Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(340) is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzyme B, and that the P4' residue (Glu(344)) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/K(m) 9.5 x 10(3) and 1.2 x 10(5) s(-1) M(-1), respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4' Glu was cleaved less efficiently (k(cat)/K(m) 5.3 x 10(4) s(-1) M(-1)). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by granzyme B (k(cat)/K(m) 7.5 x 10(5) s(-1) M(-1)) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K(m) 6.7 x 10(4) s(-1) M(-1)) and confirms that P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridge with Lys(27) of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.     

Recombinant DNA-derived forms of human alpha 1-proteinase inhibitor. Studies on the alanine 358 and cysteine 358 substituted mutants

The specificity and reactivity of human alpha 1-proteinase inhibitor has been investigated by in vitro mutagenesis of the reactive site P1 methionine 358 residue to alanine 358 and cysteine 358. A comparison of the second-order association rates of both uncharged mutants with 9 serine proteinases indicated that each reacted similarly to either the normal plasma inhibitor or to a mutant containing valine in this position (Travis, J., Owen, M., George, P., Carrell, R., Rosenberg, S., Hallewell, R. A., and Barr, P. J. (1985) J. Biol. Chem. 260, 4384-4389) when tested against either neutrophil or pancreatic elastase. However, oxidation, carboxymethylation, or aminoethylation of the cysteine mutant to yield a charged P1 residue resulted in a significant decrease in association rates with both elastolytic enzymes, and aminoethylation created an excellent trypsin and plasmin inhibitor. These results indicate that the specificity of alpha 1-proteinase inhibitor is determined in a general manner by the class of amino acid residue in the P1 position. Substitution within the same category, such as from valine to alanine or cysteine among the aliphatic hydrophobic residues, has little effect on association rates with the elastolytic enzymes tested. However, alteration from an uncharged to a charged residue may cause considerable changes in both inhibitor specificity and reactivity as noted here with the cysteine derivatives and also previously with a natural variant in which methionine 358 to arginine 358 conversion resulted in the production of a potent thrombin inhibitor (Owen, M. C., Brennan, S. O., Lewis, J. H., and Carrell, R. W. (1983) N. Engl. J. Med. 309, 694-698).     

Substrate specificity of human pancreatic elastase 2

The substrate specificity of human pancreatic elastase 2 was investigated by using a series of peptide p-nitroanilides. The kinetic constants, kcat and Km, for the hydrolysis of these peptides revealed that this serine protease preferentially hydrolyzes peptides containing P1 amino acids which have medium to large hydrophobic side chains, except for those which are disubstituted on the first carbon of the side chain. Thus, human pancreatic elastase 2 appears to be similar in peptide bond specificity to the recently described porcine pancreatic elastase 2 [Gertler, A., Weiss, Y., & Burstein, Y. (1977) Biochemistry 16, 2709] but differs significantly in specificity from porcine elastase 1. The best substrates for human pancreatic elastase 2 were glutaryl-Ala-Ala-Pro-Leu-p nitroanilide and succinyl-Ala-Ala-Pro-Met-p-nitroanilide. However, there was little difference among substrates with leucine, methionine, phenylalanine, tyrosine, norvaline, or norleucine in the P1 position. Changes in the hydrolysis rate of peptides with differing P5 residues indicate that this enzyme has an extended binding site which interacts with at least five residues of peptide substrates. The overall catalytic efficiency of human pancreatic elastase 2 is significantly lower than that of porcine elastase 1 or bovine chymotrypsin with the compounds studied.     

Kinetic studies on the interaction of alpha 1-proteinase inhibitor (Pittsburgh) with trypsin-like serine proteinases

The rates of interaction of a number of serine proteinases with a mutant form of alpha 1-proteinase inhibitor (referred to as alpha 1-proteinase inhibitor (Pittsburgh)), in which a methionine-358 to arginine-358 mutation has occurred, have been determined. An approximately 6,000-fold increase in the second order association rate constant with human thrombin was observed (48 M-1 X s-1 for the normal protein to 3.1 X 10(5) M-1 X s-1 for the arginine mutant), confirming previously observed data using bovine thrombin (Owen, M.C., Brennan, S.O., Lewis, J.H. & Carrell, R.W. (1983) New England J. Med. 309, 694-698). However, substantial increases in the rates of association with other trypsin-like enzymes were also noted, indicating that the replacement of methionine by a basic residue affects all serine proteinases with this kind of specificity. There was a marked decrease in the rates of interaction of the Pittsburgh mutant with both human neutrophil elastase and porcine pancreatic elastase, the inhibitor being converted into lower molecular mass fragments after interaction with either enzyme. Butanedione caused a substantial loss in the inhibitory activity of the arginine mutant, while having no effect on the normal protein. These data, when compared to those previously reported for differences in reaction rates between normal and oxidized alpha 1-proteinase inhibitor (Beatty, K., Bieth, J. & Travis, J. (1980) J. Biol. Chem. 255, 3931-3934), are consistent with the interpretation that the amino acid in the P1-position at the reactive site of this protein has a marked effect on determining its primary specificity.     

Human cytotoxic lymphocyte granzyme B. Its purification from granules and the characterization of substrate and inhibitor specificity

Granzyme B has been purified to homogeneity from the granules of a human cytolytic lymphocyte line, Q31, in an enzymatically active form by a three-step procedure. Q31 granzyme B hydrolyzed Na-t-butyloxycarbonyl-L-alanyl-L-alanyl-L-aspartyl (Boc-Ala-Ala-Asp) thiobenzyl ester with a kcat of 11 +/- 5 mol/s/mol enzyme and catalytic efficiency kcat/Km of 76,000 +/- 44,000 M-1 s-1. The hydrolysis of Boc-Ala-Ala-Asp thiobenzyl ester by crude Q31 Percoll fractions paralleled the tryptase activity for granule-containing fractions, which showed that granzyme B was associated with granules. When chromatographed on Sephacryl S-300, Q31 granzyme B eluted in two broad bands corresponding to dimer and monomer, both of which electrophoresed at 35 kDa in reducing NaDodSo4 polyacrylamide, and both of which showed a lag phase in assays. The lag phase in assays could be extended with 0.03 mM pepstatin. Upon elution from ion-exchange chromatography Q31 granzyme B electrophoresed at 32 kDa in reducing NaDodSO4 polyacrylamide and did not have a lag phase in assays. The amino-terminal sequence of the 32-kDa Q31 granzyme B was identical to four other human cytotoxic T-lymphocyte granzymes B in 18 of 18 positions sequenced. Purified Q31 granzyme B had a preference for substrates with Glu or Asp as the residue amino-terminal to the scissile bond; little or no activity was noted with oligopeptide substrates for trypsin-like, chymotrypsin-like, and elastase-like proteases. Human plasma alpha 1-protease inhibitor, human plasma alpha 2-protease macroglobulin, soybean and lima-bean trypsin inhibitors, bovine aprotinin, phosphoramidon, and chymostatin inhibited Q31 granzyme B. The inhibition by alpha 1-protease inhibitor was rapid enough to be of physiological significance.     

Cathepsin L inactivates alpha 1-proteinase inhibitor by cleavage in the reactive site region

The lysosomal cysteine proteinases cathepsin L and cathepsin B were examined for their effect on the neutrophil elastase inhibitory activity of human alpha 1-proteinase inhibitor (alpha 1PI). Human cathepsin L catalytically inactivated human alpha 1PI by cleavage of the bonds Glu354-Ala355 and Met358-Ser359 (the serine proteinase inhibitory site). Cathepsin B did not inactivate alpha 1PI, even when equimolar amounts of enzyme were employed. Cathepsin L is the first human proteinase shown to catalytically inactivate alpha 1PI. These findings, in conjunction with other reports, suggest that alpha 1PI contains a proteolytically sensitive region encompassing residues 350-358. Taken together with the discovery of the elastinolytic activity of cathepsin L (Mason, R. W., Johnson, D. A., Barrett, A. J., and Chapman, H. A. (1986) Biochem. J. 233, 925-927), the present findings emphasize the possible importance of cathepsin L in the pathological proteolysis of elastin and diminish the role that can be attributed to cathepsin B in such processes.     

Acid-stable low molecular mass proteinase inhibitors in human lung lavage

An acid-stable, low molecular mass proteinase inhibitor, bronchial mucus proteinase inhibitor (BMPI), has been isolated from sputum and partially characterised. A single band with a modal molecular mass of 18 700 was observed following SDS-polyacrylamide gel electrophoresis. BMPI inhibited human leukocyte elastase, cathepsin G, trypsin and chymotrypsin, but not porcine pancreatic elastase. Although BMPI had a molecular mass close to the similarly isolated inhibitor of Girard et al. (Girard, F., Tournier, J.M., Polu, J.M. & Sadoul, P. (1980), Bull. Eur. Physiopathol. Respir. 16 (Suppl.) 237-245), and although it showed immunological cross reactivity to the low molecular mass inhibitor of Kramps et al. (Kramps, J.A., Franken, C., Meyer, C.J.L.M. & Dijkman, J.H. (1981) J. Histochem. Cytochem. 29, 712-719), it was found to have an amino-acid profile different to any previously described inhibitor. BMPI was detectable in bronchoalveolar lavage fluid collected from healthy and diseased human lungs. The median molar ratio of BMPI/alpha 1-proteinase inhibitor (alpha 1 PI) observed in these lavage samples was 0.7, which is generally higher than those derived from the data of other authors. This suggests that BMPI is a different protein to those previously described, although its exact relationship to other low molecular mass proteinase inhibitors remains to be determined.     

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.     

Human and mouse granzyme M display divergent and species-specific substrate specificities

Cytotoxic lymphocyte protease GrM (granzyme M) is a potent inducer of tumour cell death and a key regulator of inflammation. Although hGrM (human GrM) and mGrM (mouse GrM) display extensive sequence homology, the substrate specificity of mGrM remains unknown. In the present study, we show that hGrM and mGrM have diverged during evolution. Positional scanning libraries of tetrapeptide substrates revealed that mGrM is preferred to cleave after a methionine residue, whereas hGrM clearly favours a leucine residue at the P1 position. The kinetic optimal non-prime subsites of both granzymes were also distinct. Gel-based and complementary positional proteomics showed that hGrM and mGrM have a partially overlapping set of natural substrates and a diverged prime and non-prime consensus cleavage motif with leucine and methionine residues being major P1 determinants. Consistent with positional scanning libraries of tetrapeptide substrates, P1 methionine was more frequently used by mGrM as compared with hGrM. Both hGrM and mGrM cleaved α-tubulin with similar kinetics. Strikingly, neither hGrM nor mGrM hydrolysed mouse NPM (nucleophosmin), whereas human NPM was hydrolysed efficiently by GrM from both species. Replacement of the putative P1'-P2' residues in mouse NPM with the corresponding residues of human NPM restored cleavage of mouse NPM by both granzymes. This further demonstrates the importance of prime sites as structural determinants for GrM substrate specificity. GrM from both species efficiently triggered apoptosis in human but not in mouse tumour cells. These results indicate that hGrM and mGrM not only exhibit divergent specificities but also trigger species-specific functions.     

Subsite specificities of granzyme M: a study of inhibitors and newly synthesized thiobenzyl ester substrates

Granzyme M is a member of a family of granule serine proteases that participate in target cell death initiated by cytotoxic lymphocytes. The enzyme is almost exclusively expressed in NK cell types. Granzyme M cleaves at the carboxy side of amino acids with long, hydrophobic side chains like Met, Leu, and Nle. To further study the substrate specificity of the enzyme, a series of peptide thiobenzyl esters was synthesized. The hydrolysis of the substrates with murine and human recombinant forms of granzyme M was observed. The results show that the enzyme has a strong preference for Pro at the P2 position and Ala, Ser, or Asp at the P3 position. These results suggest that the protein residues of the S2 and S3 subsites form important binding interactions that aid in the selection of specific natural substrates for granzyme M. A series of inhibitors was also tested with granzyme M. None of the inhibitors were effective inactivators of granzyme M, including the general serine protease inhibitor, 3,4-dichloroisocoumarin, which is usually a potent inactivator of serine proteases. This suggests that inhibition of granzyme M may be difficult. Also reported for the first time is the method utilized to isolate granzyme M used in this and previous publications. The observations in this paper will be valuable in development of new potent inhibitors for granzyme M as well as assist in determining the biological function of the enzyme.     

Conversion of the Alzheimer's beta-amyloid precursor protein (APP) Kunitz domain into a potent human neutrophil elastase inhibitor

Site-specific mutagenesis techniques have been used to construct active site variants of the Kunitz-type protease inhibitor domain present in the Alzheimer's beta-amyloid precursor protein (APP-KD). Striking alteration of its protease inhibitory properties were obtained when the putative P1 residue, arginine, was replaced with the small hydrophobic residue valine. The altered protein was no longer inhibitory toward bovine pancreatic trypsin, human Factor XIa, mouse epidermal growth factor-binding protein, or bovine chymotrypsin, all of which are strongly inhibited by the unaltered APP-KD (Sinha, S., Dovey, H. F., Seubert, P., Ward, P. J., Blacher, R. W., Blaber, M., Bradshaw, R. A., Arici, M., Mobley, W. C., and Lieberburg, I. (1990) J. Biol. Chem. 265, 8983-8985). Instead, the P1-Val-APP-KD was a potent inhibitor of human neutrophil elastase, with a Ki = 0.8 nM, as estimated by the inhibition of the activity of human neutrophil elastase measured using a chromogenic substrate. It also inhibited the degradation of insoluble elastin by the enzyme virtually stoichiometrically. Replacement of the P1' (Ala) and P2' (Met) residues of P1-Val-MKD with the corresponding residues (Ser, Ile) from alpha 1-proteinase inhibitor resulted in an inactive protein, underscoring the mechanistic differences between the serpins from the Kunitz-type protease inhibitor family. These results confirm the importance of the P1 arginine residue of APP-KD in determining inhibitory specificity, and are also the first time that a single amino acid replacement has been shown to generate a specific potent human neutrophil elastase inhibitor from a human KD sequence.     

Inhibition of neutrophil elastase by recombinant human proteinase inhibitor 9.

Proteinase inhibitor PI9 (PI9) is an intracellular 42-kDa member of the ovalbumin family of serpins that is found primarily in placenta, lung and lymphocytes. PI9 has been shown to be a fast-acting inhibitor of granzyme B in vitro, presumably through the utilization of Glu(340) as the P(1) inhibitory residue in its reactive site loop. In this report, we describe the inhibition of human neutrophil elastase by recombinant human PI9. Inhibition occurred with an overall K(i)' of 221 pM and a second-order association rate constant of 1.5 x 10(5) M(-1) s(-1), indicating that PI9 is a potent inhibitor of this serine proteinase in vitro. In addition, incubation of recombinant PI9 with native neutrophil elastase resulted in the formation of an SDS-resistant 62-kDa complex. Amino-terminal sequence analyses provided evidence that inhibition of elastase occurred through the use of Cys(342) as the reactive P(1) amino acid residue in the PI9 reactive site loop. Thus, PI9 joins its close relatives PI6 and PI8 as having the ability to utilize multiple reactive site loop residues as the inhibitory P(1) residue to expand its inhibitory spectrum.     

Inhibition of alpha 2-macroglobulin-bound trypsin by soybean trypsin inhibitor

Soybean trypsin inhibitor, a protein of Mr = 20,000, has been used to assess the degree of inaccessibility of porcine trypsin within the alpha 2-macroglobulin-trypsin complex. The interaction between alpha 2-macroglobulin-bound trypsin and the inhibitor was demonstrated by affinity chromatography and trypsin inhibition. Whereas the free trypsin-inhibitor association is very fast (k = 1.2 X 10(7) M-1 s-1), the reaction between complexed trypsin and inhibitor takes 10 h to reach equilibrium. In addition, alpha 2-macroglobulin reduces, by several orders of magnitude, the affinity of trypsin for the inhibitor. Only one of the two trypsin molecules of the ternary (trypsin)2-alpha 2-macroglobulin complex is readily accessible to soybean inhibitor. It is postulated that the recently discovered proximity of the alpha 2-macroglobulin binding sites (Pochon, F., Favaudon, V., Tourbez-Perrin, M., and Bieth, J. (1981) J. Biol. Chem. 256, 547-550) accounts for this behavior. In the light of these results it is concluded that the proteinase binding sites are localized on the alpha 2-macroglobulin surface and that the two subunits of this protein are either not identical or not symmetrically arranged.     

Interaction of chymotrypsinogens with alpha 1-protease inhibitor

In a previous report [Largman, C., Brodrick, J.W., Geokas, M.C., Sischo, W.M., & Johnson, J.H. (1979) J. Biol. Chem. 254, 8516-8523] it was demonstrated that human proelastase 2 and alpha 1-protease inhibitor react slowly to form a complex that is stable to denaturation with sodium dodecyl sulfate and beta-mercaptoethanol and that the zymogen can be recovered from the isolated complex following dissociation by hydroxylamine. The present report demonstrates that bovine chymotrypsinogen A reacts with human alpha 1-protease inhibitor in a very similar manner. The rate of complex formation was measured by two methods. In the first, the reaction was followed by determining the loss of the inhibitory activity of alpha 1-protease inhibitor as a function of time. A second-order rate constant for complex formation formation (pH 7.6, 36 degrees C) of 12.9 +/- 2.4 M-1s-1 was obtained. In the second procedure, the reaction of fluorescein isothiocyanate labeled chymotrypsinogen A with alpha 1-protease inhibitor was measured by fluorescence polarization. A second-order rate constant (pH 7.6, 37 degrees C) of 13.9 +/- 2.1 M-1s-1 was obtained. The rate of complex formation is approximately 10(-5) of that measured for the reaction of bovine chymotrypsin with alpha 1-protease inhibitor. Dissociation of the complex was not observed after dilution or the addition of excess bovine alpha-chymotrypsin. As judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments, human chymotrypsinogens I and II react with alpha 1-protease inhibitor at rates that are approximatley equivalent to that determined for bovine chymotrypsinogen A. In contrast, bovine trypsinogen reacts very slowly with alpha 1-protease inhibitor, at a rate that is at most 10(-2) of that of bovine chymotrypsinogen A. These results suggest that zymogens react with alpha 1-protease inhibitor by virtue of partially formed active sites and that the potential active-site specificity of the zymogen in part determines the rate of complex formation.     

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, VII. Determination of the amino-acid sequence of the trypsin-released inhibitor from bovine inter-alpha-trypsin inhibitor

An acid-labile proteinase inhibitor, quite similar to human inter-alpha-trypsin inhibitor, was isolated from bovine serum. An acid-resistant 30-kDa inhibitor, exhibiting properties similar to human HI-30, was also isolated. Upon limited proteolysis of both bovine inhibitors, active 14-kDa domains are released which are identical with respect to molecular mass and acid resistance. The amino-acid sequence determination of these fragments revealed a strong homology to the corresponding human inhibitor HI-14 which is characterized by two covalently linked Kunitz-type domains. The reactive-site residue is leucine in the N-terminal domain (in the human inhibitor methionine) and arginine in the C-terminal domain in both bovine and human inhibitor.     

Human pregnancy zone protein and alpha 2-macroglobulin. High-affinity binding of complexes to the same receptor on fibroblasts and characterization by monoclonal antibodies

Pregnancy zone protein (PZP) was isolated from late pregnancy serum and examined for binding to normal skin fibroblasts in culture. A high-affinity binding site on these cells is demonstrated for PZP reacted with methylamine. Experiments with alpha 2-macroglobulin (alpha 2M) and PZP, both modified by methylamine, showed this receptor to be identical to the previously characterized receptor for alpha 2M-proteinase complexes (Van Leuven, F., Cassiman, J.J., and Van den Berghe, H. (1979) J. Biol. Chem. 254, 5155-5160). With available monoclonal antibodies directed toward alpha 2M and prepared toward PZP, only a limited cross-reaction was observed. We obtained a monoclonal antibody which defines a neo-antigenic site on PZP-methylamine, completely analogous to the monoclonal antibody F2B2, which was previously shown to define a neo-antigenic site on alpha 2M complexes (Marynen, P., Van Leuven, F., Cassiman, J.J., and Van den Berghe, H. (1981) J. Immunol. 127, 1782-1786). These results provide evidence for the homologous function of alpha 2M and PZP as proteinase scavengers. The need for an extra proteinase inhibitor of the alpha 2M-type in pregnancy is discussed. The monoclonal antibodies now available will prove helpful in quantitation and eventually isolation of proteinase complexes of alpha 2M and PZP.     

Characterization of proteinase-3 (PR-3), a neutrophil serine proteinase. Structural and functional properties

Proteinase 3 (PR-3) is a human polymorphonuclear leukocyte (PMNL) serine proteinase that degrades elastin in vitro and causes emphysema when administered by tracheal insufflation to hamsters (Kao, R. C., Wehner, N. G., Skubitz, K. M., Gray, B. H., and Hoidal, J. R. (1988) J. Clin. Invest. 82, 1963-1973). We have determined the primary structure of several PR-3 peptides and have analyzed catalytic properties of the enzyme. The enzyme has considerable amino acid sequence homology with two other well characterized PMNL neutral serine proteinases, elastase and cathepsin G. Furthermore, the NH2-terminal amino acid sequence of PR-3 is identical to that of the target antigen of the anti-neutrophil cytoplasmic autoantibodies associated with Wegener's granulomatosis. PR-3 degrades a variety of matrix proteins including fibronectin, laminin, vitronectin, and collagen type IV. It shows no or minimal activity against interstitial collagens types I and III, respectively. The analysis of peptides generated by PR-3 digestion of insulin chains and the activity profile against a panel of chromogenic synthetic peptide substrates show that PR-3 prefers small aliphatic amino acids (alanine, serine, and valine) at the P1 site. The elastase-like specificity of PR-3 is consistent with its striking sequence homology to elastase at substrate binding sites. PR-3 is inhibited by alpha 1-proteinase inhibitor (ka = 8.1 x 10(6) M-1 S-1; delay time = 25 ms) and alpha 2-macroglobulin (ka = 1.1 x 10(7) M-1 S-1; delay time = 114 ms) but not by alpha 1-anti-chymotrypsin. In contrast to elastase and cathepsin G, PR-3 is not inhibited by secretory leukoprotease inhibitor and is weakly inhibited by eglin c. Thus, PR-3 is distinct from the other PMNL proteinases.     

Biosynthesis and incorporation into protein of norleucine by Escherichia coli

The methionine analog norleucine was produced during the synthesis of bovine somatotropin by Escherichia coli strain W3110G containing the recombinant plasmid pBGH1. Norleucine was generated by the leucine biosynthetic pathway from pyruvate or alpha-ketobutyrate in place of alpha-ketoisovalerate as the initial substrate. The intracellular level of norleucine was high enough to permit the analog to compete successfully with methionine for incorporation into protein. Two ways were found to prevent either the formation of norleucine or its incorporation into protein. The endogenous synthesis of norleucine was eliminated by deleting the leucine operon. The addition of sufficient methionine or 2-hydroxy-4-methylthiobutanoic acid, a precursor of methionine, to the culture medium prevented any norleucine from being incorporated into protein.     

Proteolysis of human alpha 2-macroglobulin without hydrolysis of the internal thiolesters or expression of the receptor recognition site

Proteolysis of human alpha 2-macroglobulin (alpha 2M) in the bait region is the prerequisite and necessary trigger for the trapping of the proteinase by a massive conformational change of alpha 2M. This labilization of the native conformation of alpha 2M is mediated by activation of the internal thiolesters, but the underlying mechanism is unknown. We now describe observations on proteolysis of human alpha 2M without concomitant hydrolysis of the internal thiolesters or conformational change. This proteolysis was obtained with a novel bacterial proteinase we recently used to isolate the receptor-binding domain from alpha 2M (Van Leuven, F., Marynen, P., Sottrup-Jensen, L., Cassiman, J.-J., and Van Den Berghe, H. (1986) J. Biol. Chem. 261, 11369-11373). This proteinase is not inhibited by alpha 2M, and therefore it was possible to study its effect on native alpha 2M at pH 4.5, conditions used previously to produce the receptor-binding domain (Van Leuven, F., Marynen, P., Sottrup-Jensen, L., Cassiman, J.-J., and Van Den Berghe, H. (1986) J. Biol. Chem. 261, 11369-11373). The major observations are that despite extensive proteolysis, alpha 2M largely retained its native conformation as shown by rate electrophoresis, the absence of binding of monoclonal antibody F2B2, and the incorporation of [14C]methylamine into a 145-kDa fragment of alpha 2M. Moreover, the derivative still bound trypsin to 88% of control values. Treatment of the derivative with trypsin or methylamine produced the conformational change as with intact alpha 2M, and concomitantly released the receptor-binding domain. This indicated that proteolysis at Lys1313-Glu also proceeded in native alpha 2M. At least one more major proteolysis site was deduced from the observation of a 27-kDa heat-induced fragment, the 145-kDa [14C]methylamine-labeled fragment, and from the presence of the 20-kDa receptor-binding domain. These results demonstrate indirectly the particular relation of the bait region to the internal thiolesters and illustrate further the domain-structure of alpha 2M and the expression of the receptor-recognition site by activation of the internal thiolesters.     

Studies on the substrate specificity of neutral alpha-mannosidase purified from Japanese quail oviduct by using sugar chains from glycoproteins.

The substrate specificity of neutral alpha-mannosidase purified from Japanese quail oviduct [Oku, H., Hase, S., & Ikenaka, T. (1991) J. Biochem. 110, 29-34] was analyzed by using 21 oligomannose-type sugar chains. The enzyme activated with Co2+ hydrolyzed the Man alpha 1-3 and Man alpha 1-6 bonds from the non-reducing termini of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc (M5A), but hardly hydrolyzed the Man alpha 1-2 bonds of Man9GlcNAc2. The hydrolysis rate decreased as the reducing end of substrates became more bulky: the hydrolysis rate for the pyridylamino (PA) derivative of M5A as to that of M5A was 0.8; the values for M5A-Asn and Taka-amylase A having a M5A sugar chain being 0.5 and 0.04, respectively. The end product was Man beta 1-4GlcNAc2. For the substrates with the GlcNAc structure at their reducing ends (Man5GlcNAc, Man6GlcNAc and Man9GlcNAc), the hydrolysis rate was remarkably increased: Man5GlcNAc was hydrolyzed 16 times faster than M5A, and Man2GlcNAc 40 times faster than Man9GlcNAc2. The enzyme did not hydrolyze Man alpha 1-2 residue(s) linked to Man alpha 1-3Man beta 1-4GlcNAc. The end products were as follows: [formula; see text] These results suggest that oligomannose-type sugar chains with the GlcNAc structure at their reducing ends seem to be native substrates for neutral alpha-mannosidase and the enzyme seems to hydrolyze endo-beta-N-acetylgucosaminidase digests of oligomannose-type sugar chains in the cytosol.