scholarly journals The C-terminus of factor H: monoclonal antibodies inhibit heparin binding and identify epitopes common to factor H and factor H-related proteins

1998 ◽  
Vol 331 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Wolfgang M. PRODINGER ◽  
Jens HELLWAGE ◽  
Martin SPRUTH ◽  
Manfred P. DIERICH ◽  
Peter F. ZIPFEL
Keyword(s):  
Monoclonal Antibodies ◽  
Unique Feature ◽  
Factor H ◽  
Deletion Mutants ◽  
Heparin Binding ◽  
Specific Detection ◽  
Short Consensus Repeats ◽  
C Terminus ◽  
Inhibitory Antibodies ◽  
Related Proteins

We have generated monoclonal antibodies (mAbs) specific for the C-terminus of factor H that can be used as inhibitory antibodies for heparin binding and for the specific detection of factor H and factor H-related proteins (FHRs) in plasma and triacylglycerol-rich lipoproteins. Four distinct mAbs were established: IXF9 (IgG1), VD3 (IgG2a), VIG8 (IgG1) and IIC5 (IgG1). Each reacts specifically with FHR-1 and factor H (and also with FHR-2 in the case of VIG8), but none binds to the related FHR-3 and FHR-4 proteins nor to factor H-like protein 1. By the use of deletion mutants of factor H and by comparing the reactivity with FHR-1 and FHR-2, the binding epitopes of the mAbs were identified and localized to different short consensus repeats (SCRs): mAbs IXF9 and VD3 bind to related or even identical sites within SCR 18 (factor H) and SCR 3 (FHR-1) respectively. mAbs VIG8 and IIC5 bind to different epitopes located within SCRs 19 to 20 of factor H and SCRs 4 to 5 of FHR-1 respectively. Only mAb VIG8 reacts with the corresponding SCRs 3 to 4 of FHR-2. These antibodies are useful for the detection of the corresponding proteins in biological specimens such as fractions of lipoproteins. In addition, mAb VIG8 has the unique feature of inhibiting binding of factor H to heparin. Given the recent identification of a heparin- and a C3b-binding domain within the C-terminus of factor H, these mAbs should provide useful tools for functional analysis and for the precise localization of the domain(s) required for this interaction.

The C-terminus of factor H: Monoclonal antibodies inhibit heparin binding and identify epitopes common to factor H and factor-H related proteins

1998 ◽  
Vol 35 (6-7) ◽  
pp. 393
Author(s):  
Wolfgang M. Prodinger ◽  
Jens Hellwage ◽  
Martin Spruth ◽  
Manfred P. Dierich ◽  
Peter F. Zipfel
Keyword(s):  
Monoclonal Antibodies ◽  
Factor H ◽  
Heparin Binding ◽  
C Terminus ◽  
Related Proteins

scholarly journals The possible role of factor H in complement activation-related pseudoallergy (CARPA): a failed attempt to correlate blood levels of FH with liposome-induced hypersensitivity reactions in patients with autoimmune disease

2015 ◽  
Vol 7 (1) ◽  
Author(s):  
Tamas Gyula Fülöp ◽  
Mihály Józsi ◽  
Josbert Metselaar ◽  
Gert Storm ◽  
Laszló Rosivall ◽  
...  
Keyword(s):  
Initial Data ◽  
Alternative Pathway ◽  
Technical Problem ◽  
Factor H ◽  
Blood Levels ◽  
Hypersensitivity Reactions ◽  
Short Consensus Repeats ◽  
Unexpected Outcome ◽  
Related Proteins

AbstractFactor H (FH) is a natural inhibitor of the alternative pathway (AP) of complement (C) activation, an abundant protein in blood whose reduced level has been associated with proneness for increased C activation. There are also 5 FH-related proteins (FHR), which have different impacts on C function. After brief outlines of the C system and its activation via the AP, this review focuses on FH and FHR, collecting data from the literature that suggest that reduced levels or function of FH is associated with C activation-related hypersensitivity reactions (HSRs), called C activation related pseudoallergy (CARPA). Based on such observations we initiated the measurement of FH in the blood of patients with inflammatory bowel disease (IBD) and rheumatoid arthritis (RA), and examined the correlation between FH levels and HSRs following i.v. administration of PEGylated liposomal prednisolone phosphate (PLPP). ELISA assay of FH was conducted on plasma samples before treatment, immediately after treatment and at follow-up visits up to 7 weeks, and an attempt was made to correlate the FH levels obtained with the presence or absence of HSR that occurred in five of twenty patients. However, the initial data presented here on three reactive and three non-reactive patients showed FH levels >600 μg/mL, while the normal range of FH is 2–300 μg/mL. This unexpected outcome of the test led us to realize that the ELISA we used was based on antibodies raised against the short consensus repeats (SCR) in FH, which are also present in FHR. Thus the kit cannot distinguish these proteins and we most likely measured the combined levels of FH and FHR. These initial data highlighted an unforeseen technical problem in assessing FH function when using a FH ELISA that cross reacts with FHR, information that helps in further studies exploring the role of FH in CARPA.

Factor H family proteins: on complement, microbes and human diseases

2002 ◽  
Vol 30 (6) ◽  
pp. 971-978 ◽  
Author(s):  
P. F. Zipfel ◽  
C. Skerka ◽  
J. Hellwage ◽  
S. T. Jokiranta ◽  
S. Meri ◽  
...  
Keyword(s):  
Serum Proteins ◽  
Hot Spot ◽  
Gene Mutations ◽  
Factor H ◽  
Surface Proteins ◽  
Short Consensus Repeats ◽  
Contact Points ◽  
Multiple Binding Sites ◽  
C Terminus ◽  
Control Modules

At present, the human Factor H protein family represents seven multidomain, multifunctional serum proteins. This group includes the complement and immune regulators Factor H, the Factor H-like protein 1 (FHL-1) and five Factor H-related proteins proteins (FHR-1, -2, -3, -4 and -5). Each is exclusively composed of individually folded protein domains, termed short consensus repeats (SCRs) or complement control modules. Structure-function analyses allowed the localization of the complement regulatory domain of Factor H and FHL-1 in the N-terminal region within SCRs 1–4. In addition, multiple binding sites for C3b, heparin and microbial surface proteins were localized in the N-terminus, within the middle region and also in the C-terminus of Factor H and FHL-1. Recent results show a central role for the C-terminus of Factor H, i.e. SCRs 19–20. These particular domains are conserved in all FHRs identified so far, include contact points for C3b, heparin and microbial surface proteins and represent a ‘hot-spot’ for gene mutations in patients that suffer from the Factor H-associated form of haemolytic uraemic syndrome.

Pharmacokinetics of Full Length and Two-Domain Tissue Factor Pathway Inhibitor in Combination with Heparin in Rabbits

1993 ◽  
Vol 70 (03) ◽  
pp. 454-457 ◽  
Author(s):  
Claus Bregengaard ◽  
Ole Nordfang ◽  
Per Østergaard ◽  
Jens G L Petersen ◽  
Giorgio Meyn ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Anticoagulant Activity ◽  
Fold Increase ◽  
Volume Of Distribution ◽  
Full Length ◽  
Heparin Binding ◽  
Extrinsic Pathway ◽  
C Terminus ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) is a feed back inhibitor of the initial activation of the extrinsic pathway of coagulation. In humans, injection of heparin results in a 2-6 fold increase in plasma TFPI and recent studies suggest that TFPI may be important for the anticoagulant activity of heparin. Full length (FL) TFPI, but not recombinant two-domain (2D) TFPI, has a poly cationic C-terminus showing very strong heparin binding. Therefore, we have investigated if heparin affects the pharmacokinetics of TFPI with and without this C-terminus.FL-TFPI (608 U/kg) and 2D-TFPI (337 U/kg) were injected intravenously in rabbits with and without simultaneous intravenous injections of low molecular weight heparin (450 anti-XaU/kg).Heparin decreased the volume of distribution and the clearance of FL-TFPI by a factor 10-15, whereas the pharmacokinetics of 2D-TFPI were unaffected by heparin. When heparin was administered 2 h following TFPI the recovery of FL-TFPI was similar to that found in the group receiving the two compounds simultaneously, suggesting that the releasable pool of FL-TFPI is removed very slowly in the absence of circulating heparin.

Deletions in the C-terminal coding region of the v-sis gene: dimerization is required for transformation

1986 ◽  
Vol 6 (4) ◽  
pp. 1304-1314
Author(s):  
M Hannink ◽  
M K Sauer ◽  
D J Donoghue
Keyword(s):  
Growth Factor ◽  
Gene Product ◽  
Platelet Derived Growth Factor ◽  
Deletion Mutants ◽  
Gene Products ◽  
Coding Region ◽  
Strong Connection ◽  
C Terminus ◽  
Its Gene ◽  
Transforming Gene

The v-sis gene encodes chain B of platelet-derived growth factor. However, this gene codes for additional amino acids at both the N terminus and the C terminus of its gene product which are not present in the amino acid sequence of platelet-derived growth factor. We constructed a series of deletion mutants with deletions in the v-sis gene in order to define the C-terminal limit of the v-sis gene product which is required for transformation. Deletion mutants of the v-sis gene which encoded truncated gene products up to 57 residues shorter than the v-siswt gene product were still able to transform cells. The minimal transforming region of the v-sis gene product contained six residues fewer than were present in chain B of platelet-derived growth factor. Only 10 residues, including the sequence Cys-Lys-Cys, separated the smallest transforming gene product from the largest nontransforming gene product. These cysteine residues were also important for dimerization of the v-sis gene product, since all of the nontransforming v-sis deletions were unable to form dimers when they were analyzed under nonreducing conditions. Our results suggest that there is a strong connection between transformation and dimerization.

Reactions of human antibodies with CR1 immobilised by mouse monoclonal antibody E11 Red cell phenotype Murine MAb Human anti- Absorbance Ratio Kn(a+) ] Kn(a-) E11 Kna 0.755 0.195 4:1 McC(a+) 0.538 McC(a-) E11 McC 0.136 4:1 Yk(a+) 0.315 Yk(a-) E11 Yka 0.120 26:1 Sl(a+) 0.342 Sl(a-) E11 Sla 0.074 4.6:1 Cs(a+) 0.139 Cs(a-) E11 Cs 0.108 Mapping relative positions of antigens on a specific protein When several murine monoclonal antibodies to different epitopes on the same protein are available, MAIEA can be used to study the relative position of antigens on that protein. This application of MAIEA depends on mutual inhibition of murine monoclonal antibodies and human antibodies. A negative result is obtained when human and monoclonal antibodies compete for the same epitope, or bind to very closely located epitopes, so no tri-molecular complex is produced. Several monoclonal antibodies to the Kell protein have been used in MAIEA to study the relationships of the Kell system antigens [10]. The decay accelerating factor DAF, CD55, is detected by several monoclonal antibodies. Three antibodies BRIC 230, BRIC 110 and BRIC 216 were known from competitive binding assays to bind to different short consensus repeats (SCR) [11]. So three of the four SCRs of the DAF molecule were positively identified (Table II). Strong positive reactions were observed with all three BRIC antibodies and anti-Cr3, anti-WES8, and anti-WESb showing that MAIEA is a useful techique for studying this system [12]. The results showed that Cr8, WESa, and WESb are not on the first three SCRs and must

1995 ◽  
pp. 190-190
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Molecular Complex ◽  
Specific Protein ◽  
Cell Phenotype ◽  
Binding Assays ◽  
Human Antibodies ◽  
Short Consensus Repeats ◽  
Murine Monoclonal Antibodies ◽  
Competitive Binding Assays

scholarly journals Characterization of four new monoclonal antibodies against the distal N-terminal region of PrPc

2014 ◽  
Author(s):  
Alessandro Didonna ◽  
Anja Colja Venturini ◽  
Katrina Hartman ◽  
Tanja Vranac ◽  
Vladka Curin Serbec ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Prion Protein ◽  
Biochemical Characterization ◽  
Prion Diseases ◽  
Physiological Role ◽  
Prion Infection ◽  
Promising Tool ◽  
C Terminus ◽  
N Terminus

Prion diseases are a group of fatal neurodegenerative disorders that affect humans and animals. They are characterized by the accumulation in the central nervous system of a pathological form of the host-encoded prion protein (PrPC). The prion protein is a membrane glycoprotein that consists of two domains: a globular, structured C-terminus and an unstructured N-terminus. The N-terminal part of the protein is involved in different functions in both health and disease. In the present work we discuss the production and biochemical characterization of a panel of four monoclonal antibodies (mAbs) against the distal N-terminus of PrPC using a well-established methodology based on the immunization of Prnp0/0 mice. Additionally, we show their ability to block prion (PrPSc) replication at nanomolar concentrations in a cell culture model of prion infection. These mAbs represent a promising tool for prion diagnostics and for studying the physiological role of the N-terminal domain of PrPC.

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