scholarly journals Human lysosomal α-glucosidase: functional characterization of the glycosylation sites

1993 ◽  
Vol 289 (3) ◽  
pp. 681-686 ◽  
Author(s):  
M M P Hermans ◽  
H A Wisselaar ◽  
M A Kroos ◽  
B A Oostra ◽  
A J J Reuser
Keyword(s):  
Functional Characterization ◽  
Enzyme Synthesis ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Post Translational Modification ◽  
Transport Pathway ◽  
Glycosylation Sites ◽  
Mature Enzyme ◽  
Alpha Glucosidase

N-linked glycosylation is one of the important events in the post-translational modification of human lysosomal alpha-glucosidase. Phosphorylation of mannose residues ensures efficient transport of the enzyme to the lysosomes via the mannose 6-phosphate receptor. The primary structure of lysosomal alpha-glucosidase, as deduced from the cDNA sequence, indicates that there are seven potential glycosylation sites. We have eliminated these sites individually by site-directed mutagenesis and thereby demonstrated that all seven sites are glycosylated. The sites at Asn-882 and Asn-925 were found to be located in a C-terminal propeptide which is cleaved off during maturation. Evidence is presented that at least two of the oligosaccharide side chains of human lysosomal alpha-glucosidase are phosphorylated. Elimination of six of the seven sites does not disturb enzyme synthesis or function. However, removal of the second glycosylation site at Asn-233 interferes dramatically with the formation of mature enzyme. The mutant precursor is synthesized normally and assembles in the endoplasmic reticulum, but immunoelectron microscopy reveals a deficiency of alpha-glucosidase in the Golgi complex and in the more distal compartments of the lysosomal transport pathway.

scholarly journals Characterization of iduronate sulphatase mutants affecting N-glycosylation sites and the cysteine-84 residue

1997 ◽  
Vol 326 (1) ◽  
pp. 243-247 ◽  
Author(s):  
Gilles MILLAT ◽  
Roseline FROISSART ◽  
Irène MAIRE ◽  
Dominique BOZON
Keyword(s):  
Catalytic Activity ◽  
Amino Acid Position ◽  
Cysteine Residue ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Lysosomal Storage ◽  
Post Translational Modification ◽  
Cos Cells ◽  
Conserved Sequence ◽  
Glycosylation Sites

Iduronate sulphatase (IDS) is responsible for mucopolysaccharidosis type II, a rare recessive X-linked lysosomal storage disease. The aim of this work was to evaluate the functional importance of each N-glycosylation site, and of the cysteine-84 residue. IDS mutant cDNAs, lacking one of the eight potential N-glycosylation sites, were expressed in COS cells. Although each of the potential sites was used, none of the eight glycosylation sites appeared to be essential for lysosomal targeting. Another important sulphatase co- or post-translational modification for generating catalytic activity involves the conversion of a cysteine residue surrounded by a conserved sequence C-X-P-S-R into a 2-amino-3-oxopropionic acid residue [Schmidt, Selmer, Ingendoh and von Figura (1995) Cell 82, 271–278]. This conserved cysteine, located at amino acid position 84 in IDS, was replaced either by an alanine (C84A) or by a threonine (C84T) using site-directed mutagenesis. C84A and C84T mutant cDNAs were expressed either in COS cells or in human lymphoblastoid cells deleted for the IDS gene. C84A had a drastic effect both for IDS processing and for catalytic activity. The C84T mutation produced a small amount of mature forms but also abolished enzyme activity, confirming that the cysteine residue at position 84 is required for IDS activity.

scholarly journals DeepNGlyPred: A Deep Neural Network-Based Approach for Human N-Linked Glycosylation Site Prediction

Molecules ◽  
2021 ◽  
Vol 26 (23) ◽  
pp. 7314
Author(s):  
Subash C. Pakhrin ◽  
Kiyoko F. Aoki-Kinoshita ◽  
Doina Caragea ◽  
Dukka B. KC
Keyword(s):  
Computational Prediction ◽  
Structural Features ◽  
Glycosylation Site ◽  
Protein Glycosylation ◽  
Evolutionary Information ◽  
Computational Technique ◽  
Post Translational Modification ◽  
Glycosylation Sites ◽  
Better Than

Protein N-linked glycosylation is a post-translational modification that plays an important role in a myriad of biological processes. Computational prediction approaches serve as complementary methods for the characterization of glycosylation sites. Most of the existing predictors for N-linked glycosylation utilize the information that the glycosylation site occurs at the N-X-[S/T] sequon, where X is any amino acid except proline. Not all N-X-[S/T] sequons are glycosylated, thus the N-X-[S/T] sequon is a necessary but not sufficient determinant for protein glycosylation. In that regard, computational prediction of N-linked glycosylation sites confined to N-X-[S/T] sequons is an important problem. Here, we report DeepNGlyPred a deep learning-based approach that encodes the positive and negative sequences in the human proteome dataset (extracted from N-GlycositeAtlas) using sequence-based features (gapped-dipeptide), predicted structural features, and evolutionary information. DeepNGlyPred produces SN, SP, MCC, and ACC of 88.62%, 73.92%, 0.60, and 79.41%, respectively on N-GlyDE independent test set, which is better than the compared approaches. These results demonstrate that DeepNGlyPred is a robust computational technique to predict N-Linked glycosylation sites confined to N-X-[S/T] sequon. DeepNGlyPred will be a useful resource for the glycobiology community.

scholarly journals Functional Characterization of the N-glycosylation Sites of Human Acid Sphingomyelinase by Site-Directed Mutagenesis

1997 ◽  
Vol 243 (1-2) ◽  
pp. 511-517 ◽  
Author(s):  
Klaus Ferlinz ◽  
Robert Hurwitz ◽  
Heidi Moczall ◽  
Stephanie Lansmann ◽  
Edward H. Schuchman ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Acid Sphingomyelinase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Glycosylation Sites ◽  
Human Acid

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

Identification and functional characterization of legumain in amphioxus Branchiostoma belcheri

2009 ◽  
Vol 30 (3) ◽  
pp. 177-186 ◽  
Author(s):  
Lei Teng ◽  
Hiroshi Wada ◽  
Shicui Zhang
Keyword(s):  
Functional Characterization ◽  
Specific Substrate ◽  
Glycosylation Sites ◽  
Branchiostoma Belcheri ◽  
Hen’S Egg ◽  
Hind Gut ◽  
Catalytic Dyad ◽  
Pepstatin A

Legumain has been reported from diverse sources such as plants, parasites (animals) and mammals, but little is known in the lower chordates. The present study reports the first characterization of legumain cDNA from the protochordate Branchiostoma belcheri. The deduced 435-amino-acid-long protein is structurally characterized by the presence of a putative N-terminal signal peptide, a peptidase_C13 superfamily domain with the conserved Lys123-Gly124-Asp125 motif and catalytic dyad His153 and Cys195 and two potential Asn-glycosylation sites at Asn85 and Asn270. Phylogenetic analysis demonstrates that B. belcheri legumain forms an independent cluster together with ascidian legumain, and is positioned at the base of vertebrate legumains, suggesting that B. belcheri legumain gene may represent the archetype of vertebrate legumain genes. Both recombinant legumain expressed in yeast and endogenous legumain are able to be converted into active protein of ~37 kDa via a C-terminal autocleavage at acid pH values. The recombinant legumain efficiently degrades the legumain-specific substrate Z-Ala-Ala-Asn-MCA (benzyloxycarbonyl-L-alanyl-L-alanyl-L-asparagine-4-methylcoumaryl-7-amide) at optimum pH 5.5; and the enzymatic activity is inhibited potently by iodoacetamide and N-ethylmaleimide, partially by hen's-egg white cystatin, but not by E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane], PMSF and pepstatin A. In addition, legumain is expressed in vivo in a tissue-specific manner, with main expression in the hepatic caecum and hind-gut of B. belcheri. Altogether, these results suggest that B. belcheri legumain plays a role in the degradation of macromolecules in food.

scholarly journals Functional Characterization of a Novel Promoter Element Required for an Innate Immune Response in Drosophila

2003 ◽  
Vol 23 (22) ◽  
pp. 8272-8281 ◽  
Author(s):  
Hanna Uvell ◽  
Ylva Engström
Keyword(s):  
Gene Expression ◽  
Antimicrobial Peptide ◽  
Functional Characterization ◽  
Binding Activity ◽  
Innate Immune ◽  
Site Directed Mutagenesis ◽  
Promoter Element ◽  
Peptide Gene ◽  
Inducible Genes

ABSTRACT Innate immune reactions are crucial processes of metazoans to protect the organism against overgrowth of faster replicating microorganisms. Drosophila melanogaster is a precious model for genetic and molecular studies of the innate immune system. In response to infection, the concerted action of a battery of antimicrobial peptides ensures efficient killing of the microbes. The induced gene expression relies on translocation of the Drosophila Rel transcription factors Relish, Dif, and Dorsal to the nucleus where they bind to κB-like motifs in the promoters of the inducible genes. We have identified another putative promoter element, called region 1 (R1), in a number of antimicrobial peptide genes. Site-directed mutagenesis of the R1 site diminished Cecropin A1 (CecA1) expression in transgenic Drosophila larvae and flies. Infection of flies induced a nuclear R1-binding activity that was unrelated to the κB-binding activity in the same extracts. Although the R1 motif was required for Rel protein-mediated CecA1 expression in cotransfection experiments, our data argue against it being a direct target for the Drosophila Rel proteins. We propose that the R1 and κB motifs are targets for distinct regulatory complexes that act in concert to promote high levels of antimicrobial peptide gene expression in response to infection.

scholarly journals Two N-Linked Glycans Differentially Control Maturation, Trafficking and Proteolysis, but not Activity of the IL-11 Receptor

2018 ◽  
Vol 45 (5) ◽  
pp. 2071-2085 ◽  
Author(s):  
Maria Agthe ◽  
Yvonne Garbers ◽  
Joachim Grötzinger ◽  
Christoph Garbers
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Proteolytic Processing ◽  
Biologically Active ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Post Translational Modification ◽  
Glycosylation Sites ◽  
D2 Domain

Background/Aims: The cytokine interleukin-11 (IL-11) has important pro- and anti-inflammatory functions. It activates its target cells through binding to the IL-11 receptor (IL-11R), and the IL-11/IL-11R complex recruits a homodimer of glycoprotein 130 (gp130). N-linked glycosylation, a post-translational modification where complex oligosaccharides are attached to the side chain of asparagine residues, is often important for stability, folding and biological function of cytokine receptors. Methods: We generated different IL-11R mutants via site-directed mutagenesis and analyzed them in different cell lines via Western blot, flow cytometry, confocal microscopy and proliferation assays. Results: In this study, we identified two functional N-glycosylation sites in the D2 domain of the IL-11R at N127 and N194. While mutation of N127Q only slightly affects cell surface expression of the IL-11R, mutation of N194Q broadly prevents IL-11R appearance at the plasma membrane. Accordingly, IL-11R mutants lacking N194 are retained within the ER, whereas the N127 mutant is transported through the Golgi complex to the cell surface, uncovering a differential role of the two N-glycan sequons for IL-11R maturation. Interestingly, IL-11R mutants devoid of one or both N-glycans are still biologically active. Furthermore, the IL-11RN127Q/N194Q mutant shows no inducible shedding by ADAM10, but is rather constitutively released into the supernatant. Conclusion: Our results show that the two N-glycosylation sites differentially influence stability and proteolytic processing of the IL-11R, but that N-linked glycosylation is not a prerequisite for IL-11 signaling.

Functional characterization of two anti-estradiol antibodies as deduced from modelling and site-directed mutagenesis experiments

2001 ◽  
Vol 14 (2) ◽  
pp. 99-109 ◽  
Author(s):  
Florence Bettsworth ◽  
C�line Monnet ◽  
B�n�dicte Watelet ◽  
Nicole Battail-Poirot ◽  
Bernard Gilquin ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals N-glycosylation requirements for the AT1a angiotensin II receptor delivery to the plasma membrane

1999 ◽  
Vol 339 (2) ◽  
pp. 397-405 ◽  
Author(s):  
Benoit DESLAURIERS ◽  
Cecilia PONCE ◽  
Colette LOMBARD ◽  
Renée LARGUIER ◽  
Jean-Claude BONNAFOUS ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Cellular Localization ◽  
Inositol Phosphate ◽  
Glycosylation Site ◽  
Receptor Expression ◽  
Site Directed Mutagenesis ◽  
Pharmacological Properties ◽  
Peptide Epitope ◽  
Glycosylation Sites

The purpose of this work was to investigate the role of N-glycosylation in the expression and pharmacological properties of the the rat AT1a angiotensin II (AII) receptor. Glycosylation-site suppression was carried out by site-directed mutagenesis (Asn → Gln) of Asn176 and Asn188 (located on the second extracellular loop) and by the removal of Asn4 at the N-terminal end combined with the replacement of the first four amino acids by a 10 amino acid peptide epitope (c-Myc). We generated seven possible N-glycosylation-site-defective mutants, all tagged at their C-terminal ends with the c-Myc epitope. This double-tagging strategy, associated with photoaffinity labelling, allowed evaluation of the molecular masses and immunocytochemical cellular localization of the various receptors transiently expressed in COS-7 cells. We showed that: (i) each of the three N-glycosylation sites are utilized in COS-7 cells; (ii) the mutant with three defective N-glycosylation sites was not (or was very inefficiently) expressed at the plasma membrane and accumulated inside the cell at the perinuclear zone; (iii) the preservation of two sites allowed normal receptor delivery to the plasma membrane, the presence of only Asn176 ensuring a behaviour similar to that of the wild-type receptor; and (iv) all expressed receptors displayed unchanged pharmacological properties (Kd for 125I-sarcosine1-AII; sarcosine1-AII-induced inositol phosphate production). These results demonstrate that N-glycosylation is required for the AT1 receptor expression. They are discussed in the light of current knowledge of membrane-protein maturation and future prospects of receptor overexpression for structural studies.

Effects of N224 glycosylation in Saccharomycopsis fibuligera R64 α-amylase on enzyme activity and stability

2021 ◽  
Vol 17 ◽  
Author(s):  
Yovin Sugijo ◽  
Tina Dewi Rosahdi ◽  
Fernita Puspasari ◽  
Wangsa Tirta Ismaya ◽  
Khomaini Hasan ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Enzyme Activity ◽  
Evolutionary Relationship ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Saccharomycopsis Fibuligera ◽  
Glycosylation Sites ◽  
Starch Substrate ◽  
Relationship Of

Background: The amino acid sequence of an α-amylase of the yeast Saccharomycopsis fibuligera R64 (SfamyR64) contains the two putative N-linked glycosylation sites N153 and N224. N224 is hypothetically responsible for the binding of starch substrate because it is highly conserved among SfamyR64 homologs. Objective: To test whether N224 plays a key role in enzyme activity and stability. Methods: N224Q substitution was introduced by site-directed mutagenesis. The wild type and the mutant were independently over-produced in Pichia pastoris KM71. Activity of the wild type and of the mutant were compared, and their thermal-stability was assessed using heat treatments. The evolutionary relationship of SfamyR64 with its structural homologs with different glycosylation patterns was examined. Results: Activity of the N224Q mutant was approximately 80% lower than that of the wild type. The mutant showed no activity after 10 min of pre-incubation at 50 °C, whereas the wild type SfamyR64 showed activity until 30 min of treatment. Sfamy appeared to have evolved earlier than its structural homolog. Conclusion: SfamyR64 N224 is crucial for enzyme activity and thermal stability. This glycosylation site is unique for fungal and bacterial α-amylases.

Sign in / Sign up

Export Citation Format

Share Document