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.

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 Molecular basis of the Kell (K1) phenotype

Blood ◽  
1995 ◽  
Vol 85 (4) ◽  
pp. 912-916 ◽  
Author(s):  
S Lee ◽  
X Wu ◽  
M Reid ◽  
T Zelinski ◽  
C Redman
Keyword(s):  
Amino Acid ◽  
Molecular Basis ◽  
Base Change ◽  
Glycosylation Site ◽  
Base Substitution ◽  
Blood Group System ◽  
Hemolytic Disease ◽  
High Prevalence ◽  
Low Prevalence ◽  
K Antigen

K1 (K, Kell) is a strong immunogen; its antibodies can cause severe reactions if incompatible blood is transfused and may cause hemolytic disease of the newborn in sensitized mothers. K1 is a member of the Kell blood group system, which is complex, containing over 20 different antigens. Some of the antigens are organized in allelic pairs of high and low prevalence whereas others are independently expressed. K1, which is present in 9% of the population, is antithetical to the high- prevalence K2 (k) antigen. We have determined the molecular basis of the K1/K2 polymorphism by sequencing the 19 exons of the Kell gene (KEL) of a K1/K1 person. Polymerase chain reaction was performed on genomic DNA isolated from peripheral blood and the amplified products were either directly sequenced or subcloned and sequenced. Comparisons of K1/K1 and K2/K2 DNA showed a C to T base substitution in exon 6 that predicts a threonine to methionine change at amino acid residue 193. This amino acid substitution occurs at a consensus N-glycosylation site (Asn. X. Thr) and probably prevents N-glycosylation, leading to a change in phenotype. The C to T substitution creates a Bsm I restriction enzyme site, which was tested in 42 different samples to confirm that this base change identifies the K1/K1 genotype. This test differentiates genotypes, K1/K1, K2/K2, and the K1/K2 heterozygote and should prove useful in the prenatal diagnosis of K1-related hemolytic disease of the newborn.

scholarly journals Molecular Basis for Resistance of Acanthamoeba Tubulins to All Major Classes of Antitubulin Compounds

2007 ◽  
Vol 52 (3) ◽  
pp. 1133-1135 ◽  
Author(s):  
Fiona L. Henriquez ◽  
Paul R. Ingram ◽  
Stephen P. Muench ◽  
David W. Rice ◽  
Craig W. Roberts
Keyword(s):  
Amino Acid ◽  
Binding Sites ◽  
Molecular Basis ◽  
Eukaryotic Cells ◽  
Inhibitor Binding ◽  
Critical Amino Acid

ABSTRACT Tubulin is essential to eukaryotic cells and is targeted by several antineoplastics, herbicides, and antimicrobials. We demonstrate that Acanthamoeba spp. are resistant to five antimicrotubule compounds, unlike any other eukaryote studied so far. Resistance correlates with critical amino acid differences within the inhibitor binding sites of the tubulin heterodimers.

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 Rabbit DNase I: purification from urine, immunological and proteochemical characterization, nucleotide sequence, expression in tissues, relationships with other mammalian DNases I and phylogenetic analysis

1997 ◽  
Vol 325 (2) ◽  
pp. 465-473 ◽  
Author(s):  
Toshihiro YASUDA ◽  
Haruo TAKESHITA ◽  
Tamiko NAKAJIMA ◽  
Osamu HOSOMI ◽  
Yoshimitsu NAKASHIMA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Parotid Gland ◽  
Signal Sequence ◽  
Dnase I ◽  
Glycosylation Site ◽  
Enzymic Activity ◽  
Actin Binding ◽  
Immunological Properties ◽  
Apparent Homogeneity ◽  
I Gene

DNase I from rabbit urine was purified approx. 3600-fold to apparent homogeneity with a 41% yield by affinity chromatography utilizing DNA–cellulose; the purity of the final preparation was assessed by SDS/PAGE, lack of contamination by other nucleases and production of a monospecific antibody against the enzyme. Although the proteochemical and enzymological properties of the purified enzyme resembled those of other mammalian DNases I, the enzymic activity of rabbit DNase I was less efficiently inhibited by monomeric actin than was that of human DNase I, probably due to substitution of an amino acid residue involved in actin binding (Tyr-65 to Phe). The effects of specific antibodies to human, rabbit and rat DNases I on the activities of the corresponding purified enzymes revealed that human DNase I lies between the rat and rabbit enzymes with regard to its immunological properties. An 1158 bp full-length cDNA encoding rabbit DNase I was constructed from the total RNA of rabbit pancreas using a combination of reverse transcriptase-PCR and rapid amplification of cDNA ends, followed by sequencing. This identified a 17- or 21-amino-acid signal sequence, with the mature enzyme containing 260 amino acids and a single N-glycosylation site at Asn-18. The amino acid sequence deduced from the cDNA sequence exactly matched that determined proteochemically from the purified enzyme up to residue 20. A systematic survey of DNase I distribution as measured by both enzymic activity and DNase I gene transcripts in 12 rabbit tissues showed the pancreas and parotid gland to produce equivalent levels, higher than those in other tissues. Enzymic activity and DNase I gene expression levels in each tissue correlated well. The results of phylogenetic and sequence identity analysis, immunological properties and tissue-distribution patterns of DNase I indicated a closer relationship between the rabbit and human enzymes than for other mammalian DNases I. Furthermore, differences between the enzymic activities expressed in mammalian parotid gland and pancreas suggest that the distribution of DNase I in mammalian tissue is species-specific.

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