Comparative properties of human alpha-1-proteinase inhibitor glycosylation variants

Variant forms of human alpha-1-proteinase inhibitor (alpha-1-PI), obtained by the treatment of human Hep G2 cells with specific inhibitors of glycosylation were tested for both inhibitory activity and heat stability. All were found to have the same second-order association rate with human neutrophil elastase, indicating a lack of importance of the carbohydrate moiety. In contrast, incompletely glycosylated forms of alpha-1-PI were found to be heat sensitive relative to the mature protein, suggesting a role for carbohydrate in protein stabilization.     

Affinity chromatography of alpha-1-protease inhibitor using Sepharose-4B-bound anhydrochymotrypsin

Sepharose 4B-bound bovine anhydrochymotrypsin (AnhCT), a catalytically inactive form of chymotrypsin, was shown to be effective for retaining active alpha-1-protease inhibitor (alpha-1-PI, also alpha-1-antitrypsin) from human plasma, while showing no measurable affinity for oxidized or protease complexed alpha-1-PI, or for most other plasma proteins. alpha-1-PI eluted from this resin with 0.1 M chymostatin retained full activity against trypsin, chymotrypsin, and elastase. In addition to alpha-1-PI, AnhCT-Sepharose binds a limited number of other plasma proteins. Using monospecific antisera to plasma protease inhibitors, one of these proteins was identified as inter-alpha-trypsin inhibitor, and it was recoverable in active form. Therefore, an AnhCT-Sepharose 4B resin has been demonstrated to be of value for isolating active forms of alpha-1-PI from solutions, and may also be useful for the isolation of inter-alpha-trypsin inhibitor.     

Alpha-1-proteinase inhibitor is a heparin binding serpin: molecular interactions with the Lys rich cluster of helix-F domain

Alpha-1-proteinase (alpha-1-PI) inhibitor is the major circulating serine protease inhibitor in humans. The porcine elastase and trypsin inhibitory activity of human and ovine alpha-1-PI is activated several fold in the presence of anti-coagulant heparin. The activation is allosteric and appears to be characterized by two steps of binding; a weak followed by a strong binding. The Kass for ovine and human alpha-1-PI inhibition of porcine pancreatic elastase was increased approximately 45 fold and 38 fold respectively. Using a combinatorial approach of multiple sequence alignment, surface topology, chemical modification and tryptic peptide mapping to identify the sequence of the heparin bound peptide; we demonstrate that heparin binds to the lysyl rich region of the F-helix of alpha-1-PI, which differs from that of heparin-antithrombin (AT) interactions. Molecular docking prediction using the MEDock algorithm approximates the three positively charged lysines (K154, K155, K174) of human alpha-1-PI in this interaction. This heparin alpha-1-PI interaction has been exploited to develop an affinity purification method, which can be used universally to obtain homogenous preparations of mammalian alpha-1-PIs useful for augmentation therapy. Collectively, all these findings imply that alpha-1-PI has a major role in regulating extra cellular protease activity and the physiological activator is heparin.     

Human alpha-1-antichymotrypsin: purification and properties.

Human alpha-1-antichymotrypsin has been purified to homogeneity by the following sequential steps--(a) ammonium sulfate fractionation; (b) chromatography on Cibacron Blue Sepharose at pH 7.0; and (c) chromatography on SP-Sephadex C-50 at pH 5.5. The inhibitor has a molecular weight near 68,000 and contains approximately 26% carbohydrate alpha-1-Antichymotrypsin has an amino-terminal arginine and a carboxy-terminal glycine. It also has some homology with alpha-1-PI based on amino-terminal sequence analysis of both proteins. Complexes of alpha-1-antichymotrypsin with human chymotrypsin and human leukocyte cathepsin G are stable in sodium dodecyl sulfate and have molecular weights near 90,000 suggesting 1:1 complex formation on a molar basis between inhibitor and enzyme.     

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.     

Purification and biochemical characterization of ovine alpha-1-proteinase inhibitor: mechanistic adaptations and role of Phe350 and Met356

The glycoprotein alpha-1-proteinase inhibitor (alpha-1-PI) is a member of the serpin super family that causes rapid and irreversible inhibition of redundant serine protease activity. A homogenous preparation of ovine alpha-1-PI, a 60 kDa protein was obtained by serially subjecting ovine serum to 40-70% (NH(4))(2)SO(4) precipitation, Blue Sepharose, size-exclusion, and concanavalin-A chromatography. Extensive insights into the trypsin, chymotrypsin, and elastase interaction with ovine alpha-1-PI, point towards the involvement of Phe(350) besides the largely conserved Met(356) in serine protease recognition and consequent inhibition. The N-terminal of C-terminal peptides cleaved on interaction with elastase, trypsin, and chymotrypsin prove the presence of diffused sub-sites in the vicinity of Met(356) and the strategically positioned Pro anchored peptide stretch. Further, human alpha-1-PI is more thermolabile compared to ovine alpha-1-PI, higher thermolability is mainly attributed to poorer glycosylation. The enzymatic deglycosylation of human and ovine alpha-1-PI results in diminished thermostability of the inhibitors, with sharp decrease in thermal transition temperatures but retaining their inhibitory potency. Homology modeling of the deduced amino acid sequence of ovine alpha-1-PI using the human alpha-1-PI template has been used to explain the observed inhibitor-protease interactions.     

Bi- and tri-antennary human transferrin glycopeptides and their affinities for the hepatic lectin specific for asialo-glycoproteins

Glycopeptides were isolated from a proteolytic digest of human transferrin. After mild acid hydrolysis the desialylated glycopeptides were labelled by the galactose oxidase/NaB(3)H(4) procedure and then fractionated by Sephadex-gel filtration or by anion-exchange chromatography. Either technique allowed separation of the two heterosaccharide chains (designated glycan I and glycan II) previously described for this protein by Spik, Vandersyppe, Fournet, Bayard, Charet, Bouquelet, Strecker & Montreuil (1974) (in Actes du Colloque Internationale No. 221 vol. 1, pp. 483-499). Subsequent chromatography on Sepharose-concanavalin A separated fractions containing different quantities of carbohydrates for each glycan, as indicated by analyses. The isolated glycan fractions were then tested for their abilities to bind to the immobilized rabbit hepatic lectin. Our studies suggest that either glycan can have a bi- or tri-antennary structure. Desialylated biantennary glycans I and II did not bind to the hepatic lectin. Desialylated triantennary glycan I was slightly retarded by the hepatic lectin, whereas the triantennary glycan II consisted of equal quantities of a retarded and a bound type. Desialylated triantennary glycan II was totally displaced from the hepatic lectin by using a buffer containing 0.05m-EDTA. The results suggest that greater structural heterogeneity exists in the carbohydrate moiety of human transferrin than was previously envisaged. Such heterogeneity could be reflected in several molecular forms of human transferrin, which, after desialylation, differ significantly in their affinities for the hepatic lectin.     

Structures of the carbohydrate moiety attached to one site in the first domain of turkey ovomucoid: elucidation by 1H-n.m.r. spectroscopy

1H-N.m.r. spectroscopy was used to elucidate the primary structures of the carbohydrate moiety attached to asparagine at residue 53 in the first domain of turkey ovomucoid, a serine proteinase inhibitor. The carbohydrate moiety is a heterogeneous mixture of three structurally closely related complex-type oligosaccharides. Of the total carbohydrate moiety, 61% is tetra-antennary with terminal galactose and with an intersecting N-acetylglucosamine, and containing an additional N-acetylglucosamine (10') attached to mannose (4'). Another 23% is tri-antennary with terminal galactose and with an intersecting N-acetylglucosamine. The remaining 16% is tri-antennary with terminal galactose (6 and 8 only), with an intersecting N-acetylglucosamine.     

Proteinase-proteinase inhibitor complex in rats under oxidative stress caused by administration of cobalt chloride

Mechanisms of proteinase-inhibitor proteinase system response was estimated following of cobalt chloride injection. The increase proteinase activity, which led to significant decrease of alpha-2-macroglobulin (alpha-2-MG) level was established that indicated to the removal of the proteinase in complex with alpha-2-MG from the organism. Increase of alpha-1-proteinase inhibitor (alpha-1-PI) trypsin-inhibitory activity in the kidneys testify about removal of oxidative alpha-1-PI.     

Purification and properties of two different α1-protease inhibitors from mouse plasma

Two similar but distinct forms of α1-protease inhibitor (α1-PI) have been isolated and purified 120-fold to homogeneity from the plasma of female, white Swiss (Ha/ICR) mice. The two inhibitors can be separated by chromatography on DEAE-cellulose using a shallow NaCl gradient at pH 8.9 for elution. Because of their differing specificities for elastase and trypsin we have labeled the two inhibitors α1-PI(E) and α1-PI(T), respectively. The apparent M_r for both proteins, as estimated by gel exclusion chromatography, is approximately 53,000 daltons. However by polyacrylamide gel electrophoresis in the presence of SDS, α1-PI(T) has an apparent m_r of 65,000 while the apparent m_r of α1-PI(E) is 55,000. These results suggest differences in charge and carbohydrate composition. The two mouse inhibitors also have different AT-terminal amino acids. Like human α1-PI the mouse inhibitors form stable complexes with proteases. However they differed from human α1-PI in that they were not found to neutralize either human thrombin or plasmin. While α1-PI(E) inhibits bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase, α1-PI(T) is an effective inhibitor only of trypsin. Plasma levels of α1-PI(E) increase significantly 24 h after stimulation of the acute phase reaction while those of α1-PI(T) do not. Our data suggest that α1-PI(E) and α1-PI(T) are products of different genes.     

The use of monoclonal antibodies to distinguish several chemically modified forms of human alpha 1-proteinase inhibitor.

The purpose of our investigation was to obtain monoclonal antibodies that could distinguish three forms of alpha 1-proteinase inhibitor (alpha 1-PI): native alpha 1-PI, N-chlorosuccinimide-oxidized alpha 1-PI (Ox-alpha 1-PI) and proteolytically modified alpha 1-PI (alpha 1-PI). Three specific monoclonal antibodies were characterized as to their binding properties. By using the Bio-Dot assay, it was found that all three forms of alpha 1-PI were capable of binding to antibody 6D4-6-18, that only Ox-alpha 1-PI, but not native alpha 1-PI or alpha 1-PI, could bind to antibody 6C7-5, and that alpha 1-PI and a complex between alpha 1-PI and trypsin uniquely were not able to bind to antibody 5C12-8-7. Thus it was concluded that it is possible to use monoclonal antibodies with different epitopic specificities to distinguish two chemically modified forms of alpha 1-PI from the native protein.     

Isolation and characterization of alpha-1-proteinase inhibitor from goat plasma

Alpha-1-proteinase inhibitor (alpha-1-PI) was isolated from goat plasma by salt fractionation, and chromatography on a DEAE-cellulose column. The inhibitor was found to be homogeneous by gel chromatography, SDS-PAGE and PAGE.Mr values by gel filtration (57 kDa), and by SDS-PAGE (52 kDa), under reducing conditions were nearly the same suggesting that the inhibitor consists of a single polypeptide chain. It contained 13.8% neutral hexose but no sialic acid residue. The values of isoionic pH, and extinction coefficient at 278 nm were 4.84, and 4.6, respectively. Fluorescence spectral properties showed tryptophan residues in the inhibitor. Solvent perturbation difference spectra suggested 74% exposure of the tryptophan residues in the native molecule. Gel filtration behaviour of the inhibitor was consistent with a Stokes radius of 3.16 nm, diffusion coefficient of 7.02 X 10(-7) cm2-sec-1 and a frictional ratio of 1.24 suggesting asymmetry and/or excessive hydration of the inhibitor molecule. Goat alpha-1-PI, unlike human alpha-1-PI was found to be potent inhibitor of bovine trypsin but a poor inhibitor of porcine pancreatic elastase. It was virtually devoid of antichymotryptic activity.     

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.     

Neutrophil-mediated solubilization of the subendothelial matrix: oxidative and nonoxidative mechanisms of proteolysis used by normal and chronic granulomatous disease phagocytes

Both normal and chronic granulomatous disease (CGD) neutrophils were able to degrade the subendothelial matrix secreted by human endothelial cells via an elastase-dependent process. In the absence of the plasma antiproteinase, alpha-1-proteinase inhibitor (alpha-1-PI), normal neutrophils protect their released elastase from inactivation by using the chlorinated oxidants hypochlorous acid and endogenous N-chloroamines to suppress the antiproteinase's activity. In contrast, CGD neutrophils were unable to generate either class of chlorinated oxidant or to inactivate the porcine pancreatic elastase inhibitory capacity of alpha-1-PI unless the cells were supplemented with exogenous hydrogen peroxide. Despite the reliance of normal neutrophils on chlorinated oxidants to inactivate alpha-1-PI, neutrophils triggered in the presence of agents that block the generation of these reactive species continued to degrade the subendothelial matrix at a suppressed but significant rate in the presence of a 50-fold excess of the antiproteinase. The continued solubilization of the matrix by normal neutrophils was not due to the incomplete inhibition of oxidant generation because triggered CGD neutrophils were also able to degrade the matrix in the presence of excess alpha-1-PI. If CGD neutrophils were stimulated in the presence of an exogenous source of H2O2 and alpha-1-PI, the proteolytic potential of the cells was identical to that observed with normal stimulated neutrophils. We conclude that normal neutrophils can enhance their ability to degrade the subendothelial matrix by oxidatively protecting elastase from inactivation by alpha-1-PI but both normal and CGD neutrophils possess non-oxidatively linked mechanisms for sequestering and using elastase to mediate proteolytic effects in the presence of native antiproteinase.     

Localization of neutral N-linked carbohydrate chains in pig zona pellucida glycoprotein ZPC.

Zona pellucida, a transparent envelope surrounding the mammalian oocyte, plays important roles in fertilization and consists of three glycoproteins; ZPA, ZPB and ZPC. In pig, neutral complex-type N-linked chains obtained from a ZPB/ZPC mixture possess sperm-binding activity. We have recently reported that among neutral N-linked chains triantennary and tetraantennary chains have a sperm-binding activity stronger than that of diantennary chains. Triantennary and tetraantennary chains are localized at the second of the three N-glycosylation sites of ZPB. In this study, we focused on the localization of neutral N-linked chains in ZPC. ZPB and ZPC can not be separated from each other unless the acidic N-acetyllactosamine regions of their carbohydrate chains are removed by endo-beta-galactosidase digestion. A large part of the acidic N-linked chains becomes neutral by the digestion, but the main neutral N-linked chains are not susceptible to the enzyme. N-glycanase digestion indicated that ZPC has three N-glycosylation sites. Three glycopeptides each containing one of the N-glycosylation sites were obtained by tryptic digestion of ZPC and the N-glycosylation sites were revealed as Asn124, Asn146 and Asn271. The carbohydrate structures of the neutral N-linked chains from each glycopeptide were characterized by two-dimensional sugar mapping analysis taking into consideration the structures of the main, intact neutral N-linked chains of ZPB/ZPC mixture reported previously. Triantennary and tetraantennary chains were found mainly at Asn271 of ZPC, whereas diantennary chains were present at all three N-glycosylation sites. Thus, ZPC has tri-antennary and tetra-antennary chains as well as ZPB, but the localization of the chains is different from that in ZPB.     

Isolation and structure determination of the intact sialylated N-linked carbohydrate chains of recombinant human follitropin expressed in Chinese hamster ovary cells

Biologically active recombinant human follitropin has been expressed in Chinese hamster ovary cells. The carbohydrate chains of the recombinant glycoprotein hormone were enzymatically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The oligosaccharides were separated from the N-deglycosylated protein by gel-permeation chromatography on Bio-Gel P-100, and fractionated by a combination of FPLC on Mono Q and HPLC on Lichrosorb-NH2. The structures of the carbohydrate chains were determined by 500- or 600-MHz 1H-NMR spectroscopy. The following types of carbohydrates occur: monosialylated diantennary (10%), disialylated diantennary (43%), disialylated tri-antennary (5%), trisialylated tri-antennary (13%), trisialylated tri'-antennary (8%), and tetrasialylated tetraantennary (12%) N-acetyllactosamine type of carbohydrate chains, all bearing exclusively alpha 2-3-linked N-acetylneuraminic acid (Neu5Ac). Previously, for pituitary follitropin mono-, di-, tri-, tri'-, and tetra-antennary oligosaccharides containing alpha 2-3- as well as alpha 2-6-linked Neu5Ac residues were reported. The bisecting GlcNAc residues present in native follitropin were not detected in the recombinant glycoprotein. Of the oligosaccharides 29% have an alpha 1-6-linked Fuc residue at the asparagine-bound GlcNAc, whereas this amount is about 50% in pituitary follitropin. In some of the tri-, tri'- and tetra-antennary oligosaccharide fractions small amounts (less than 5%) of compounds were detected having one or more additional N-acetyllactosamine units.     

Isolation and properties of oxidized alpha-1-proteinase inhibitor from human rheumatoid synovial fluid

Human alpha-1-proteinase inhibitor (α-1-PI) from synovial fluid has been isolated to near 90% purity. The preparation has a molecular weight near 52,000, contains 3.5 residues of methionine sulfoxide, and an amino terminal glutamine residue. Sequence studies indicate that the first 17 residues, normally present in plasma α-1-PI, are missing from this protein. The inhibitor did not form a complex with porcine pancreatic elastase but, instead, was converted to a lower molecular weight form. Sequence studies on the latter indicated that two methionyl residues, one at the P₁ reactive site and the other at P₈, had been oxidized. These data confirm the fact that oxidized α-1-PI may be formed $\text{in vivo}$, presumably by the action of myeloperoxidase. This latter effect may alter the proteinase-proteinase inhibitor balance in tissues so that excess proteolysis and abnormal tissue degradation may occur.     

Isolation and characterization of alpha1-proteinase inhibitor from common carp (Cyprinus carpio) seminal plasma

Using a three-step procedure, we purified (79 and 51.6-fold to homogeneity) and characterized the two isoforms (a and b) of alpha1-proteinase inhibitor-like protein from carp seminal plasma. The isoforms have molecular masses of 55.5 and 54.0 kDa, respectively. These inhibitors formed SDS-stable complexes with cod and bovine trypsin, chymotrypsin and elastase. The thirty-three amino acids within the reactive loop SLPDTVILNRPFLVLIVEDTTKSILFMGKITNP were identified for isoform b. The same first ten amino acids were obtained for isoform a, and this sequence revealed 100% homology to carp alpha1-proteinase inhibitor (alpha1-PI) from perimeningeal fluid. Both isoforms of alpha1-PI are glycoproteins and their carbohydrate content was determined to be 12.6 and 12.1% for a and b, respectively. Our results indicated that alpha1-PI is one of the main proteins of carp seminal plasma. Using polyclonal anti-alpha1-PI antibodies, alpha1-PI was for the first time localized to the carp testis. The presence of alpha1-PI in testis lobules and in the area surrounding spermatides suggests that this inhibitor may be involved in the maintenance of testis connective tissue integrity, control of spermatogenesis or protection of tissue and spermatozoa against unwanted proteolysis. Since similar alpha1-PI has been identified in rainbow trout semen it can be suggested that the presence of alpha1-PI in seminal plasma is a common feature of cyprinid and salmonid fish.     

Acute phase mediator oncostatin M regulates affinity of alpha1-protease inhibitor for concanavalin A in hepatoma-derived but not lung-derived epithelial cells

Quantitative changes in plasma protein concentrations during tissue injury or inflammation (acute phase response) are often accompanied by specific alterations in the carbohydrate moieties of these proteins. The glycosylation changes comprise alterations in the type of branching of the carbohydrate structures as revealed by modulated reactivity of acute phase glycoproteins with the lectin concanavalin A. Interestingly, inflammation-induced changes in the glycosylation of acute phase proteins have been shown to affect the functional properties of these proteins. In this study we demonstrate that synthesis of acute phase protein alpha(1)-PI, the controlling inhibitor of neutrophil elastase, is significantly up-regulated in hepatic and lung-derived epithelial cells by the inflammatory mediator oncostatin M. Although oncostatin M markedly altered the concanavalin A reactivity of hepatic alpha(1)-PI, lung-derived epithelial cells did not change the pattern of alpha(1)-PI glycan branching upon stimulation with oncostatin M. These results indicate that inflammation-induced changes in glycosylation of alpha(1)-PI may have different impacts on functional properties of liver and lung-synthesized alpha(1)-PI.     

Qualitative studies of lung lavage alpha 1-proteinase inhibitor

A method is described which enables identification of the molecular size of alpha 1-proteinase inhibitor (alpha 1-PI) in biological fluids. This technique when applied to bronchoalveolar lavage fluids clearly demonstrates alpha 1-PI in three molecular forms; the native molecule (Mr approximately equal to ++54 000), a partially proteolysed form (Mr approximately equal to 49 000) and in a form suggestive of a complex with enzyme (Mr approximately equal to 82 000). Samples showing the presence of native alpha 1-PI inhibited more porcine pancreatic elastase than samples where no native alpha 1-PI was seen or where the predominant form was partially proteolysed alpha 1-PI (p less than 0.01). Although the predominant band of alpha 1-PI was more frequently the partially proteolysed form in current smokers (p less than 0.01), there was no clear difference in the inhibitory function of alpha 1-PI between current smokers and non-smokers and those with and without airflow obstruction.