The positions of the disulfide bonds and the glycosylation site in a lectin of the acorn barnacle Megabalanus rosa

ABSTRACT Monoclonal antibodies (MAbs) directed against epitopes in the V2 domain of human immunodeficiency virus type 1 gp120 often possess neutralizing activity, but these generally are highly type specific, neutralize only laboratory isolates, or have low potency. The most potent of these is C108g, directed against a type-specific epitope in HXB2 and BaL gp120s, which is glycan dependent and, in contrast to previous reports, dependent on intact disulfide bonds. This epitope was introduced into two primary Envs, derived from a neutralization-sensitive (SF162) and a neutralization-resistant (JR-FL) isolate, by substitution of two residues and, for SF162, addition of an N-linked glycosylation site. C108g effectively neutralized both variant Envs with considerably higher potency than standard MAbs against the V3 and CD4-binding domains and the broadly neutralizing MAbs 2G12 and 2F5. These amino acid substitutions also introduced the epitope recognized by a second V2-specific MAb, 10/76b, but this MAb possessed potent neutralizing activity only in the absence of the glycan required for C108g reactivity. In contrast to other gp120-specific neutralizing MAbs, C108g did not block binding of soluble Env proteins to either the CD4 or the CCR5 receptor, but studies with a fusion-arrested Env indicated that C108g neutralized at a step preceding the one blocked by the gp41-specific MAb, 2F5. These results indicate that the V1/V2 domain possesses targets that mediate potent neutralization of primary viral isolates via a novel mechanism and suggest that inclusion of carbohydrate determinants into these epitopes may help overcome the indirect masking effects that limit the neutralizing potency of antibodies commonly produced after infection.

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Positions of disulfide bonds and N-glycosylation site in juvenile hormone binding protein

Archives of Biochemistry and Biophysics ◽

10.1016/j.abb.2003.10.019 ◽

2004 ◽

Vol 421 (2) ◽

pp. 260-266 ◽

Cited By ~ 15

Author(s):

Janusz Dębski ◽

Aleksandra Wysłouch-Cieszyńska ◽

Michał Dadlez ◽

Krystyna Grzelak ◽

Barbara Kłudkiewicz ◽

...

Keyword(s):

Juvenile Hormone ◽

Disulfide Bonds ◽

Binding Protein ◽

Glycosylation Site ◽

Hormone Binding ◽

Juvenile Hormone Binding Protein ◽

Hormone Binding Protein

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Coupled Site-Directed Mutagenesis/Transgenesis Identifies Important Functional Domains of the Mouse Agouti Protein

Genetics ◽

10.1093/genetics/144.1.255 ◽

1996 ◽

Vol 144 (1) ◽

pp. 255-264 ◽

Cited By ~ 2

Author(s):

William L Perry ◽

Takuro Nakamura ◽

Deborah A Swing ◽

Lisa Secrest ◽

Bryn Eagleson ◽

...

Keyword(s):

Disulfide Bonds ◽

Coat Color ◽

Signal Sequence ◽

Biological Activities ◽

Normal Function ◽

Glycosylation Site ◽

Site Directed Mutagenesis ◽

Functional Domains ◽

Basic Domain

Abstract The agouti locus encodes a novel paracrine signaling molecule containing a signal sequence, an N-linked glycosylation site, a central lysine-rich basic domain, and a C-terminal tail containing 10 cysteine (Cys) residues capable of forming five disulfide bonds. When overexpressed, agouti causes a number of pleiotropic effects including yellow coat and adult-onset obesity. Numerous studies suggest that agouti causes yellow coat color by antagonizing the binding of a-melanocyte-stimulating hormone (α-MSH) to the a-MSH-(melanocortin-1) receptor. With the goal of identifying functional domains of agouti important for its diverse biological activities, we have generated 14 agouti mutations by in vitro sitedirected mutagenesis and analyzed these mutations in transgenic mice for their effects on coat color and obesity. These studies demonstrate that the signal sequence, the N-linked glycosylation site, and the C-terminal Cys residues are important for full biological activity, while at least a portion of the lysine-rich basic domain is dispensable for normal function. They also show that the same functional domains of agouti important in coat color determination are important for inducing obesity, consistent with the hypothesis that agouti induces obesity by antagonizing melanocortin binding to other melanocortin receptors.

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Cryo-electron microscopy of two-dimensional crystals of reduced type B botulinum neurotoxin

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100123052 ◽

1992 ◽

Vol 50 (1) ◽

pp. 530-531

Author(s):

P. F. Flicker ◽

V.S. Kulkarni ◽

J. P. Robinson ◽

G. Stubbs ◽

B. R. DasGupta

Keyword(s):

Disulfide Bonds ◽

Clostridium Botulinum ◽

Structural Changes ◽

Two Dimensional ◽

Type B ◽

Single Chain ◽

Muscle Paralysis ◽

Cryo Electron Microscopy ◽

Disulfide Linkages ◽

Intracellular Action

Botulinum toxin is a potent neurotoxin produced by Clostridium botulinum. The toxin inhibits release of neurotransmitter, causing muscle paralysis. There are several serotypes, A to G, all of molecular weight about 150,000. The protein exists as a single chain or or as two chains, with two disulfide linkages. In a recent investigation on intracellular action of neurotoxins it was reported that type B neurotoxin can inhibit the release of Ca++-activated [3H] norepinephrine only if the disulfide bonds are reduced. In order to investigate possible structural changes in the toxin upon reduction of the disulfide bonds, we have prepared two-dimensional crystals of reduced type B neurotoxin. These two-dimensional crystals will be compared with those of the native (unreduced) type B toxin.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647041 ◽

1988 ◽

Vol 60 (02) ◽

pp. 255-261 ◽

Cited By ~ 81

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.

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The Influence of Glycosylation on the Catalytic and Fibrinolytic Properties of Pro-Urokinase

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646314 ◽

1992 ◽

Vol 68 (05) ◽

pp. 539-544 ◽

Cited By ~ 22

Author(s):

Catherine Lenich ◽

Ralph Pannell ◽

Jack Henkin ◽

Victor Gurewich

Keyword(s):

Escherichia Coli ◽

Mammalian Cell ◽

Human Gene ◽

Culture Media ◽

Mammalian Cell Culture ◽

Glycosylation Site ◽

Clot Lysis ◽

Cell Culture Media ◽

E Coli ◽

The Uk

SummaryWe previously found that human pro-UK expressed in Escherichia coli is more active in fibrinolysis than recombinant human pro-UK obtained from mammalian cell culture media. To determine whether this difference is related to the lack of glycosylation of the E. coli product, we compared the activity of E. coli-derived pro-UK [(-)pro-UK] with that of a glycosylated pro-UK [(+)pro-UK] and of a mutant of pro-UK missing the glycosylation site at Asn-302 [(-) (302) pro-UK]. The latter two pro-UKs were obtained by expression of the human gene in a mammalian cell. The nonglycosylated pro-UKs were activated by plasmin more efficiently (≈2-fold) and were more active in clot lysis (1.5-fold) than the (+)pro-UK. Similarly, the nonglycosylated two-chain derivatives (UKs) were more active against plasminogen and were more rapidly inactivated by plasma inhibitors than the (+)UK.These findings indicate that glycosylation at Asn-302 influences the activity of pro-UK/UK and could be the major factor responsible for the enhanced activity of E. coli-derived pro-UK.

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Specific Cross-reaction of IgG Anti-phospholipid Antibody with Platelet Glycoprotein IIIa

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650238 ◽

1996 ◽

Vol 75 (01) ◽

pp. 168-174 ◽

Cited By ~ 13

Author(s):

Shigeru Tokita ◽

Morio Arai ◽

Naomasa Yamamoto ◽

Yasuhiro Katagiri ◽

Kenjiro Tanoue ◽

...

Keyword(s):

Platelet Aggregation ◽

Heparan Sulfate ◽

Disulfide Bonds ◽

Dextran Sulfate ◽

Human Platelets ◽

Platelet Glycoprotein ◽

Activated Platelets ◽

Fibrinogen Binding ◽

Dose Dependent Fashion ◽

Glycoprotein Iiia

SummaryTo study the pathological functions of anti-phospholipid (anti-PL) antibodies, we have analyzed their effect on platelet function. We identified an IgG anti-PL mAb, designated PSG3, which cross-reacted specifically with glycoprotein (GP) IIIa in human platelets and inhibited platelet aggregation. PSG3 bound also to certain polyanionic substances, such as double-stranded DNA, heparan sulfate, dextran sulfate and acetylated-LDL, but not to other polyanionic substances. The binding of PSG3 to GPIIIa was completely inhibited by heparan sulfate and dextran sulfate, indicating that PSG3 recognizes a particular array of negative charges expressed on both GPIIIa and the specified polyanionic substances. Since neither neuraminidase- nor endoglycopeptidase F-treatment of GPIIIa had any significant effect on the binding of PSG3, this array must be located within the amino acid sequence of GPIIIa but not in the carbohydrate moiety. Reduction of the disulfide bonds in GPIIIa greatly reduced its reactivity, suggesting that the negative charges in the epitope are arranged in a particular conformation. PSG3 inhibited platelet aggregation induced by either ADP or collagen, it also inhibited fibrinogen binding to activated platelets in a dose-dependent fashion. PSG3, however, did not inhibit the binding of GRGDSP peptide to activated platelets. These results suggest that the PSG3 epitope on GPIIIa contains a particular array of negative charges, and possibly affects the fibrinogen binding to GPIIb/IIIa complex necessary for platelet aggregation.

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Divergence entropy to characterize the stability in selected enzymes – The role of disulfide bonds in respect to the structure of hydrophobic core

10.3390/ecea-2-b009 ◽

2015 ◽

Author(s):

Irena Roterman ◽

Mateusz Banach ◽

Leszek Konieczny ◽

Barbara Kalinowska

Keyword(s):

Disulfide Bonds ◽

Hydrophobic Core ◽

The Stability

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Nglyc: A Random Forest Method for Prediction of N-Glycosylation Sites in Eukaryotic Protein Sequence

Protein and Peptide Letters ◽

10.2174/0929866526666191002111404 ◽

2020 ◽

Vol 27 (3) ◽

pp. 178-186 ◽

Cited By ~ 2

Author(s):

Ganesan Pugalenthi ◽

Varadharaju Nithya ◽

Kuo-Chen Chou ◽

Govindaraju Archunan

Keyword(s):

Random Forest ◽

Protein Sequence ◽

Glycosylation Site ◽

Computational Method ◽

The Other ◽

Eukaryotic Protein ◽

Random Forest Method ◽

Glycosylation Sites ◽

Human And Mouse ◽

Better Than

Background: N-Glycosylation is one of the most important post-translational mechanisms in eukaryotes. N-glycosylation predominantly occurs in N-X-[S/T] sequon where X is any amino acid other than proline. However, not all N-X-[S/T] sequons in proteins are glycosylated. Therefore, accurate prediction of N-glycosylation sites is essential to understand Nglycosylation mechanism. Objective: In this article, our motivation is to develop a computational method to predict Nglycosylation sites in eukaryotic protein sequences. Methods: In this article, we report a random forest method, Nglyc, to predict N-glycosylation site from protein sequence, using 315 sequence features. The method was trained using a dataset of 600 N-glycosylation sites and 600 non-glycosylation sites and tested on the dataset containing 295 Nglycosylation sites and 253 non-glycosylation sites. Nglyc prediction was compared with NetNGlyc, EnsembleGly and GPP methods. Further, the performance of Nglyc was evaluated using human and mouse N-glycosylation sites. Results: Nglyc method achieved an overall training accuracy of 0.8033 with all 315 features. Performance comparison with NetNGlyc, EnsembleGly and GPP methods shows that Nglyc performs better than the other methods with high sensitivity and specificity rate. Conclusion: Our method achieved an overall accuracy of 0.8248 with 0.8305 sensitivity and 0.8182 specificity. Comparison study shows that our method performs better than the other methods. Applicability and success of our method was further evaluated using human and mouse N-glycosylation sites. Nglyc method is freely available at https://github.com/bioinformaticsML/ Ngly.

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Intelligent Techniques Analysis for Glycosylation Site Prediction

Current Bioinformatics ◽

10.2174/1574893615666210108094847 ◽

2021 ◽

Vol 15 ◽

Author(s):

Alhassan Alkuhlani ◽

Walaa Gad ◽

Mohamed Roushdy ◽

Abdel-Badeeh M. Salem

Keyword(s):

Machine Learning ◽

Prediction Models ◽

Cell Interaction ◽

Glycosylation Site ◽

Machine Learning Classification ◽

Site Prediction ◽

Glycosylation Sites ◽

Wide Range ◽

Feature Extraction And Selection ◽

Computational Intelligent

Background: Glycosylation is one of the most common post-translation modifications (PTMs) in organism cells. It plays important roles in several biological processes including cell-cell interaction, protein folding, antigen’s recognition, and immune response. In addition, glycosylation is associated with many human diseases such as cancer, diabetes and coronaviruses. The experimental techniques for identifying glycosylation sites are time-consuming, extensive laboratory work, and expensive. Therefore, computational intelligence techniques are becoming very important for glycosylation site prediction. Objective: This paper is a theoretical discussion of the technical aspects of the biotechnological (e.g., using artificial intelligence and machine learning) to digital bioinformatics research and intelligent biocomputing. The computational intelligent techniques have shown efficient results for predicting N-linked, O-linked and C-linked glycosylation sites. In the last two decades, many studies have been conducted for glycosylation site prediction using these techniques. In this paper, we analyze and compare a wide range of intelligent techniques of these studies from multiple aspects. The current challenges and difficulties facing the software developers and knowledge engineers for predicting glycosylation sites are also included. Method: The comparison between these different studies is introduced including many criteria such as databases, feature extraction and selection, machine learning classification methods, evaluation measures and the performance results. Results and conclusions: Many challenges and problems are presented. Consequently, more efforts are needed to get more accurate prediction models for the three basic types of glycosylation sites.

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