Real-time Biospecific Interaction Analysis Research Articles

Engineering and characterization of a bispecific HER2 × EGFR‐binding affibody molecule

HER2 (human epidermal-growth-factor receptor-2; ErbB2) and EGFR (epidermal-growth-factor receptor) are overexpressed in various forms of cancer, and the co-expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2-binding affibody molecule was genetically fused in-frame with a bivalent EGFR-binding affibody molecule via a (G4S)3 [(Gly4-Ser)3]-encoding gene fragment. The encoded 30 kDa affibody construct (ZHER2)2-(G4S)3-(ZEGFR)2, with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow-cytometric analysis, the latter using HER2- and EGFR-overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real-time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR-overexpressing cells and soluble HER2; and (iii) a cell-cell interaction analysis where the bs affibody molecule bound HER2-overexpressing SKBR-3 cells and EGFR-overexpressing A-431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed.

Prevention of venous thrombosis by preoperative glycyrrhizin infusion in a rat model

BackgroundGlycyrrhizin is an agent with the capacity to bind to selectin molecules expressed on vascular endothelial cells and potentially prevent the adherence of neutrophils to the vascular endothelial surface. It has been found to prevent intravenous thrombus formation. MethodsVenous thrombosis was induced in male rats by ligation of the inferior vena cava (IVC) for 6 h. Before the ligation, the study rats were given intravenous injections of glycyrrhizin through the IVC. After 6 h of venous ligation, the rats were sacrificed and the IVC segments were harvested. Thrombus within the IVC was collected to measure the wet weight. Gene expression of P-, L-, and E-selectin was detected by reverse transcriptase polymerase chain reaction using extracts of mRNA from the IVC vein wall. As baseline controls, IVC samples without ligation were harvested immediately after laparotomy. Neutrophil adhesion to the luminal surface of IVC was assessed on histological sections stained with hematoxylin and eosin. Blood samples were collected through the IVC proximal to the ligation after 6 h to estimate activated partial thromboplastin time (APTT) and prothrom-bin time (PT). To investigate the effect of glycyrrhizin on binding capacity of P-selectin to human neutrophils, real-time biospecific interaction analysis was performed with the Biacore 2000 system. ResultsThe mean weight of thrombus in the glycyrrhizintreated group was 12.9 ± 11.1 mg, which is significantly lower than that of the saline-treated control group (21.3 ± 12.5 mg). The expression level of P- and L-selectin mRNA in both saline- and glycyrrhizin-treated groups was significantly higher than that of the baseline control. Histological studies of cross sections of IVC showed significantly fewer neutrophils adhering to the luminal surface with glycyrrhizin treatment than in the saline-treated controls. There was no significant difference in the values of coagulation parameters with or without glycyrrhizin treatment. In vitro analysis showed that glycyrrhizin caused a dose-dependent reduction of neutrophils binding to immobilized recombinant P-selectin. ConclusionsPreoperative treatment with glycyrrhizin is potentially useful for preventing venous thrombosis by suppressing the adherence of neutrophils to the venous endothelium during the initial phase of thrombus formation without reducing coagulation capacity and the subsequent risk for increased bleeding.

All individual domains of staphylococcal protein A show Fab binding.

The interactions between the individual domains (E, D, A, B and C) of staphylococcal protein A (SPA) and Fc and Fab regions of human immunoglobulins were studied using real-time biospecific interaction analysis. An engineered domain Z, similar to fragment B but with a single glycine to alanine amino acid substitution, was also included in the study. The domains were expressed in Escherichia coli, affinity purified and immobilised onto sensor chip surfaces in a directed manner using a unique C-terminal cysteine residue engineered into the recombinant proteins. All domains bound to a recombinant human IgG1 Fc fragment with similar strength. For the first time, binding to human Fab was demonstrated for all native SPA domains, using both polyclonal F(ab')2 and a recombinant scFv fragment as reagents. Interestingly, the engineered Z domain showed a considerably lower affinity for Fab as compared to the native domains.

Epitope mapping of four novel CD44 monoclonal antibodies using surface plasmon resonance and soluble CD44.

CD44 is a ubiquitous multistructural and multifunctional cell surface adhesion molecule. The molecular diversity of this glycoprotein is generated by both post-translational modification and the differential use of alternatively spliced exons which play a critical role in determining the exact conformation of the molecule. CD44 isoforms are found in many tissues and in soluble form in plasma. Soluble CD44 was purified by a two-step purification combining ion exchange and immuno-affinity chromatography. Our aim was to check that all the known antigenic epitopes are present on sCD44, which could thus be used for the mapping of new anti-CD44 antibodies. Competitive binding assays using reference antibodies and novel anti-CD44 monoclonal antibodies were performed by real-time biospecific interaction analysis. Reference mAbs identified on soluble CD44 the three distinct epitopes previously defined using red blood cell membrane CD44. From the four novel CD44 mAbs, two mAbs (NaM198-6B5, NaM198-10B4) mapped to epitope group 1, whereas the others (NaM10-8F4, NaM77-9D6) mapped to epitope group 2. Immunopurified sCD44 obtained from the plasma of healthy donors appears to be a usable tool for the mapping of anti-CD44 mAbs.

Structural and Functional Characteristics of the B-domain-deleted Recombinant Factor VIII Protein, r-VIII SQ

Recombinant factor VIII SQ (r-VIII SQ), ReFacto, is a recombinant factor VIII product similar to the smallest active factor VIII protein found in plasma-derived factor VIII (p-VIII) concentrates. The protein comprises two polypeptide chains of 80 and 90 kDa and lacks the major part of the heavily glycosylated B-domain i.e. amino acids Gln744 to Ser1637. r-VIII SQ retains six potential glycosylation sites for N-linked oligosaccharides at asparagine residues 41, 239, 582, 1685, 1810 and 2118. We describe a thorough comparison of the characteristics of r-VIII SQ with those of p-VIII. The primary and secondary structures of r-VIII SQ were in good agreement with that of B-domain-deleted p-VIII (p-VIII-LMW) as shown by SDS-PAGE, Western blotting with antifactor VIII antibodies, tryptic mapping, amino acid sequence analysis and circular dichroism spectroscopy. A few divergences also existed. Thus r-VIII SQ was shown to contain a small amount of the single chain primary translation product of 170 kDa and also the product specific sequence of 14 amino acids, the SQ-link, in the C-terminal end of the 90 kDa chain. It was shown that r-VIII SQ had a high specific activity of about 14,000 IU VIII:C/mg as determined by use of a chromogenic substrate assay. The r-VIII SQ protein was comparable to p-VIII forms with a retained B-domain, in terms of potency measured by a chromogenic substrate or a two-stage clotting assay, in interactions with thrombin, and with activated protein C (APC) in combination with Protein S. The ability of r-VIII SQ to participate as a cofactor in factor Xa generation in a mixture of factors IXa and X, phospholipid and calcium was in conformity with that of p-VIII. Furthermore r-VIII SQ had a good binding capacity for phospholipid vesicles and von Willebrand factor (vWF) as shown in gel filtration studies. The same kinetics in binding to von Willebrand factor was found for r-VIII SQ and p-VIII as determined by real-time biospecific interaction analysis (BIA) with use of the BIAcore instrument. The apparent association rate constant was 4 x 10(6) M(-1)s(-1). Two dissociation rate constants were found, 1 X 10(-2)s(-1) and 4 x 10(-4)s(-1). The results extend the present knowledge that the factor VIII B-domain is dispensable for the factor VIII cofactor function in hemostasis.

Surface plasmon resonance biosensors as a tool in antibody engineering

Modern gene technology combined with efficient microbial expression systems provides tools to produce antibodies with reduced functional size and improved binding properties as well as antibody fusions or novel antibodies. Surface plasmon resonance based biosensors, which measure antigen–antibody interactions in real-time, can be used for a diverse characterization of the modified antibodies. To date, the majority of published work originates from real-time biospecific interaction analysis based on the BIAcore ® instruments. This article describes the range of applications in antibody engineering in which BIAcore has been applied.

One-step assay for the determination of free protein S antigen in plasma using real-time biospecific interaction analysis.

Real-time biospecific interaction analysis based on optical detection by surface plasmon resonance was used to develop an accurate one-step method for the direct measurement of free protein S in human plasma. This assay was validated, compared with classical immunological methods and shown to be suitable for the routine clinical diagnosis of protein S deficiency. The method relies on the specific capture of free protein S directly from plasma by a monoclonal antibody (mAb), 34G2, immobilized on a sensor chip surface. A calibration curve was established with serial dilutions of standard plasma (working range 5-50%) and a linear relationship was found to exist between the relative response in resonance units (RU) and the concentration of free protein S expressed as percentage plasma dilution (r = 0.99). The specificity of the assay was confirmed using purified human protein S and polyethylene glycol treated plasma. In addition, it could be demonstrated that no dissociation of C4b-BP-protein S complexes occurred under the chosen experimental conditions. The technique was reproducible with inter-assay, intra-assay and inter-sensor chip variation coefficients of 1.5-5.4%, 2-3.1% and 4.4-4.9%, respectively, as evaluated in two different plasma samples. Since all tests are automatic and long series of analyses can be performed with the same sensor chip, the method was applied to the determination of free protein S antigen in plasma from 20 normal blood donors and 38 thrombophilic patients. Results displayed excellent correlation with those of free protein S enzyme-linked immunosorbent assay (r = 0.99) and rocket immunoelectrophoresis of polyethylene glycol-treated plasma (r = 0.93).

Capture of Single-Stranded DNA Assisted by Oligonucleotide Modules

Real-time biospecific interaction analysis was employed to monitor direct capture of a hepatitis C virus (HCV) derived polymerase chain reaction (PCR) product by nucleic acid hybridization. Different formats for hybridization were used to study the interaction between a single-stranded HCV PCR product and capture oligonucleotides immobilized on a sensor chip via streptavidin–biotin chemistry. By employing a prehybridization step in solution with nonbiotin oligonucleotides complementary to the single-stranded target and adjacent to the immobilized probe, a significant capture was achieved in comparison to the low capture efficiency obtained using single immobilized probes (9–36 mer). High capture efficiencies were also observed when shorter immobilized probes were used in combination with strings of adjacently positioned prehybridized probes (i.e., modules). Interestingly, the introduction of single nucleotide gaps between prehybridized and/or immobilized probes dramatically reduced the capture efficiency. These results suggest that flexible systems for capture could be designed from libraries of short oligonucleotides (9 mers) used in module fashion, taking advantage of stacking interactions between the oligonucleotides. The potential applications of such oligonucleotide-assisted capture systems are discussed.

Beta-2-glycoprotein I Dependent Lupus Anticoagulants Form Stable Bivalent Antibody Beta-2-Glycoprotein I Complexes on Phospholipid Surfaces

The precise mechanism by which Beta-2-glycoprotein I (beta2-GPI-) dependent lupus anticoagulants lengthen phospholipid-dependent clotting reactions is still poorly understood. In order to study this, murine monoclonal antibodies (moabs) against human beta2GPI were raised. Eight of the 21 anti-beta2GPI moabs, obtained from 2 fusions, fulfilled the criteria for lupus anticoagulant (LA) activity as tested with a variety of sensitive screening assays and confirmatory tests. Seven moabs did not influence any clotting test. The LA positive moabs were found to compete for similar or closely spaced epitopes on immobilized beta2GPI. Two moabs with potent LA activity (moabs 22 F 6 and 22 B 3) and 1 moab without LA activity (moab 16 B 3) were selected to study the interaction between antibody, beta2GPI and phospholipid. Interactions were investigated by real-time biospecific interaction analysis (BIA) based on plasmon surface resonance technology on a BIA-core instrument using a sensor chip coated with phospholipid. When 22 F 6, the moab with the most pronounced LA activity, was allowed to interact with the phospholipid surface at concentrations between 0 and 400 nmol/l, no appreciable binding could be detected. Likewise, no binding could be measured when beta2GPI at concentrations between 0 and 400 nmol/l was passed over the phospholipid coated sensor chip. Combinations of beta2GPI and 22 F 6 resulted in significant binding. Similar results were obtained with 22 B 3, another moab with LA activity. A LA negative Moab, 16 B 3, did not cause binding of antibody-beta2GPI complexes. Fab' fragments, derived from moab 22 F 6, inhibited the binding of beta2GPI-22 F 6 and beta2GPI-22 B 3 in a concentration dependent way, indicating that only bivalent beta2GPI-antibody complexes bind with high affinity to phospholipids. Fab' fragments, derived from moab 22 F 6, also inhibited the LA effect of moabs 22 F 6 and 22 B 3 in diluted plasma. In summary, these experiments indicate that the beta2GPI-dependent LA effect depends on the formation of bivalent beta2GPI-antibody complexes on phospholipid surfaces.

All individual domains of staphylococcal protein A show Fab binding

The interactions between the individual domains (E, D, A, B and C) of staphylococcal protein A (SPA) and Fc and Fab regions of human immunoglobulins were studied using real-time biospecific interaction analysis. An engineered domain Z, similar to fragment B but with a single glycine to alanine amino acid substitution, was also included in the study. The domains were expressed in Escherichia coli, affinity purified and immobilised onto sensor chip surfaces in a directed manner using a unique C-terminal cysteine residue engineered into the recombinant proteins. All domains bound to a recombinant human IgG1 Fc fragment with similar strength. For the first time, binding to human Fab was demonstrated for all native SPA domains, using both polyclonal F(ab′) 2 and a recombinant scFv fragment as reagents. Interestingly, the engineered Z domain showed a considerably lower affinity for Fab as compared to the native domains.

Biochemical and functional characterization of recombinant von Willebrand factor produced on a large scale.

Recombinant von Willebrand factor (r-vWF) was produced in serum-free medium on a large scale in recombinant Chinese hamster ovary cells and was purified from fermentation supernatant by a combination of anion exchange chromatography and heparin affinity chromatography. Heparin affinity chromatography yielded r-vWF polymers of different degrees of multimerization. r-vWF was analysed by qualitative and quantitative functional analysis. We could show that while binding of r-vWF to platelets did not depend on multimerization of the molecule, ristocetin-induced platelet aggregation, binding to collagen and binding to heparin correlated directly with the extent of multimerization. Binding of recombinant coagulation factor VIII (r-FVIII) to r-vWF was studied by real-time biospecific interaction analysis and surface plasmon technology. The data indicated that binding of r-FVIII did not depend on r-vWF multimerization. Real-time biospecific interaction analysis suggested a potential stoichiometry of 2 to 2.5 r-vWF subunits per r-FVIII molecule. Kinetic analysis of the r-vWF-r-FVIII interaction gave a binding rate constant of 3 x 10(6) M-1 s-1 and an association constant of 2.5 x 10(9) M-1. Reaction of r-vWF with carbohydrate-specific lectins demonstrated that r-vWF contained a high proportion of N-glycans composed of mannose, galactose, glucose, N-acetylglucosamine and terminal sialic acid. Carbohydrate moities were covalently bound to the protein structure and were quantitatively removed from r-vWF only after protein denaturation. The results demonstrated that r-vWF produced on large scale under serum-free culture conditions exhibited qualitative and quantitative functional properties comparable to human plasma-derived vWF.

Functional epitope mapping of insulin-like growth factor I (IGF-I) by anti-IGF-I monoclonal antibodies.

Based on a collection of monoclonal antibodies (mAb) against insulin-like growth factor I (IGF-I), we have defined the IGF-I epitopes involved in the interaction with IGF-binding proteins (IGFBP) and IGF-I receptors. We have also characterized the ability of these antibodies to block IGF-I-induced survival of the IL-3-dependent Ba/F3 cell line. More than 140 hybridomas secreting IGF-I-specific mAb were characterized, of which 28 were studied in detail. They display apparent affinity constants ranging from less than 10(6) to 10(10) M-1 and varying crossreactivity with IGF-II, including 2 mAb with higher affinity for IGF-II than for IGF-I. None crossreact with insulin or any other growth factor tested. Using both enzyme immunoassays and real-time biospecific interaction analysis, we have identified 8 epitopic clusters related to the primary structure of IGF-I, according to mAb reactivity to synthetic peptides, proteolytic fragments of IGF-I, and various IGF-I mutants. The mAb panel also was used to map the IGF domains implicated in the interaction with IGFBP and IGF-I receptors. An IGF-I domain has been identified that remains exposed after IGF-I binding to IGFBP-1 or to IGFBP-3, which is recognized by 6 different mAb. The mAb in this group also bind IGF-I, when complexed to the type-1 IGF receptor on the murine pro-B cell line Ba/F3, and BALB/c 3T3 fibroblasts overexpressing the human receptor. Finally, IGF-I-promoted survival can be blocked with mAb specific for target epitopes, and their potential use in tumor cell growth control is discussed.

Functional Epitope Mapping of Insulin-Like Growth Factor I (IGF-I) by Anti-IGF-I Monoclonal Antibodies

Based on a collection of monoclonal antibodies (mAb) against insulin-like growth factor I (IGF-I), we have defined the IGF-I epitopes involved in the interaction with IGF-binding proteins (IGFBP) and IGF-I receptors. We have also characterized the ability of these antibodies to block IGF-I-induced survival of the IL-3-dependent Ba/F3 cell line. More than 140 hybridomas secreting IGF-I-specific mAb were characterized, of which 28 were studied in detail. They display apparent affinity constants ranging from less than 106 to 1010m−1 and varying crossreactivity with IGF-II, including 2 mAb with higher affinity for IGF-II than for IGF-I. None crossreact with insulin or any other growth factor tested. Using both enzyme immunoassays and real-time biospecific interaction analysis, we have identified 8 epitopic clusters related to the primary structure of IGF-I, according to mAb reactivity to synthetic peptides, proteolytic fragments of IGF-I, and various IGF-I mutants. The mAb panel also was used to map the IGF domains implicated in the interaction with IGFBP and IGF-I receptors. An IGF-I domain has been identified that remains exposed after IGF-I binding to IGFBP-1 or to IGFBP-3, which is recognized by 6 different mAb. The mAb in this group also bind IGF-I, when complexed to the type-1 IGF receptor on the murine pro-B cell line Ba/F3, and BALB/c 3T3 fibroblasts overexpressing the human receptor. Finally, IGF-I-promoted survival can be blocked with mAb specific for target epitopes, and their potential use in tumor cell growth control is discussed.

Suppression by platelet factor 4 of the myogenic activity of basic fibroblast growth factor.

The effect of platelet factor 4 (PF4) on myoblast cultures with or without basic fibroblast growth factor (bFGF) or other growth factors was investigated in the present in vitro experiments, with reference to bFGF binding to myoblast membrane fraction. When PF4 was added to the culture medium 1 day after myoblast cultivation, the nuclei of both myoblasts and myotubes were markedly reduced in number in a dose-dependent manner, whereas the inhibitory effect of PF4 on myoblast development was not observed when PF4 was added to the culture medium 3, 7, or 14 days after myoblast cultivation. In contrast, bFGF significantly increased the numbers of myoblast and myotube nuclei. When bFGF and PF4 were simultaneously added to the culture medium, PF4 abolished the facilitatory effects of bFGF on myogenesis. The real-time biospecific interaction analysis (BLA) core system showed that the myoblast membrane fraction at 1 day after cultivation contains bFGF-binding elements which are blocked by PF4 in a dose-dependent manner. Moreover, [126I]-bFGF binding experiments indicated the existence of both high and low affinity binding sites on myoblast membranes, although the high affinity binding sites decreased in number and the dissociation constant increased in value as the culture period was prolonged. Among the six other growth factors examined, acidic fibroblast growth factor and platelet-derived growth factor-BB stimulated myogenesis, and their effects were blocked by PF4 treatment. These findings suggest that: 1) PF4 inhibits myoblast proliferation and myotube formation only for a limited initial period of cultivation, possibly because of the time-dependent down-regulation of high affinity bFGF receptors: and 2) PF4 may be used as a tool to investigate the function of endogenous heparin-binding growth factors upregulated transiently at a certain developmental stage or in case of tissue damage and repair, even though it is not monospecific to bFGF.

Detection of mutations in PCR products from clinical samples by surface plasmon resonance.

Two different strategies for scanning and screening of mutations in polymerase chain reaction (PCR) products by hybridization analysis are described, employing real-time biospecific interaction analysis (BIA) for detection. Real-time BIA was used to detect differences in hybridization responses between PCR products and different 17-mer oligonucleotide probes. For the analysis using a biosensor instrument, two different experimental formats were investigated based on immobilization of either biotinylated PCR products or oligonucleotide probes onto a sensor chip. Applied on the human tumour suppressor p53 gene, differences in hybridization levels for full-match and mismatch situations employing both formats allowed the detection of point mutations in exon 6 PCR products, derived from a breast tumour biopsy sample. In addition, a mutant sample sequence could be detected in a 50/50 background of wild type exon 6 sequence. The suitability of the different formats for obtaining a regenerable system and a high throughput of samples is discussed.

Interactions between Staphylokinase, Plasmin(ogen), and Fibrin: STAPHYLOKINASE DISCRIMINATES BETWEEN FREE PLASMINOGEN AND PLASMINOGEN BOUND TO PARTIALLY DEGRADED FIBRIN

Staphylokinase (STA), a protein of bacterial origin, induces highly fibrin-specific thrombolysis both in human plasma in vitro and in pilot clinical trials. Using fluorescence microscopy, we investigated the spatial distribution of fluorescein isothiocyanate (FITC)-labeled STA during lysis of a plasma clot and its binding to purified fibrin clots in the presence or in the absence of plasmin(ogen). STA highly accumulated in a thin superficial layer of the lysing plasma clot following the distribution of plasminogen (Pg) during lysis. Experiments with purified fibrin clots revealed that STA binds to Pg bound to partially degraded fibrin but not to Pg bound to intact fibrin. Binding of FITC-labeled STA to various forms of plasmin(ogen) in a buffer solution was studied by measuring fluorescence anisotropy. The binding constant for Glu-Pg was estimated as 7.4 microM and for Lys-Pg as 0.28 microM; for active-site blocked plasmin the binding constant was less than 0.05 microM. The much lower affinity of STA for Glu-Pg compared with that for active site-blocked plasmin was mainly due to a lower association rate constant, as assessed by real time biospecific interaction analysis. Gel filtration of a mixture of STA with a molar excess of Glu-Pg demonstrated that STA migrated as an unbound 18-kDa protein when activation of Pg into plasmin was precluded by inhibitors of plasmin. When gel-filtered under the same conditions with plasmin, STA migrated in complex with plasmin with an apparent molecular mass of 100 kDa. Confocal fluorescence microscopy finally demonstrated that when FITC-labeled STA was added to plasma before clotting, it did not bind to fibrin fibers during the first minutes (lag phase), although Pg bound to the fibers moderately. Then, both Pg and STA started to accumulate on the fibers progressively, followed by complete lysis of the clot. In conclusion, our results imply that, when STA is added to plasma, only a small percentage associates with Pg. In contrast, STA binds strongly to plasmin and to Pg, which is bound to partially degraded fibrin. These findings add a new mechanism to the known explanations for the inefficient Pg activation by STA in plasma and specify the mechanism for fibrin-dependent activation of Pg.

EFFECT OF MULTIMERIZATION OF HUMAN AND RECOMBINANT VON WILLEBRAND FACTOR ON PLATELET AGGREGATION, BINDING TO COLLAGEN AND BINDING OF COAGULATION FACTOR VIII

The smallest circulating von Willebrand factor (vWF) molecule is a dimer composed of two identical subunits containing binding sites for heparin, collagen, platelet glycoproteins and coagulation factor VIII (FVIII). Interdimeric disulfide linking leads to multimers composed of up to 40 dimers. vWF serves as a carrier of FVIII and is required for normal interactions of platelets with the subendothelium of the injured vessel wall. Von Willebrand factor was purified from human plasma cryoprecipitate and fermentation supernatant of recombinant CHO cells by anion exchange chromatography. Heparin affinity chromatography was used to isolate vWF polymers of different degree of multimerization. Analysis of collagen binding and platelet aggregation revealed that these activities increase with increasing degree of multimerization of vWF. Binding of FVIII to vWF was studied by real-time biospecific interaction analysis and surface plasmon technology. The binding data showed that the binding of FVIII is independent of vWF multimerization. Using recombinant FVIII and recombinant vWF, real-time biospecific interaction analysis resulted in a potential stoichiometry of 2 to 2.5 vWF-subunits per bound FVIII molecule. The kinetic analysis of the vWF-FVIII interaction resulted in a binding rate constant of about 3 × 10 6 M −1 s −1 and an equilibrium dissociation constant of about 0.4 × 10 −9 M.

Cloning, sequencing and functional overexpression of the Streptococcus equisimilis H46A gapC gene encoding a glyceraldehyde-3-phosphate dehydrogenase that also functions as a plasmin(ogen)-binding protein. Purification and biochemical characterization of the protein.

We previously identified DNA sequences involved in the function of the complex promoter of the streptokinase gene from Streptococcus equisimilis H46A, a human serogroup C strain known to express this gene at a high level. As a prerequisite to understanding possible mechanisms that control the balance between the plasminogen activating and plasmin(ogen) binding capacities of H46A, we describe here its gapC gene encoding glyceraldehyde-3-phosphate dehydrogenase (GraP-DH, EC 1.2.1.12), a glycolytic enzyme apparently transported to the cell surface where it functions as a plasmin(ogen).binding protein. The gapC gene was cloned and sequenced and found to code for a 336-amino-acid polypeptide (approximately 35.9 kDa) exhibiting 94.9% sequence identity to the Plr protein from Streptococcus pyogenes shown by others to be capable of plasmin binding [Lottenberg, R., Broder, C. C., Boyle, M. D., Kain, S. J., Schroeder, B. L. & Curtiss, R. III (1992) J. Bacteriol. 174, 5204-5210]. To study the properties of the GapC protein, its gene was inducibly overexpressed in Escherichia coli from QIAexpress expression plasmids to yield the authentic GapC or (His)6GapC carrying a hexahistidyl N-terminus to permit affinity purification. Both proteins were functionally active, exhibiting specific GraP-DH activities of about 80 kat/mol (approximately 130 U/mg) after purification. Their binding parameters [association (ka) and dissociation (kd) rate constants, and equlibrium dissociation constants (Kd = kd/ka)] for the interaction with human Gluplasminogen and plasmin were determined by real-time biospecific interaction analysis using the Pharmacia BIAcore instrument. For comparative purposes, the commercial GraP-DH from Bacillus stearothermophilus (BstGraP-DH), a nonpathogenic organism, was included in these experiments. The Kd values for binding of plasminogen to GapC, (His)6GapC and BstGraP-DH were 220 nM, 260 nM and 520 nM, respectively, as compared to 25 nM, 17 nM and 98 nM, respectively, for the binding to plasmin. These data show that both the zymogen and active enzyme possess low-affinity binding sites for the gapC gene product and that the hexahistidyl terminus does not affect its function. Prior limited treatment with plasmin enhanced the subsequent plasminogen binding capacity of all three GraP-DHs, presumably by the exposure of new C-terminal lysine residues for binding to the zymogen.

Detection of a Physical and Functional Interaction between Csk and Lck Which Involves the SH2 Domain of Csk and Is Mediated by Autophosphorylation of Lck on Tyrosine 394

The COOH-terminal Src kinase (Csk) is responsible for the phosphorylation of the conserved, negative regulatory, carboxyl-terminal tyrosine of most of the Src family protein tyrosine kinases. Up to now, no stable binding of Csk to Src kinases has been detected. We therefore decided to analyze this interaction using two systems which allow detection of transient interaction. We produced and purified recombinant proteins in the glutathione S-transferase prokaryotic expression system. First, using real-time biospecific interaction analysis (BIAcore(TM)), we detected in vitro a specific interaction between Csk and one of its substrates Lck, a lymphocyte-specific member of the Src family. This interaction requires the autophosphorylation of Lck on tyrosine 394 (the phosphorylation of which is correlated with an increase of the kinase activity) and involves a functional Csk SH2 domain. Second, using the yeast two-hybrid system, we confirmed in vivo the physical interaction between Csk and Lck. Furthermore, in vitro we showed that autophosphorylation of Lck on tyrosine 394 enhances the phosphorylation of Lck by Csk on the negative regulatory site, tyrosine 505, suggesting that activated Lck serves preferentially as substrate for Csk. These findings might explain the mechanism(s) by which Csk interacts with most of Src kinases to down-regulate their kinase activity.

Kinetics of internalization and subcellular binding sites for T 3 in mouse liver

The intracellular fate of radiolabeled T 3 taken up by mice hepatocytes in vivo was determined at specific time intervals (2–120 min) after injection by quantitative electron microscopic radioautography. Injection of a 200-fold excess of unlabeled T 3 together with [ 125I]-T 3 resulted in a more than 90% inhibition of radioactivity detected in hepatocytes. A simple grain density (GD) analysis of radioautograms revealed that a specific labeling (GD > 1) was displayed by only five cell compartments: the plasma membrane, lipid droplets, mitochondria, nuclear envelope and nuclear matrix whereas other compartments were not labeled. Labeled compartments showed distinct changes in the pattern of labeling over time: the plasma membrane was labeled only 2 min after T 3 injection, whereas labeling of the nuclear envelope was high at 2 min, decreased at 15 min and progressively increased to maximal measured levels at 120 min. After a lag time of 30 min, nuclear matrix labeling increased progressively with time. Mitochondrial labeling was found to be specific at any time point studied but showed no change over time. These ultrastructural data have been confirmed in vitro by the interaction of T 3 with plasma membrane, nuclear membrane, nuclear matrix and mitochondria by real-time biospecific interaction analysis in a BIAcore ™ system. These results demonstrate that T 3 binds to hepatocytes before internalization, is transported both to mitochondria and to the nuclear envelope and translocated into the nuclear matrix.