Binding of recombinant interleukin-1 beta to the third complement component and alpha 2-macroglobulin after activation of serum by immune complexes

Abstract Activation of human normal serum with tetanus/antitetanus immune complexes (TAT-IC) resulted in increased binding of 125I-labeled interleukin-1 beta (IL-1 beta) to serum factors, as opposed to untreated serum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography showed labeling of two large molecular mass factors of an apparent molecular weight (Mr) of 200,000 and 400,000, respectively. These complexes could be dissociated by reduction. No complexes were formed when reducing compounds were added to serum-TAT-IC-125I-IL-1 beta mixtures. Complex formation was largely prevented by alkylating compounds. Molecular sieve chromatography of TAT-IC-activated serum confirmed that 125I-IL-1 beta became bound to high Mr serum proteins. Fractions containing high molecular 125I-IL-1 serum protein complexes partially retained IL-1- like activity since they induced proliferation of an IL-1-dependent murine T helper (D10G4) cell lineage. The 125I-IL-1 beta binding factors could be immunoprecipitated from TAT-IC-activated serum 125I-IL- 1 beta solutions by antisera to alpha 2-macroglobulin (alpha 2M) or to the third complement component (C3). SDS-PAGE of the immunoprecipitates showed radioactive bands corresponding to the expected Mr resulting from complex formation between 125I-IL-1 beta and these two proteins. Treatment of purified plasma alpha 2M and C3 with trypsin or activation with methylamine, which causes cleavage of the internal thiol ester and the appearance of free thiol groups in these proteins, mediated binding of 125I-IL-1 beta to alpha 2M and C3b. The results suggest that cleavage of the internal thiol ester in C3 and alpha 2M makes these plasma proteins susceptible to binding of 125I-IL-1 beta and that free thiol groups do play a role in the formation of 125I-IL-1 beta plasma protein complexes. Activated C3 and alpha 2M may function as IL-1 beta carrier proteins in biologic fluids, in addition to their other physiologic roles.

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Binding of recombinant interleukin-1 beta to the third complement component and alpha 2-macroglobulin after activation of serum by immune complexes

Blood ◽

10.1182/blood.v75.12.2388.bloodjournal75122388 ◽

1990 ◽

Vol 75 (12) ◽

pp. 2388-2395

Author(s):

W Borth ◽

A Urbanski ◽

R Prohaska ◽

M Susanj ◽

TA Luger

Keyword(s):

Complex Formation ◽

Immune Complexes ◽

Protein Complexes ◽

Interleukin 1 ◽

Complement Component ◽

Interleukin 1 Beta ◽

Thiol Groups ◽

Thiol Ester ◽

The Third ◽

Sds Page

Activation of human normal serum with tetanus/antitetanus immune complexes (TAT-IC) resulted in increased binding of 125I-labeled interleukin-1 beta (IL-1 beta) to serum factors, as opposed to untreated serum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography showed labeling of two large molecular mass factors of an apparent molecular weight (Mr) of 200,000 and 400,000, respectively. These complexes could be dissociated by reduction. No complexes were formed when reducing compounds were added to serum-TAT-IC-125I-IL-1 beta mixtures. Complex formation was largely prevented by alkylating compounds. Molecular sieve chromatography of TAT-IC-activated serum confirmed that 125I-IL-1 beta became bound to high Mr serum proteins. Fractions containing high molecular 125I-IL-1 serum protein complexes partially retained IL-1- like activity since they induced proliferation of an IL-1-dependent murine T helper (D10G4) cell lineage. The 125I-IL-1 beta binding factors could be immunoprecipitated from TAT-IC-activated serum 125I-IL- 1 beta solutions by antisera to alpha 2-macroglobulin (alpha 2M) or to the third complement component (C3). SDS-PAGE of the immunoprecipitates showed radioactive bands corresponding to the expected Mr resulting from complex formation between 125I-IL-1 beta and these two proteins. Treatment of purified plasma alpha 2M and C3 with trypsin or activation with methylamine, which causes cleavage of the internal thiol ester and the appearance of free thiol groups in these proteins, mediated binding of 125I-IL-1 beta to alpha 2M and C3b. The results suggest that cleavage of the internal thiol ester in C3 and alpha 2M makes these plasma proteins susceptible to binding of 125I-IL-1 beta and that free thiol groups do play a role in the formation of 125I-IL-1 beta plasma protein complexes. Activated C3 and alpha 2M may function as IL-1 beta carrier proteins in biologic fluids, in addition to their other physiologic roles.

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C-Reactive Protein Serum Levels as an Internal Load Indicator of Sprints in Competitive Football Matches

International Journal of Sports Medicine ◽

10.1055/a-0985-4464 ◽

2019 ◽

Vol 40 (12) ◽

pp. 762-767

Author(s):

Pedro Jatene ◽

Gustavo Silva dos Santos ◽

Daniel Leite Portella

Keyword(s):

Interleukin 6 ◽

Interleukin 1 ◽

Future Research ◽

Interleukin 1 Beta ◽

Moderate Correlation ◽

C Reactive Protein ◽

Internal Load ◽

Reactive Protein ◽

The Third ◽

Cortisol Ratio

AbstractThis study compared internal load variable dynamics across three consecutive football matches and investigated its relationship with the number of sprints performed by players. Twenty-three male players had blood and salivary samples collected for hormonal concentration (testosterone, cortisol, and testosterone-cortisol ratio), and serum analysis (interleukin-6, interleukin-1-beta, and c-reactive-protein), respectively. Sprints were measured through Global Position System devices. Testosterone and testosterone-cortisol-ratio presented a decreasing behavior up to the second match, and all other indicators presented an increasing behavior during the same period, c-reactive-protein was the only indicator observed to significantly rise up to the third match as well (0.38±0.02 mg/L; 0.49±0.05 mg/L; 0.69±0.05 mg/L; 0.89±0.08 mg/L). C-reactive-protein showed strong correlations with sprints in the second and third matches (p<0.01, r=0.71 and 0.79), and weak-to-moderate in the first one (p<0.05, r=0.59). Interleukin-6 and interleukin-1-beta presented weak-to-moderate correlation in every match (p<0.05, r=0.48 to 0.51; r=0.51 to 0.55) while testosterone-cortisol ratio presented weak-to-moderate correlation only in the third one (p<0.05, r=0.42). Multilevel linear regression showed that c-reactive-protein had a higher R2 than other biomarker in any regression model (R2=0.624; p<0.001). Therefore, c-reactive-protein can be a valid and reliable indicator of sprinting in competitive football. Future research should explore longer periods of monitoring and/or others external load variables so that other behaviors may arise to knowledge.

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Intramolecular domain dynamics regulate synaptic MAGUK protein interactions

eLife ◽

10.7554/elife.41299 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 9

Author(s):

Nils Rademacher ◽

Benno Kuropka ◽

Stella-Amrei Kunde ◽

Markus C Wahl ◽

Christian Freund ◽

...

Keyword(s):

Complex Formation ◽

Protein Interactions ◽

Hippocampal Neurons ◽

Protein Complexes ◽

Pdz Domain ◽

Domain Architecture ◽

Protein Subunit ◽

Functional Changes ◽

The Third ◽

Domain Dynamics

PSD-95 MAGUK family scaffold proteins are multi-domain organisers of synaptic transmission that contain three PDZ domains followed by an SH3-GK domain tandem. This domain architecture allows coordinated assembly of protein complexes composed of neurotransmitter receptors, synaptic adhesion molecules and downstream signalling effectors. Here we show that binding of monomeric CRIPT-derived PDZ3 ligands to the third PDZ domain of PSD-95 induces functional changes in the intramolecular SH3-GK domain assembly that influence subsequent homotypic and heterotypic complex formation. We identify PSD-95 interactors that differentially bind to the SH3-GK domain tandem depending on its conformational state. Among these interactors, we further establish the heterotrimeric G protein subunit Gnb5 as a PSD-95 complex partner at dendritic spines of rat hippocampal neurons. The PSD-95 GK domain binds to Gnb5, and this interaction is triggered by CRIPT-derived PDZ3 ligands binding to the third PDZ domain of PSD-95, unraveling a hierarchical binding mechanism of PSD-95 complex formation.

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Intramolecular domain dynamics regulate synaptic MAGUK protein interactions

10.1101/462358 ◽

2018 ◽

Author(s):

Nils Rademacher ◽

Benno Kuropka ◽

Stella-Amrei Kunde ◽

Markus C. Wahl ◽

Christian Freund ◽

...

Keyword(s):

Complex Formation ◽

Protein Interactions ◽

Protein Complexes ◽

Pdz Domain ◽

Domain Architecture ◽

Cytoskeletal Proteins ◽

Protein Subunit ◽

Functional Changes ◽

The Third ◽

Domain Dynamics

AbstractPSD-95 MAGUK family scaffold proteins are multi-domain organisers of synaptic transmission that contain three PDZ domains followed by an SH3-GK domain tandem. This domain architecture allows coordinated assembly of protein complexes composed of neurotransmitter receptors, synaptic adhesion molecules, cytoskeletal proteins and downstream signalling effectors. Here we show that binding of monomeric PDZ3 ligands to the third PDZ domain of PSD-95 induces functional changes in the intramolecular SH3-GK domain assembly that influence subsequent homotypic and heterotypic complex formation. We identify PSD-95 interactors that differentially bind to the SH3-GK domain tandem depending on its conformational state. Among these interactors we further establish the heterotrimeric G protein subunit Gnb5 as a PSD-95 complex partner at dendritic spines. The PSD-95 GK domain binds to Gnb5 and this interaction is triggered by PDZ3 ligands binding to the third PDZ domain of PSD-95, unraveling a hierarchical binding mechanism of PSD-95 complex formation.

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Western Blot Analyses of Prekallikrein and its Activation Products in Human Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648157 ◽

1991 ◽

Vol 65 (04) ◽

pp. 382-388 ◽

Cited By ~ 9

Author(s):

Dulce Veloso ◽

Robert W Colman

Keyword(s):

Complex Formation ◽

Western Blot ◽

Human Plasma ◽

Contact System ◽

Plasma Activation ◽

Normal Plasma ◽

Sds Page ◽

Activation Products ◽

Major Antigen ◽

Formation Of Complexes

SummaryPrekallikrein (PK), a zymogen of the contact system, and its activation products, kallikrein (KAL), KAl-inhibitor complexes and fragments containing KAL epitope(s) have been detected in human plasma by immunoblotting with a monoclonal anti-human plasma PK antibody, MAb 13G1L. Detection of antigen-MAb 13G11 complexes with peroxidase-conjugated anti-IgG showed that the two variants of PK (85- and 88-kDa) are the only major antigen species in normal, non-activated plasma. Upon plasma activation with kaolin, the intensity of the PK bands decreased with formation of complexes of KAL with CL inhibitor (C1 INH) and α2-rrtzcroglobulin (α2M) identical to those formed by the purified proteins. Immunoblots of normal plasma showed good correlation between the PK detected and the amount of plasma assayed. Increasing amounts of KAL incubated with a constant volume of PK-deficient plasma showed increasing amounts of KAL and of KAL-C1 INH and KAL-α2M complexes. Complexes of KALantithrombin III (ATIII) and the ratio of KALα2M/ KAL-CL INH were higher in activated CL INH-deficient plasmas than in activated normal plasmas. Protein resolution by 3-12% gradient SDS-PAGE and epitope detection with [125I]MAb 13G11 showed four KALα2M species and a 45-kDa fragment(s) in both surface-activated normal plasma and complexes formed by purified KAL and α2M. Immunoblots of activated plasma also showed bands at the position of KALCL INH and KALATIII complexes. When α1-antitrypsin Pittsburgh (cα1-AT, Pitts) was added to plasma before activation, KAL-α1-ALPitts was the main complex. The non-activated normal plasma revealed only an overloaded PK band. This is the first report of an antibody that recognizes KAL epitope(s) in KAL-α2M, KALATIII and KALa1-α1Pitts complexes and in the 45-kDa fragment(s). Therefore, MAb 13G11 should be useful for studying the structure of these complexes as well as the mechanism of complex formation. In addition, immunoblotting with MAb 13G11 would allow detection of KAl-inhibitor complexes in patient plasmas as indicators of activation of the contact system.

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