Chemical cross-linking leads to two high molecular mass aggregates of rat alpha 1 beta 1 integrin differing in their conformation but not in their composition.

In order to detect protein interactions of the collagen/laminin receptor alpha 1 beta 1 integrin, covalent chemical cross-linking was performed with the homo-bifunctional, amine reactive reagents DSS (disuccinimidylsuberate) and DSP (dithiobis(succinimidylpropionate)). After cross-linking of the 190 kDa rat alpha 1 integrin subunit, immunoblotting revealed two additional, immunoreactive, high molecular mass complexes (M(r) 240/290 k). Generation of the 240/290 kDa aggregates depended on the presence of the intact tertiary protein structure. As shown with immunoaffinity purified proteins, the 240/290 kDa aggregates consist exclusively of alpha 1 and beta 1 integrin subunits. No other cross-linked proteins associated with the alpha 1 or beta 1 subunit were detected. In contrast to the non-cross-linkable alpha 1 beta 1 integrin, the 240/290 kDa aggregates presumably represent active forms of the adhesion receptor, because both bound in vitro to collagen I and IV. This ability of alpha 1 beta 1 integrin to cross-link and produce two additional high molecular mass forms is shared by rat alpha 9 beta 1 integrin. Thus, the cross-linking approach directly indicates that beta 1 integrins occur in different conformations caused by variations in the folding and/or spatial arrangement of their subunits.

Rat dipeptidyl peptidase IV (DPP IV) exhibits endopeptidase activity with specificity for denatured fibrillar collagens.

Dipeptidyl peptidase IV (DPP IV, CD 26) is an integral membrane serine protease exhibiting a well characterized exopeptidase activity. The present study shows that DPP IV also possesses a novel gelatinase activity and therefore endopeptidase activity, which was directly demonstrated by gelatin zymography. Protease inhibitor profile analysis showed that the endo- and exopeptidase activities of DPP IV share a common active site. Substrate specificity was detected for denatured collagen types I, II, III and V suggesting that DPP IV might contribute to collagen trimming and metabolism. On the basis of these data we propose that DPP IV and the recently sequenced gelatinolytic seprase (FAPalpha) represent a new subfamily of gelatinolytic integral membrane serine proteases.

Novel antibody coating of a magnetizable solid phase for use in enzyme immunoassays.

Novel kinds of magnetizable particles have been prepared using the interaction between the complexing groups of phosphonic acid and polyvalent metal ions on the surface of Fe3O4 particles. After modification of monoclonal and polyclonal antibodies by the bifunctional chelating agent 2-(4-diazophenyl)-1-hydroxyethane-1,1-bisphosphonic acid, antibodies were immobilized at a ratio of 5-10 mg antibody molecules per ml Fe3O4 particles without loss of immunological reactivity. Very sensitive and fast immunoenzymometric assays for the quantitative determination of human interferon-alpha 1 and mouse immunoglobulins were developed using such particles. The advantages of the method include immobilization of proteins on magnetizable carriers without chemical modification of the carrier and the short assay time compared to conventional immunoenzymometric assays.

Inhibition of urokinase activity and prevention of urokinase receptor binding by monoclonal antibodies.

Two murine monoclonal antibodies produced against human urokinase-type plasminogen activator were characterized with respect to their antigen-binding specificity and their effects on urokinase activity and urokinase receptor binding. One of the antibodies binds to the protease domain of urokinase (Kass = 2.1 X 10(7) M-1). Antibody binding inhibits catalysis of plasminogen activation. It does not, however, affect amidolytic activity of urokinase towards the chromogenic substrate D-Val-Leu-Arg-p-nitroanilide. The antibody thus appears to interfere with plasminogen binding without directly affecting catalytically active amino acid residues of the enzyme. The other antibody binds to the aminoterminal fragment of urokinase (Kass = 1.0 X 10(7) M-1) and prevents binding of the enzyme to high affinity receptors on human granulocytes. Binding of this antibody neither influences plasminogen activation nor the amidolytic activity of urokinase. Both antibodies are potentially useful for the further analysis and manipulation of urokinase function.

Effect of von Willebrand Factor and its proteolytic fragments on kinetics of interaction between the light and heavy chains of human factor VIII.

It was previously shown that vWF increases the rate of divalent cation-mediated fVIII reconstitution from isolated light chain (LCh) and heavy chain (HCh) subunits. We examined the effect of vWF on kinetic parameters for interaction between LCh and HCh in the presence of Ca2+ and Mn2+ ions, the most effective mediators of fVIII reconstitution from isolated subunits, and determined the minimal structural portion of vWF able to enhance fVIII formation. We found that affinity (Kd) for LCh/HCh binding mediated by Ca2+ and Mn2+ was 91 and 34.9 nM in the absence of vWF and 15.5 and 5.6 nM in its presence. This decrease of Kd resulted from a sixfold increase of the association rate constant (k(on)) for this interaction. The value of the dissociation rate constant (k(off)) for LCh/HCh complex was lower in the presence of Mn2+ (k(off) 4.6x 10(-6) s(-1)) than Ca2+ (k(off) 8.4 x 10(-6) s(-1)) but in both cases vWF had no effect on k(off). This indicates that at physiological concentration of 1 nM the rate of fVIII inactivation via dissociation to subunits would be entirely determined by the k(off) value, and it should not depend on the presence of vWF. Indeed, our experiments demonstrated that vWF did not have any effect on the rate of fVIII inactivation resulting from its dissociation to subunits at the physiological concentrations of the fVIII and vWF proteins. We identified the minimal portion of the vWF molecule, able to enhance reconstitution of fVIII from isolated subunits. Only vWF large proteolytic N-terminal homodimeric fragment SPIII (vWF residues 1-1365), but not small monomeric N-terminal fragment SPIII-T4 (1-272), both of which are known to contain a major fVIII binding site, was able to support reconstitution of fVIII activity from isolated LCh and HCh subunits in the presence of Mn2+ or Ca2+. The effect of SPIII on the LCh/HCh association was similar to that of vWF, because both proteins identically increased of the value of k(on) and did not alter the k(off) value.

Characterization of molecular aggregates of alpha 1 beta 1-integrin and other rat liver membrane proteins by combination of size-exclusion chromatography and chemical cross-linking.

Many membrane proteins display their biological activity in molecular aggregates of interacting counterparts. The analysis of these aggregates remains difficult; especially intermolecular complexes of membrane proteins tend to dissociate or artificially aggregate during detergent extraction out of membranes. Thus, the existence of protein aggregates was investigated by two approaches. First, after modest detergent extraction, the presence of three well characterized rat liver membrane proteins, alpha 1 beta 1-integrin, dipeptidyl aminopeptidase IV (DPP IV) and cell-CAM 105 (CAM = cell adhesion molecule), in aggregates could be demonstrated when investigated by size-exclusion chromatography (SEC) under non-denaturating conditions. However, the applied detergents partially influenced the resolution of the separation reducing the ability to discriminate between native and artificial protein aggregates. To circumvent these problems, a second approach based on covalent cross-linking of native protein complexes by dithiobis(succinimidylpropionate) was combined with the performance of denaturating SEC. Under such optimized some high-molecular-mass complexes of all model proteins consisting of unknown components could also be detected. Taken together, non-denaturating SEC and chemical cross-linking in combination with denaturating SEC represent methodological approaches for the characterization of protein aggregates.

Use of surface plasmon resonance for studies of protein-protein and protein-phospholipid membrane interactions. Application to the binding of factor VIII to von Willebrand factor and to phosphatidylserine-containing membranes.

The surface plasmon resonance phenomenon is used for real time measurements of protein-protein and protein-membrane interactions. In the present study two surface plasmon resonance-based binding assays permitting study of the interaction of coagulation factor VIII (fVIII) with von Willebrand factor (vWf) and phospholipid have been developed. These interactions of fVIII are required for maintenance of fVIII concentration in circulation and for the assembly of the functional factor Xase complex, respectively. With these binding assays, the role of the light chain (LCh) in fVIII binding to vWf and to immobilized phospholipid monolayers and intact vesicles containing 25% phosphatidylserine (PS) and 4% PS was examined. The finding that Kd of LCh binding to vWf (3.8 nM) is 9.5 times higher than that of fVIII (0.4 nM), indicates that the heavy chain (HCh) is required for the maximal affinity of fVIII for vWf. In contrast, affinities of LCh for 25/75 PS/phosphatidylcholine (PC) monolayers and 4/76/20 PSPC-phosphatidylethanolamine (PE) vesicles are similar to that of fVIII, indicating that LCh is solely responsible for these interactions. It was also examined how removal of the acidic region affects the binding affinity of the remaining part of LCh for vWf and phospholipid. It was demonstrated that the loss of the LCh acidic region upon thrombin cleavage leads to an 11 and 160-fold increase in the dissociation rate constant (k(off) value) and a 165 and 1500-fold increase in the Kd value of the binding of fVIII fragment A3-C1-C2 to vWf compared to that of LCh and fVIII, respectively. In contrast, the binding affinity of A3-C1-C2 for PS-containing membranes was 8-11-fold higher than that of LCh. Possible conformational change(s) in C2 domain upon removal of the acidic region were studied using anti-fVIII monoclonal antibody NMC-VIII/5 with an epitope within the C2 domain of LCh as a probe. The determined lower binding affinity of A3-C1-C2 for NMC-VIII/5 immobilized to a sensor chip than that of LCh, indicates that these conformational changes do occur.

Cultured dermal papilla cells of the rat vibrissa follicle. Proliferative activity, adhesion properties and reorganization of the extracellular matrix in vitro.

The dermal papilla of the mammalian hair follicle plays an important role in regulating and controlling the hair cycle. Distinct functional stages of dermal papilla cells (DPC) are involved in this process, thus suggesting that the dermal papilla is a highly specialized suborgan of the pilosebaceous unit. The aim of the present study was to investigate the functional properties of cultured DPC in various assays and to compare their functional properties with those of dermal fibroblasts (DFB). In monolayer cell cultures DPC showed an aggregative growth pattern, different to that of DFB, and lower proliferation rates, as compared to the controls. Adhesion assays performed using a 51[Cr]labeling method showed strong adhesion of both cell populations to collagen types I and IV, fibronectin and laminin, but DPC in vitro showed significantly higher adhesiveness to collagen type IV, a major component of the basement membrane of dermal papillae in vivo. The capacity of DPC to reorganize extracellular matrix components, as measured by gel contraction with three-dimensional collagen type I lattices, proved to be significantly lower than that of DFB and, moreover, DPC lysed the collagen lattices completely after 48 h in culture. The functional differences between DPC and DFB were paralleled by higher surface expression and synthesis levels of the beta 1, alpha 1, and alpha 5 chains of integrin adhesion receptors in DPC, as detected by fluorescence-activated cell-sorter analysis and radioimmunoprecipitation. These findings provide evidence that DPC are a highly specialized cell population, which clearly differs from another mesenchymal cell type, DFB. After their isolation and cultivation in vitro, DPC still preserve functional properties related to important steps of cell-matrix interaction involved in the hair cycle.

Enzymatic quantification of cell-matrix and cell-cell adhesion.

Adhesion assays are powerful tools to investigate the adhesive properties of cells. The quantification of cell adhesion enables determination of the capacity of cells to stick to a target, screening for novel adhesion involved binding molecules, exploration of structure-function relationships of adhesion molecules, evaluation of adhesion targets, and examination of compounds interfering with cell adhesion. Thus, quantification of cell adhesion needs simple and reliable methods that might be applied for both research and diagnostic purposes. This review presents methodological principles of enzymatic approaches for quantification of cell adhesion. In particular, the advantages of exogenous cell labelling with horseradish peroxidase are described.

Analysis of protein aggregates by combination of cross-linking reactions and chromatographic separations.

Chemical cross-linking provides a method that covalently bridges near-neighbour associations within proteins and protein aggregates. Combined with chromatographic separations and protein-chemical methods, it may be used to localize and to investigate three-dimensional relations as present under natural conditions. This paper reviews the chemistry and application of cross-linking reagents and the development of combination experimental approaches in view of chromatographic separations and cross-linking reactions. Investigations of homooligomeric and heterooligomeric protein associations as well as conformational analysis are presented.

The cysteine-rich region of dipeptidyl peptidase IV (CD 26) is the collagen-binding site.

A remarkable property of the integral glycoprotein dipeptidyl peptidase IV (DPP IV, CD 26) is its affinity to proteins of the extracellular matrix (ECM). By in vitro binding assays we have shown that DPP IV binds to collagens; preferentially to the collagens I and III, which are both characterized by the formation of large triplehelical domains. No binding of DPP IV to laminin or fibronectin could be observed. Within collagen I, the alpha 1(I) chain was found to be the most prominent binding ligand of DPP IV. A monoclonal anti DPP IV antibody (13.4) specifically inhibited the interaction of DPP IV with collagen I. Peptide mapping and N-terminal sequencing revealed that the corresponding epitope of mAb 13.4 is located in the cysteine-rich domain of DPP IV. We therefore conclude that the putative collagen binding site of DPP IV is different from the region of the catalytic site containing the exopeptidase activity, which is located at the C-terminal portion of the molecule.

Localization of integrin beta 1, alpha 1, alpha 5 and alpha 9 subunits in the rat testis.

Very late activation (VLA, beta 1; alpha 1; alpha 5, alpha 9) integrins were studied by immunoblotting and immunohistochemistry in the testes of sexually mature rats. All integrin subunits were present in membrane fractions of homogenized testes. Immunohistochemistry revealed that the anti beta 1 antibody recognized peritubular cells and the basement membrane of blood vessels. Immunoreactivity was also demonstrated in the lamina propria, basement membrane, and the basal cytoplasm of Sertoli cells. In elongating spermatids, beta 1 integrin was localized to the acrosome. The alpha 1 subunit was expressed in peritubular cells and in the lamina propria. In the adluminal compartment, round spermatids were stained diffusely for the alpha 1 subunit. Immunoreactivity for alpha 1 integrin was found additionally in the acrosomes of elongating spermatids shortly before their release into the seminiferous tubule lumen. The alpha 5 subunit was expressed in the acrosomes of elongating spermatids as well as in their distal cytoplasm during stages III-VI; the cytoplasmic lobes of elongate spermatids and/or residual bodies also appeared to be immunostained in seminiferous tubules at stages VII-VIII. The alpha 9 subunit was immunolocalized only in the basement membrane and in peritubular cells. These data suggest that integrins are involved in spermatogenesis, in particular in the process of spermatid maturation.

Chemical cross-linking detects different conformational arrangements of platelet integrin alpha IIb beta III (gpIIb/IIIa).

Treatment of Triton X-100 solubilized platelet membrane with the homobifunctional cross-linker dithiobis(succinimidyl propionate) resulted in covalent cross-linking of the platelet integrin alpha IIb beta 3 (gpIIb/IIIa), the fibrinogen receptor, into three high-molecular-mass complexes with apparent M of 200, 220 and 240 k. Generation of these cross-linked alpha IIb beta 3 aggregates depended on the presence of a native receptor structure and was not influenced by Ca2+ ions and other experimental conditions. Immunoblotting analysis of purified 200/220/240 kDa aggregates revealed that they were made up exclusively by alpha IIb and beta 3 integrin chains in roughly stoichiometric amounts. We therefore conclude that alpha IIb beta 3 integrin occurs in different conformations in the platelet membrane that can be directly detected by chemical cross-linking.

Distribution and quantification of alpha 1-integrin subunit in rat organs.

The alpha 1 beta 1-integrin is known to be a receptor for collagen and laminin mediating cell-matrix interactions. A monoclonal antibody, 33.4, which specifically inhibits the alpha 1-integrin-mediated in vitro cell-collagen binding of rat hepatocytes and hepatoma-derived A-cells (Löster et al., 1994), was used to purify by immunoaffinity chromatography the alpha 1-integrin subunit from rat liver in large quantities for inducing a polyclonal antiserum. In immunoblot analysis on membrane extracts of several rat organs this polyclonal antiserum recognized only a 190 kDa-band, suggesting that it is highly specific for the alpha 1-integrin subunit. A sandwich-ELISA with monoclonal antibody 33.4 and the polyclonal antiserum against the alpha 1-integrin subunit, respectively, enabled the quantitative expression pattern of the alpha 1-integrin subunit to be studied in different rat organs. With the exceptions of brain (not detectable) and muscle (low concentration), the alpha 1-integrin subunit was detectable in almost all organs of the digestive, respiratory and urogenital system as well as in lymphatic organs. The highest relative concentrations of alpha 1-integrin subunit were found in uterus, lung and spleen, whereas in seminal vesicle, stomach, parotid gland, epididymis, kidney and liver only modest concentrations were evident. The organ distribution and localization of alpha 1-integrin subunit were studied by immunohistochemistry with monoclonal and polyclonal antibodies. Immunoreactivity was present in the plasma membranes of all smooth muscle cells, vascular endothelial cells of many organs and fibrocyte-fibroblast sheaths in the heart and kidney. Since these cells are in close contact with collagen-containing basal membranes as well as reticular fibrils, strong evidence exists that in rat tissues the alpha 1-integrin subunit is expressed at sites where collagen is present and might be involved in vivo in cell-collagen binding.

Cell-collagen adhesion is inhibited by monoclonal antibody 33.4 against the rat alpha 1-integrin subunit.

A monoclonal antibody (mAb 33.4) is described which inhibits the adhesion and spreading of an adherent cell line (A-cells), established from Morris hepatoma 7777 and isolated hepatocytes on collagen IV but not on laminin, fibronectin, and vitronectin. mAb 33.4 retains its immunological activity after immobilization and covalent cross-linking to Protein G-Sepharose and is therefore a suitable tool for the preparative equimolar purification of two proteins with M(r) of 130 and 190 kDa from detergent-solubilized membrane fractions by immunoaffinity chromatography. The 190-kDa protein was identified as rat alpha 1-integrin subunit by N-terminal amino acid sequencing, while the 130-kDa protein was specifically stained by a beta 1-integrin subunit-specific antiserum. In immunoblot analysis mAb 33.4 recognized the 190-kDa band, suggesting that it is specific for the rat alpha 1-integrin subunit. The epitope recognized by mAb 33.4 is conformation-dependent because the staining in immunoblots was very strong if the SDS-PAGE was performed in the absence of reducing agents. The expression of alpha 1 beta 1-integrin in sinusoidal hepatocyte membrane domains of liver sections is shown by mAb 33.4 and antisera raised against the rat alpha 1- and beta 1-integrin subunits.

Purification of monoclonal antibodies by hydroxylapatite HPLC and size exclusion HPLC.

Monoclonal antibodies of both the IgG and the IgM type were purified by hydroxylapatite HPLC (HA-HPLC) under very mild conditions. The IgM type antibodies, which were isolated from ascites fluid and separated from other proteins also by means of size exclusion HPLC. It was shown that the most frequently observed disadvantage of HA-HPLC, that is the relative short life of the columns (P. Steffen (1989) GIT Fachz. Lab., Suppl. 3/89 (Chromatogr.), 50-90), is due to microbial contamination rather than lower mechanical stability. In order to monitor column performance, a test was developed based on the use of standard proteins under isocratic separation conditions. This allows a direct comparison between the respective performances of columns made from different materials, hydroxylapatite or fluoroapatite, from different sources and with different particle sizes. A problem which often occurs with HA-HPLC in the case of IgM antibody isolation, namely precipitation of the antibodies at low salt concentrations at the beginning of a chromatographic run, was avoided by adding sodium chloride to both separation buffers.

High-performance membrane chromatography of serum and plasma membrane proteins.

Porous discs made of poly(glycidyl methacrylate) were used for high-performance membrane chromatography (HPMC) of proteins. In model experiments, separations of standard proteins by anion-exchange HPMC using a DEAE disc were carried out. The influences of sample distribution and disc diameter and thickness on separation performance were studied. The separation disc allowed a scaling-up from analytical (diameter 10 mm) to semi-preparative (diameter 50 mm) dimensions. In an application study, separations with anion-exchange and affinity HPMC were carried out using different complex samples such as rat serum and plasma membrane proteins. In all experiments the results on poly(glycidyl methacrylate) discs were comparable to those achieved on adequate high-performance liquid chromatographic (HPLC) columns. However, the separations on HPMC discs could be carried out faster than corresponding separations on HPLC columns. The pressure drop on the discs was low even at high flow-rates. The experiments show that the poly(glycidyl methacrylate) discs used are especially suitable for the isolation of proteins and other biopolymers which occur in a diluted state in complex mixtures.

Immunenzymometric assay for the heart specific glycogen phosphorylase BB in human serum using monoclonal antibodies.

An enzyme-linked immunosorbent assay for the determination of the human glycogen phosphorylase isoenzyme BB (GP BB) using two murine monoclonal antibodies was developed. A series of hybridoma clones producing monoclonal antibodies to GP BB were obtained by the standard lymphocyte hybridoma technique. Two of the selected clones synthesizing monoclonal antibodies, which recognize different epitopes, were employed for the immunenzymometric assay. The first monoclonal antibody was immobilized on microtiter plates and the bound glycogen phosphorylase BB was detected with the second monoclonal antibody conjugated with horse radish peroxidase. This assay enables a specific and sensitive measurement of GP BB in the range of 0.5-150 ng/ml phosphate-buffered saline containing 0.5% bovine serum albumin in less than 3 hours. The lower limit in human serum amounts about 3 ng/ml. Preliminary data obtained with human sera from patients after aorto-coronary artery bypass surgery are demonstrated.

Transforming growth factor-beta (TGF-beta) stimulates the expression of beta1 integrins and adhesion by rat mesangial cells.

Transforming growth factor-beta (TGF-beta) has been implicated in the pathogenesis of mesangial matrix (MM) accumulation in human and experimental glomerulonephritis. To clarify molecular mechanisms responsible for pathological MM deposition, we examined the effect of TGF-beta on the production of beta1 integrins and on adhesion function of rat mesangial cells (MC). In immunoprecipitation experiments using [35S]methionine-labeled MC, stimulation of MC with TGF-beta for 48 h resulted in an increase in the synthesis of alpha1beta1 (collagen/laminin receptor) and alpha5beta1 (fibronectin receptor) integrins accompanied by increases in the synthesis of their ligands, collagen type I (collagen I), and fibronectin. A time-dependent increase in beta1, alpha1 integrin subunit mRNA peaking 48 h after exposure to TGF-beta was shown by Northern blot analysis. After 48 h of treatment with TGF-beta, MC displayed significant increases in adhesion to fibronectin, collagen I, and laminin as compared to untreated MC. Anti-beta1 antiserum significantly inhibits MC adhesion to fibronectin, collagen I, and laminin. Anti-alpha1 subunit antibody very strongly inhibited adhesion to collagen I and laminin, but not to fibronectin. Synthetic peptides containing RGD sequences specifically blocked adhesion to fibronectin. These data suggest that TGF-beta may promote MM deposition by increasing MC synthesis of both matrix proteins and beta1 integrins which facilitate binding of these proteins to the MC surface and thus enhance their incorporation into MM.

Quantification of cell-matrix and cell-cell adhesion using horseradish peroxidase.

A simple, universal, and rapid enzymatic method for the quantitative determination of cell adhesion in 96-well cell culture plates has been established. The assay is based on cellular steady-state endocytosis, which is used to label cells with horseradish peroxidase (HRP) prior to adhesion. Subsequently, attached cells can be detected by a simple enzymatic reaction, in which the accumulated HRP catalyzes dye formation from a colorless hydrogen donor, e.g., o-phenylenediamine, in the presence of hydrogen peroxide. As demonstrated with different cell lines and test systems, the method can be used to quantify cell-matrix as well as cell-cell interactions and allows a very sensitive quantification of adherent cells. The HRP label is nontoxic and does not affect the adhesion properties of tested cell lines; the quantity of dye formed is proportional to the number of adherent cells. Furthermore, the assay represents an alternative method to isotopic cell labeling, e.g., with 51Cr, which is usually used for quantifying cell-cell interactions.