scholarly journals N-linked glycosylation of native and recombinant cauliflower xyloglucan endotransglycosylase 16A

2003 ◽  
Vol 375 (1) ◽  
pp. 61-73 ◽  
Author(s):  
Hongbin HENRIKSSON ◽  
Stuart E. DENMAN ◽  
Iain D. G. CAMPUZANO ◽  
Pia ADEMARK ◽  
Emma R. MASTER ◽  
...  
Keyword(s):  
Glycosyl Hydrolase ◽  
General Structure ◽  
Hydrolytic Activity ◽  
Glycosylation Site ◽  
Enzymic Activity ◽  
Sequence Motif ◽  
Supporting Evidence ◽  
Xyloglucan Endotransglycosylase ◽  
Conserved Sequence ◽  
High Mannose

The gene encoding a XET (xyloglucan endotransglycosylase) from cauliflower (Brassica oleracea var. botrytis) florets has been cloned and sequenced. Sequence analysis indicated a high degree of similarity to other XET enzymes belonging to glycosyl hydrolase family 16 (GH16). In addition to the conserved GH16 catalytic sequence motif EIDFE, there exists one potential N-linked glycosylation site, which is also highly conserved in XET enzymes from this family. Purification of the corresponding protein from extracts of cauliflower florets allowed the fractionation of a single, pure glycoform, which was analysed by MS techniques. Accurate protein mass determination following the enzymic deglycosylation of this glycoform indicated the presence of a high-mannose-type glycan of the general structure GlcNAc2Man6. LC/MS and MS/MS (tandem MS) analysis provided supporting evidence for this structure and confirmed that the glycosylation site (underlined) was situated close to the predicted catalytic residues in the conserved sequence YLSSTNNEHDEIDFEFLGNRTGQPVILQTNVFTGGK. Heterologous expression in Pichia pastoris produced a range of protein glycoforms, which were, on average, more highly mannosylated than the purified native enzyme. This difference in glycosylation did not influence the apparent enzymic activity of the enzyme significantly. However, the removal of high-mannose glycosylation in recombinant cauliflower XET by endoglycosidase H, quantified by electrospray-ionization MS, caused a 40% decrease in the transglycosylation activity of the enzyme. No hydrolytic activity was detected in native or heterologously expressed BobXET16A, even when almost completely deglycosylated.

scholarly journals Molecular basis of N-acetylglucosaminyltransferase I deficiency in Arabidopsis thaliana plants lacking complex N-glycans

2005 ◽  
Vol 387 (2) ◽  
pp. 385-391 ◽  
Author(s):  
Richard STRASSER ◽  
Johannes STADLMANN ◽  
Barbara SVOBODA ◽  
Friedrich ALTMANN ◽  
Josef GLÖSSL ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Amino Acid ◽  
Heterologous Expression ◽  
Molecular Cloning ◽  
Molecular Basis ◽  
Insect Cells ◽  
Glycosylation Site ◽  
Enzymic Activity ◽  
Critical Amino Acid ◽  
High Mannose

GnTI (N-acetylglucosaminyltransferase I) is a Golgi-resident enzyme essential for the processing of high-mannose to hybrid and complex N-glycans. The Arabidopsis thaliana cgl mutant lacks GnTI activity and as a consequence accumulates oligomannosidic structures. Molecular cloning of cgl GnTI cDNA revealed a point mutation, which causes a critical amino acid substitution (Asp144→Asn), thereby creating an additional N-glycosylation site. Heterologous expression of cgl GnTI in insect cells confirmed its lack of activity and the use of the N-glycosylation site. Remarkably, introduction of the Asp144→Asn mutation into rabbit GnTI, which does not result in the formation of a new N-glycosylation site, led to a protein with strongly reduced, but still detectable enzymic activity. Expression of Asn144 rabbit GnTI in cgl plants could partially restore complex N-glycan formation. These results indicate that the complete deficiency of GnTI activity in cgl plants is mainly due to the additional N-glycan, which appears to interfere with the proper folding of the enzyme.

scholarly journals Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula×tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris

2005 ◽  
Vol 390 (1) ◽  
pp. 105-113 ◽  
Author(s):  
Åsa M. Kallas ◽  
Kathleen Piens ◽  
Stuart E. Denman ◽  
Hongbin Henriksson ◽  
Jenny Fäldt ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Specific Activity ◽  
Glycosylation Site ◽  
Populus Tremula ◽  
Site Directed Mutagenesis ◽  
Glycoside Hydrolase Family ◽  
Sequence Motif ◽  
Hybrid Aspen ◽  
Xyloglucan Endotransglycosylase ◽  
Wild Type

The cDNA encoding a xyloglucan endotransglycosylase, PttXET16A, from hybrid aspen (Populus tremula×tremuloides) has been isolated from an expressed sequence tag library and expressed in the methylotrophic yeast Pichia pastoris. Sequence analysis indicated a high degree of similarity with other proteins in the XTH (xyloglucan transglycosylase/hydrolase) gene subfamily of GH16 (glycoside hydrolase family 16). In addition to the conserved GH16 catalytic sequence motif, PttXET16A contains a conserved N-glycosylation site situated proximal to the predicted catalytic residues. MS analysis indicated that the recombinant PttXET16A expressed in P. pastoris is heterogeneous due to the presence of variable N-glycosylation and incomplete cleavage of the α-factor secretion signal peptide. Removal of the N-glycan by endoglycosidase H treatment did not influence the catalytic activity significantly. Similarly, site-directed mutagenesis of Asn93 to serine to remove the N-glycosylation site resulted in an enzyme which was comparable with the wild-type enzyme in specific activity and thermal stability but had clearly reduced solubility. Hydrolytic activity was detected neither in wild-type PttXET16A before or after enzymatic deglycosylation nor in PttXET16A N93S (Asn93→Ser) mutant.

The Influence of Carbohydrate Structure on the Clearance of Recombinant Tissue-Type Plasminogen Activator

1988 ◽  
Vol 60 (02) ◽  
pp. 255-261 ◽  
Author(s):  
A Hotchkiss ◽  
C J Refino ◽  
C K Leonard ◽  
J V O'Connor ◽  
C Crowley ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Sodium Periodate ◽  
Glycosylation Site ◽  
Tissue Type ◽  
Carbohydrate Structure ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
High Mannose ◽  
Oligosaccharide Structures

SummaryModification of the carbohydrate structures of recombinant tissue-type plasminogen activator (rt-PA) can increase or decrease its rate of clearance in rabbits. When rt-PA was treated with sodium periodate to oxidize carbohydrate residues, the rate of clearance was decreased from 9.6 ± 1.9 ml min−1 kg−1 to 3.5 ± 0.6 ml min−1 kg−1 (mean ± SD, n = 5). A similar change in the clearance of rt-PA was introduced by the use of endo-β-N-acetyl- glucosaminidase H (Endo-H), which selectively removes high mannose asparagine-linked oligosaccharides; the clearance of Endo-H-treated rt-PA was 5.0 ± 0.5 ml min−1 kg−1. A mutant of rt-PA was produced with an amino acid substitution at position 117 (Asn replaced with Gin) to remove a potential glycosylation site that normally contains a high mannose structure. The clearance of this material was also decreased, similar to the periodate and Endo-H-treated rt-PA. Conversely, when rt-PA was produced in the CHO 15B cell line, which can produce only high mannose oligosaccharide structures on glycoproteins, the clearance was increased by a factor of 1.8. These results demonstrate that the removal of rt-PA from the blood depends significantly upon the nature of its oligosaccharide structures.

scholarly journals The Crystal Structure of Shikimate Dehydrogenase (AroE) Reveals a Unique NADPH Binding Mode

2003 ◽  
Vol 185 (14) ◽  
pp. 4144-4151 ◽  
Author(s):  
Sheng Ye ◽  
Frank von Delft ◽  
Alexei Brooun ◽  
Mark W. Knuth ◽  
Ronald V. Swanson ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Binding Pocket ◽  
Binding Mode ◽  
Sequence Motif ◽  
Binary Complex ◽  
Shikimate Dehydrogenase ◽  
Conserved Residues ◽  
Conserved Sequence ◽  
Nicotinamide Mononucleotide ◽  
Reversible Reduction

ABSTRACT Shikimate dehydrogenase catalyzes the NADPH-dependent reversible reduction of 3-dehydroshikimate to shikimate. We report the first X-ray structure of shikimate dehydrogenase from Haemophilus influenzae to 2.4-Å resolution and its complex with NADPH to 1.95-Å resolution. The molecule contains two domains, a catalytic domain with a novel open twisted α/β motif and an NADPH binding domain with a typical Rossmann fold. The enzyme contains a unique glycine-rich P-loop with a conserved sequence motif, GAGGXX, that results in NADPH adopting a nonstandard binding mode with the nicotinamide and ribose moieties disordered in the binary complex. A deep pocket with a narrow entrance between the two domains, containing strictly conserved residues primarily contributed by the catalytic domain, is identified as a potential 3-dehydroshikimate binding pocket. The flexibility of the nicotinamide mononucleotide portion of NADPH may be necessary for the substrate 3-dehydroshikimate to enter the pocket and for the release of the product shikimate.

Functional importance of a conserved sequence motif in FhaC, a prototypic member of the TpsB/Omp85 superfamily

FEBS Journal ◽  
2010 ◽  
Vol 277 (22) ◽  
pp. 4755-4765 ◽  
Author(s):  
Anne-Sophie Delattre ◽  
Bernard Clantin ◽  
Nathalie Saint ◽  
Camille Locht ◽  
Vincent Villeret ◽  
...  
Keyword(s):  
Sequence Motif ◽  
Functional Importance ◽  
Conserved Sequence

scholarly journals The alpha subunit of the Saccharomyces cerevisiae oligosaccharyltransferase complex is essential for vegetative growth of yeast and is homologous to mammalian ribophorin I.

1995 ◽  
Vol 128 (4) ◽  
pp. 525-536 ◽  
Author(s):  
S Silberstein ◽  
P G Collins ◽  
D J Kelleher ◽  
P J Rapiejko ◽  
R Gilmore
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Vegetative Growth ◽  
Functional Characterization ◽  
Glycosylation Site ◽  
Alpha Subunit ◽  
Protein Sequence Analysis ◽  
Exogenous Lipid ◽  
Microsomal Membranes ◽  
High Mannose

Oligosaccharyltransferase mediates the transfer of a preassembled high mannose oligosaccharide from a lipid-linked oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (alpha-zeta), two of which are glycoproteins (alpha and beta). The beta and delta subunits of the oligosaccharyltransferase are encoded by the WBP1 and SWP1 genes. Here we describe the functional characterization of the OST1 gene that encodes the alpha subunit of the oligosaccharyltransferase. Protein sequence analysis revealed a significant sequence identity between the Saccharomyces cerevisiae Ost1 protein and ribophorin I, a previously identified subunit of the mammalian oligosaccharyltransferase. A disruption of the OST1 locus was not tolerated in haploid yeast showing that expression of the Ost1 protein is essential for vegetative growth of yeast. An analysis of a series of conditional ost1 mutants demonstrated that defects in the Ost1 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins at both the permissive and restrictive growth temperatures. Microsomal membranes isolated from ost1 mutant yeast showed marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Microsomal membranes isolated from the ost1 mutants contained elevated amounts of the Kar2 stress-response protein.

A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes

Nature ◽  
1986 ◽  
Vol 319 (6049) ◽  
pp. 154-158 ◽  
Author(s):  
Harinder Singh ◽  
Ranjan Sen ◽  
David Baltimore ◽  
Phillip A. Sharp
Keyword(s):  
Transcriptional Control ◽  
Sequence Motif ◽  
Nuclear Factor ◽  
Immunoglobulin Genes ◽  
Conserved Sequence

scholarly journals Viral DNA Synthesis Defects in Assembly-Competent Rous Sarcoma Virus CA Mutants

2001 ◽  
Vol 75 (1) ◽  
pp. 242-250 ◽  
Author(s):  
Tina M. Cairns ◽  
Rebecca C. Craven
Keyword(s):  
Dna Synthesis ◽  
Rous Sarcoma Virus ◽  
Virus Assembly ◽  
Virus Infectivity ◽  
Sequence Motif ◽  
Viral Dna ◽  
Conserved Sequence ◽  
Rous Sarcoma ◽  
Core Proteins ◽  
Sarcoma Virus

ABSTRACT The major structural protein of the retroviral core (CA) contains a conserved sequence motif shared with the CA-like proteins of distantly related transposable elements. The function of this major region of homology (MHR) has not been defined, in part due to the baffling array of phenotypes in mutants of several viruses and the yeast TY3. This report describes new mutations in the CA protein of Rous sarcoma virus (RSV) that were designed to test whether these different phenotypes might indicate distinct functional subdomains in the MHR. A comparison of 25 substitutions at 10 positions in the RSV conserved motif argues against this possibility. Most of the replacements destroyed virus infectivity, although either of two lethal phenotypes was obtained depending on the residue introduced. At most of the positions, one or more replacements (generally the more conservative substitutions) caused a severe replication defect without having any obvious effects on virus assembly, budding, Gag-Pol and genome incorporation, or protein processing. The mutant particles exhibited a defect in endogenous viral DNA synthesis and showed increased sensitivity of the core proteins to detergent, indicating that the mutations interfere with the formation and/or activity of the virion core. The distribution of these mutations across the MHR, with no evidence of clustering, suggests that the entire region is important for a critical postbudding function. In contrast, a second class of lethal substitutions (those that destroyed virus assembly and release) consists of alterations that are expected to cause severe effects on protein structure by disruption either of the hydrophobic core of the CA carboxyl-terminal domain or of the hydrogen bond network that stabilizes the domain. We suggest that this duality of phenotypes is consistent with a role for the MHR in the maturation process that links the two parts of the life cycle.

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