Purification and characterization of N-glycanase, a concanavalin A binding protein from jackbean (Canavalia ensiformis)

Sheldon, S. Philip; Keen, Nicholas; Bowles, J. Dianna

doi:10.1042/bj3300013

Cited by 14 publications

(4 citation statements)

References 53 publications

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“…In this purification procedure, the lectin-affinity chromatography increased the b-D-xylosidase specific activity by approximately a factor of 7 (Table I). Since Con A Sepharose is normally used for the purification of glycoproteins (Farooqi et al, 1997;Sheldon et al, 1998), this result suggests that the three purified enzymes with b-D-xylosidase activity are glycosylated.…”

Section: Purification Of Enzymes That Exhibit B-d-xylosidase Activitymentioning

confidence: 88%

Purification and Characterization of Enzymes Exhibiting β-d-Xylosidase Activities in Stem Tissues of Arabidopsis

et al. 2004

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This work describes the purification and characterization of enzymes that exhibit b-D-xylosidase activity in stem tissues of Arabidopsis. This is the first detailed investigation that concerns the characterization of catalytic properties and sequence identity of enzymes with b-D-xylosidase activities in a dicotyledonous plant. Three different enzymes, ARAf, XYL4, and XYL1 with apparent molecular masses of 75, 67, and 64 kD, respectively, were purified to homogeneity. ARAf was identified as a putative a-L-arabinofuranosidase, and XYL4 and XYL1 as putative b-D-xylosidases using matrix-assisted laser-desorption ionization time of flight. ARAf belongs to family 51 and XYL4 and XYL1 to family 3 of glycoside hydrolases. ARAf and XYL1 have highest specificity for p-nitrophenyl-a-L-arabinofuranoside and XYL4 for p-nitrophenyl-b-D-xylopyranoside and natural substrates such as xylobiose and xylotetraose. XYL4 was shown to release mainly D-Xyl from oat spelt xylan, rye arabinoxylan, wheat arabinoxylan, and oligoarabinoxylans. ARAf and XYL1 can also release D-Xyl from these substrates but less efficiently than XYL4. Moreover, they can also release L-Ara from arabinoxylans and arabinan. Overall, the results indicate that XYL4 possesses enzymatic specificity characteristic for a b-D-xylosidase, while ARAf and XYL1 act as bifunctional a-L-arabinofuranosidase/b-D-xylosidases. Analysis of the activity of these three enzymes in stem tissues at different stages of development has shown that young stems possess the highest activities for all three enzymes in comparison to the activities of the enzymes present in stems at older stages of development. High enzyme activities are most likely related to the necessary modifications of cell wall structure occurring during plant growth. Plant cells are surrounded by an extracellular matrix known as the cell wall. The regulation of cell wall morphology and composition is important for the determination of cell size and shape, cellular interactions with the environment, mechanical resistance, and defense against pathogen attacks (Reiter, 2002). The cell wall is also involved in plant growth and development (Stolle-Smits et al., 1999;Obel et al., 2002). In addition, although cell walls consist mainly of polysaccharides broadly classified as celluloses, hemicelluloses, and pectins, their constitution varies, not only from plant to plant, but also in different tissues of the same plant (Heredia et al., 1995;Cosgrove, 1997;Popper and Fry, 2003). A coordinated series of biochemical processes occur during plant development resulting in the biosynthesis and degradation of cell wall components. Consequently, numerous enzymes must be implicated in these processes (Heredia et al., 1995;Cosgrove, 1997;Stolle-Smits et al., 1999;Obel et al., 2002;Reiter, 2002;Popper and Fry, 2003). However, to date, little information is available concerning such enzymes, their mode of action, and their physiological role. Increased research interest has focused on enzymes involved in the metabolism of hemicelluloses in ...

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Section: Purification Of Enzymes That Exhibit B-d-xylosidase Activitymentioning

confidence: 88%

Purification and Characterization of Enzymes Exhibiting β-d-Xylosidase Activities in Stem Tissues of Arabidopsis

et al. 2004

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“…Terriglobus strains can, in contrast to other isolates from the phylum Acidobacteria , also grow under mildly acidic conditions [ 25 ]. Most acidic PNGases were described to have a pH optimum between 4.0 and 5.0 [ 15 , 16 , 26 , 27 ], which is probably based on the slightly acidic environment in vacuoles or their extracellular environment after secretion [ 15 , 28 ].…”

Section: Discussionmentioning

confidence: 99%

Discovery and characterization of a novel extremely acidic bacterial N-glycanase with combined advantages of PNGase F and A

Wang

Cai

et al. 2014

Bioscience Reports

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Peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidases [PNGases (peptide N-glycosidases), N-glycanases, EC 3.5.1.52] are essential tools in the release of N-glycans from glycoproteins. We hereby report the discovery and characterization of a novel bacterial N-glycanase from Terriglobus roseus with an extremely low pH optimum of 2.6, and annotated it therefore as PNGase H+. The gene of PNGase H+ was cloned and the recombinant protein was successfully expressed in Escherichia coli. The recombinant PNGase H+ could liberate high mannose-, hybrid- and complex-type N-glycans including core α1,3-fucosylated oligosaccharides from both glycoproteins and glycopeptides. In addition, PNGase H+ exhibited better release efficiency over N-glycans without core α1,3-fucose compared with PNGase A. The facile expression, non-glycosylated nature, unusual pH optimum and broad substrate specificity of this novel type of N-glycanase makes recombinant PNGase H+ a versatile tool in N-glycan analysis.

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“…The studies from Ivessa's laboratory on truncated ribophorin I have raised the possibility that the cleavage of the N-glycans may actually take place in the lumen of the ER prior to retrotranslocation [70,102]. Moreover, the site-specific N-deglycosylation of diglycosylated ovalbumin can be attributed to an ER situated N-glycanase [96], and a particulate PNGase is also believed to convert concanavalin A to an active lectin by cleaving the N-glycan of the newly synthesized protein [107,108]. Direct examination of the polymannose oligosaccharides in mouse T-lymphoma cells in which accelerated degradation of the TCR a-subunit takes place indicated that there was a reduction in their release into the cytosol in the presence of proteasome inhibitors [36].…”

Section: N-deglycosylation Of Glycoproteins During Quality Control: Amentioning

confidence: 99%

Role of N -linked polymannose oligosaccharides in targeting glycoproteins for endoplasmic reticulum-associated degradation

Spiro

2004

Cellular and Molecular Life Sciences (CMLS)

138

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Misfolded or incompletely assembled multisubunit glycoproteins undergo endoplasmic reticulum-associated degradation (ERAD) regulated in large measure by their N-linked polymannose oligosaccharides. In this quality control system lectin interaction with Glc(3)Man(9)GlcNAc(2) glycans after trimming with endoplasmic reticulum (ER) alpha-glucosidases and alpha-mannosidases sorts out persistently unfolded glycoproteins for N-deglycosylation and proteolytic degradation. Monoglucosylated (Glc(1)Man(9)GlcNAc(2)) glycoproteins take part in the calnexin/calreticulin glucosylation-deglucosylation cycle, while the Man(8)GlcNAc(2) isomer B product of ER mannosidase I interacts with EDEM. Proteasomal degradation requires retrotranslocation into the cytosol through a Sec61 channel and deglycosylation by peptide: N-glycosidase (PNGase); in alternate models both PNGase and proteasomes may be either free in the cytosol or ER membrane-imbedded/attached. Numerous proteins appear to undergo nonproteasomal degradation in which deglycosylation and proteolysis take place in the ER lumen. The released free oligosaccharides (OS) are transported to the cytosol as OS-GlcNAc(2) along with similar components produced by the hydrolytic action of the oligosaccharyltransferase, where they together with OS from the proteasomal pathway are trimmed to Man(5)GlcNAc(1) by the action of cytosolic endo-beta- N-acetylglucosaminidase and alpha-mannosidase before entering the lysosomes. Some misfolded glycoproteins can recycle between the ER, intermediate and Golgi compartments, where they are further processed before ERAD. Moreover, properly folded glycoproteins with mannose-trimmed glycans can be deglucosylated in the Golgi by endomannosidase, thereby releasing calreticulin and permitting formation of complex OS. A number of regulatory controls have been described, including the glucosidase-glucosyltransferase shuttle, which controls the level of Glc(3)Man(9)GlcNAc(2)-P-P-Dol, and the unfolded protein response, which enhances synthesis of components of the quality control system.

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Purification and characterization of N-glycanase, a concanavalin A binding protein from jackbean (Canavalia ensiformis)

Cited by 14 publications

References 53 publications

Purification and Characterization of Enzymes Exhibiting β-d-Xylosidase Activities in Stem Tissues of Arabidopsis

Purification and Characterization of Enzymes Exhibiting β-d-Xylosidase Activities in Stem Tissues of Arabidopsis

Discovery and characterization of a novel extremely acidic bacterial N-glycanase with combined advantages of PNGase F and A

Role of N -linked polymannose oligosaccharides in targeting glycoproteins for endoplasmic reticulum-associated degradation

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