Development and evaluation of an ELISA for quantification of human alpha-1-proteinase inhibitor in complex biological mixtures

Human alpha-1-proteinase inhibitor(1) (alpha(1)-PI) is the most abundant serine protease inhibitor in plasma. Its major function is inhibition of neutrophil elastase in lungs. alpha(1)-PI deficiency may result in severe, ultimately fatal emphysema. Three plasma-derived (pd-) alpha(1)-PI products are licensed in the US for replacement therapy of deficient patients. The recombinant versions (r-alpha(1)-PI), proposed as alternatives to pd-alpha(1)-PI products, have been under intensive investigation. For accurate determination of alpha(1)-PI from different sources and in various forms, there is an obvious need for reliable standardized assays for alpha(1)-PI quantification and potency measurements. As a part of our multi-step research focused on alpha(1)-PI structure-function investigation, we have established a simple and reproducible double-sandwich ELISA based on commercially available polyclonal antibodies. The developed ELISA allows the quantification of both pd-alpha(1)-PI and r-alpha(1)-PI in various complex matrices. A validation of the ELISA was performed with the working range of the assay (3.1-50 ng/ml) established on the bases of the following parameters: linearity (3-100 ng/ml, r(2)=0.995); accuracy (87.3-114.6% recovery); intra-assay precision (%CV, 2.8%); inter-assay plate-to-plate precision (3.9% per day and 4.1% day-to-day); detection limit (1.10 ng/ml); and quantification limit (3.34 ng/ml). The analytical performance of the alpha(1)-PI ELISA indicates that this assay can be used for monitoring concentration levels of alpha(1)-PI in multi-component biological matrices, based on the following: (a) quantification of r-alpha(1)-PI in various fermentation mixtures (E. coli and A. niger); (b) investigation of alpha(1)-PI enzymatically digested in the conditions of harsh fungal proteolysis; (c) evaluation of thermally polymerized alpha(1)-PI; (d) quantification of alpha(1)-PI in human serum; and (e) comparative quantification of alpha(1)-PI in commercially available products.     

Expression screening of bacterial libraries of recombinant alpha-1 proteinase inhibitor variants for candidates with thrombin inhibitory capacity

Exhaustive mutagenesis studies of the reactive centre loop (RCL), a key structural component of proteins belonging to the serpin superfamily of protease inhibitors, are complicated by the size of the RCL, serpin conformational complexity, and, for most serpins, the lack of a serpin-dependent phenotype of expressing cells. Here, we describe a thrombin capture assay that distinguished thrombin-inhibitory recombinant human alpha-1 proteinase inhibitor (API M358R) from non-inhibitory API variants in Escherichia coli lysates prepared from either single clones or pools. Binding of API proteins in the lysates to thrombin immobilized on microtiter plate wells was quantified via colour generated by a peroxidase-coupled anti-API antibody. Bacterial expression plasmids encoding inhibitory API M358R were mixed 1:99 with plasmids encoding non-inhibitory API T345R/M358R and the resulting library screened in pools of 10. All above-background signals arising from pools or subsequently re-probed single clones were linked to the presence of plasmids encoding API M358R. Screening of a portion of another expression library encoding hypervariable API with all possibilities at codons 352–358 also yielded only novel, thrombin-inhibitory variants. Probing a smaller library expressing all possible codons at Ala347 yielded the wild type, 6 different functional variants, one partially active variant, and two variants with no thrombin-inhibitory activity. API antigen levels varied considerably less among Ala347 variants than activity levels, and comparison of rate constants of inhibition of purified API variants to their corresponding thrombin capture assay lysate values was used to establish the sensitivity and specificity of the assay. The results indicate that the approach is sufficiently robust to correctly identify functional versus non-functional candidates in API expression libraries, and could be of value in systematically probing structure/function relationships not only in the API RCL, but also in that of other serpins.     

Identification of alpha-1-proteinase inhibitor-containing cells in pancreatic islets

Summary By a regular immunoperoxidase method, alpha-1-proteinase inhibitor (Api) was demonstrated in pancreatic islets in individuals with and without genetic deficiency of Api. Subsequently a double immunoperoxidase method, with two different chromogens (diaminobenzidine-brown and 4-chloro-1-naphthol-blue), was applied on the same tissue section in order to identify cells containing Api and cells secreting polypeptide hormones. Api-positive cells and hormone-secreting cells were found to be mutually exclusive indicating that Api is synthesized by previously unrecognized islet cell. The population of Api cells was significantly higher in persons with genetic deficiency than in other individuals, implying a possible compensatory hyperplasia of those cells triggered by a low level of circulating Api.     

The molecular stoichiometry of trypsin inhibition by human alpha-1-proteinase inhibitor

The stoichiometry of interaction of human alpha-1-proteinase inhibitor with porcine trypsin has been determined using a highly purified preparation of inhibitor. In contrast to the reports of others, one mole of alpha-1-proteinase inhibitor was found to inhibit two moles of trypsin. Disc gel electrophoresis indicates that the 2:1 complex is preferentially formed even when free alpha-1-proteinase inhibitor is still present.     

Heme binding to human alpha-1 proteinase inhibitor

Background

Heme is a unique prosthetic group of various hemoproteins that perform diverse biological functions; however, in its free form heme is intrinsically toxic in vivo. Due to its potential toxicity, heme binding to plasma proteins is an important safety issue in regard to protein therapeutics derived from human blood. While heme binding by hemopexin, albumin and α₁-microglobulin has been extensively studied, the role of other plasma proteins remains largely unknown.

Methods

We examined two acute-phase plasma proteins, haptoglobin (Hp) and alpha-1 proteinase inhibitor (α₁-PI) for possible interactions with heme and bilirubin (BR), the final product of heme degradation, using various techniques: UV/Vis spectroscopy, fluorescence, circular dichroism (CD), and surface plasmon resonance (SPR).

Results

According to our data, Hp exhibits a very weak association with both heme and BR; α₁-PI's affinity to BR is also very low. However, α₁-PI's affinity to heme (K_D 2.0 × 10^− 8 M) is of the same order of magnitude as that of albumin (1.26 × 10^− 8 M). The data for α₁-PI binding with protoporphyrin IX (PPIX) suggest that the elimination of the iron atom from the porphyrin structure results in almost 350-fold lower affinity (K_D 6.93 × 10^− 6 M), thus indicating that iron is essential for the heme coordination with the α₁-PI.

Conclusions

This work demonstrates for the first time that human α₁-PI is a heme binding protein with an affinity to heme comparable to that of albumin.

General significance

Our data may have important implications for safety and efficacy of plasma protein therapeutics.     

Inactivation of alpha-1-proteinase inhibitor in serum by stimulated human polymorphonuclear leucocytes. Evidence for a myeloperoxidase-dependent mechanism

Triggered polymorphonuclear leucocytes (PMNL) can decrease the elastase inhibitory capacity of serum by inactivating the main inhibitor of elastase alpha-1-proteinase inhibitor (alpha-1-PI). Maximal inactivation occurs with stimuli that release myeloperoxidase from PMNL along with hydrogen peroxide. Specific protection of alpha-1-PI function is obtained with antioxidants that interfere with this system. PMNL that are activated with phorbol myristate acetate release hydrogen peroxide but not myeloperoxidase, and only inactivate alpha-1-PI in the presence of exogenously-added PMNL-derived supernatants which contain this enzyme. Cell-free inactivation requires both active enzyme and hydrogen peroxide, and is greatest at pH 6.2, the pH optimum for myeloperoxidase-catalysed inactivation of alpha-1-PI. This data supports the notion that leucocyte myeloperoxidase may act to suppress the antiprotease screen afforded by alpha-1-PI by generating hypochlorous acid in the presence of chloride and respiratory burst-derived hydrogen peroxide, and in the microenvironment of lowered pH associated with degranulation. Pulmonary emphysema seems to be associated with an imbalance between elastase and its inhibitors at the lung surface. PMNL are likely to play an important role in the pathogenesis of emphysema since they contain both elastase, which can solubilize connective tissue elastin, and the constituents of an oxidative system which can inactivate the most important antielastase, alpha-1-PI.     

Characterization of two cDNA clones encoding isozymes of the F1F0-ATPase inhibitor protein of rice mitochondria

Two cDNA clones encoding F₁F₀-ATPase inhibitor proteins, which are loosely associated with the F₁ part of the mitochondrial F₁F₀-ATPase, were characterized from rice (Oryza sativa L. cv. Nipponbare). A Northern hybridization showed that the two genes (designated as IF ₁ -1 and IF ₁ -2) are transcribed in all the organs examined. However, the steady-state mRNA levels varied among organs. A comparison of the deduced amino acid sequences of the two IF ₁ genes and the amino acid sequence of the mature IF₁ protein from potato revealed that IF₁-1 and IF₁-2 have N-terminal extensions with features that are characteristic of a mitochondrial targeting signal. To determine the subcellular localization of the gene products, the IF₁-1 or IF₁-2 proteins were fused in frame to the green fluorescent protein (GFP) or the fused GFP-β-glucuronidase, and expressed transiently in onion or dayflower epidermal cells. Localized fluorescence was detected in mitochondria, confirming that the two IF₁ proteins are targeted to mitochondria. Received: 9 July 1999 / Accepted: 17 August 1999     

Antizyme and antizyme inhibitor activities influence cellular responses to polyamine analogs

Summary. Close structural analogs of spermidine and spermine, polyamine mimetics, are potential chemotheraputic agents as they depress cellular polyamines required for tumor growth. Specific mimetic analogs stimulate synthesis of the regulatory protein antizyme (AZ), which not only inactivates the initial enzyme in polyamine biosynthesis but also inhibits cellular uptake of polyamines. The role of AZ induction in influencing cellular uptake of representative analogs was investigated using three analogs produced by Cellgate Inc., CGC-11047, CGC-11102, and CGC-11144, which exhibit markedly distinct AZ-inducing potential. An inverse correlation was noted between the AZ-inducing activity of a compound and the steady-state levels accumulated in cells. As some tumor cells over express AZI as a means of enhancing the polyamines required for aggressive growth, analog sensitivity was examined in transgenic CHO cells expressing exogenous antizyme inhibitor protein (AZI). Although AZI over expression increased cell sensitivity to analogs, the degree of this affect varied with the analog used.     

Immunohistochemical demonstration of proteinase inhibitor alpha-1-antichymotrypsin in normal human central nervous system

The presence of alpha-1-antichymotrypsin, a serine proteinase inhibitor with a high affinity for cathepsin G, is demonstrated in the normal human central nervous system (CNS) by immunohistochemical techniques. Paraffin-embedded normal human CNS tissue from five adult, two fetal, one neonatal and three newborn autopsies were stained with monospecific rabbit antibodies to human alpha-1-antichymotrypsin using biotinylated goat anti-rabbit antibodies and an avidinbiotin-peroxidase complex. Positive immunostaining was seen in neurons and glial cells in the cerebral cortex, basal ganglia, hippocampus, cerebellum, brainstem, and spinal cord of the adults. The epithelium of the adult choroid plexus had the most intense staining in apical granular organelles corresponding in position to lysosomes or secretory granules. Ependymal cells, particularly those near the choroid plexus, were immunostained. The fetal CNS had no alpha-1-antichymotrypsin staining. Limited staining of choroid plexus, ependyma, and frontal lobe was found in the newborns. Immunostaining in the neonatal temporal lobe was only found in the choroid-plexus epithelium. These observations establish a widespread distribution of this proteinase inhibitor in the normal human CNS. Developmental regulation of this inhibitor in the human CNS is also indicated.     

Recombinant therapeutic proteins: production platforms and challenges

Since the approval of insulin in 1982, more than 120 recombinant drug substances have been approved and become available as extremely valuable therapeutic options. Exact copying of the most common human form is no longer a value per se, as challenges, primarily related to the pharmacokinetics of artificial recombinant drugs, can be overcome by diverging from the original. However, relatively minor changes in manufacturing or packaging may impact safety of therapeutic proteins. A major achievement is the development of recombinant proteins capable of entering a cell. Such drugs open up completely new opportunities by targeting intracellular mechanisms or by substituting intracellularly operating enzymes. Concerns that protein variants would cause an intolerable immune response turned out to be exaggerated. Although most recombinant drugs provoke some immune response, they are still well tolerated. This knowledge might result in a change in attitude towards antibody formation, i.e., neutralizing antibody activity (in vitro) may be overcome by dosing consistently on the basis of antibody titers and not only on body weight. As with other drugs, efficacy and safety of therapeutic proteins have to be demonstrated in clinical studies, and superiority over available products has to be proven instead of just claimed.     

Cytokine regulatory effects on alpha-1 proteinase inhibitor expression in NOD mouse islet endothelial cells

Human microvascular islet endothelial cells (IEC) exhibit specific morphological and functional characteristics that differ from endothelia derived from other organs. One of these characteristics is the expression of alpha-1 proteinase inhibitor (Api). In this study, we observed its expression in nonobese diabetic (NOD) mouse IEC, in relation to the occurrence of type 1 diabetes and in response to cytokines, namely IL-1 beta and IL-10. In addition, IL-10-deficient NOD mice as well as IL-10 transgenic NODs were studied. Results have demonstrated that Api expression is: (i) highly specific for IEC in NOD mouse islets, as for humans; (ii) linked to the occurrence of early type 1 diabetes, and iii) strongly modulated by Th1 and Th2 cytokines. In fact, Api mRNA found in pre-diabetic NOD animals is significantly reduced when they become hyperglycemic and disappears by 25 weeks of age, when mice are diabetic. Moreover, Api mRNAs are never seen in nondiabetic controls. Furthermore, in cultured NOD IEC, Api expression is downregulated by the addition of IL-1 beta and is upregulated by IL-10; it is always absent in IL-10-deficient NOD mice and overexpressed in IL-10 transgenic NODs, thus further supporting that this cytokine upregulates Api expression.     

Phenylalanine ammonia-lyase modified with polyethylene glycol: Potential therapeutic agent for phenylketonuria

Summary. Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by the defects in the phenylalanine hydroxylase (PAH) gene. Individuals homozygous for defective PAH alleles show elevated levels of systemic phenylalanine and should be under strict dietary control to reduce the risk of neuronal damage associated with high levels of plasma phenylalanine. Researchers predict that plant phenylalanine ammonia-lyase (PAL), which converts phenylalanine to nontoxic t-cinnamic acid, will be an effective therapeutic enzyme for the treatment of PKU. The problems of this potential enzyme therapy have been the low stability in the circulation and the antigenicity of the plant enzyme. Recombinant PAL originated from parsley (Petroselinum crispum) chemically conjugated with activated PEG₂ [2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine] showed greatly enhanced stability in the circulation and was effective in reducing the plasma concentration of phenylalanine in the circulation of mice. PEG-PAL conjugate will be an effective therapeutic enzyme for the treatment of PKU.     

Non-irradiation-derived reactive oxygen species (ROS) and cancer: therapeutic implications

Summary. Owing to their chemical reactivity, radicals have cytocidal properties. Destruction of cells by irradiation-induced radical formation is one of the most frequent interventions in cancer therapy. An alternative to irradiation-induced radical formation is in principle drug-induced formation of radicals, and the formation of toxic metabolites by enzyme catalysed reactions. Although these developments are currently still in their infancy, they nevertheless deserve consideration. There are now numerous examples known of conventional anti-cancer drugs that may at least in part exert cytotoxicity by induction of radical formation. Some drugs, such as arsenic trioxide and 2-methoxy-estradiol, were shown to induce programmed cell death due to radical formation. Enzyme-catalysed radical formation has the advantage that cytotoxic products are produced continuously over an extended period of time in the vicinity of tumour cells. Up to now the enzymatic formation of toxic metabolites has nearly exclusively been investigated using bovine serum amine oxidase (BSAO), and spermine as substrate. The metabolites of this reaction, hydrogen peroxide and aldehydes are cytotoxic. The combination of BSAO and spermine is not only able to prevent tumour cell growth, but prevents also tumour growth, particularly well if the enzyme has been conjugated with a biocompatible gel. Since the tumour cells release substrates of BSAO, the administration of spermine is not required. Combination with cytotoxic drugs, and elevation of temperature improves the cytocidal effect of spermine metabolites. The fact that multidrug resistant cells are more sensitive to spermine metabolites than their wild type counterparts makes this new approach especially attractive, since the development of multidrug resistance is one of the major problems of conventional cancer therapy.     

Galanin as a modulator of anxiety and depression and a therapeutic target for affective disease

Summary. Galanin is a 29 amino-acid (30 in humans) neuropeptide with a close functional relationship with neurotransmitter systems implicated in the pathophysiology and treatment of depression and anxiety disorders. In rodent models of depression-related behavior, treatment with galanin or compounds with agonist actions at galanin receptors has been shown to affect depression-related behaviors and the behavioral and neurochemical effects of antidepressants. Treatment with clinically efficacious antidepressants alters galanin and galanin receptor gene expression in rodents. Rodent anxiety-like behaviors appear to be modulated by galanin in a complex manner, with studies showing either increases, decreases and no effects of galanin treatments and galanin mutations on anxiety-like behavior in various tasks. One concept to emerge from this literature is that galanin recruitment during extreme behavioral and physiological provocations such as stress and opiate withdrawal may serve to attenuate negative emotional states caused by noradrenergic hyperactivation. The specific galanin receptor subtypes mediating the anxiety- and depression-related effects of galanin remains to be determined, with evidence supporting a possible contribution of GalR1, GalR2 and GalR3. While our understanding of the role of galanin as a modulator of emotion remains at an early stage, recent progress in this rapidly evolving field raise possibility of that galanin may represent a target for the development of novel antidepressant and anxiolytic drug treatments.     

A study of the effects of altering the sites for N-glycosylation in alpha-1-proteinase inhibitor variants M and S.

alpha-1-Proteinase inhibitor (A1Pi) is a monomeric secreted protein glycosylated at asparagines 46, 83, and 247. For this study cDNAs for M (normal) and S (Glu264-->Val) variants of A1Pi were altered by site-directed mutagenesis to produce the combinations of single, double, and triple mutants that can be generated by changing the codons normally specifying these Asn residues to encode Gln. The fates of the mutant proteins were followed in transiently transfected COS-1 cells. All variants with altered glycosylation sites are secreted at reduced rates, are partially degraded, accumulate intracellularly, and some form Nonidet P-40-insoluble aggregates. The carbohydrate attached at Asn83 seems to be of particular importance to the export of both A1PiM and A1PiS from the endoplasmic reticulum. All mutations affecting glycosylation of A1PiS notably reduce secretion, cause formation of insoluble aggregates, and influence degradation of the altered proteins. The variant of A1PiS missing all three glycosylation sites is poorly secreted, is incompletely degraded, and accumulates in unusual perinuclear vesicles. These studies show that N-linked oligosaccharides in A1Pi are vital to its efficient export from the endoplasmic reticulum and that the consequences of changing the normal pattern of glycosylation vary depending upon the sites altered and the variant of A1Pi bearing these alterations.     

Hepatocyte uptake of alpha 1-proteinase inhibitor-trypsin complexes in vitro: evidence for a shared uptake mechanism for proteinase complexes of alpha 1-proteinase inhibitor and antithrombin III

In vivo clearance studies have indicated that the clearance of proteinase complexes of the homologous serine proteinase inhibitors alpha 1-proteinase inhibitor and antithrombin III occurs via a specific and saturable pathway located on hepatocytes. In vitro hepatocyte-uptake studies with antithrombin III-proteinase complexes confirmed the hepatocyte uptake and degradation of these complexes, and demonstrated the formation of a disulfide interchange product between the ligand and a cellular protein. We now report the results of in vitro hepatocyte uptake studies with alpha 1-proteinase inhibitor-trypsin complexes. Trypsin complexes of alpha 1-proteinase inhibitor were prepared and purified to homogeneity. Uptake of these complexes by hepatocytes was time and concentration-dependent. Competition experiments with alpha 1-proteinase inhibitor, alpha 1-proteinase inhibitor-trypsin, and antithrombin III-thrombin indicated that the proteinase complexes of these two inhibitors are recognized by the same uptake mechanism, whereas the native inhibitor is not. Uptake studies were performed at 37 degrees C with 125I-alpha 1-proteinase inhibitor-trypsin and analyzed by sodium dodecyl sulfate-gel electrophoresis in conjunction with autoradiography. These studies demonstrated time-dependent uptake and degradation of the ligand to low molecular weight peptides. In addition, there was a time-dependent accumulation of a high molecular weight complex of ligand and a cellular protein. This complex disappeared when gels were performed under reducing conditions. The sole cysteine residue in alpha 1-proteinase inhibitor was reduced and alkylated with iodoacetamide. Trypsin complexes of the modified inhibitor were prepared and purified to homogeneity. Uptake and degradation studies demonstrated no differences in the results obtained with this modified complex as compared to unmodified alpha 1-proteinase inhibitor-trypsin complex. In addition, the high molecular weight disulfide interchange product was still present on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized cells. Clearance and clearance competition studies with alpha 1-proteinase inhibitor-trypsin, alkylated alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin, and anti-thrombin III-factor IXa further demonstrated the shared hepatocyte uptake mechanism for all these complexes.