scholarly journals Quantitative glycoproteomics reveals new classes of STT3A- and STT3B-dependent N-glycosylation sites

2019 ◽  
Vol 218 (8) ◽  
pp. 2782-2796 ◽  
Author(s):  
Natalia A. Cherepanova ◽  
Sergey V. Venev ◽  
John D. Leszyk ◽  
Scott A. Shaffer ◽  
Reid Gilmore
Keyword(s):  
Protein Translocation ◽  
Site Occupancy ◽  
Glycosylation Site ◽  
Wild Type ◽  
New Class ◽  
Cysteine Rich Protein ◽  
Glycosylation Sites ◽  
Flanking Sequences ◽  
Dependent Site ◽  
Mutant Cells

Human cells express two oligosaccharyltransferase complexes (STT3A and STT3B) with partially overlapping functions. The STT3A complex interacts directly with the protein translocation channel to mediate cotranslational glycosylation, while the STT3B complex can catalyze posttranslocational glycosylation. We used a quantitative glycoproteomics procedure to compare glycosylation of roughly 1,000 acceptor sites in wild type and mutant cells. Analysis of site occupancy data disclosed several new classes of STT3A-dependent acceptor sites including those with suboptimal flanking sequences and sites located within cysteine-rich protein domains. Acceptor sites located in short loops of multi-spanning membrane proteins represent a new class of STT3B-dependent site. Remarkably, the lumenal ER chaperone GRP94 was hyperglycosylated in STT3A-deficient cells, bearing glycans on five silent sites in addition to the normal glycosylation site. GRP94 was also hyperglycosylated in wild-type cells treated with ER stress inducers including thapsigargin, dithiothreitol, and NGI-1.

Plasma membrane delivery of the gastric H,K-ATPase: the role of β-subunit glycosylation

2003 ◽  
Vol 285 (4) ◽  
pp. C968-C976 ◽  
Author(s):  
O. Vagin ◽  
S. Denevich ◽  
G. Sachs
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Glycosylation Site ◽  
Β Subunit ◽  
Wild Type ◽  
Α Subunit ◽  
Hek 293 ◽  
Glycosylation Sites ◽  
293 Cells

The factors determining trafficking of the gastric H,K-ATPase to the apical membrane remain elusive. To identify such determinants in the gastric H,K-ATPase, fusion proteins of yellow fluorescent protein (YFP) and the gastric H,K-ATPase β-subunit (YFP-β) and cyan fluorescent protein (CFP) and the gastric H,K-ATPase α-subunit (CFP-α) were expressed in HEK-293 cells. Then plasma membrane delivery of wild-type CFP-α, wild-type YFP-β, and YFP-β mutants lacking one or two of the seven β-subunit glycosylation sites was determined using confocal microscopy and surface biotinylation. Expression of the wild-type YFP-β resulted in the plasma membrane localization of the protein, whereas the expressed CFP-α was retained intracellularly. When coexpressed, both CFP-α and YFP-β were delivered to the plasma membrane. Removing each of the seven glycosylation sites, except the second one, from the extracellular loop of YFP-β prevented plasma membrane delivery of the protein. Only the mutant lacking the second glycosylation site (Asn103Gln) was localized both intracellularly and on the plasma membrane. A double mutant lacking the first (Asn99Gln) and the second (Asn103Gln) glycosylation sites displayed intracellular accumulation of the protein. Therefore, six of the seven glycosylation sites in the β-subunit are essential for the plasma membrane delivery of the β-subunit of the gastric H,K-ATPase, whereas the second glycosylation site (Asn103), which is not conserved among the β-subunits from different species, is not critical for plasma delivery of the protein.

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.

scholarly journals Restoration of virulence of escape mutants of H5 and H9 influenza viruses by their readaptation to mice

2005 ◽  
Vol 86 (10) ◽  
pp. 2831-2838 ◽  
Author(s):  
Irina A. Rudneva ◽  
Natalia A. Ilyushina ◽  
Tatiana A. Timofeeva ◽  
Robert G. Webster ◽  
Nikolai V. Kaverin
Keyword(s):  
Amino Acid ◽  
Amino Acid Change ◽  
Glycosylation Site ◽  
Influenza Viruses ◽  
Phenotypic Characterization ◽  
Escape Mutant ◽  
Wild Type ◽  
Glycosylation Sites ◽  
Escape Mutants ◽  
The One

Antigenic mapping of the haemagglutinin (HA) molecule of H5 and H9 influenza viruses by selecting escape mutants with monoclonal anti-HA antibodies and subjecting the selected viruses to immunological analysis and sequencing has previously been performed. The viruses used as wild-type strains were mouse-adapted variants of the original H5 and H9 isolates. Phenotypic characterization of the escape mutants revealed that the amino acid change in HA that conferred resistance to a monoclonal antibody was sometimes associated with additional effects, including decreased virulence for mice. In the present study, the low-virulence H5 and H9 escape mutants were readapted to mice. Analysis of the readapted variants revealed that the reacquisition of virulence was not necessarily achieved by reacquisition of the wild-type HA gene sequence, but was also associated either with the removal of a glycosylation site (the one acquired previously by the escape mutant) without the exact restoration of the initial wild-type amino acid sequence, or, for an H5 escape mutant that had no newly acquired glycosylation sites, with an additional amino acid change in a remote part of the HA molecule. The data suggest that such ‘compensating’ mutations, removing the damaging effects of antibody-selected amino acid changes, may be important in the course of influenza virus evolution.

scholarly journals Human α-galactosidase A: glycosylation site 3 is essential for enzyme solubility

1998 ◽  
Vol 332 (3) ◽  
pp. 789-797 ◽  
Author(s):  
Yiannis A. IOANNOU ◽  
Ken M. ZEIDNER ◽  
Marie E. GRACE ◽  
Robert J. DESNICK
Keyword(s):  
Endoplasmic Reticulum ◽  
Site Occupancy ◽  
Glycosylation Site ◽  
Enzymic Activity ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Consensus Sequences ◽  
Hybrid Type ◽  
Type Site ◽  
And Function

Human α-galactosidase A (EC 3.2.1.22; α-Gal A) is the homodimeric glycoprotein that hydrolyses the terminal α-galactosyl moieties from glycolipids and glycoproteins. The type, site occupancy and function of the N-linked oligosaccharide chains on this lysosomal hydrolase were determined. Endoglycosidase treatment of the purified recombinant enzyme and mutagenesis studies indicated that three (Asn-139, Asn-192 and Asn-215) of the four potential N-glycosylation consensus sequences were occupied by complex, high-mannose and hybrid-type oligosaccharides respectively. When expressed in COS-1 cells, glycoforms with glycosylation site 1 or 2 obliterated had more than 70% of wild-type activity, and both glycoforms were secreted. In contrast, the glycoform with only site 3 eliminated had decreased activity (less than 40%); little, if any, was secreted. Expressed mutant glycoforms in which site 3 and site 1 or 2 were obliterated had little, if any, intracellular or secreted enzymic activity, and immunofluorescence microscopy revealed that the expressed mutant glycoforms were retained in the endoplasmic reticulum, presumably where they were degraded. Thus glycosylation at site 3 was crucial to the formation of soluble, active enzyme, as well as transport to the lysosome. Absence of the site 3 hybrid-type oligosaccharide exposed an adjacent, normally protected, hydrophobic region, resulting in aggregation of the enzyme polypeptide in the endoplasmic reticulum. In support of this concept, endoglycosidase H-treated enzyme or mannose-terminated enzyme expressed in Autographa californica cells also aggregated when concentrated, emphasizing that site 3 occupancy by a hybrid-type oligosaccharide was required for enzyme solubility.

scholarly journals Involvement of oligomerization, N-glycosylation and sialylation in the clearance of cholinesterases from the circulation

1995 ◽  
Vol 311 (3) ◽  
pp. 959-967 ◽  
Author(s):  
C Kronman ◽  
B Velan ◽  
D Marcus ◽  
A Ordentlich ◽  
S Reuveny ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Half Life ◽  
Sharp Decrease ◽  
Life Time ◽  
Absolute Number ◽  
Glycosylation Site ◽  
Protein Glycosylation ◽  
Wild Type ◽  
Attachment Sites ◽  
Glycosylation Sites

The possible role of post-translational modifications such as subunit oligomerization, protein glycosylation and oligosaccharide processing on the circulatory life-time of proteins was studied using recombinant human acetylcholinesterase (rHuAChE). Different preparations of rHuAChE containing various amounts of tetramers, dimers and monomers are cleared at similar rates from the circulation, suggesting that oligomerization does not play an important role in determining the rate of clearance. An engineered rHuAChE mutant containing only one N-glycosylation site was cleared from the circulation more rapidly than the wild-type triglycosylated enzyme. On the other hand, hyperglycosylated mutants containing either four or five occupied N-glycosylation sites, analagous to those present on the slowly cleared fetal bovine serum acetylcholinesterase (FBS-AChE), were also cleared more rapidly from the bloodstream than the wild-type species. Furthermore, the two different tetraglycosylated mutants were cleared at different rates while the pentaglycosylated mutant exhibited the most rapid clearance profile. These results imply that though the number of N-glycosylation sites plays a role in the circulatory life-time of the enzyme, the number of N-glycan units in itself does not determine the rate of clearance. When saturating amounts of asialofetuin were administered together with rHuAChE, the circulatory half-life of the enzyme was dramatically increased (from 80 min to 19 h) and was found to be similar to that displayed by plasma-derived cholinesterases while desialylation of these enzymes caused a sharp decrease in the circulatory half-life to approximately 3-5 min. Determination of the average number of sialic acid residues per enzyme subunit of the five different N-glycosylation species generated, revealed that the rate of clearance is not a function of the absolute number of appended sialic acid moieties but rather of the number of unoccupied sialic acid attachment sites per enzyme molecule. Specifically, we demonstrate an inverse-linear relationship between the number of vacant sialic acid attachment sites and the values of the enzyme residence time within the bloodstream.

scholarly journals Role of Site-Specific Glycosylation in the I-Like Domain of Integrin β1 in Small Extracellular Vesicle-Mediated Malignant Behavior and FAK Activation

2021 ◽  
Vol 22 (4) ◽  
pp. 1770 ◽  
Author(s):  
Lin Cao ◽  
Yurong Wu ◽  
Xiuxiu Wang ◽  
Xiang Li ◽  
Zengqi Tan ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Biological Function ◽  
Extracellular Vesicle ◽  
Wild Type ◽  
Mcf7 Cells ◽  
Integrin Β1 ◽  
Site Specific ◽  
Glycosylation Sites ◽  
Mutant Cells

Integrin β1 plays an essential role in the crosstalk between tumor cells and their microenvironment. Aberrant N-glycosylation of integrin β1 was documented to alter integrin β1 expression, dimerization, and biological function. However, the biological function of site-specific N-glycosylation of integrin β1 on extracellular vesicles is not fully understood. In this study, we mutated putative N-glycosylation sites in different domains of integrin β1. Removal of the N-glycosylation sites on the I-like domain of integrin β1 (termed the Δ4–6 β1 mutant) suppressed focal adhesion kinase (FAK) signaling, cell migration, and adhesion compared with other β1 mutants. Cell adhesion, migration, and activation of FAK were suppressed in recipient MCF7 cells co-cultured with Δ4–6 mutant cells and treated with small extracellular vesicles (sEVs) from Δ4–6 mutant cells. Notably, the wild-type and β1 mutant were both present in sEVs, and could be transferred to recipient cells via sEVs, resulting in changes of cell behavior. Our findings demonstrate the important roles of N-glycosylation of the I-like domain of integrin β1. Moreover, the vesicular Δ4–6 β1 mutant can regulate integrin-mediated functions in recipient cells via sEVs.

scholarly journals SEC62 encodes a putative membrane protein required for protein translocation into the yeast endoplasmic reticulum.

1989 ◽  
Vol 109 (6) ◽  
pp. 2653-2664 ◽  
Author(s):  
R J Deshaies ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Alpha Helix ◽  
Wild Type ◽  
Cytosol Fraction ◽  
Arginine Residues ◽  
Mutant Cells

Yeast sec62 mutant cells are defective in the translocation of several secretory precursor proteins into the lumen of the endoplasmic reticulum (Rothblatt et al., 1989). The deficiency, which is most restrictive for alpha-factor precursor (pp alpha F) and preprocarboxypeptidase Y, has been reproduced in vitro. Membranes isolated from mutant cells display low and labile translocation activity with pp alpha F translated in a wild-type cytosol fraction. The defect is unique to the membrane fraction because cytosol from mutant cells supports translocation into membranes from wild-type yeast. Invertase assembly is only partly affected by the sec62 mutation in vivo and is nearly normal with mutant membranes in vitro. A potential membrane location for the SEC62 gene product is supported by evaluation of the molecular clone. DNA sequence analysis reveals a 32-kD protein with no obvious NH2-terminal signal sequence but with two domains of sufficient length and hydrophobicity to span a lipid bilayer. Sec62p is predicted to display significant NH2- and COOH-terminal hydrophilic domains on the cytoplasmic surface of the ER membrane. The last 30 amino acids of the COOH terminus may form an alpha-helix with 14 lysine and arginine residues arranged uniformly about the helix. This domain may allow Sec62p to interact with other proteins of the putative translocation complex.

scholarly journals Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70.

1993 ◽  
Vol 120 (1) ◽  
pp. 95-102 ◽  
Author(s):  
J L Brodsky ◽  
S Hamamoto ◽  
D Feldheim ◽  
R Schekman
Keyword(s):  
Protein Translocation ◽  
Soluble Fraction ◽  
Wild Type ◽  
Peptide Processing ◽  
E Coli ◽  
Hsc 70 ◽  
Soybean Phospholipids ◽  
Mutant Cells ◽  
Dnak Protein ◽  
Wild Type Yeast

We reconstituted prepro-alpha-factor translocation and signal peptide processing using a yeast microsomal detergent soluble fraction formed into vesicles with soybean phospholipids. Reconstituted translocation required ATP, and was deficient when sec63 and kar2 (BiP) mutant cells were used as a source of membranes. Normal translocation was observed with vesicles reconstituted from a mixture of pure wild-type yeast BiP and a soluble fraction of kar2 mutant membranes. Two other heat-shock cognate (hsc) 70 homologs, yeast cytosolic hsc70 (Ssalp) and E. coli dnaK protein did not replace BiP. Conversely, BiP was not active under conditions where translocation into native ER vesicles required cytosolic hsc70. We conclude that cytosolic hsc70 and BiP serve noninterchangeable roles in polypeptide translocation, possibly because distinct, asymmetrically oriented membrane proteins are required to recruit each protein to opposing surfaces of the ER membrane.

scholarly journals Lecithin:cholesterol acyltransferase: role of N-linked glycosylation in enzyme function

1993 ◽  
Vol 294 (3) ◽  
pp. 879-884 ◽  
Author(s):  
K O ◽  
J S Hill ◽  
X Wang ◽  
R McLeod ◽  
P H Pritchard
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Consensus Sequence ◽  
Fold Increase ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Lecithin:Cholesterol Acyltransferase ◽  
Mutant Proteins ◽  
Glycosylation Sites

Lecithin:cholesterol acyltransferase (LCAT; phosphatidylcholine-sterol acyltransferase, EC 2.3.1.43) is a glycoprotein which is responsible for the formation of cholesteryl ester in plasma. The carbohydrate content has been estimated to be approx. 25% of the total LCAT mass, and four potential N-linked glycosylation sites have been predicted at residues 20, 84, 272 and 384 of the LCAT protein sequence. In the present study, we have examined which of these sites are utilized and how the N-glycosylation affects the secretion and function of the enzyme. Site-directed mutagenesis was performed to eliminate the glycosylation consensus sequence at each of the four potential sites, and the mutant proteins were expressed in COS cells. The amount of each mutant LCAT secreted was similar to that of the wild-type enzyme but the molecular mass was decreased by 3-4 kDa. The specific activity of each mutant LCAT was significantly different from the wild-type; however, the magnitude and direction of the change depended on the glycosylation site mutagenized. Loss of carbohydrate at position 20, 84 or 272 resulted in a decrease in the specific activity of the mutant enzymes by 18%, 82%, and 62% respectively. In contrast, the mutant protein without glycosylation at position 384 displayed a 2-fold increase in enzyme activity. In addition, a quadruple mutant was constructed such that all four potential glycosylation sites were eliminated. The amount of the unglycosylated LCAT secreted into the culture medium was less than 10% of the wild-type level and the specific activity of this enzyme was decreased to 5% of that of the wild type. The results demonstrate that all four potential N-glycosylation sites in LCAT are used and the presence of carbohydrate at each site has diverse effects on the enzyme activity.

scholarly journals A Cytosolic Reductase Pathway is Required for Complete N-Glycosylation of an STT3B-Dependent Acceptor Site

2021 ◽  
Author(s):  
Marcel van Lith ◽  
Marie Anne Pringle ◽  
Bethany Fleming ◽  
Giorgia Gaeta ◽  
Jisu Im ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Protein Translocation ◽  
Site Occupancy ◽  
Secretory Protein ◽  
Glycosylation Site ◽  
Redox Conditions ◽  
Translation System ◽  
Acceptor Site ◽  
Post Translational Modification

AbstractN-linked glycosylation of proteins entering the secretory pathway is an essential post-translational modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here we use an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could also be mimicked by the addition of a membrane impermeable reducing agent. The identified hypoglycosylated acceptor site is adjacent to a cysteine involved in a short range disulfide bond, which has been shown to be dependent on the STT3B-containing oligosaccharyl transferase. We also show that efficient glycosylation at this site is dependent on the STT3A-containing oligosaccharide transferase. Our results provide further insight into the important role of the ER redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.

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