Isolation and properties of recombinant DNA produced variants of human alpha 1-proteinase inhibitor

Using the glyceraldehyde-3-phosphate dehydrogenase promoter, nonglycosylated human alpha 1-proteinase inhibitor, representing 10% of the soluble cell protein, has been synthesized in yeast. Two forms of this protein were isolated with one being analogous to the human plasma protein and the other having the amino acid valine replacing methionine at position 358 (the P1 position). Both proteins were more sensitive to heat inactivation than the plasma form, and both had shorter half-lives in rabbits. These differences were presumably due to the absence of carbohydrate. Each protein could bind neutrophil elastase at a rate only slightly slower than that of human plasma alpha 1-proteinase inhibitor. However, the valine variant was stable to oxidation, while the P1 methionine-containing protein was readily inactivated. The specificity of alpha 1-proteinase inhibitor (methionine) was identical to that of the plasma form; however, the valine form could only effectively bind to neutrophil or pancreatic elastase, "trypsin-like" serine proteinases not being inactivated at all. These data indicate the potential importance of mutant forms of proteinase inhibitors, produced by recombinant DNA technology, as therapeutic agents for the inactivation of excess proteinases of a specific type in tissues.     

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.     

Inhibitory activity and conformational transition of alpha 1-proteinase inhibitor variants.

Several variants of alpha 1-proteinase inhibitor (alpha 1-PI) were investigated by spectroscopic methods and characterized according to their inhibitory activity. Replacement of Thr345 (P14) with Arg in alpha 1-PI containing an Arg residue in position 358 (yielding [Thr345----Arg, Met358----Arg]alpha 1-PI) results in complete loss of its inhibitory activity against human alpha-thrombin; whereas an exchange of residue Met351 (P8) by Glu [( Met351----Glu, Met358----Arg]alpha 1-PI) does not alter activity. [Thr345----Arg, Met358----Arg]alpha 1-PI is rapidly cleaved by thrombin, while [Met358----Arg]alpha 1-PI and [Met351----Glu, Met358----Arg]alpha 1-PI form stable proteinase-inhibitor complexes. The stability of [Thr345----Arg, Met358----Arg]alpha 1-PI against guanidinium chloride denaturation is significantly enhanced compared to wild-type alpha 1-PI, and does not change after cleavage, resembling ovalbumin, a serpin with no inhibitory activity, from which the Thr345----Arg amino acid exchange had been derived. [Met351----Glu, Met358----Arg]alpha 1-PI and [Met358----Arg]alpha 1-PI resemble the wild-type protein in this respect. The CD spectra of intact and cleaved alpha 1-PI variants do not compare well with the wild-type protein, probably reflecting local structural differences. Insertion of a synthetic peptide, which corresponds to residues Thr345----Met358 of human alpha 1-PI, leads to the formation of binary complexes with all variants having the characteristic features of the binary complex between peptide and wild-type protein.     

Inactivation of human thrombin in the presence of human alpha1-proteinase inhibitor.

Both the clotting and esterase activities of thrombin are inhibited by alpha1-proteinase inhibitor (alpha1-antitrypsin). The inhibition is a time-and temperature-dependent reaction which is proportional to the molar ratio of thrombin to inhibitor. Both the active-site serine residue of thrombin and the reactive-site lysine residue of alpha1-proteinase inhibitor are involved. alpha1-Proteinase inhibitor forms a 1:1 complex with thrombin that is comparable with the complex formed with trypsin and other proteinases. Incubation of the inhibitor with excess of thrombin, however, results in inactivation of nearly all the enzyme, even though only as much complex is formed as alpha1-proteinase inhibitor present. A portion of the remaining thrombin apparently aggregates. These results suggest that the mechanism for inhibition of thrombin may not be exactly the same as for trypsin, which is inhibited only to the extent to which complex is formed.     

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.     

Inactivation of human alpha 1-proteinase inhibitor by thiol proteinases.

Human plasma alpha1 proteinase inhibitor is the body's principal modulator of serine proteinases (such as those released from phagocytic cells). Cysteine-active-site proteinases, which are not inhibited, have now been found to inactivate this important inhibitor by proteolytic cleavage of a scissile peptide bond. Papain carries out this inactivation catalytically, whereas cathepsin B1 acts stoicheiometrically. Thus thiol proteinases could easily disrupt the delicately regulated balance between serine proteinases and alpha1 proteinase inhibitor.     

The identification of epitopic sites in human alpha 1-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.     

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.     

Inactivation of human plasma alpha 1-proteinase inhibitor by human PMN leucocyte collagenase

Highly purified human polymorphonuclear leucocyte collagenase cleaved human alpha-1-proteinase inhibitor (alpha 1-PI) at the carboxyl site of Phe352 (P7). The inhibitor was thereby rapidly inactivated and generated a primary degradation product as shown by reverse-phase HPLC and N-terminal sequencing. Prolonged incubation of the modified inhibitor with polymorphonuclear leucocyte collagenase led to the generation of a secondary degradation product with additional cleavage at the carboxyl site of Pro357 (P2).     

Interactions of the complex between human urinary trypsin inhibitor and human leukocyte elastase with alpha 1-proteinase inhibitor and alpha 2-macroglobulin

The urinary trypsin inhibitor was recently shown to inhibit human leukocyte elastase. Complexes of human urinary trypsin inhibitor with human leukocyte elastase or human trypsin were produced and subjected to gel filtration. The complexes were found to be sufficiently stable up to 24 h incubation (at least 70% recovery). When human serum was added, elastase and trypsin dissociated from the urinary trypsin inhibitor and associated with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The addition of alpha 1-proteinase inhibitor to a complex of urinary trypsin inhibitor and leukocyte elastase caused a rapid dissociation of the complex (kdiss = 3.2 X 10(-2) s-1).     

Studies on the interactions of human pancreatic elastase 2 with human alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin.

Several intermediates in the reaction of 2-methylglutamate with glutamate decarboxylase from Escherichia coli were detected by stopped-flow spectrophotometry and by rapid-scanning spectrophotometry after conventional mixing. Structures were assigned to intermediates on the basis of kinetic and spectral evidence. In the early stages of the reaction an intermediate with the properties expected of a geminal diamine accumulated significantly. Changes consistent with the conversion of this species into the external aldimine were also observed. The course of product formation was determined and linked with spectral changes taking place in the bound coenzyme. The effect of the minor decarboxylation-dependent transamination that accompanies the major reaction was analysed.     

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.     

Stoichiometry of the interaction of human plasma alpha 1-proteinase inhibitor with subtilisin BPN'

Interaction of human plasma alpha 1-proteinase inhibitor (alpha 1PI) with subtilisin BPN' was assessed by spectrophotometric determination of the inhibitory capacity and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). During the course of incubation of the enzyme and the inhibitor (E : I = 1 : 7.5) at pH 8.0 about 17% of the enzyme activity which had been inhibited initially was regenerated, indicating a temporary type of inhibition. The results of the titration experiments indicate that 9.8 mol of the inhibitor is required to inhibit 1 mol of the enzyme completely. However, patterns of 5% disc SDS-PAGE under non-reducing conditions revealed only an equimolar complex (Mr80K) of alpha 1PI with the enzyme and no other higher Mr component than the native inhibitor (Mr 56K). On the other hand, complete dissociation of the complex occurred under reducing conditions, producing an enzymatically modified inhibitor. When 5 21% gradient slab SDS-PAGE was employed, no complex formation was observed under either reducing or non-reducing conditions. With the gradient gel system, dissociation of the equimolar complex produced different forms of the inhibitor, that is, regeneration of an intact alpha 1PI under non-reducing conditions and an enzymatically modified form under reducing conditions. All these results indicate that the complex formed between subtilisin BPN' and human alpha 1PI is not so stable as that of the inhibitor with bovine chymotrypsin and that no covalent bond may be involved in the complex formation. The results also indicate that human alpha 1PI is not an effective inhibitor of subtilisin BPN' and behaves like a substrate for the enzyme.     

Proteolytic inactivation of alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin by oxidatively activated human neutrophil metalloproteinases.

Human neutrophils use the H2O2-myeloperoxidase-chloride system to generate chlorinated oxidants capable of activating metalloproteinase zymogens that hydrolyze not only native and denatured collagens, but also the serine proteinase inhibitor (serpin) alpha 1-proteinase inhibitor (alpha 1 PI). To identify the metalloenzyme that hydrolyzes and inactivates alpha 1 PI, neutrophil releasates were chromatographed over gelatin-Sepharose and divided into fractions containing either progelatinase or procollagenase. The gelatinase-containing fraction cleaved alpha 1 PI in a manner inhibitable by native type V, but not type I, collagen. Conversely, while the collagenase-containing fraction also cleaved alpha 1 PI, this activity was inhibited by type I, but not type V, collagen. Because type I and V collagens are competitive substrates for collagenase and gelatinase, respectively, each of the metalloproteinase zymogens were purified to apparent homogeneity and examined for alpha 1 PI-hydrolytic activities. Both purified gelatinase and collagenase inactivated alpha 1PI by hydrolyzing the serpin within its active-site loop at the Phe352-Leu353 and Pro357-Met358 bonds, albeit with distinct kinetic properties. Furthermore, purified collagenase, but not gelatinase, cleaved a second serpin, alpha 1-antichymotrypsin, by hydrolyzing the Ala362-Leu363 bond within its active-site loop. These data demonstrate that human neutrophils use chlorinated oxidants to activate collagenolytic metalloproteinases whose substrate specificities can be extended to members of the serpin superfamily.     

Modification of the isoinhibitors of human serum alpha 1-proteinase inhibitor (alpha 1-antitrypsin) by pancreatic proteases

Due to the action of a serum protease, the two most cathodal isoinhibitors of the alpha 1-proteinase inhibitor (alpha 1-PI) are cleaved at the Gly5-Asp6 bond and lack two negative charges. In spite of this, these can bind trypsin and chymotrypsin, showing that the N-terminal pentapeptide is not indispensable for inhibition function. Pancreatic proteases also cleave a bond near the N-terminus in alpha 1-PI, resulting in a loss of two negative charges and a corresponding cathodal shift in the electrofocusing behavior of the isoinhibitors. Trypsin cleaves isoinhibitors near the N-terminus at a large inhibitor excess and unless an additional cleavage takes place, at least two of the new isoinhibitors remain active. An additional cleavage(s), most likely at a distance of 30-40 residues from the C-terminus results in a corresponding decrease of the molecular mass and a loss of inhibition function. Although the C-terminal cleavage peptide does separate from the protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it remains associated with it under conditions of polyacrylamide gel isoelectric focusing. Chymotrypsin also cleaved alpha 1-PI near the N-terminus but this could be observed only at protease excess and the modified isoinhibitors did not form complexes with chymotrypsin. The molecular polymorphism of alpha 1-PI is partly explained by the absence of the N-terminal pentapeptide from some of the isoinhibitors.     

Purification and some properties of two proteinases from Crotalus adamanteus venom that inactivate human alpha 1-proteinase inhibitor.

Two proteinases (proteinases I and II) have been purified from Crotalus adamanteus venom to the stage of electrophoretic homogeneity and proteinase II has been crystallized. The proteinase differ slightly in molecular weight and amino acid composition. Both are metalloenzymes requiring Zn2+ or Ca2+, or both; neither requires thiol compounds for activation. The proteinases are free of esterolytic activity against benzoly-L-arginine ethyl ester and benzoyl--tyrosine ethyl ester. Proteinase II cleaves the oxidized B chain of insulin at the bonds Phe1-Val2, His5-Leu6, His10-Leu11, Ala14-Leu15, Leu15-Tyr16, and Tyr-16-Leu17. Digestion of polylsine and polyarginine by proteinase II liberates products ranging from dodecapeptides to hexapeptides. Proteinases I and II catalytically inactive human plasma alpha 1-proteinase inhibitor (54,000 daltons). Electrophoretic analysis of the reaction of proteinase II with alpha 1-proteinase inhibitor reveals that an inactivated inhibitor species of 50,000 daltons is formed, and a peptide of 4,000 daltons is released. The gradual disappearance of the native inhibitor results in the corresponding loss of inhibitory activity against trypsin and chymotrypsin.