scholarly journals IgG Charge: Practical and Biological Implications

Antibodies ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 24 ◽  
Author(s):  
Danlin Yang ◽  
Rachel Kroe-Barrett ◽  
Sanjaya Singh ◽  
Thomas Laue
Keyword(s):  
Negative Charge ◽  
Fundamental Property ◽  
Protein Protein Interaction ◽  
Physiological Properties ◽  
Systematic Exploration ◽  
Dependent Protein ◽  
Interaction Curves ◽  
A Charge ◽  
Phosphate Buffered Saline ◽  
Selection Of

Practically, IgG charge can contribute significantly to thermodynamic nonideality, and hence to solubility and viscosity. Biologically, IgG charge isomers exhibit differences in clearance and potency. It has been known since the 1930s that all immunoglobulins carry a weak negative charge in physiological solvents. However, there has been no systematic exploration of this fundamental property. Accurate charge measurements have been made using membrane confined electrophoresis in two solvents (pH 5.0 and pH 7.4) on a panel of twelve mAb IgGs, as well as their F(ab’)2 and Fc fragments. The following observations were made at pH 5.0: (1) the measured charge differs from the calculated charge by ~40 for the intact IgGs, and by ~20 for the Fcs; (2) the intact IgG charge depends on both Fv and Fc sequences, but does not equal the sum of the F(ab)’2 and Fc charge; (3) the Fc charge is consistent within a class. In phosphate buffered saline, pH 7.4: (1) the intact IgG charges ranged from 0 to −13; (2) the F(ab’)2 fragments are nearly neutral for IgG1s and IgG2s, and about −5 for some of the IgG4s; (3) all Fc fragments are weakly anionic, with IgG1 < IgG2 < IgG4; (4) the charge on the intact IgGs does not equal the sum of the F(ab’)2 and Fc charge. In no case is the calculated charge, based solely on H+ binding, remotely close to the measured charge. Some mAbs carried a charge in physiological salt that was outside the range observed for serum-purified human poly IgG. To best match physiological properties, a therapeutic mAb should have a measured charge that falls within the range observed for serum-derived human IgGs. A thermodynamically rigorous, concentration-dependent protein–protein interaction parameter is introduced. Based on readily measured properties, interaction curves may be generated to aid in the selection of proteins and solvent conditions. Example curves are provided.

scholarly journals IgG Charge

2018 ◽  
Author(s):  
Danlin Yang ◽  
Rachel Kroe-Barrett ◽  
Sanjaya Singh ◽  
Thomas Laue
Keyword(s):  
Negative Charge ◽  
Fundamental Property ◽  
Physiological Properties ◽  
Systematic Exploration ◽  
A Charge ◽  
Phosphate Buffered Saline ◽  
Thermodynamic Nonideality

It has been known since the 1930&rsquo;s that all immunoglobulins carry a weak negative charge in physiological solvents. However, there has been no systematic exploration of this fundamental property. Accurate charge measurements have been made using membrane confined electrophoresis in two solvents (pH 5.0 and pH 7.4) on a panel of twelve mAb IgGs, as well as their F(ab&rsquo;)2 and Fc fragments. The following observations were made at pH 5.0: 1) the measured charge differs from the calculated charge by ~40 for the intact IgGs, and by ~20 for the Fcs; 2) the intact IgG charge depends on both Fv and Fc sequences, but does not equal the sum of the F(ab)&rsquo;2 and Fc charge; 3) the Fc charge is consistent within a class. In phosphate buffered saline, pH 7.4: 1) the intact IgG charges ranged from 0 to -13; 2) the F(ab&rsquo;)2 fragments are nearly neutral for IgG1s and IgG2s, and about -5 for some of the IgG4s; 3) all Fc fragments are weakly anionic, with IgG1 &lt; IgG2 &lt; IgG4; 4) the charge on the intact IgGs does not equal the sum of the F(ab&rsquo;)2 and Fc charge. In no case is the calculated charge, based on H+ binding, remotely close to the measured charge. The charge on IgGs in physiological solvent is sufficiently small to minimize its contribution to thermodynamic nonideality. Some of the mAbs carried a charge in physiological salt that was outside the range observed for serum-purified human poly IgG. To best match physiological properties, a therapeutic mAb should have a measured charge that falls within the range observed for serum-derived human IgGs.

Histidine-Mediated Intramolecular Electrostatic Repulsion for Controlling pH-Dependent Protein–Protein Interaction

2019 ◽  
Vol 14 (12) ◽  
pp. 2729-2736 ◽  
Author(s):  
Hideki Watanabe ◽  
Chuya Yoshida ◽  
Ayako Ooishi ◽  
Yasuto Nakai ◽  
Momoko Ueda ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Electrostatic Repulsion ◽  
Protein Protein Interaction ◽  
Dependent Protein ◽  
Ph Dependent

A gel-membrane model of glomerular charge and size selectivity in series

2001 ◽  
Vol 280 (3) ◽  
pp. F396-F405 ◽  
Author(s):  
Maria Ohlson ◽  
Jenny Sörensson ◽  
Börje Haraldsson
Keyword(s):  
Negative Charge ◽  
Careful Analysis ◽  
Size Selectivity ◽  
Gel Structure ◽  
Experimental Conditions ◽  
Ion Interactions ◽  
In Series ◽  
A Charge ◽  
Glomerular Barrier

We have analyzed glomerular sieving data from humans, rats in vivo, and from isolated perfused rat kidneys (IPK) and present a unifying hypothesis that seems to resolve most of the conflicting results that exist in the literature. Particularly important are the data obtained in the cooled IPK, because they allow a variety of experimental conditions for careful analysis of the glomerular barrier; conditions that never can be obtained in vivo. The data strongly support the classic concept of a negative charge barrier, but separate components seem to be responsible for charge and size selectivity. The new model is composed of a dynamic gel and a more static membrane layer. First, the charged gel structure close to the blood compartment has a charge density of 35–45 meq/l, reducing the concentration of albumin to 5–10% of that in plasma, due to ion-ion interactions. Second, the size-selective structure has numerous functional small pores (radius 45–50 Å) and far less frequent large pores (radius 75–115 Å), the latter accounting for 1% of the total hydraulic conductance. Both structures are required for the maintenance of an intact glomerular barrier.

scholarly journals Proteomic Analysis of Rhesus Macaque Brain Explants Treated With Borrelia burgdorferi Identifies Host GAP-43 as a Potential Factor Associated With Lyme Neuroborreliosis

2021 ◽  
Vol 11 ◽  
Author(s):  
Lianbao Li ◽  
Lisha Luo ◽  
Taigui Chen ◽  
Wenjing Cao ◽  
Xin Xu ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Bioinformatics Analysis ◽  
Interaction Network ◽  
Lyme Neuroborreliosis ◽  
Differentially Expressed Proteins ◽  
Protein Protein Interaction ◽  
Potential Marker ◽  
Potential Factor ◽  
Phosphate Buffered Saline ◽  
Protein Protein Interaction Network

BackgroundLyme neuroborreliosis (LNB) is one of the most dangerous manifestations of Lyme disease, but the pathogenesis and inflammatory mechanisms are not fully understood.MethodsCultured explants from the frontal cortex of rhesus monkey brain (n=3) were treated with live Borrelia burgdorferi (Bb) or phosphate-buffered saline (PBS) for 6, 12, and 24 h. Total protein was collected for sequencing and bioinformatics analysis. In addition, changes in protein expression in the explants over time following Bb treatment were screened.ResultsWe identified 1237 differentially expressed proteins (DEPs; fold change ≥1.5 or ≤0.67, P-value ≤0.05). One of these, growth-associated protein 43 (GAP-43), was highly expressed at all time points in the explants. The results of the protein-protein interaction network analysis of DEPs suggested that GAP-43 plays a role in the neuroinflammation associated with LNB. In HMC3 cells incubated with live Bb or PBS for 6, 12, and 24 h, real-time PCR and western blot analyses confirmed the increase of GAP-43 mRNA and protein, respectively.ConclusionsElevated GAP-43 expression is a potential marker for LNB that may be useful for diagnosis or treatment.

scholarly journals Rapid alteration of protein phosphorylation during postmortem: implication in the study of protein phosphorylation

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Yifan Wang ◽  
Yanchong Zhang ◽  
Wen Hu ◽  
Shutao Xie ◽  
Cheng-Xin Gong ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Glycogen Synthase ◽  
Post Translational Modification ◽  
Dependent Protein Kinase ◽  
Regular Time ◽  
Liver And Kidney ◽  
Dependent Protein ◽  
Gsk 3Β ◽  
Phosphate Buffered Saline ◽  
The Brain

Abstract Protein phosphorylation is an important post-translational modification of proteins. Postmortem tissues are widely being utilized in the biomedical studies, but the effects of postmortem on protein phosphorylation have not been received enough attention. In the present study, we found here that most proteins in mouse brain, heart, liver and kidney were rapidly dephosphorylated to various degrees during 20 sec to 10 min postmortem. Phosphorylation of tau at Thr212 and glycogen synthase kinase 3β (GSK-3β) at Ser9 was reduced by 50% in the brain with 40 sec postmortem, a regular time for tissue processing. During postmortem, phosphorylation of cAMP-dependent protein kinase (PKA) and AMP activated kinase (AMPK) was increased in the brain, but not in other organs. Perfusion of the brain with cold or room temperature phosphate-buffered saline (PBS) also caused significant alteration of protein phosphorylation. Cooling down and maintaining mouse brains in the ice-cold buffer prevented the alteration effectively. This study suggests that phosphorylation of proteins is rapidly changed during postmortem. Thus, immediate processing of tissues followed by cooling down in ice-cold buffer is vitally important and perfusion has to be avoided when protein phosphorylation is to be studied.

scholarly journals Active Ca2+/Calmodulin-Dependent Protein Kinase IIγB Impairs Positive Selection of T Cells by Modulating TCR Signaling

2005 ◽  
Vol 175 (2) ◽  
pp. 656-664 ◽  
Author(s):  
Maureen A. McGargill ◽  
Leslie L. Sharp ◽  
Jack D. Bui ◽  
Stephen M. Hedrick ◽  
Sébastien Calbo
Keyword(s):  
T Cells ◽  
Protein Kinase ◽  
Positive Selection ◽  
Tcr Signaling ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Selection Of

scholarly journals Platelet sarco/endoplasmic reticulum Ca2+ATPase isoform 3b and Rap 1b: interrelation and regulation in physiopathology

1998 ◽  
Vol 332 (1) ◽  
pp. 173-181 ◽  
Author(s):  
Christine LACABARATZ-PORRET ◽  
Elisabeth CORVAZIER ◽  
Tünde KOVÀCS ◽  
Régis BOBE ◽  
Raymonde BREDOUX ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Human Platelets ◽  
Spontaneously Hypertensive ◽  
Protein Protein Interaction ◽  
Reverse Transcription Pcr ◽  
Pathological Model ◽  
Dependent Protein ◽  
Pre Treatment ◽  
Wistar Kyoto

Platelet Ca2+ signalling involves intracellular Ca2+ pools, whose content is controlled by sarco/endoplasmic reticulum Ca2+ATPases (SERCAs). Among these, a key role is played by the inositol trisphosphate-sensitive Ca2+ pool, associated with the SERCA 3b isoform. We have investigated the control of this Ca2+ pool through the cAMP-dependent phosphorylation of the GTP-binding protein, Rap (Ras-proximate) 1b. We first looked for this Ca2+ pool target of regulation by studying the expression of the different SERCA and Rap 1 proteins in human platelets and various cell lines, by Western blotting and reverse transcription-PCR. Since co-expression of Rap 1b and SERCA 3b was obtained, we looked for their protein–protein interaction as a function of the cAMP-dependent phosphorylation of Rap 1b. Co-immunoprecipitations of SERCA 3b and Rap 1b proteins were found in the absence of phosphorylation, induced by the catalytic subunit of the cAMP-dependent protein kinase (csPKA). In contrast, upon pre-treatment of platelet membranes with csPKA, the SERCA 3b dissociated from the Rap 1b protein, in agreement with a role of its phosphorylated state in their interaction. Finally, we looked for adaptation of this complex in a platelet pathological model of hypertension. We investigated the expression of both proteins, as well as the cAMP-dependent phosphorylation of Rap 1b and SERCA 3b activity in platelets from control normotensive Wistar-Kyoto rats and from spontaneously hypertensive rats (SHRs). A decrease in SERCA 3b activity was associated with a decrease in Rap 1b endogenous phosphorylation in SHR platelets, consistent with a functional role in the regulation of the SERCA 3b-associated Ca2+ pool.

scholarly journals Dynamic rewiring of the human interactome by interferon signalling

2019 ◽  
Author(s):  
Craig H. Kerr ◽  
Michael A. Skinnider ◽  
Angel M. Madero ◽  
Daniel D.T. Andrews ◽  
R. Greg Stacey ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Interaction Network ◽  
Cell Types ◽  
Antiviral Response ◽  
Type I ◽  
Protein Protein Interactions ◽  
Human Interactome ◽  
Viral Pathogens ◽  
Protein Protein Interaction ◽  
Dependent Protein

ABSTRACTBackgroundThe type I interferon (IFN) response is an ancient pathway that protects cells against viral pathogens by inducing the transcription of hundreds of IFN-stimulated genes (ISGs). Transcriptomic and biochemical approaches have established comprehensive catalogues of ISGs across species and cell types, but their antiviral mechanisms remain incompletely characterized. Here, we apply a combination of quantitative proteomic approaches to delineate the effects of IFN signalling on the human proteome, culminating in the use of protein correlation profiling to map IFN-induced rearrangements in the human protein-protein interaction network.ResultsWe identified >27,000 protein interactions in IFN-stimulated and unstimulated cells, many of which involve proteins associated with human disease and are observed exclusively within the IFN-stimulated network. Differential network analysis reveals interaction rewiring across a surprisingly broad spectrum of cellular pathways in the antiviral response. We identify IFN-dependent protein-protein interactions mediating novel regulatory mechanisms at the transcriptional and translational levels, with one such interaction modulating the transcriptional activity of STAT1. Moreover, we reveal IFN-dependent changes in ribosomal composition that act to buffer ISG protein synthesis.ConclusionsOur map of the IFN interactome provides a global view of the complex cellular networks activated during the antiviral response, placing ISGs in a functional context, and serves as a framework to understand how these networks are dysregulated in autoimmune or inflammatory disease.

Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assays (ELISA) for the Measurement of Vitamin K-Dependent Protein S: The Effect of Antibody Immunoreactivity on Plasma Protein S Antigen Determinations

1992 ◽  
Vol 67 (06) ◽  
pp. 631-638 ◽  
Author(s):  
Silvana Vigano D’Angelo ◽  
Sandra Tombesi ◽  
Santica Marcovina ◽  
Alberto Albertini ◽  
Patrizia Della Valle ◽  
...  
Keyword(s):  
Protein S ◽  
Plasma Samples ◽  
Potential Influence ◽  
Polyclonal Igg ◽  
Dependent Protein ◽  
Capture Antibody ◽  
Antigen Determination ◽  
Sample Dilution ◽  
Selection Of

SummaryTwo monoclonal antibodies (Mabs) specifically directed to human protein S (PS) - named 5E9E9 and 3B10.25 - were produced and their properties compared to those of 2 previously characterized anti-PS-Mabs (HPS-2 and S10). 3B10.25, similar to S10, was directed to the calcium-free conformation of PS and had virtually identical affinity for free and C4b-binding protein (C4b-BP)-bound PS; 5E9E9 similar to HPS-2, had no calcium-dependency and was selectively directed to free PS. All Mabs were equally reactive to freshly purified and thrombin-cleaved PS. To evaluate the influence of C4b-BP bound PS on PS antigen determinations, ELISA systems employing the four Mabs individually as capture antibody (Ab) and peroxidase-conjugated polyclonal anti-PS IgG as detecting Ab were developed and compared to immunoelectrophoresis (EIA) and to an ELISA employing polyclonal anti-PS IgG as capture and detecting Ab, in the determination of PS in purified systems and in plasma. With all the ELISAs there was parallelism of dilution cuiyes obtained with normal plasma and purified PS; however, supplementation of plasma with purified C4b-BP resulted in loss of parallelism when employing the Mabs directed to free PS as capture Ab. Influence of high C4b-BP on PS antigen determinations was confirmed in a series of plasma samples from patients with C4b-BP levels ranging from 70% to over 200%. Compared to the values obtained with the S10- or 3B10.25 - based ELISAs - which were similar despite a 10-fold difference in sample dilution -plasma PS was underestimated by the ELISAs employing 5E9E9 or HPS-2 while it was overestimated by EIA. In addition, plasma PS and C4b-BP levels were significantly correlated only when it was measured by EIA or the ELISAs employing S10, 3B10.25 or polyclonal IgG. These results highlight the potential influence of high C4b-BP on plasma PS antigen determination. Accurate measurement of PS by ELISA requires selection of antibodies with identical affinity for all plasma PS forms.

scholarly journals Selection of In Vivo Predictive Dissolution Media Using Drug Substance and Physiological Properties

2020 ◽  
Vol 22 (2) ◽  
Author(s):  
Deanna M. Mudie ◽  
Nasim Samiei ◽  
Derrick J. Marshall ◽  
Gregory E. Amidon ◽  
Christel A.S. Bergström
Keyword(s):  
Drug Substance ◽  
Physiological Properties ◽  
Selection Of
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