Differential inhibition of serine proteinases by rabbit alpha 1-proteinase inhibitors F and S

Inhibition of six serine proteinases (bovine trypsin and chymotrypsin, equine leucocyte proteinases type 1 and 2A, porcine pancreatic elastase type III and rabbit plasmin) by rabbit alpha 1-proteinase inhibitors F and S was studied. In each case examined, the F form reacted more rapidly. The number of moles of an enzyme inhibited by one mole of alpha 1-proteinase inhibitor in a complete reaction (molar inhibitory capacity) ranged from 0.26 (leucocyte proteinase type 1) to 1.01 (trypsin). More significantly, however, the molar inhibitory capacities of both alpha 1-proteinase inhibitors differed for the same enzymes. The highest F/S inhibitory ratio was recorded with chymotrypsin (1.88), and the lowest with elastase (0.69). These differences in molar inhibitory capacities are likely to reflect the dual nature of the reaction between the inhibitor and a proteinase, that is, either complex formation or inactivation of alpha 1-proteinase inhibitor without enzyme inhibition. No evidence was obtained to suggest that differential reactivity and differential inhibitory capacity are interdependent. The observations are consistent with the view that rabbit alpha 1-proteinase inhibitors F and S are closely related yet functionally distinct proteins.     

Deglycosylation of alpha 1-proteinase inhibitor by endo-beta-N-acetylglucosaminidase F

The glycosidase endo-beta-N-acetylglucosaminidase F (endo F) from Flavobacterium meningosepticum was used for the deglycosylation of rat alpha 1-proteinase inhibitor (alpha 1 PI). alpha 1 PI containing three oligosaccharide side chains of the complex type was isolated from rat serum or from the medium of rat hepatocyte primary cultures. High-mannose-type alpha 1 PI or hybrid-type alpha 1 PI was isolated from the media of hepatocytes treated with 1-deoxymannojirimycin or swainsonine, respectively. The susceptibility of complex-type alpha 1 PI to endo F was studied in the presence of various detergents. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate and octyl glucopyranoside turned out to be most effective. In the absence of detergents, digestion of alpha 1 PI with high concentrations of endo F and/or long times of incubation led to the formation of alpha 1 PI with one and two oligosaccharide side chains. In the presence of 0.5% octyl glucopyranoside, the major cleavage products were unglycosylated alpha 1 PI and alpha 1 PI carrying one carbohydrate side chain. In contrast to the complex-type alpha 1 PI, the high-mannose type can be totally deglycosylated by endo F even in the absence of detergents. The susceptibility of the hybrid-type alpha 1 PI to endo F is between that of the complex and the high-mannose types.     

Differential inhibitory properties of dog and human alpha 1-proteinase inhibitors

Dog alpha 1-proteinase inhibitor (alpha 1-PI) was found to be an effective inhibitor of bovine chymotrypsin and also of porcine pancreatic elastase as in the case of human inhibitor. The dog inhibitor inactivated both proteinases at a molar ratio of 1:1. However, compared to the human inhibitor, dog alpha 1-PI was a relatively poor inhibitor of bovine trypsin. The association rate constants (kass) of the interactions of dog alpha 1-PI with bovine chymotrypsin and with porcine elastase were determined to be 6.9 +/- 0.3 X 10(6) M-1 s-1 and 6.4 +/- 0.1 X 10(5) M-1 s-1, respectively. These values are 1.3- and 2.7-fold higher than the corresponding values for the human inhibitor. On the other hand, kass for the dog inhibitor with bovine trypsin (2.6 +/- 0.3 X 10(4)M-1 s-1) was found to be about 5 times smaller than that of the human inhibitor.     

Synthesis and catabolism of rabbit alpha 1-antitrypsins F and S.

The metabolic relationship between the two major forms of rabbit alpha 1-antitrypsin, F and S, was investigated by using labeling techniques in vivo and in vitro. After the injection of [14C]leucine, the S/F specific-radioactivity ratio showed characteristic changes with time: at 1 h, the ratio was high (1.2-1.4), but by later times (5-7h) it decreased to a value of approx. 1.1. Two different techniques were used to purify alpha 1-antitrypsin for labelling with iodine. The half-lives of the differentially labelled and simultaneously injected F- and S-forms were 68.1 (+/- 7.6 S.D) and 55.3 (+/- 8.1 S.D)h respectively. Combined electrophoretic and gamma-spectrometric studies provided no evidence for metabolic interconversion of the alpha 1-antitrypsin forms in the circulation. These observations suggest that rabbit alpha 1-antitrypsins F and S are, despite their close chemical composition and immunological identity, metabolically independent proteins. Therefore the possibility is raised that alpha 1-antitrypsin synthesis in rabbits is controlled by two autosomal genes or two sets of such genes.     

Patterns of divergence during evolution of alpha 1-proteinase inhibitors in mammals

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.      

Studies on the interaction between leukocyte elastase, antileukoproteinase and the plasma proteinase inhibitors alpha 1-proteinase inhibitor and alpha 2-macroglobulin

The dominating inhibitor of leukocyte elastase in human respiratory tract secretions is a low molecular mass inhibitor, designated antileukoproteinase. An equimolar antileukoproteinase-elastase complex was produced and subjected to gel filtration after differing time intervals and was found to be stable. On addition to human serum, however, elastase dissociated from antileukoproteinase and formed a complex with alpha 1-proteinase inhibitor. A small amount of elastase was also found bound to alpha 2-macroglobulin. Antileukoproteinase was capable of inhibiting elastase bound to alpha 2-macroglobulin. This inhibition was more complete and more rapid when the alpha 2-macroglobulin-elastase complex was in a molar ratio of 1:1 than in a ratio of 1:2.     

Modeling the intact form of the alpha 1-proteinase inhibitor

The structure of the intact form of the serpin alpha 1-proteinase inhibitor has been modeled based on the assumption that the central strand s4A of the six-stranded beta-sheet A of the cleaved inhibitor is not incorporated into the sheet of intact alpha 1-proteinase inhibitor. This strand was removed from its position in the center of the sheet by suitable rotations about the backbone dihedrals of Lys343 using molecular graphics. The resulting structure was then annealed using molecular dynamics (MD) while applying progressive distance restraints to the reactive peptide bond (Met358-Ser359) for 50 ps. During this time, the disrupted beta-sheet reformed to create a five-stranded beta-sheet with strands 3 and 5 in a parallel arrangement. This change and accompanying structural rearrangements are largely confirmed by the X-ray structure of plakalbumin, whose structure reflects the overall structure of intact serpins. The successful modeling experiment demonstrates the utility of MD for making gross structural predictions based on related structures. The binding loop of the intact form is modeled to allow docking with serine proteinases, in particular thrombin, which most highly constrains the possible conformations of the binding loop.     

Different effects of the glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine on the glycosylation of rat alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein.

The glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine were used to inhibit oligosaccharide processing in primary cultures of rat hepatocytes. Their effect on the glycosylation of alpha 1-proteinase inhibitor (alpha 1PI) and alpha 1-acid glycoprotein (alpha 1AGP) was studied. Of the three glucosidase inhibitors examined, 1-deoxynojirimycin inhibited not only oligosaccharide trimming but also glycosylation de novo of newly synthesized proteins, resulting in the formation of alpha 1PI with two and three (normally carrying three) and alpha 1AGP with two to five (normally carrying six) oligosaccharide side chains. In the presence of the glucosidase inhibitors, glucosylated high-mannose-type oligosaccharides accumulated. Whereas most of the endoglucosaminidase-H-sensitive oligosaccharides formed in the presence of 1-deoxynojirimycin contained only one glucose residue, N-methyl-1-deoxynojirimycin and castanospermine led mainly to the formation of oligosaccharides with three glucose residues. None of the three glucosidase inhibitors completely prevented the formation of complex-type oligosaccharides. Thus, in their presence, alpha 1PI and alpha 1AGP with a mixture of both high-mannose and complex-type oligosaccharides were secreted.     

Construction and expression of alpha 1-proteinase inhibitor mutants and the effects of these mutations on secretion of the variant inhibitors

Human alpha 1-proteinase inhibitor (A1Pi) deficiency, associated with the Z variant A1Pi gene, results from defective secretion of the inhibitor from the liver and appears to be a direct consequence of replacement of Glu342 with Lys. To investigate the effect of the amino acid occupying position 342 on secretion of A1Pi, we have used oligonucleotide-directed mutagenesis of A1Pi cDNA to randomly change the codon specifying this amino acid. Since replacement of Glu342 by Lys leads to a change in the predicted secondary structure for this protein, we also tested the possibility that defective secretion of A1PiZ is the result of this type of alteration. For this purpose, site-directed mutagenesis was used to produce sequences encoding A1Pi retaining Glu342 but predicted to have A1PiZ type secondary structure. The effects of 10 different amino acids occupying position 342 on the secretion of A1Pi were determined by pulse-chase experiments and by enzyme-linked immunosorbent assay of medium from transiently transfected COS cells. Results of these studies show that secretion of A1Pi is most efficient when position 342 is occupied by a negatively charged amino acid, efficient but somewhat less so when occupied by a neutral amino acid, and least efficient when a positively charged residue is present. The mutation designed to alter secondary structure had no effect on the secretion of A1Pi. As indicated by immunofluorescence microscopy and mobility of intracellular A1Pi on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lowered secretion is accompanied by accumulation of A1Pi in the endoplasmic reticulum of the transfected cells. These results are compatible with the ideas that secretion of A1Pi is directly influenced by the amino acid occupying position 342, that a positively charged amino acid in this position is especially detrimental to secretion of this protein, and that the rate-limiting step in the secretion of the altered forms is transport from endoplasmic reticulum to Golgi.     

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.     

Involvement of hyposialylated immunoglobulins in the inactivation of alpha 1-proteinase inhibitor

The elastase inhibitory capacity of alpha 1-proteinase inhibitor (alpha 1-PI) was measured, using a direct and reproducible method, with phagocytic cells maintained in the tissue culture plate through the assay. The oxidative inactivation of alpha 1-PI is known to be mediated by the action of myeloperoxidase (MPO). The fact that hyposialylated IgG (hs IgG) induce the release of MPO prompted us to investigate the effects of such hs IgG on the inhibitory capacity of alpha 1-PI. The results show that 1-PI inactivation was observed only when phagocytic cells were activated by aggregated hs IgG, and not by unaggregated hs IgG. These observations indicate that hyposialylation should be completed by aggregation to perpetuate the oxidative reactions characteristic of inflammatory diseases.     

A novel mode of polymerization of alpha1-proteinase inhibitor

Patients homozygous for the Z mutant form of alpha1-proteinase inhibitor (alpha1-PI) have an increased risk for the development of liver disease because of the accumulation in hepatocytes of inclusion bodies containing linear polymers of mutant alpha1-PI. The most widely accepted model of polymerization proposes that a linear, head-to-tail polymer forms by sequential insertion of the reactive center loop (RCL) of one alpha1-PI monomer between the central strands of the A beta-sheet of an adjacent monomer. This model derives primarily from two observations: peptides that are homologous with the RCL insert into the A beta-sheet of alpha1-PI monomer and this insertion prevents alpha1-PI polymerization. Normal alpha1-PI monomer does not spontaneously polymerize; however, here we show that the disulfide-linked dimer of normal alpha1-PI spontaneously forms linear polymers in buffer. The monomers within this dimer are joined head-to-head. Thus, the arrangement of monomers in these polymers must be different from that predicted by the loop-A sheet model. Therefore, we propose a new model for alpha1-PI polymer. In addition, polymerization of disulfide-linked dimer is not inhibited by the presence of the peptide even though dimer appears to interact with the peptide. Thus, RCL insertion into A beta-sheets may not occur during polymerization of this dimer.     

In vivo catabolism of alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin and alpha 2-macroglobulin-methylamine

The clearances of 125I-labeled alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin and alpha 2-macroglobulin-methylamine (CH3NH2) were compared in our previously described mouse model. alpha 1-Proteinase inhibitor-trypsin cleared with a t 1/2 of 20 min, antithrombin III-thrombin of 7 min and 125I-labeled alpha 2-macroglobulin-methylamine of 2 min. Competition studies were performed to determine whether one or several pathways clear these three ligands. The clearance of 125I-labeled alpha 1-proteinase inhibitor-trypsin and 125I-labeled antithrombin III-thrombin was blocked by large molar excesses of either ligand, but not by alpha 2-macroglobulin-methylamine. The clearance of 125I-labeled alpha 2-macroglobulin-methylamine can be blocked by a large molar excesses of unlabeled alpha 2-macroglobulin-methylamine but not by alpha 1-proteinase inhibitor-trypsin. These studies demonstrate that the clearance of alpha 1-proteinase inhibitor-trypsin complexes is independent of alpha 2-macroglobulin-methylamine and utilizes the same pathway which is involved in the clearance of antithrombin III-thrombin complexes.     

Differences between the binding of trypsin and chymotrypsin by alpha 1-proteinase inhibitor

Complexes of alpha 1-proteinase inhibitor with proteases were examined by SDS-PAGE in 7.5% polyacrylamide gel and in a gel gradient. While the inhibitor-chymotrypsin complex was stable under both sets of conditions, the inhibitor-trypsin complex quantitatively dissociated under the second set of conditions, indicating that trypsin, unlike chymotrypsin, is not linked covalently to the inhibitor. Although the inhibitor sustained at least two discrete cleavages by trypsin, its overall recovery after dissociation was 100%. Due to an increased rate of autolytic breakdown in the presence of the inhibitor, the recovery of trypsin after dissociation was appreciably less than 100%. Based on these observations, a new theory of trypsin inhibition by alpha 1-proteinase inhibitor is proposed. This method is suitable for the examination of other inhibition systems as well.     

High-level production and isolation of human recombinant alpha 1-proteinase inhibitor in yeast

The cDNA coding for mature human alpha 1-proteinase inhibitor (alpha 1-PI) has been inserted into a variety of yeast expression vectors. Yeast cells transformed with these plasmids were then assayed for the production of mature, unglycosylated alpha 1-PI. The production level is optimal when the recombinant plasmid carries the TDH promoter, the complete 2mu and the leu2D selection marker. Biologically active recombinant alpha 1-PI can be purified either analytically, by affinity chromatography using a monoclonal antibody, or on a large scale, by a procedure involving precipitation of high-Mr yeast material with polyethylene glycol 3300 followed by successive chromatography on DEAE-agarose, Zn-chelate agarose, kappa-chain agarose, heparin-agarose and aminohexyl-agarose.     

Conformational stability of the covalent complex between elastase and alpha 1-proteinase inhibitor

The equilibrium unfolding-refolding process of the elastase-alpha 1-proteinase inhibitor complex, induced by guanidinium chloride, was followed by spectroscopic methods. A reversible transition with a midpoint at 2.04 +/- 0.04 M guanidinium chloride was observed by fluorescence. This transition was attributed to elastase on the basis of circular dichroism and uv absorption difference data obtained for the covalent complex and for the free proteins. The conformational stability of elastase in the complex was analyzed considering the approximation of a two-state transition. The free energy of denaturation delta GH2O was 4.2 kcal.mol-1 for complexed elastase compared to 10.5 kcal.mol-1 for the free enzyme. Such a decrease in the stability of elastase suggests that, after forming the covalent complex with the inhibitor, the enzyme undergoes not only the expected local modifications of the active site, but also an extensive structural reorganization.     

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.