgroup 2 includes mammalian Golgi alpha1,2-Mannosidases IA, IB, and IC that trim Man(9)GlcNAc(2) to Man(5)GlcNAc(2) via Man(8)GlcNAc(2) isomers A and C. The structure of the catalytic domain of the subgroup 2 alpha1,2-Mannosidase from Penicillium citrinum has been determined by molecular replacement at 2.2-A resolution. The fungal alpha1,2-Mannosidase is an (alphaalpha)(7)-helix barrel, very similar to the subgroup 1 yeast (Vallee, F., Lipari, F., Yip, P., Sleno, B., Herscovics, A., and Howell, P. L. (2000) EMBO J. 19, 581-588) and human (Vallee, F., Karaveg, K., Herscovics, A., Moremen, K. W., and Howell, P. L. (2000) J. Biol. Chem. 275, 41287-41298) ER enzymes. The location of the conserved acidic residues of the catalytic site and the binding of the inhibitors, kifunensine and 1-deoxymannojirimycin, to the essential calcium ion are conserved in the fungal enzyme. However, there are major structural differences in the oligosaccharide binding site between the two alpha1,2-Mannosidase subgroups. In the subgroup 1 enzymes, an arginine residue plays a critical role in stabilizing the oligosaccharide substrate. In the fungal alpha1,2-Mannosidase this arginine is replaced by glycine. This replacement and other sequence variations result in a more spacious carbohydrate binding site. Modeling studies of interactions between the yeast, human and fungal enzymes with different Man(8)GlcNAc(2) isomers indicate that there is a greater degree of freedom to bind the oligosaccharide in the active site of the fungal enzyme than in the yeast and human ER alpha1,2-Mannosidases.
-
structure and function of class i α1 2 Mannosidases involved in glycoprotein synthesis and endoplasmic reticulum quality control
Biochimie, 2001Co-Authors: Annette HerscovicsAbstract:Abstract Class I α1,2-Mannosidases (glycosylhydrolase family 47) are conserved through eukaryotic evolution. This protein family comprises three subgroups distinguished by their enzymatic properties. The first subgroup includes yeast ( Saccharomyces cerevisiae ) and human α1,2-Mannosidases of the endoplasmic reticulum that primarily form Man 8 GlcNAc 2 isomer B from Man 9 GlcNAc 2 . The second subgroup includes mammalian Golgi α1,2-Mannosidases, as well as enzymes from insect cells and from filamentous fungi, that trim Man 9 GlcNAc 2 to Man 8 GlcNAc 2 isomers A and/or C intermediates toward the formation of Man 5 GlcNAc 2 . Yeast and mammalian proteins of the third subgroup have no enzyme activity with Man 9 GlcNAc 2 as substrate. The members of subgroups 1 and 3 participate in endoplasmic reticulum quality control and promote proteasomal degradation of misfolded glycoproteins. The yeast endoplasmic reticulum α1,2-Mannosidase has served as a model for structure-function studies of this family. Its structure was determined by X-ray crystallography as an enzyme-product complex. It consists of a novel (αα) 7 barrel containing the active site that includes essential acidic residues and calcium. The structures of the subgroup 1 human endoplasmic reticulum α1,2-Mannosidase and of a subgroup 2 fungal α1,2-Mannosidase were determined by molecular replacement. Comparison of the enzyme structures is providing some insight into the reasons for their different specificities.
-
a novel er α Mannosidase like protein accelerates er associated degradation
EMBO Reports, 2001Co-Authors: Nobuko Hosokawa, Linda O Tremblay, Annette Herscovics, Ikuo Wada, Kiyotaka Hasegawa, Tetuya Yorihuzi, Kazuhiro NagataAbstract:The quality control mechanism in the endoplasmic reticulum (ER) discriminates correctly folded proteins from misfolded polypeptides and determines their fate. Terminally misfolded proteins are retrotranslocated from the ER and degraded by cytoplasmic proteasomes, a mechanism known as ER-associated degradation (ERAD). We report the cDNA cloning of Edem, a mouse gene encoding a putative type II ER transmembrane protein. Expression of Edem mRNA was induced by various types of ER stress. Although the luminal region of ER degradation enhancing α-Mannosidase-like protein (EDEM) is similar to class I α1,2-Mannosidases involved in N-glycan processing, EDEM did not have enzymatic activity. Overexpression of EDEM in human embryonic kidney 293 cells accelerated the degradation of misfolded α1-antitrypsin, and EDEM bound to this misfolded glycoprotein. The results suggest that EDEM is directly involved in ERAD, and targets misfolded glycoproteins for degradation in an N-glycan dependent manner.
Khanita Karaveg - One of the best experts on this subject based on the ideXlab platform.
-
substrate recognition and catalysis by gh47 α Mannosidases involved in asn linked glycan maturation in the mammalian secretory pathway
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Yong Xiang, Khanita Karaveg, Kelley W. MoremenAbstract:Maturation of Asn-linked oligosaccharides in the eukaryotic secretory pathway requires the trimming of nascent glycan chains to remove all glucose and several mannose residues before extension into complex-type structures on the cell surface and secreted glycoproteins. Multiple glycoside hydrolase family 47 (GH47) α-Mannosidases, including endoplasmic reticulum (ER) α-Mannosidase I (ERManI) and Golgi α-Mannosidase IA (GMIA), are responsible for cleavage of terminal α1,2-linked mannose residues to produce uniquely trimmed oligomannose isomers that are necessary for ER glycoprotein quality control and glycan maturation. ERManI and GMIA have similar catalytic domain structures, but each enzyme cleaves distinct residues from tribranched oligomannose glycan substrates. The structural basis for branch-specific cleavage by ERManI and GMIA was explored by replacing an essential enzyme-bound Ca2+ ion with a lanthanum (La3+) ion. This ion swap led to enzyme inactivation while retaining high-affinity substrate interactions. Cocrystallization of La3+-bound enzymes with Man9GlcNAc2 substrate analogs revealed enzyme–substrate complexes with distinct modes of glycan branch insertion into the respective enzyme active-site clefts. Both enzymes had glycan interactions that extended across the entire glycan structure, but each enzyme engaged a different glycan branch and used different sets of glycan interactions. Additional mutagenesis and time-course studies of glycan cleavage probed the structural basis of enzyme specificity. The results provide insights into the enzyme catalytic mechanisms and reveal structural snapshots of the sequential glycan cleavage events. The data also indicate that full steric access to glycan substrates determines the efficiency of mannose-trimming reactions that control the conversion to complex-type structures in mammalian cells.
-
substrate recognition and catalysis by gh47 α Mannosidases involved in asn linked glycan maturation in the mammalian secretory pathway
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Yong Xiang, Khanita Karaveg, Kelley W. MoremenAbstract:Maturation of Asn-linked oligosaccharides in the eukaryotic secretory pathway requires the trimming of nascent glycan chains to remove all glucose and several mannose residues before extension into complex-type structures on the cell surface and secreted glycoproteins. Multiple glycoside hydrolase family 47 (GH47) α-Mannosidases, including endoplasmic reticulum (ER) α-Mannosidase I (ERManI) and Golgi α-Mannosidase IA (GMIA), are responsible for cleavage of terminal α1,2-linked mannose residues to produce uniquely trimmed oligomannose isomers that are necessary for ER glycoprotein quality control and glycan maturation. ERManI and GMIA have similar catalytic domain structures, but each enzyme cleaves distinct residues from tribranched oligomannose glycan substrates. The structural basis for branch-specific cleavage by ERManI and GMIA was explored by replacing an essential enzyme-bound Ca2+ ion with a lanthanum (La3+) ion. This ion swap led to enzyme inactivation while retaining high-affinity substrate interactions. Cocrystallization of La3+-bound enzymes with Man9GlcNAc2 substrate analogs revealed enzyme–substrate complexes with distinct modes of glycan branch insertion into the respective enzyme active-site clefts. Both enzymes had glycan interactions that extended across the entire glycan structure, but each enzyme engaged a different glycan branch and used different sets of glycan interactions. Additional mutagenesis and time-course studies of glycan cleavage probed the structural basis of enzyme specificity. The results provide insights into the enzyme catalytic mechanisms and reveal structural snapshots of the sequential glycan cleavage events. The data also indicate that full steric access to glycan substrates determines the efficiency of mannose-trimming reactions that control the conversion to complex-type structures in mammalian cells.
-
energetics of substrate binding and catalysis by class 1 glycosylhydrolase family 47 α Mannosidases involved inn glycan processing and endoplasmic reticulum quality control
Journal of Biological Chemistry, 2005Co-Authors: Khanita Karaveg, Kelley W. MoremenAbstract:Abstract Nascent glycoproteins are subject to quality control in the lumen of the endoplasmic reticulum (ER) where they can either be effectively folded with the aid of a collection of ER chaperones or they can be targeted for disposal in a process known as ER-associated degradation. Initiation of the ER disposal process involves selective trimming of N-glycans by ER α-Mannosidase I and subsequent recognition by the ER degradation-enhancing α-Mannosidase-like protein family of lectins, both members of glycosylhydrolase family 47. The kinetics and energetics of substrate binding and catalysis by members of this family were investigated here by the analysis of wild type and mutant forms of human ER α-Mannosidase I. The contributions of several amino acid residues and an enzyme-associated Ca2+ ion to substrate binding and catalysis were demonstrated by a combination of surface plasmon resonance and enzyme kinetic analyses. One mutant, E330Q, shown previously to alter general acid function within the catalytic site, resulted in an enzyme that possessed increased glycan binding affinity but compromised glycan hydrolysis. This mutant protein was used in a series of glycan binding studies with a library of mannose-containing ligands to examine the energetics of Man9GlcNAc2 substrate interactions. These studies provide a framework for understanding the nature of the unusual substrate interactions within the family 47 Mannosidases involved in glycan maturation and ER-associated glycoprotein degradation.
-
energetics of substrate binding and catalysis by class 1 glycosylhydrolase family 47 α Mannosidases involved inn glycan processing and endoplasmic reticulum quality control
Journal of Biological Chemistry, 2005Co-Authors: Khanita Karaveg, Kelley W. MoremenAbstract:Abstract Nascent glycoproteins are subject to quality control in the lumen of the endoplasmic reticulum (ER) where they can either be effectively folded with the aid of a collection of ER chaperones or they can be targeted for disposal in a process known as ER-associated degradation. Initiation of the ER disposal process involves selective trimming of N-glycans by ER α-Mannosidase I and subsequent recognition by the ER degradation-enhancing α-Mannosidase-like protein family of lectins, both members of glycosylhydrolase family 47. The kinetics and energetics of substrate binding and catalysis by members of this family were investigated here by the analysis of wild type and mutant forms of human ER α-Mannosidase I. The contributions of several amino acid residues and an enzyme-associated Ca2+ ion to substrate binding and catalysis were demonstrated by a combination of surface plasmon resonance and enzyme kinetic analyses. One mutant, E330Q, shown previously to alter general acid function within the catalytic site, resulted in an enzyme that possessed increased glycan binding affinity but compromised glycan hydrolysis. This mutant protein was used in a series of glycan binding studies with a library of mannose-containing ligands to examine the energetics of Man9GlcNAc2 substrate interactions. These studies provide a framework for understanding the nature of the unusual substrate interactions within the family 47 Mannosidases involved in glycan maturation and ER-associated glycoprotein degradation.
-
human edem2 a novel homolog of family 47 glycosidases is involved in er associated degradation of glycoproteins
Glycobiology, 2005Co-Authors: Steven W Mast, Khanita Karaveg, Krista Diekman, Ann Davis, Richard N Sifers, Kelley W. MoremenAbstract:In the endoplasmic reticulum (ER), misfolded proteins are retrotranslocated to the cytosol and degraded by the proteasome in a process known as ER-associated degradation (ERAD). Early in this pathway, a proposed lumenal ER lectin, EDEM, recognizes misfolded glycoproteins in the ER, disengages the nascent molecules from the folding pathway, and facilitates their targeting for disposal. In humans there are a total of three EDEM homologs. The amino acid sequences of these proteins are different from other lectins but are closely related to the Class I Mannosidases (family 47 glycosidases). In this study, we characterize one of the EDEM homologs from Homo sapiens, which we have termed EDEM2 (C20orf31). Using recombinantly generated EDEM2, no -1,2 Mannosidase activity was observed. In HEK293 cells, recombinant EDEM2 is localized to the ER where it can associate with misfolded 1-antitrypsin. Overexpression of EDEM2 accelerates the degradation of misfolded 1-antitrypsin, indicating that the protein is involved in ERAD.
G J Davies - One of the best experts on this subject based on the ideXlab platform.
-
structure and function of bs164 beta Mannosidase frombacteroides salyersiaethe founding member of glycoside hydrolase family gh164
Journal of Biological Chemistry, 2020Co-Authors: Zachary Armstrong, G J DaviesAbstract:Recent work exploring protein sequence space has revealed a new glycoside hydrolase (GH) family (GH164) of putative Mannosidases. GH164 genes are present in several commensal bacteria, implicating these genes in the degradation of dietary glycans. However, little is known about the structure, mechanism of action, and substrate specificity of these enzymes. Herein we report the biochemical characterization and crystal structures of the founding member of this family (Bs164) from the human gut symbiont Bacteroides salyersiae. Previous reports of this enzyme indicated that it has alpha-Mannosidase activity, however, we conclusively show that it cleaves only beta-mannose linkages. Using NMR spectroscopy, detailed enzyme kinetics of WT and mutant Bs164, and multiangle light scattering we found that it is a trimeric retaining beta-Mannosidase, that is susceptible to several known Mannosidase inhibitors. X-ray crystallography revealed the structure of Bs164, the first known structure of a GH164, at 1.91 A resolution. Bs164 is composed of three domains: a (beta/alpha)8 barrel, a trimerization domain, and a beta-sandwich domain, representing a previously unobserved structural-fold for beta-Mannosidases. Structures of Bs164 at 1.80-2.55 A resolution in complex with the inhibitors noeuromycin, mannoimidazole, or 2,4-dinitrophenol 2-deoxy-2-fluoro-mannoside reveal the residues essential for specificity and catalysis including the catalytic nucleophile (Glu-297) and acid/base residue (Glu-160). These findings further our knowledge of the mechanisms commensal microbes use for nutrient acquisition.
-
structure and function of bs164 beta Mannosidase frombacteroides salyersiaethe founding member of glycoside hydrolase family gh164
Journal of Biological Chemistry, 2020Co-Authors: Zachary Armstrong, G J DaviesAbstract:: Recent work exploring protein sequence space has revealed a new glycoside hydrolase (GH) family (GH164) of putative Mannosidases. GH164 genes are present in several commensal bacteria, implicating these genes in the degradation of dietary glycans. However, little is known about the structure, mechanism of action, and substrate specificity of these enzymes. Herein we report the biochemical characterization and crystal structures of the founding member of this family (Bs164) from the human gut symbiont Bacteroides salyersiae. Previous reports of this enzyme indicated that it has α-Mannosidase activity, however, we conclusively show that it cleaves only β-mannose linkages. Using NMR spectroscopy, detailed enzyme kinetics of WT and mutant Bs164, and multiangle light scattering we found that it is a trimeric retaining β-Mannosidase, that is susceptible to several known Mannosidase inhibitors. X-ray crystallography revealed the structure of Bs164, the first known structure of a GH164, at 1.91 A resolution. Bs164 is composed of three domains: a (β/α)8 barrel, a trimerization domain, and a β-sandwich domain, representing a previously unobserved structural-fold for β-Mannosidases. Structures of Bs164 at 1.80-2.55 A resolution in complex with the inhibitors noeuromycin, mannoimidazole, or 2,4-dinitrophenol 2-deoxy-2-fluoro-mannoside reveal the residues essential for specificity and catalysis including the catalytic nucleophile (Glu-297) and acid/base residue (Glu-160). These findings further our knowledge of the mechanisms commensal microbes use for nutrient acquisition.
Wolfram Tempel - One of the best experts on this subject based on the ideXlab platform.
-
structure of mouse golgi alpha Mannosidase ia reveals the molecular basis for substrate specificity among class 1 family 47 glycosylhydrolase alpha1 2 Mannosidases
Journal of Biological Chemistry, 2004Co-Authors: Wolfram Tempel, Khanita Karaveg, J P Rose, B C Wang, Kelley W. MoremenAbstract:Abstract Three subfamilies of mammalian Class 1 processing α1,2-Mannosidases (family 47 glycosidases) play critical roles in the maturation of Asn-linked glycoproteins in the endoplasmic reticulum (ER) and Golgi complex as well as influencing the timing and recognition for disposal of terminally unfolded proteins by ER-associated degradation. In an effort to define the structural basis for substrate recognition among Class 1 Mannosidases, we have crystallized murine Golgi Mannosidase IA (space group P212121), and the structure was solved to 1.5-A resolution by molecular replacement. The enzyme assumes an (αα)7 barrel structure with a Ca2+ ion coordinated at the base of the barrel similar to other Class 1 Mannosidases. Critical residues within the barrel structure that coordinate the Ca2+ ion or presumably bind and catalyze the hydrolysis of the glycone are also highly conserved. A Man6GlcNAc2 oligosaccharide attached to Asn515 in the murine enzyme was found to extend into the active site of an adjoining protein unit in the crystal lattice in a presumed enzyme-product complex. In contrast to an analogous complex previously isolated for Saccharomyces cerevisiae ER Mannosidase I, the oligosaccharide in the active site of the murine Golgi enzyme assumes a different conformation to present an alternate oligosaccharide branch into the active site pocket. A comparison of the observed protein-carbohydrate interactions for the murine Golgi enzyme with the binding cleft topologies of the other family 47 glycosidases provides a framework for understanding the structural basis for substrate recognition among this class of enzymes.
-
structure of mouse golgi alpha Mannosidase ia reveals the molecular basis for substrate specificity among class 1 family 47 glycosylhydrolase alpha1 2 Mannosidases
Journal of Biological Chemistry, 2004Co-Authors: Wolfram Tempel, Khanita Karaveg, J P Rose, B C Wang, Kelley W. MoremenAbstract:Abstract Three subfamilies of mammalian Class 1 processing α1,2-Mannosidases (family 47 glycosidases) play critical roles in the maturation of Asn-linked glycoproteins in the endoplasmic reticulum (ER) and Golgi complex as well as influencing the timing and recognition for disposal of terminally unfolded proteins by ER-associated degradation. In an effort to define the structural basis for substrate recognition among Class 1 Mannosidases, we have crystallized murine Golgi Mannosidase IA (space group P212121), and the structure was solved to 1.5-A resolution by molecular replacement. The enzyme assumes an (αα)7 barrel structure with a Ca2+ ion coordinated at the base of the barrel similar to other Class 1 Mannosidases. Critical residues within the barrel structure that coordinate the Ca2+ ion or presumably bind and catalyze the hydrolysis of the glycone are also highly conserved. A Man6GlcNAc2 oligosaccharide attached to Asn515 in the murine enzyme was found to extend into the active site of an adjoining protein unit in the crystal lattice in a presumed enzyme-product complex. In contrast to an analogous complex previously isolated for Saccharomyces cerevisiae ER Mannosidase I, the oligosaccharide in the active site of the murine Golgi enzyme assumes a different conformation to present an alternate oligosaccharide branch into the active site pocket. A comparison of the observed protein-carbohydrate interactions for the murine Golgi enzyme with the binding cleft topologies of the other family 47 glycosidases provides a framework for understanding the structural basis for substrate recognition among this class of enzymes.
