Human Α1-proteinase Inhibitor Research Articles

Human α1-proteinase inhibitor (Respreeza®) in α1-antitrypsin deficiency emphysema: a profile of its use in the EU

Augmentation therapy with human α1-proteinase inhibitor (Respreeza®) is an effective and generally well tolerated treatment option for patients with severe α1-antitrypsin deficiency. In the 2-year RAPID trial, intravenous Respreeza 60 mg/kg once weekly was associated with a trend towards reduced mean annual rate of lung density loss (as measured by computed tomography) at total lung capacity (TLC) and functional residual capacity (FRC) combined, and at FRC alone, with a significant reduction seen at TLC alone. The continued efficacy of Respreeza in slowing the progression of emphysema was demonstrated in a 2-year open-label extension of the RAPID trial. Although lung density loss was slowed when treatment was initiated later in the disease course, lung density loss prior to treatment was not recovered.

Human α1-proteinase inhibitor (Respreeza®) in α1-antitrypsin deficiency emphysema: a guide to its use in the EU

Augmentation with intravenous infusions of human α1-proteinase inhibitor [Respreeza® (EU); Zemaira® (USA)] slows the progression of emphysema in patients with severe α1-antitrypsin (AAT)-deficiency emphysema. In the randomized, double-blind RAPID trial in patients with severe AAT-deficiency emphysema, Respreeza®/Zemaira® significantly reduced the mean annual rate of lung density loss based on computed tomography imaging at total lung capacity (TLC), but not at functional residual capacity (FRC) or combined TLC + FRC measurements. Respreeza®/Zemaira® is generally well tolerated, with a generally similar tolerability profile to that of placebo.

AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α1-Proteinase Inhibitor.

α1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based on biochemical data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found in WT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo polymerization showing a surprising diversity of polymer topography.

Single amino acid substitutions in recombinant plant-derived human α1-proteinase inhibitor confer enhanced stability and functional efficacy

BackgroundHuman α1-proteinase inhibitor (α1-PI) is the most abundant serine protease inhibitor in the blood and the heterologous expression of recombinant α1-PI has great potential for possible therapeutic applications. However, stability and functional efficacy of the recombinant protein expressed in alternate hosts are of major concern. MethodsFive variants of plant-expressed recombinant α1-PI protein were developed by incorporating single amino acid substitutions at specific sites, namely F51C, F51L, A70G, M358V and M374I. Purified recombinant α1-PI variants were analyzed for their expression, biological activity, oxidation-resistance, conformational and thermal stability by DAC-ELISA, porcine pancreatic elastase (PPE) inhibition assays, transverse urea gradient (TUG) gel electrophoresis, fluorescence spectroscopy and far-UV CD spectroscopy. ResultsUrea-induced unfolding of recombinant α1-PI variants revealed that the F51C mutation shifted the mid-point of transition from 1.4M to 4.3M, thus increasing the conformational stability close to the human plasma form, followed by F51L, A70G and M374I variants. The variants also exhibited enhanced stability for heat denaturation, and the size-reducing substitution at Phe51 slowed down the deactivation rate ~5-fold at 54°C. The M358V mutation at the active site of the protein did not significantly affect the conformational or thermal stability of the recombinant α1-PI but provided enhanced resistance to oxidative inactivation. ConclusionsOur results suggest that single amino acid substitutions resulted in improved stability and oxidation-resistance of the plant-derived recombinant α1-PI protein, without inflicting the inhibitory activity of the protein. General significanceOur results demonstrate the significance of engineered modifications in plant-derived recombinant α1-PI protein molecule for further therapeutic development.

Transgenic chickpea expressing a recombinant human α1-proteinase inhibitor (α1-PI) driven by a seed-specific promoters from the common bean Phaseolus vulgaris (L.)

The 5′ regulatory sequences of seed-storage protein genes, arcelin 5-I (Arc), phaseolin (Phas) of common bean (Phaseolus vulgaris L.) and legumin (Leg) gene of Cicer arietinum (L.) were evaluated for seed-specific expression of β-glucuronidase (uidA) and recombinant human α1-proteinase inhibitor (α1-PI) in transgenic chickpea. Histochemical assay of transformed plants developed with seed-specific promoters, showed GUS expression in seeds and not in other plant tissues. Fluorometric assay of β-glucuronidase revealed that phaseolin promoter with arcelin 5′ UTR (pPAG) resulted into maximum GUS activity in seeds, followed by somatic tissues and minimal expression in leaves. The expression profile of GUS driven by different seed-specific promoters in chickpea, are in order phaseolin > arcelin > legumin respectively. RT-PCR analysis confirmed that transcripts of β-glucuronidase and α1-PI genes, driven by the phaseolin promoter are stable in chickpea and their accumulation is confined into the seed tissues. Analysis of α1-PI expression in seeds of transgenic chickpea was performed by direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA), residual porcine pancreatic elastase activity assay and Western immunoblotting. Results of DAC-ELISA showed that modified α1-PI gene driven by the phaseolin promoter with arcelin 5′ UTR showed maximum accumulation of α1-PI up to 1.95 μg mg−1 of fresh weight in seeds. Biological activity of recombinant α1-PI, confirmed by elastase inhibition assays exhibiting 0.264 μg mg−1 of active protein. Western immunoblot analysis confirmed the molecular mass of plant-expressed recombinant α1-PI of molecular mass ~50 kDa in the seeds of transgenic chickpea.

Differential subcellular targeting of recombinant human α1-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants

The response of protein accumulation site on yield, biological activity and in planta stability of therapeutic recombinant human α1-proteinase inhibitor (α1-PI) was analyzed via targeting to different subcellular locations, like endoplasmic reticulum (ER), apoplast, vacuole and cytosol in leaves of transgenic tomato plants. In situ localization of the recombinant α1-PI protein in transgenic plant cells was monitored by immunohistochemical staining. Maximum accumulation of recombinant α1-PI in T0 and T1 transgenic tomato plants was achieved from 1.5 to 3.2% of total soluble protein (TSP) by retention in ER lumen, followed by vacuole and apoplast, whereas cytosolic targeting resulted into degradation of the protein. The plant-derived recombinant α1-PI showed biological activity for elastase inhibition, as monitored by residual porcine pancreatic elastase (PPE) activity assay and band-shift assay. Recombinant α1-PI was purified from transgenic tomato plants with high yield, homogeneity and biological activity. Purified protein appeared as a single band of ∼48–50kDa on SDS-PAGE with pI value ranging between 5.1 and 5.3. Results of mass spectrometry and optical spectroscopy of purified recombinant α1-PI revealed the structural integrity of the recombinant protein comparable to native serum α1-PI. Enzymatic deglycosylation and lectin-binding assays with the purified recombinant α1-PI showed compartment-specific N-glycosylation of the protein targeted to ER, apoplast and vacuole. Conformational studies based on urea-induced denaturation and circular dichroism (CD) spectroscopy revealed relatively lower stability of the recombinant α1-PI protein, compared to its serum counterpart. Pharmacokinetic evaluation of plant derived recombinant and human plasma-purified α1-PI in rat, by intravenous route, revealed significantly faster plasma clearance and lower area under curve (AUC) of recombinant protein. Our data suggested significance of protein sorting sequences and feasibility to use transgenic plants for the production of stable, glycosylated and biologically active recombinant α1-PI for further therapeutic applications.

Determination of site-specific glycan heterogeneity on glycoproteins

The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco)peptides are analyzed by capillary/nano-liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). If required, specific glycopeptide enrichment steps, such as hydrophilic interaction liquid chromatography (HILIC), can also be performed. Particular emphasis is placed on data interpretation and the determination of site-specific glycan heterogeneity. The described workflow takes approximately 3-5 d, including sample preparation and data analysis. The data obtained from analyzing released glycans of rHuEPO and IgG, described in the second protocol of this series (10.1038/nprot.2012.063), provide complementary detailed glycan structural information that facilitates characterization of the glycopeptides.

Effect of point mutations in translation initiation context on the expression of recombinant human α1-proteinase inhibitor in transgenic tomato plants

The functional and biological significance of translation initiation context sequence in determining high-level expression of modified synthetic human alpha(1)-proteinase inhibitor (alpha(1)-PI) gene was documented in stable transgenic tomato plants. Context sequence of initiator ATG codon derived from statistical analysis of databases was identified as taaA(A/C)aATGGCt in highly expressed dicot plant genes. Removal of initiator ATG context sequence reduced the expression of recombinant alpha(1)-PI protein to fourfolds. The mutation of consensus base at +4 position to a pyrimidine either alone or with substitution at -3 position eliminated most of the alpha(1)-PI expression, while mutation at -3 alone resulted in about sevenfold reduction. The presence of steady-state levels of alpha(1)-PI transcript in transgenic plants indicated that the variation in expression is entirely due to the point mutations incorporated in translation initiation context. These results indicated the significance of conserved nucleotide sequence around initiator ATG codon in augmenting post-transcriptional events and high-level expression of heterologous genes in transgenic plants.

Identification and Role of the Homodimerization Interface of the Glycosylphosphatidylinositol-anchored Membrane Type 6 Matrix Metalloproteinase (MMP25)

The membrane type (MT) 6 matrix metalloproteinase (MMP) (MMP25) is a glycosylphosphatidylinositol-anchored matrix metalloproteinase (MMP) that is highly expressed in leukocytes and in some cancer tissues. We previously showed that natural MT6-MMP is expressed on the cell surface as a major reduction-sensitive form of M(r) 120, likely representing enzyme homodimers held by disulfide bridges. Among the membrane type-MMPs, the stem region of MT6-MMP contains three cysteine residues at positions 530, 532, and 534 which may contribute to dimerization. A systematic site-directed mutagenesis study of the Cys residues in the stem region shows that Cys(532) is involved in MT6-MMP dimerization by forming an intermolecular disulfide bond. The mutagenesis data also suggest that Cys(530) and Cys(534) form an intramolecular disulfide bond. The experimental observations on cysteines were also investigated by computational studies of the stem peptide, which validate these proposals. Dimerization is not essential for transport of MT6-MMP to the cell surface, partitioning into lipid rafts or cleavage of alpha-1-proteinase inhibitor. However, monomeric forms of MT6-MMP exhibited enhanced autolysis and metalloprotease-dependent degradation. Collectively, these studies establish the stem region of MT6-MMP as the dimerization interface, an event whose outcome imparts protease stability to the protein.

Production, purification, and characterization of human α1 proteinase inhibitor from Aspergillus niger

Human alpha one proteinase inhibitor (alpha1-PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28 degrees C. Eight milligrams per liter of active alpha1-PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant alpha1-PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MS(n), and GC-MS. The MALDI and NSI- full MS spectra of permethylated N-glycans revealed that the N-glycans of both variants contain a series of high-mannose type glycans with 5-20 hexose units. Monosaccharide analysis showed that these were composed of N-acetylglucos-amine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non-reducing position. The Galactofuranose-containing high-mannnose type N-glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger.

Expression of human α1-proteinase inhibitor in Aspergillus niger

BackgroundHuman α1-proteinase inhibitor (α1-PI), also known as antitrypsin, is the most abundant serine protease inhibitor (serpin) in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, α1-PI is available for replacement therapy as an FDA licensed plasma-derived (pd) product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant α1-PI (r-α1-PI) could provide an attractive alternative. Although r-α1-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation.ResultsWe have explored the possibility of expressing the gene for human α1-PI in the filamentous fungus Aspergillus niger (A. niger), a system reported to be capable of providing more "mammalian-like" glycosylation patterns to secretable proteins than commonly used yeast hosts. Our expression strategy was based on fusion of α1-PI with a strongly expressed, secreted leader protein (glucoamylase G2), separated by dibasic processing site (N-V-I-S-K-R) that provides in vivo cleavage. SDS-PAGE, Western blot, ELISA, and α1-PI activity assays enabled us to select the transformant(s) secreting a biologically active glycosylated r-α1-PI with yields of up to 12 mg/L. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis further confirmed that molecular mass of the r-α1-PI was similar to that of the pd-α1-PI. In vitro stability of the r-α1-PI from A. niger was tested in comparison with pd-α1-PI reference and non-glycosylated human r-α1-PI from E. coli.ConclusionWe examined the suitability of the filamentous fungus A. niger for the expression of the human gene for α1-PI, a medium size glycoprotein of high therapeutic value. The heterologous expression of the human gene for α1-PI in A. niger was successfully achieved to produce the secreted mature human r-α1-PI in A. niger as a biologically active glycosylated protein with improved stability and with yields of up to 12 mg/L in shake-flask growth.

Production of human α1 proteinase inhibitor from Aspergillus niger

Production of human α1 proteinase inhibitor from Aspergillus niger

Reporter gene construct containing 1.4-kB alpha 1-proteinase inhibitor promoter confers expression in the cornea of transgenic mice.

A 1.4-kb human alpha1-proteinase inhibitor (alpha1-PI) 5'-flanking sequence fused to the E. coli LacZ gene was used to generate transgenic mice. The 1.4-kb alpha 1-PI fragment was found to target LacZ expression preferentially in the epithelium and stroma of the mouse cornea, and moderately or weakly in white blood cells and a few other tissues, such as the skin and brain. This finding implies that the alpha 1-PI promoter may offer an option for targeting foreign genes in both the epithelial and stromal layers of the cornea in future transgenic experiments.

Purification and N-Terminal Amino Acid Sequence of Sheep Neutrophil Cathepsin G and Elastase

Sheep cathepsin G (CG) and neutrophil elastase (NE) were isolated from a crude leukocyte membrane preparation by elastin-Sepharose 4B and CM-Sepharose 4B chromatography, followed by native preparative PAGE. The N-termini of CG and NE were sequenced to 24 and 20 residues, showing 96 and 85% identity with human CG and NE, respectively. During SDS–PAGE, sheep CG and NE migrated parallel to human CG and NE and have apparent molecular masses of 28 and 26 kDa, respectively. Following incubation of sheep CG and NE with human α1-antichymotrypsin and α1-proteinase inhibitor, complexes with apparent molecular masses of 89 and 81 kDa respectively were observed by SDS–PAGE. Polyclonal antibodies to human CG and NE cross-reacted with purified sheep CG and NE, respectively. These results indicate that sheep neutrophils contain CG and elastase that are analogous to human CG and NE in terms of molecular mass, reactivity with endogenous inhibitors, immunocross-reactivity, and N-terminal sequence.

Oxidation Regulates the Inflammatory Properties of the Murine S100 Protein S100A8

The myeloid cell-derived calcium-binding murine protein, S100A8, is secreted to act as a chemotactic factor at picomolar concentrations, stimulating recruitment of myeloid cells to inflammatory sites. S100A8 may be exposed to oxygen metabolites, particularly hypochlorite, the major oxidant generated by activated neutrophils at inflammatory sites. Here we show that hypochlorite oxidizes the single Cys residue (Cys41) of S100A8. Electrospray mass spectrometry and SDS-polyacrylamide gel electrophoresis analysis indicated that low concentrations of hypochlorite (40 microM) converted 70-80% of S100A8 to the disulfide-linked homodimer. The mass was 20,707 Da, 92 Da more than expected, indicating additional oxidation of susceptible amino acids (possibly methionine). Phorbol 12-myristate 13-acetate activation of differentiated HL-60 granulocytic cells generated an oxidative burst that was sufficient to efficiently oxidize exogenous S100A8 within 10 min, and results implicate involvement of the myeloperoxidase system. Moreover, disulfide-linked dimer was identified in lung lavage fluid of mice with endotoxin-induced pulmonary injury. S100A8 dimer was inactive in chemotaxis and failed to recruit leukocytes in vivo. Positive chemotactic activity of recombinant Ala41S100A8 indicated that Cys41 was not essential for function and suggested that covalent dimerization may structurally modify accessibility of the chemotactic hinge domain. Disulfide-dependent dimerization may be a physiologically significant regulatory mechanism controlling S100A8-provoked leukocyte recruitment.

Properties of the His57–Asp102Dyad of Rat Trypsin D189S in the Zymogen, Activated Enzyme, and α1-Proteinase Inhibitor Complexed Forms

Structural and biochemical studies suggest that serpins induce structural rearrangements in their target serine-proteinases. Previous NMR studies of the complex between a serpin, α1-proteinase inhibitor, and a mutant of recombinant rat trypsin (the Asp189to Ser mutant, D189S, which is much more stable than wild-type rat trypsin against autoproteolysis) provided information about the state of catalytic residues in this complex: the hydrogen bond between Asp102and His57remains intact in the complex, and spectral properties of His57are more like those of the zymogen than of the activated enzyme (G. Kaslik,et al.,1997,Biochemistry36, 5455–5464). Here we report the protonation and exchange behavior of His57of recombinant rat trypsin D189S in three states: the zymogen, the active enzyme, and the complex with human α1-proteinase inhibitor and compare these with analogous behavior of His57of bovine chymotrypsinogen and α-chymotrypsin. In these studies the pKaof His57has been determined from the pH dependence of the1H NMR signal from the Hδ1proton of histidine in the Asp102–His57dyad, and a measure of the accessibility of this part of the active site has been obtained from the rate of appearance of this signal following its selective saturation. The activation of rat trypsinogen D189S (zymogen, pKa= 7.8 ± 0.1; Hill coefficient = 0.86 ± 0.05) decreased the pKaof His57by 1.1 unit and made the protonation process cooperative (active enzyme, pKa= 6.7 ± 0.1; Hill coefficient = 1.37 ± 0.08). The binding of α1-proteinase inhibitor to trypsin D189S led to an increase in the pKavalue of His57to a value higher than that of the zymogen and led to negative cooperativity in the protonation process (complex, pKa= 8.1 ± 0.1; Hill coefficient = 0.70 ± 0.08), as was observed for the zymogen. In spite of these differences in the pKaof His57in the zymogen, active enzyme, and α1-proteinase inhibitor complex, the solvent exchange lifetime of the His57Hδ1proton was the same, within experimental error, in all three states (lifetime = 2 to 12.5 ms). The linewidth of the1H NMR signal from the Hδ1proton of His57was relatively sharp, at temperatures between 5 and 20°C at both low pH (5.2) and high pH (10.0), in spectra of bovine α-chymotrypsin, recombinant rat trypsin D189S, and the complex between rat trypsin D189S and human α1-proteinase inhibitor; however, in spectra of the complex between α-chymotrypsin and human α1-proteinase inhibitor, the peak was broader and could be well-resolved only at the lower temperature (5°C).

Cloning and functional expression of the murine homologue of proteinase 3: implications for the design of murine models of vasculitis

Anti-neutrophil cytoplasmic autoantibodies recognizing conformational epitopes (c-ANCA) of proteinase 3 (PR3) from azurophil granules are a diagnostic hallmark in Wegener's granulomatosis (WG). Because a functional PR3 homologue has not been identified in rodents, it is difficult to assess immunopathological responses in rats or mice immunized with patients' derived c-ANCA or human PR3. Here we report the full length cDNA cloning and functional expression of murine PR3 in HMC-1 cells. Recombinant murine PR3 shows highly similar substrate specificities towards synthetic peptides and is inhibited by human α1-proteinase inhibitor like human PR3. However, neither human c-ANCA, rabbit sera nor mouse monoclonal antibodies to human PR3 recognize the murine homologue. Consequently, it is unlikely that disease observed in mice after immunization with c-ANCA or human PR3 is caused by pathogenic antibodies directed against mouse PR3. Recombinant human-mouse chimaeric variants will be a valuable new tool to localize the disease-specific immunodominant epitopes in human PR3.

Comparison of the effects of purified human α1-antichymotrypsin and α1-proteinase inhibitor on NK cytotoxicity: only α1-proteinase inhibitor inhibits natural killing

While an inhibitory effect on natural killer (NK) cell activity was demonstrated with partially purified α 1Achy, neither highly purified α 1Achy from two healthy donors nor from one patient with giant-cell arteritis, which carries more highly branched glycans, inhibited the NK. cytotoxicity. Our purification procedure, based on immunoaffinity chromatography and gel filtration, was not in question since the pure α 1-proteinase inhibitor ( α 1PI) prepared in our laboratory by using a similar procedure continued to inhibit the NK cytotoxicity. If an inhibitory effect not related to antiprotease activity occurs with α 1PI, it is surprising that it is not shared by α 1Achy which, like α 1PI, belongs to the serpin family and which possesses a strong structural homology with α 1PI. Our finding that α 1PI is able to affect human NK cytotoxicity while α 1Achy (even with more highly branched glycans) is unable to suggests that events controlling NK activity may involve other enzymes than chymotrypsin-like enzymes.

Changes in serum level and affinity for concanavalin A of human α1-proteinase inhibitor in severe burn patients: relationship to natural killer cell activity

In serum from five patients with severe burns, alpha 1-proteinase inhibitor (alpha 1-PI) was analyzed and then isolated by immunosorption chromatography. By Con A-Sepharose chromatography alpha 1-PI was separated into two types of fractions: the first containing the Con A-non-reactive isoforms and the second containing the Con A-reactive isoforms. The increase of alpha 1-PI serum level in burn patients is associated on the fifth day after the burn with a significant shift toward species enriched in bi-antennary oligosaccharides (Con A-reactive isoforms). This latter change passed very quickly and ten days after the burn, whereas the alpha 1-PI serum level was still high, the difference in proportions of Con A-reactive and non-reactive isoforms was not statistically significant. With respect to the difference in oligosaccharide structure, it appeared that the glycan moiety was involved in the inhibitory effect on natural killer cell activity. At the same concentration, purified alpha 1-PI and retained alpha 1-PI isoforms had an equal effect, whereas the non-retained alpha 1-PI isoforms were more efficient (P less than or equal to 0.01). Purified alpha 1-PI and its isoforms inhibited the natural killer cell activity in a dose-dependent manner.

The identification of epitopic sites in human α1-proteinase inhibitor

Human alpha 1-proteinase inhibitor (alpha 1-PI) yielded nine fragments on cleavage with CNBr. The amino acid sequences of these fragments were determined. Three of these CNBr-cleavage fragments, namely fragment I (residues 64-220), fragment II (residues 243-351) and fragment III (residues 1-63), were found to bind rabbit polyclonal antibodies against chemically oxidized alpha 1-PI and mouse polyclonal antibodies against native alpha 1-PI by the Bio-Dot method (enzyme-linked immunosorbent assay on nitrocellulose). These fragments, I, II and III, inhibited by 60%, 25% and 5% respectively the binding between alpha 1-PI and the rabbit antibodies. Fragments I, II and III were subjected to proteolytic digestion, and 15, ten and five peptides were obtained from these fragments respectively. Only four of these peptides showed binding to the mouse antibodies against native alpha 1-PI. These were residues 40-63, 79-86, 176-206 and 299-323. A panel of monoclonal antibodies was prepared by conventional hybridoma technology, with chemically oxidized alpha 1-PI as the antigen. The ability of the monoclonal antibodies to bind native alpha 1-PI and CNBr-cleavage fragments I-III was determined. The monoclonal antibodies fell into three categories. Most (over 90%) belonged to group I, which was capable of binding alpha 1-PI and only fragment I. Antibodies in groups II and III bound alpha 1-PI and either fragment II or fragment III respectively. The ability of the peptides derived from proteolytic digestion of fragments I, II and III to bind three monoclonal antibodies representing each of the three groups was determined. Among all the peptides tested, only one (residues 176-206) derived from fragment I showed binding to the antibodies from group I, one (residues 299-323) derived from fragment II showed binding to the antibodies from group II, and one (residues 40-63) from fragment III showed binding to the antibodies from group III. Each of these three peptides also inhibited the binding between alpha 1-PI and the corresponding monoclonal antibodies. From these data we concluded that at least four epitopic regions (residues 40-63, 79-86, 176-206 and 299-323) were present in alpha 1-PI. Specific monoclonal antibodies to three of these sites were obtained.