Differential binding properties of human pregnancy zone protein- and alpha2-macroglobulin-proteinase complexes to low-density lipoprotein receptor-related protein.

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein strongly related to alpha2-macroglobulin (alpha2-M). Both alpha-macroglobulins (alpha-Ms) covalently bind proteinases, which is accompanied by the exposure of carboxy terminal receptor recognition domains important for the rapid clearance from the circulation and tissues. It is accepted that the molecule responsible for the clearance of alpha2-M- and PZP-proteinase complexes is the low-density lipoprotein receptor-related protein (LRP). Although both alpha-M-proteinase complexes bind to the same receptor, differences in the binding properties have been reported. In addition, although it is known that the binding of alpha2-M-proteinase complexes to LRP can be blocked by Ni2+, the effect on PZP-proteinase has never been examined. In order to investigate differences in the binding properties of both alpha-Ms to the receptor, we purified LRP from human placenta by affinity chromatography and then analyzed the specificity and affinity of binding of alpha2-M- and PZP-proteinase complexes to the receptor by enzyme immunoassay. Our results clearly established that although both alpha-M-proteinase complexes specifically bind to LRP, PZP-chymotrypsin complexes bind to the receptor with lesser apparent affinity (Kd approximately equal 320 nM) than alpha2-M-chymotrypsin complexes (Kd approximately equal 40 nM). We also demonstrated that Ni2+ blocks the binding of alpha2-M-chymotrypsin complexes, but not PZP-chymotrypsin complexes, to LRP. These data suggest that the binding to LRP involves conformational differences between both alpha-Ms in a region immediately upstream of the carboxy terminal receptor recognition domain. The possibility that PZP-proteinase complexes interact with other receptors not available to alpha2-M-proteinase complexes could be considered.

Pregnancy zone protein-tissue-type plasminogen activator complexes bind to low-density lipoprotein receptor-related protein (LRP).

Tissue-type plasminogen activator (t-PA), is a serine proteinase that catalyzes the initial and rate-limiting step in the fibrinolytic cascade. Its plasma activity is determined by the rate of release into the bloodstream, the rate of inhibition by plasminogen-activator inhibitor type 1 (PAI-1) and the rate of hepatic clearance. Two receptor systems contribute to the clearance of t-PA: the mannose receptor and the low-density lipoprotein receptor-related protein (LRP) that removes free t-PA as well as t-PA-PAI-1 complexes from the blood. During pregnancy a significant rise in the plasma levels of pregnancy zone protein (PZP) is observed, while alpha(2)-macroglobulin (alpha(2)-M) remains constant. Interestingly, the fibrinolytic activity is decreased during this period. In this context, we have recently demonstrated the in vitro formation of PZP-t-PA complexes. Here, we purified LRP from human placenta by affinity chromatography and then analyzed the binding specificity and affinity of PZP-proteinase complexes to the receptor by enzyme immunoassay (EIA). Our results clearly established that the binding of PZP-t-PA complexes to LRP was specific, saturable, and with K(d) = 337 +/- 31 nM. Moreover, by using the same EIA, we further observed that this binding was inhibited by receptor-associated protein. These data suggest that PZP, by binding to t-PA and promoting its clearance via LRP, might contribute in vivo to the downregulation of the fibrinolytic activity during pregnancy.

Stabilization of homogeneous preparations of pregnancy zone protein lyophilized in the presence of saccharose. Structural and functional studies.

Human pregnancy zone protein (PZP) is a macromolecule of 360 kDa, organized as a disulfide-linked homodimer of two 180 kDa subunits, with an amino acid sequence and structure remarkably similar to that of human alpha2-Macroglobulin. Homogeneous PZP samples undergo fast aging forming oligomeric aggregates of high molecular weight. This aged PZP loses its ability to interact with proteinases and consequently, non-recognition of receptors occurs. In the present work, we assessed the effect of saccharose on the stability of native PZP on lyophilized samples kept for a long period of time. Herein, we demonstrate that the addition of 0.25 M saccharose to homogeneous PZP and further lyophilization is enough to prevent aging and preserve functional activity for more than 1 year. Hence, high quality samples, in terms of purity, stability and functional activity will allow to develop biochemical studies in order to know the PZP role in physiological and pathological states where the protein levels are increased, such as pregnancy and tumoral disorders.

Novel immunogenicity of Thomsen-Friedenreich disaccharide obtained by a molecular rotation on its carrier linkage.

The alpha-anomeric Galbeta1-3GalNAc, called Thomsen-Friedenreich disaccharide (TFD), is overexpressed in epithelial cancer cells by aberrant O-glycosylation. TFD is also the main ligand of Agaricus bisporus lectin (ABL), a reversible noncytotoxic inhibitor of proliferation of epithelial cell lines. In order to obtain anti-TFD antibody response with a fine carbohydrate-binding specificity similar to that of ABL, we designed an immunogen of TFD with a molecular rotation on its carrier linkage that exposes more GalNAc than Gal, since ABL recognizes GalNAc more than Gal in TFD. The synthesis was accomplished by C-6 oxidation of Gal from TFD or its alpha-benzyl derivative (BzlalphaTFD), followed by reductive amination between the C-6 aldehyde yielded and the available amine of protein. Mice immunized with TFD-KLH (keyhole limpet hemocyanin) or BzlalphaTFD-KLH produced antibodies which were then analyzed by ELISA against several target antigens. Both immunogens raised anti-KLH antibody titers; however, TFD-KLH did not raise anti-TFD antibodies showing low TFD immunogenicity. In contrast, BzlalphaTFD-KLH gave much higher anti-TFD antibody response, indicating that benzyl residue helps improve anti-carbohydrate immune response. When IgG and IgM anti-TFD antibodies were analyzed by competitive ELISA using TFD-related carbohydrates as inhibitors, a high specificity to TFD as well as an enhanced binding to GalNAc over Gal were observed. The axial C-4 hydroxyl group of GalNAc interacted with IgG anti-TFD antibody, as evidenced by the lack of inhibitory activity of GlcNAc in contrast to GalNAc. These findings indicate that the anti-TFD antibodies have fine carbohydrate-binding specificity more similar to ABL than to other TFD-binding proteins that stimulate proliferation of epithelial cell lines.

Structural requirements of carbohydrates to bind Agaricus bisporus lectin.

Galbeta1-3GalNAc (T-disaccharide) and related molecules were assayed to describe the structural requirements of carbohydrates to bind Agaricus bisporus lectin (ABL). Results provide insight into the most relevant regions of T-disaccharide involved in the binding of ABL. It was found that monosaccharides bind ABL weakly indicating a more extended carbohydrate-binding site as compared to those involvedin the T-disaccharide specific lectins such as jacalin and peanut agglutinin. Lacto-N-biose (Galbeta1-3GlcNAc) unlike T-disaccharide, is unable to inhibit the ABL interaction, thus showing the great importance of the position of the axial C-4 hydroxyl group of GalNAc in T-disaccharide. This finding could explain the inhibitory ability of Galbeta1-6GlcNAc and lactose because C-4 and C-3 hydroxyl groups of reducing Glc, respectively, occupy a similar position as reported by conformational analysis. From the comparison of different glycolipids bearing terminal T-disaccharide bound to different linkages, it can be seen than ABL binding is even more impaired by an adjacent C-6 residual position than by the anomeric influence of T-disaccharide. Furthermore, the addition of beta-GlcNAc to the terminal T-disaccharide in C-3 position of Gal does not affect the ABL binding whereas if an anionic group such as glucuronic acid is added to C-3, the binding is partially affected. These findings demonstrate that ABL holds a particular binding nature different from that of other T-disaccharide specific lectins.

Interaction of human tissue plasminogen activator (t-PA) with pregnancy zone protein: a comparative study with t-PA-alpha2-macroglobulin interaction.

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein strongly related to alpha2-macroglobulin (alpha2-M). Interactions of tissue plasminogen activator (t-PA) with PZP and alpha2-M were both investigated in vitro and the complexes were analyzed by polyacrylamide gel electrophoresis (PAGE). The results demonstrated that PZP-t-PA complex formation was evident within 1 h of incubation, whereas alpha2-M-t-PA complexes were formed after 18 h. Conclusions were supported by the following evidence: (i) PZP and alpha2-M complexes revealed changes of the mobility rate in non-denaturing PAGE, similar to those observed with alpha-Ms-chymotrypsin; (ii) both PZP and alpha2-M formed complexes of molecular size >360 kDa by SDS-PAGE, in accordance with the covalent binding of t-PA, which was previously reported for other proteinases; and (iii) PZP underwent a specific cleavage of the bait region with appearence of fragments of 85-90 kDa as judged by reducing SDS-PAGE. In contrast, the proteolytic attack on alpha2-M was found to occur more slowly, requiring several hours of incubation with t-PA for generation of an appreciable amount of fragments of 85-90 kDa. The appearance of free SH-groups of alpha-Ms was further investigated by titration with 5, 5'-dithiobis(2-nitrobenzoic acid). The maximal level of SH-groups raised was 3.9 mol/mol of PZP and 3.5 mol/mol of alpha2-M, indicating approximately one SH-group for each 180-kDa subunit. Finally, t-PA activity in PZP-t-PA complex was evaluated by measuring the hydrolysis of the chromogenic substrate Flavigen t-PA. Our results revealed that prolongation of the incubation period of this complex increased t-PA-mediated hydrolysis of Flavigen t-PA until a plateau was reached, approximately between 60 and 120 min. The present study suggests that PZP, by binding to t-PA, may contribute to the control of the activity of proteinases derived from fibrinolytic systems.

A procedure for human pregnancy zone protein (and human alpha 2-macroglobulin) purification using hydrophobic interaction chromatography on phenyl-sepharose CL-4B column.

In the present work we describe a procedure for the purification of human pregnancy zone protein (PZP) from pooled late pregnancy plasma by using hydrophobic interaction chromatography (HIC) on a phenyl-Sepharose column. The HIC step allowed the complete isolation of haptoglobins and the partial separation of human alpha 2-macroglobulin (alpha 2-M) from a protein fraction containing PZP previously obtained by a DEAE-Sephacel chromatography. Pure and native PZP, with a recovery of nearly 25% and biological activity of protease-binding, was obtained by two definitive final steps consisting of zinc-chelate and size-filtration chromatographies. Moreover, we further present an alternative procedure for the purification of alpha 2-M from the same pregnancy plasma, based on the differential elution of PZP and alpha 2-M from the HIC. This purification step gave rise to a highly purified product with a recovery of 10%. This differential elution could be explained by differences in surface hydrophobicity observed between both proteins. In addition, considering the different hydrophobic properties exhibited by native PZP and PZP-protease complexes, HIC on phenyl-Sepharose column could also be used for separating both conformational states of PZP.

Agaricus bisporus lectin binds mainly O-glycans but also N-glycans of human IgA subclasses.

The primary interaction between purified Agaricus bisporus lectin (ABL) and human IgA subclasses was studied by ABL-affinity chromatography, dot blot assay and competitive enzyme-lectin assay, considering that ABL could be an alternative tool for detection of IgA1 O-glycans. Both secretory IgA subclasses bound to ABL-Sepharose and the IgA2 subclass (which contains only N-glycans) was recovered with a high degree of purity when NH4OH was used as eluent. ABL-Ig interaction was also observed by dot blot assays using ABL-peroxidase against monoclonal IgA1 k Pan, IgA2m(1)k Gir, IgA2m(2)k Bel, secretory IgA2 and normal IgG (also contains only N-glycans). When these immunoglobulins were enzymatically treated with peptide N-glycosidase F (N-glycan hydrolysis), the ABL-IgA2 and -IgG interaction did not occur while IgA1 maintained a high degree of interaction with ABL. Also, the ABL-IgA interaction was observed by competitive enzyme-lectin assay, and when IgA1 subclass was treated with endo-alpha-N-acetylgalactosaminidase for O-glycans hydrolysis, the reactivity with ABL was very low. We conclude that the complementary use of ABL and peptide N-glycosidase F could be a useful tool to assess the O-glycosylation state of human IgA1 subclass, which is of relevant importance in the effector functions of immunoglobulins.

Inhibitory effects of human alpha 2-macroglobulin on Trypanosoma cruzi epimastigote proteinases.

The inactivation of Trypanosoma cruzi proteinases by human alpha 2-macroglobulin (alpha 2-M), a major plasma proteinase inhibitor was studied. Evidences regarding the interaction between alpha 2-M and proteolytic enzymes contained in crude cell-free extracts of T. cruzi were derived from electrophoretic and enzymatic assays. The former showed conformational and structural changes occurring in alpha 2-M, as judged by the appearance of transformed 'fast' form on native PAGE; generation of bands of approximately 90 kDa on reduced SDS-PAGE and formation of covalent complexes enzyme-inhibitor on SDS-PAGE. On the other hand, the total proteolytic activity on azocasein dropped significantly in the presence of alpha 2-M, although partial activity was still maintained. The proteinases detected as a double band of 44 and 53 kDa on gelatin SDS-PAGE were also inhibited by alpha 2-M. Results suggest that the study of specific interactions between alpha 2-M and T. cruzi-proteinases, probably with cruzipain, could be biologically important in the fate of T. cruzi-infection and Chagas' disease.

Differential reactivity of Agaricus bisporus lectin with human IgA subclasses in gel precipitation.

The interaction between purified Agaricus bisporus lectin and several human proteins was studied using the Ouchterlony double diffusion and immunoelectrophoresis techniques. Only one precipitation line was observed with normal human serum, normal human colostrum, IgA1 myeloma serum, both serum monoclonal and secretory IgA1 and monoclonal IgD. No reaction was observed with monoclonal and secretory IgA2, IgG, IgM, alpha 2 macroglobulin or pregnancy-associated alpha 2 glycoprotein. These results were confirmed by hemagglutination inhibition assays when IgA1, IgA2 and IgD were tested. On the basis of this reactivity, ABL could be a useful tool for distinguishing and isolating human IgA subclasses.

Parameters affecting the adsorption of ligands to polyvinyl chloride plates in enzyme immunoassays.

In the present work, we studied the efficacy of three blocking agents (HSA, BSA and OVA) in the inhibition of non-specific binding to PVC plates. According to the inhibition data, 1% OVA was the most effective blocking agent. On the other hand, the presence of detergents in all of the blocking solutions drastically decreased the percent inhibition of the non-specific binding. Furthermore, the effect of ligand concentration on adsorption and the kinetics of ligand adsorption to PVC plates were also investigated. Ligand adsorption is a linear function of input up to a limit (around 8.70 ng/mm2) where saturation is reached. The rate of adsorption of pure human IgG to PVC plates was proportionally increased with the temperature, as shown by proportional rate constants almost 2 times faster at 37 degrees C than at 4 degrees C. These results have practical implications for investigators using PVC for immunoassays and should be taken into consideration when designing such assays.

A novel functional assay of protein C in human plasma and its comparison with amidolytic and anticoagulant assays.

A simple and fast method for the quantitative determination of protein C activity in plasma is here described. The first step consists in the conversion of protein C in the test sample into activated protein C by means of an activator isolated from Southern Copperhead venom. Subsequently, the degradation of factor Va, in presence of protein C-deficient plasma, is measured by the prolongation of the prothrombin time which is proportional to the amount of protein C in the sample. The dose-response curve showed a linear relationship from 6 to 150% protein C activity and the inter- and intra-assay reproducibility was 3.5% and 5.6% respectively. In normal subjects, a mean of protein C level of 98 +/- 15% of normal pooled plasma was found. Comparison with the anticoagulant assay in samples of patients with oral anticoagulant, liver cirrhosis, disseminated intravascular coagulation and severe preeclampsia revealed an excellent correlation (r = 0.94, p less than 0.001). Also, a similar correlation (r = 0.93, p less than 0.001) existed between amidolytic assay and the method here proposed for all the samples studied without including the oral anticoagulant group. These results allowed us to infer that this method evaluates the ability of protein C to interact with protein S, phospholipids, calcium ions and factor Va.

Prothrombin time in the monitoring of oral anticoagulant: a comparison of results using different thromboplastins.

The objective of this report is to compare the prothrombin time performed with thromboplastin of different tissues and species in patients under oral anticoagulant therapy as well as the way of expressing the results. The results showed that the ISI of the thromboplastin of human and rabbit brain are very close to the IRP BCT/253 (1.2 vs 1.1) and RBT/79 (1.3 vs 1.4), respectively. In contrast, the rabbit lung thromboplastin showed the greatest differences in the ISI values (1.6 vs 1.4) and in the CV (6.1%). The authors found significant statistical differences with the results of the prothrombin time as expressed in activity percentage (p less than 0.001) in three plasma pools of patients under different oral anticoagulant level for all thromboplastins studied. However, if the results are expressed in terms of INR, the values obtained are almost the same. The results here reported would demonstrate that the prothrombin time as INR allows the use of only one scheme for oral anticoagulant control when the thromboplastin reagent is calibrated according to the recommendations of the WHO.

Serum pregnancy-associated alpha 2-glycoprotein levels in the evolution of hepatitis B virus infection.

Serum pregnancy-associated alpha 2-glycoprotein (alpha 2-PAG) levels were evaluated in a follow-up study of patients with hepatitis B virus (HBV) infection and compared with biochemical and virological parameters. In a study of 25 patients with acute hepatitis, an association was found between high alpha 2-PAG values, ALT levels, and HBsAg in 20 patients (80%) (P less than 0.05), 18 recovered completely, and 2 had a protracted course. In five patients serum alpha 2-PAG levels were similar to those in the control group. On the other hand, eight (100%) chronic persistent HBV patients showed high levels of alpha 2-PAG (P less than 0.05) during the study period, and these levels correlated well with inflammatory activity and failure of HBsAg elimination. There were no significant differences in alpha 2-PAG values between asymptomatic HBsAg carriers and controls. Serial analysis of alpha 2-PAG, in correlation with viral markers, biochemical parameters, and histological data, would contribute to the ability to predict the final outcome of HBV infection.

Serologic study by competitive enzyme-linked immunosorbent assay of pregnancy-associated alpha 2-glycoprotein in hepatitis B virus infection.

Serum levels of pregnancy-associated alpha 2-glycoprotein (alpha 2-PAG) in patients with hepatitis B virus (HBV) infection were compared with those of healthy male subjects used as controls by a competitive enzyme-linked immunosorbent assay (ELISA). Assay parameters were optimized, and minimal detectable concentration was 100 ng/ml. The alpha 2-PAG levels in 22/26 acute HBV patients showed a very significant statistical difference when compared with controls (x2 = 19.93, p less than 0.0005). On the other hand, 8/8 chronic persistent HBV patients showed high levels with a range between 51 to 200 ug/ml (x2 = 18.16, p less than 0.0005). There was no significant difference between asymptomatic HBsAg carriers and controls. Although alpha 2-PAG apparently exhibits immunosuppressive properties similar to other factors present in HBV infection, follow-up studies are needed to elucidate its role in the natural evolution of this disease.

Influence of specificity and affinity of antibodies on the kinetics of a sandwich enzyme immunoassay for human IgG.

In comparative experiments, we desired to determine the influence of specificity and affinity of various combinations of antibodies on the kinetics of a "sandwich" enzyme immunoassay for human IgG. Some antibodies were immobilized and others labeled with glucose oxidase. When antibodies employed were heterogeneous in specificity and affinity, long periods of time were required to reach equilibrium, especially on the second step of the assay. In contrast, when antibodies were separated into populations with specificity towards two different antigenic sites, and each one was used as immunoadsorbent and labeled, time was significantly reduced. Finally, when the assay took place--the last antibodies being selected by their high affinities--the rate of the reaction improved even more. We assume selection of antibodies by specificity and affinity by two antigenic sites as an essential requirement to improve performance of these assays, independently of the antigen and marker used.