Monoclonal Antibodies Directed to the Calcium-Free Conformation of Human Protein S

1989 ◽  
Vol 62 (02) ◽  
pp. 708-714 ◽  
Author(s):  
Santica Marcovina ◽  
Raffaella Coppola ◽  
Carla Valsecchi ◽  
Adele Zoppo ◽  
Cecilia Gelfi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Protein ◽  
Antigenic Site ◽  
Fluid Phase ◽  
Protein S ◽  
Human Protein ◽  
Sephadex Column ◽  
Sds Page ◽  
Igg1 Subclass ◽  
Sepharose 4B

SummaryFour mouse hybridomas secreting monoclonal antibodies specific for human protein S (PS) have been generated. The antibodies, all of the IgG1 subclass, were designated S2, S3, S8, and S10. In a fluid phase radioimmunoassay, the binding of monoclonal antibodies to PS was about 30% greater in the presence of EDTA and totally inhibited in presence of Ca2+. Using the same technique, we performed displacement curves of 125I-labeled PS by purified PS, thrombin-cleaved PS, normal plasma, plasma from a patient on warfarin therapy, and plasma from a patient with no free PS and only PS bound to C4b-binding protein. The slopes of the curves show that the monoclonal antibodies reacted equally with all the tested forms of PS indicating that the antigenic site(s) to which the monoclonal antibodies are directed are present and exposed in free and bound PS, in thrombin-cleaved PS, and in the coumarin form of the protein. Each EDTA-dependent antibody, immobilized on Sepharose 4B-CNBr was used to purify PS from the barium citrate-absorbed, ammonium sulphate-soluble fraction of plasma. The fraction eluted from the immunoabsorbent with a buffer containing 4 mmol/1 CaCl2 and analysed by SDS-PAGE, contained two bands, one migrating with conventionally purified PS and the other with purified C4b-binding protein. Homogeneous PS was obtained by chromatography of the barium citrate absorbate on a DEAE-Sephadex column. The protein peak containing the bulk of PS was subsequently applied to the immunoadsorbent and eluted with 4 mmol/1 CaCl2. These studies demonstrate that EDTA-dependent monoclonal antibodies can simplify the isolation of PS from human plasma.

EDTA-DEPENDENT MONOCLONAL ANTIBODIES TO HUMAN PROTEIN S

1987 ◽  
Author(s):  
S Marcovina ◽  
R Coppola ◽  
M P Protti ◽  
C Gelfi ◽  
P M Mannucci
Keyword(s):  
Monoclonal Antibodies ◽  
Ascitic Fluid ◽  
Solid Phase ◽  
Protein A ◽  
Fluid Phase ◽  
Protein S ◽  
The Other ◽  
Human Protein ◽  
Sephadex Column ◽  
Plot Analysis

Splenocytes from a Balb/c mouse immunized with purified human protein S (PS) were fused with murine hybridoma cell line SP2/0-Agl4 and cultured in Iscove's medium without addition of fetal bovine serum. Hybrid supernatants were screened for the presence of specific antibodies by solid-phase ELISA and cloned by the limiting dilution technique. Pour clones, named S2, S3, S8, and S10, were selected, recloned several times, and injected intraperitoneally into Balb/c mice for the production of antibody-rich ascitic fluid. The monoclonal antibodies (Mabs), all of IgGl subclass with k light chain, were purified from ascitic fluid by Protein-A chromatography. The specificity of Mabs was controlled by the immunoblotting technique: the Mabs appeared to react only with two plasma proteins, one with a MW of about 70.000 dal tons comigrating with purified PS, and the other with a MW >300.000 da that is likely to be the C4b-binding protein-PS complex. No interaction has been observed with PS-depleted plasma. As tested by a fluid phase radio immunoassay, the binding of Mabs to PS was significantly higher in the presence of EDTA while was totally inhibited in the presence of Ca2+. Scatchard plot analysis of the binding between 125I-PS and Mabs showed that the binding affinity of the antibodies ranged from 108 to 109 1/mol. Each EDTA-dependent Mab was then immobilized on Sepharose 4B-CNBr and used to purify PS from barium precipitation of citrated plasma. The fraction eluted with 2 mmol of CaCl2 from the immunoadsorbent appeared to contain only two proteins when stained with Cocmassie Blue. By immuno blotting, all Mabs reacted with both proteins, one comigrating with purified PS and the other with a MW >300.000. Essentially homogeneous PS, free from the high MW component, was obtained when the barium citrate adsorbate was applied to a DEAE-Sephadex column and the protein peack containing the balk of PS was sussequently applied to the immunoadsorbent and eluted with 2 mmol CaCl2.In summary, we have described an unusual set of EDTA-dependent monoclonal antibodies to PS and their use for the purification of homogeneous protein S from human plasma.

scholarly journals Degradation of human complement component C4b in the presence of the C4b-binding protein-protein S complex

1983 ◽  
Vol 209 (3) ◽  
pp. 857-863 ◽  
Author(s):  
B Dahlbäck ◽  
B Hildebrand
Keyword(s):  
Binding Sites ◽  
Binding Protein ◽  
Sodium Dodecyl ◽  
Fluid Phase ◽  
Protein S ◽  
Factor I ◽  
Haemolytic Assay ◽  
Dependent Protein ◽  
Molecular Weight Form ◽  
Human Complement Component

Vitamin K-dependent protein S and the higher-molecular-weight form of C4b-binding protein (C4bp-high) interact, forming a 1:1 complex with a KD of approx. 1×10(-7) M [Dahlbäck (1983) Biochem. J. 209, 847-856]. In the present study the effect of protein S on the degradation of C4b by Factor I (C3b inactivator) and C4bp was investigated both in fluid phase and on cell surfaces, with the use of highly purified components. Fluid-phase degradation of C4b was monitored on sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis, and the effect on surface-bound C4b was estimated by haemolytic assay. No effect of protein S could be demonstrated in any of the systems used. Thus, although bound to C4bp, protein S is neither involved in, nor does it affect, the interaction between C4bp and C4b. This indicates that the binding sites on the C4bp molecule for protein S and for C4b are independent and different.

MONOCLONAL ANTIBODIES TO HUMAN PROTEIN S AND C4b-BINDING PROTEIN

1987 ◽  
Author(s):  
Martin Hessing ◽  
Joost C M Meijers ◽  
Jan A van Mourik ◽  
Bonno N Bouma
Keyword(s):  
Monoclonal Antibodies ◽  
Complex Formation ◽  
Binding Protein ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Thrombin Cleavage ◽  
Prolonged Aptt ◽  
Acid Domain ◽  
A Minor

Protein S (PS) circulates in plasma both free and in reversible association with the complement component C4b-binding protein (C4bp). Only free PS is functional as a cofactor for activated protein C (APC). Cleavage of PS by thrombin at a site near the r-carboxyglutamic acid domain is associated with a loss of cofactor activity. This may be a control mechanism for the anticoagulant activity of APC. These observations led us to investigate the role of C4bp and thrombin in the regulation of PS. Complex formation between purified PS and C4bp was studied in plasma and in a system with purified components. 125I-labeled PS was first incubated with either C4bp or citrated plasma and then subjected to polyacrylamide gelelectrophoresis in the absence of SDS. The formation of the C4bp-PS complex in plasma and in the purified system was demonstrated by autoradiography. Crossed immuno-electrophoresis using an antiserum against PS was performed in the presence of 8 mM EDTA. Human citrated plasma showed two precipitin peaks. Free PS migrated rapidly in the first dimension, whereas the C4bp-PS complex was just anodal to the application slot. The addition of C4bp to either plasma or purified PS resulted in the disappearance of the free PS peak and an increase of the slower migrating peak. The effect of purified C4bp on the PS-cofactor function of APC was studied in citrated plasma. The prolongation of the APTT induced by the addition of APC could be inhibited by the addition of increasing amounts of C4bp. Monoclonal antibodies to PS and C4bp were prepared and characterized. The monoclonal antibodies to either PS or C4bp did not block the complex formation between and PS, as was demonstrated by dot blotting of C4bp with 125I-PS and agarose gelelectrophoresis followed by Western blotting. Three out of 7 monoclonal antibodies to PS did not detect PS after thrombin cleavage on an immunoblot after non-reduced SDS polyacrylamide gelelectrophoresis. These 3 antibodies gave a significant shortening of the prolonged APTT induced by the addition of APC to normal plasma, indicating that these monoclonals inhibited the cofactor function of PS. The other 4 monoclonals to PS that did detect PS after thrombin cleavage on an immunoblot, gave only a minor inhibition of the PS cofactor function.

One-step sandwich enzyme immunoassays for human C4b-binding protein (C4BP) and protein S-C4BP complex using monoclonal antibodies

1995 ◽  
Vol 234 (1-2) ◽  
pp. 115-125 ◽  
Author(s):  
Hironori Tamei ◽  
Takumi Hoshino ◽  
Shin'ichi Yoshida ◽  
Tatsuya Hayashi ◽  
Kazushi Iwata ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Protein ◽  
Protein S ◽  
Enzyme Immunoassays ◽  
One Step

Two site-specific radioimmunoassays which demonstrate the presence of proteolytically modified insulin-like growth factor-binding protein-3 in the circulation

1993 ◽  
Vol 137 (2) ◽  
pp. 321-328 ◽  
Author(s):  
S. C. Cwyfan Hughes ◽  
J. A. H. Wass ◽  
J. M. P. Holly
Keyword(s):  
Growth Factor ◽  
Binding Protein ◽  
Antigenic Site ◽  
Binding Domain ◽  
Insulin Like Growth Factor ◽  
Factor Binding ◽  
Sds Page ◽  
Igf Binding ◽  
Harsh Conditions ◽  
Igfbp 3

ABSTRACT The presence of proteolytic activity in the circulation directed against insulin-like growth factor binding protein-3 (IGFBP-3) was originally described in pregnancy but has subsequently been described in a number of catabolic and other pathological conditions. However, detection of this proteolytically modified IGFBP-3 has been demonstrated, in most cases, following separation using SDS-PAGE and this has led to speculation that its occurrence may be an artefact of the harsh conditions employed. Using two sitespecific antisera, one of which recognizes as its antigenic site a region of IGFBP-3 which is close to that of the IGF-binding domain, we have developed two radioimmunoassays which, when compared, can reveal alterations in the IGF-binding domain of IGFBP-3. The presence of IGFBP-3 modified by the circulating protease is denoted in this system by a divergence in the IGFBP-3 levels observed; this divergence has been shown to coincide with protease activity as determined by Western ligand blotting. The use of these assays has confirmed that the IGF-binding site of IGFBP-3 undergoes proteolytic modification in the circulation during a number of pathologies. Journal of Endocrinology (1993) 137, 321–328

Expression and Characterization of Recombinant Human Protein S in Heterologous Cells - Studies of the Interaction of Amino Acid Residues Leu-608 to Glu-612 with Human C4b-Binding Protein

1992 ◽  
Vol 67 (05) ◽  
pp. 526-532 ◽  
Author(s):  
Glenn T G Chang ◽  
Hans K Ploos van Amstel ◽  
Martin Hessing ◽  
Pieter H Reitsma ◽  
Rogier M Bertina ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Recombinant Protein ◽  
Plasma Protein ◽  
Binding Protein ◽  
Protein S ◽  
Full Length ◽  
Human Protein ◽  
Amino Acid Residues ◽  
Amino Terminal

SummaryMouse C127 epithelioid cells were genetically engineered to produce biologically active ³-carboxylated human protein S. A full length human protein S cDNA was cloned into a bovine papilloma virus (BPV) based shuttle vector under the transcriptional control of the Moloney murine sarcoma virus enhancer and the mouse metallothionein promoter. Stable expression was obtained in transfected C127 cells. Expression of ³-carboxylated protein S was dependent on the presence of vitamin K in the culture medium. Protein sequence analysis showed that recombinant and plasma protein S have the same amino terminal sequence. Analysis of specific post-translationally modified amino acids shows that recombinant protein S is fully ³-carboxylated and fully p-hydroxylated. Immunoblotting analysis using polyclonal and monoclonal antibodies shows that recombinant protein S has a slightly higher molecular weight than plasma protein S. After N-Glycanase treatment, identical molecular weights are observed for recombinant and plasma protein S, indicating that the difference is caused by differences in the N-linked carbohydrate side chains. Recombinant protein S also demonstrates normal cofactor activity for activated protein C in a clotting assay. Binding studies with the complement component, C4b-binding protein (C4BP), shows that recombinant protein S binds to C4BP with the same apparent affinity as plasma protein S. Two variant molecules are also tested for their binding to C4BP. The first variant has a replacement of amino acid residue leu-608 by val and was designated B variant. The second variant has three alterations, at positions 609, 611 and 612 where the acidic amino acid residues asp, asp and glu were replaced by asn, asn and gin, respectively and this variant was designated C variant. The binding of these variants to C4BP was the same as wild type recombinant protein S. This suggests that amino acid residues leu-608, asp-609, asp-611 and glu-612 are not essential for binding of the intact full length protein to C4BP.

BINDING SITE OF VITAMIN K-DEPENDENT PROTEIN S ON C4b-BINDING PROTEIN

1987 ◽  
Author(s):  
K Suzuki ◽  
J Nishioka ◽  
H Kusumoto ◽  
Y Deyashiki
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Disulfide Bonds ◽  
Protein C ◽  
Binding Protein ◽  
Gel Filtration ◽  
Activated Protein C ◽  
Protein S ◽  
Cofactor Activity ◽  
Carboxyl Terminal

Protein S, a cofactor for activated protein C, reversibly complexes with a regulatory complement component C4b-binding protein (C4bp) in plasma. In plasma of patients with congenital protein S deficiency, most protein S exists as a complex with C4bp, which has no cofactor activity. C4bp (Mw 550,000) is composed of approximately seven subunits with Mw 75,000 which are linked by disulfide bonds near the carboxy1-terminus. We report here about the complex formation between protein S and C4bp particularly on the binding site of protein S on C4bp molecule. Protein S and C4bp were purified from human plasma. Seventeen mouse monoclonal antibodies against C4bp were prepared. Chymotrypsin-digested C4bp was separated on gel filtration into a fragment with Mw 160,000 derived from the carboxyl-terminal core of the intact C4bp and fragments with Mw 48,000 from the amino-terminus. The carboxy1-terminal fragment with Mw 160,000 was found to be composed of approximately seven polypeptides with Mw 25,000, which were linked by disulfide bonds.The experiments using these fragments and the monoclonal antibodies showed that: (1) Protein S bound not only to the intact C4bp, but also to the fragment with Mw 160,000. (2) The fragment with Mw 160,000 inhibited the binding of protein S to C4bp, but the fragment with Mw 48,000 did not. (3) One of the seventeen monoclonal antibodies blocked the inhibition of C4bp on the cofactor activity of protein S. (4) This antibody inhibited C4bp binding to protein S. (5) The antibody bound to the fragment with Mw 160,000. Based on these results, protein S was suggested to lose its cofactor activity for activated protein C by binding to the carboxyl-terminal core of C4bp where seven subunits are linked by disulfide bonds.

Monoclonal antibodies to human plasma Protein X alias complement S-protein

1985 ◽  
Vol 5 (4) ◽  
pp. 343-352 ◽  
Author(s):  
Dieter Jenne ◽  
Ferdinand Hugo ◽  
Sucharit Bhakdi
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Protein ◽  
Antithrombin Iii ◽  
Plasma Concentrations ◽  
Fluid Phase ◽  
Gel Chromatography ◽  
S Protein ◽  
Sds Page ◽  
Protein X ◽  
Terminal Complement

Protein X alias complement S-protein was isolated by dissociation from purified XCSb-9 (fluid-phase terminal C5b-9) complexes with 250 mM deoxycholate and subsequent sucrose density gradient centrifugation and Sephacryl gel chromatography. Polyclonal rabbit and monoclonal mouse antibodies were used to preliminarily characterize the protein in human serum and plasma. In plasma, Protein X yielded a symmetrical immuno-precipitate of α2-mobility in a crossed immunoelectrophoresis assay. However, a second immunoprecipitate of Oh-mobility was observed when serum was analysed; this precipitate represented Protein X in complex with antithrombin-III. The co-precipitation of Protein X with serum antithrombin-III was exploited for establishing a simple screening test for unequivocal identification of monocJonal anti – Protein X antibodies. SDS-PAGE immunoblotting with monoclonal antibodies showed that Protein X exhibits pronounced microheterogeneity, migrating as a diffuse moiety of approx. Mr 80–90 000. Additionally, a small amount of polymeric aggregates appear to be present in plasma. Reduction of disulfide bonds led to liberation of a polypeptide of approx. 15 K as discerned by two-dimensional SDS-PAGE immunoblotting. Protein X is not cleaved to lower molecular weight entities during the process of blood coagulation or during formation of fluid-phase terminal complement complexes. The plasma concentrations in healthy adults were in the range of 500–700 pg/ml. The availability of methods for isolating Protein X and raising monoclonal antibodies will facilitate further studies on the dual role of this protein in the terminal complement and coagulation cascades.

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