Pseudomonas aeruginosa exotoxin A: effects of mutating tyrosine-470 and tyrosine-481 to phenylalanine

Directed mutagenesis was used to probe the functions of Tyr-470 and Tyr-481 of Pseudomonas aeruginosa exotoxin A (ETA) with respect to cytotoxicity, ADP-ribosylation of elongation factor 2 (EF-2), and NAD-glycohydrolase activity. Both of these residues lie in the active site cleft, close to Glu-553, a residue believed to play a direct role in catalysis of ADP-ribosylation of EF-2. Substitution of Tyr-470 with Phe caused no change in any of these activities, thus eliminating the possibility that the phenolic hydroxyl group of Tyr-470 might be directly involved in catalysis. Mutation of Tyr-481 to Phe caused an approximately 10-fold reduction in NAD:EF-2 ADP-ribosyltransferase activity and cytotoxicity but no change in NAD-glycohydrolase activity. The latter mutation did not alter the KM of NAD in the NAD-glycohydrolase reaction, which suggests that the phenolic hydroxyl of Tyr-481 does not participate in NAD binding. We hypothesize that the phenolic hydroxyl of Tyr-481 may be involved in the interaction of the toxin with substrate EF-2.     

Identification of the carboxyl-terminal amino acids important for the ADP-ribosylation activity of Pseudomonas exotoxin A

The ADP-ribosylation domain of Pseudomonas exotoxin A (PE) has been identified to reside in structural domain III (residues 405-613) and a portion of domain Ib (residues 385-404) of the molecule (Hwang, J., FitzGerald, D. J., Adhya, S., and Pastan, I. (1987) Cell 48, 129-136). To further determine the carboxyl end region essential for ADP-ribosylation activity, we constructed sequential deletions at the carboxyl-terminal of PE. Our results show that a clone with a deletion of the carboxyl-terminal amino acid residues from Arg-609 to Lys-613 and replaced with Arg-Asn retained wild-type PE ADP-ribosylation activity. Deletion of the terminal amino acid residues from Ala-596 to Lys-613 and replaced with Val-Ile-Asn reduced ADP-ribosylation activity by 75%, while deletions of 36 or more amino acids from the carboxyl terminus completely lose their ADP-ribosylation activity. These modified PEs were also examined for their ability to block PE cytotoxicity. Our results shown that modified PEs which lost their ADP-ribosylation activity correspondingly lost their cytotoxicity. Furthermore, extracts containing PE fragments without ADP-ribosylation activity were able to block the cytotoxic activity of intact PE. Our results thus indicate that carboxyl-terminal amino acids in the Ser-595 region are crucial for ADP-ribosylation activity and, consequently, cytotoxicity of PE. The modified PEs which have lost their ADP-ribosylation activity may also be a route to new PE vaccines.     

A recombinant form of Pseudomonas exotoxin directed at the epidermal growth factor receptor that is cytotoxic without requiring proteolytic processing.

Pseudomonas exotoxin A is composed of three structural domains that mediate cell recognition (I), membrane translocation (II), and ADP-ribosylation (III). Within the cell, the toxin is cleaved within domain II to produce a 37-kDa carboxyl-terminal fragment, containing amino acids 280-613, which is translocated to the cytosol and causes cell death. In this study, we constructed a mutant protein (PE37), composed of amino acids 280-613 of Pseudomonas exotoxin A, which does not require proteolysis to translocate. PE37 was targeted specifically to cells with epidermal growth factor receptors by inserting transforming growth factor-alpha (TGF-alpha) after amino acid 607 near the carboxyl terminus of Pseudomonas exotoxin A. PE37/TGF-alpha was very cytotoxic to cells with epidermal growth factor receptors. It was severalfold more cytotoxic than a derivative of full-length Pseudomonas exotoxin A containing TGF-alpha in the same position, probably because the latter requires intracellular proteolytic processing to exhibit its cytotoxicity, and proteolytic processing is not 100% efficient. Deletion of 2, 4, or 7 amino acids from the amino terminus of PE37/TGF-alpha greatly diminished cytotoxic activity, indicating the need for a proper amino-terminal sequence. In addition, a mutant containing an internal deletion of amino acids 314-380 was minimally active, indicating that other regions of domain II are also required for the cytotoxic activity of Pseudomonas exotoxin A.     

Purification and characterization of an erythroid cell-specific factor that binds the murine alpha- and beta-globin genes.

An erythroid cell-specific nuclear factor that binds tightly to a sequence motif (5'-GATAAGGA-3') shared by many erythroid cell-specific promoters was purified to homogeneity by DNA sequence affinity chromatography. Visualization of the purified factor, which we term EF-1, showed a simple pattern comprising a polypeptide doublet with Mrs of 18,000 and 19,000. We confirmed that these species account for EF-1-binding activity by eluting the polypeptides from sodium dodecyl sulfate-polyacrylamide gels and renaturing the appropriate binding activity. Using the purified polypeptides, we mapped seven factor-binding sites that are dispersed across the murine alpha- and beta-globin genes. The murine alpha-globin gene is flanked by at least two EF-1-binding sites. One site is centered at nucleotide (nt) -180 (with respect to the alpha-globin cap site). A fivefold-weaker site is located downstream of the alpha-globin poly(A) addition site, at nt +1049. We mapped five EF-1-binding sites near the murine beta-globin gene. The strongest site was centered at nt -210. Four additional sites were centered at nt -266 (adjacent to the binding site of a factor present in both murine erythroleukemia and Raji cells), -75 (overlapping the beta-globin CCAAT box), +543 (within the second intervening sequence), and -111.     

Properties of chimeric toxins with two recognition domains: interleukin 6 and transforming growth factor alpha at different locations in Pseudomonas exotoxin.

Pseudomonas exotoxin (PE) is a potent cytotoxic agent that is composed of 613 amino acids arranged into three major domains. We have previously identified two positions where ligands can successfully be placed in PE to direct it to cells with specific surface receptors. One site is at the amino terminus and the other is close to but not at the C-terminus. To examine the possibility of constructing oncotoxins with two different recognition elements that will bind to two different receptors, we have placed cDNAs encoding either transforming growth factor alpha (TGF alpha) or interleukin 6 (IL6) at the 5' end of a PE gene and also inserted a cDNA encoding TGF alpha near the 3' end of the PE gene. The plasmids encoding these chimeric toxins were expressed in Escherichia coli and the chimeric proteins purified to near homogeneity. In all the new toxins, the TGF alpha near the C-terminus was inserted after amino acid 607 of PE and followed by amino acids 604-613 so that the correct PE C-terminus (REDLK) was preserved. For each chimera, the toxin portion was either PE4E, in which the cell binding domain (domain Ia) is mutated, PE40, in which domain Ia is deleted, or PE38, in which domain Ia and part of domain Ib are deleted. These derivatives of PE do not bind to the PE receptor and allow 607, 355, or 339 amino acids, respectively, between the two ligands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional analysis of exotoxin A-related protein of Pseudomonas aeruginosa lacking residues 225-412

The crystal structure of the exotoxin A (ETA) of Pseudomonas aeruginosa showed that this protein is folded into three distinct domains. Domain I (Ia and Ib), the amino-terminal domain, is the receptor-binding domain of ETA and domain III, the carboxy-terminal domain, is responsible for the ADP-ribosyl transferase activity of the toxin. To elucidate the function(s) of domains 1b and II in the intoxication process and to define the region of the domain III necessary for ADP-ribosylating activity, a defined deletion in the structural gene of P. aeruginosa ETA encompassing residues 225-412 was constructed and an ETA-related product DeID, (from which all of domains II and Ib were deleted) was expressed. The ETA-related protein did not penetrate sensitive cells, but retained the same specific activity to ADP-ribosylate elongation factor-2 as wild-type toxin. This suggests that domain II is necessary to allow toxin internalization by sensitive cells and that the absence of domain Ib does not interfere with enzymic activity. The domain strictly involved in ADP-ribosylation activity encompasses residues 412-613.     

Expression and secretion of the S-1 subunit and C180 peptide of pertussis toxin in Escherichia coli.

The structural gene of the S-1 subunit of pertussis toxin (rS-1) and the catalytic C180 peptide of the S-1 subunit (C180 peptide) were independently subcloned downstream of the tac promoter in Escherichia coli. Both constructions included DNA encoding for the predicted leader sequence of the S-1 subunit which was inserted between the tac promoter and the structural gene. E. coli containing the plasmids encoding for rS-1 and C180 peptide produced a peptide that reacted with anti-pertussis toxin antibody and had a molecular weight corresponding to that of the cloned gene; some degradation of rS-1 was observed. Extracts of E. coli containing plasmids encoding for rS-1 and the C180 peptide possessed ADP-ribosyltransferase activity. Subcellular fractionation showed that both rS-1 and the C180 peptide were present in the periplasm, indicating that E. coli recognized the pertussis toxin peptide leader sequence. The protein sequence of the amino terminus of the C180 peptide was identical to that of authentic S-1 subunit produced by Bordetella pertussis, which showed that E. coli leader peptidase correctly processed the pertussis toxin peptide leader sequence. Two single amino acid substitutions at residue 26 (C180I-26) and residue 139 (C180S-139) which were previously shown to reduce ADP-ribosyltransferase activity were introduced into the C180 peptide. C180I-26 possessed approximately 1% of the NAD-glycohydrolase activity of the C180 peptide, suggesting that tryptophan 26 functions in the interaction of NAD with the C180 peptide. In contrast, C180S-139 possessed essentially the same level of NAD-glycohydrolase activity as the C180 peptide, suggesting that glutamic acid 139 does not function in the interaction of NAD but plays a role in a later step in the ADP-ribosyltransferase reaction.     

Immunochemical analysis of Pseudomonas aeruginosa exotoxin A. Analysis of the His426 determinant.

This study describes a combined immunochemical and genetic approach defining a site on Pseudomonas aeruginosa exotoxin A (ETA) which is critical to the ADP-ribosyltransferase (ADPRT) activity of the toxin. The sequential epitope of a monoclonal antibody (TO-1) which binds to domain III (residues 405-613), containing the ADPRT activity of ETA, has been defined using a series of synthetic peptides. This epitope spans residues 422-432 which composes the major alpha-helical segment of domain III and includes His426 which has previously been shown to be essential for ADPRT activity (Wozniak, D.J., Hsu, L.-Y., and Galloway, D. R. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8880-8884). The critical His426 residue which projects into a major cleft becomes exposed when the ETA protein is in an ADPRT-active configuration. Since the TC-1 mAb does not block the binding of NAD+, it is possible that the alpha-helix site containing the TC-1 epitope and the His426 residue is associated with the interaction between ETA and its elongation factor 2 substrate.     

Polypeptide domains of ADP-ribosyltransferase obtained by digestion with plasmin

Proteolysis by plasmin inactivates bovine ADP-ribosyltransferase; therefore, enzymatic activity depends exclusively on the intact enzyme molecule. The transferase was hydrolyzed by plasmin to four major polypeptides, which were characterized by affinity chromatography and N-terminal sequencing. Based on the cDNA sequence for human ADP-ribosyltransferase enzyme [Uchida, K., Morita, T., Sato, T., Ogura, T., Yamashita, R., Noguchi, S., Suzuki, H., Nyunoya, H., Miwa, M., & Sugimura, T. (1987) Biochem. Biophys. Res. Commun. 148, 617-622], a polypeptide map of the bovine enzyme was constructed by superposing the experimentally determined N-terminal sequences of the isolated polypeptides on the human sequence deduced from its cDNA. Two polypeptides, the N-terminal peptide (Mr 29,000) and the polypeptide adjacent to it (Mr 36,000), exhibited binding affinities toward DNA, whereas the C-terminal peptide (Mr 56,000), which accounts for the rest of the transferase protein, bound to the benzamide-Sepharose affinity matrix, indicating that it contains the NAD+-binding site. The fourth polypeptide (Mr 42,000) represents the C-terminal end of the larger C-terminal fragment (Mr 56,000) and was formed by a single enzymatic cut by plasmin of the polypeptide of Mr 56,000. The polypeptide of Mr 42,000 still retained the NAD+-binding site. The plasmin-catalyzed cleavage of the polypeptide of Mr 56,000-42,000 was greatly accelerated by the specific ligand NAD+. Out of a total of 96 amino acid residues sequenced here, there were only 6 conservative replacements between human and bovine ADP-ribosyltransferase.     

Biochemical analysis of CRM 66. A nonfunctional Pseudomonas aeruginosa exotoxin A

Pseudomonas aeruginosa exotoxin A (ETA) is an ADP-ribosyltransferase which inactivates protein synthesis by covalently attaching the ADP-ribose portion of NAD+ onto eucaryotic elongation factor 2 (EF-2). A direct biochemical comparison has been made between ETA and a nonenzymatically active mutant toxin (CRM 66) using highly purified preparations of each protein. The loss of ADP-ribosyltransferase activity and subsequent cytotoxicity have been correlated with the presence of a tyrosine residue in place of a histidine at position 426 in CRM 66. In the native conformation, CRM 66 demonstrated a limited ability (by a factor or at least 100,000) to modify EF-2 covalently and lacked in vitro and in vivo cytotoxicity, yet CRM 66 appeared to be normal with respect to NAD+ binding. Upon activation with urea and dithiothreitol, CRM 66 lost ADP-ribosyltransferase activity entirely yet CRM 66 retained the ability to bind NAD+. Replacement of Tyr-426 with histidine in CRM 66 completely restored cytotoxicity and ADP-ribosyltransferase activity. These results support previous findings from this laboratory (Wozniak, D. J., Hsu, L.-Y., and Galloway, D. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8880-8884) which suggest that the His-426 residue of ETA is not involved in NAD+ binding but appears to be associated with the interaction between ETA and EF-2.     

A dipeptide insertion in domain I of exotoxin A that impairs receptor binding

Deletions within the structural exotoxin A gene of 27 or 119 amino acids in domain I of the mature polypeptide, or of 88 or 105 amino acids in domains I and II, resulted in the synthesis of exotoxin A (ETA) polypeptides that were not secreted from Pseudomonas aeruginosa hosts but were localized in the cell membrane. Insertions of a hexanucleotide sequence, either pCGAGCT or pCGAATT, at TaqI sites within the gene resulted in variant exotoxin A polypeptides which were secreted normally. pCGAGCT causes insertion of either Glu-Leu or Ser-Ser in the amino acid sequence of the toxin, while pCGAATT causes insertion of either Glu-Phe or Asn-Ser dipeptides. Although the cytotoxicity of eight variants was unimpaired, that of four others was reduced, and one variant which had a Glu-Phe insert between residues 60 and 61 (ETA-60EF61) was 500-fold less cytotoxic than wild-type exotoxin A. Purified ETA-60EF61 dissociated much faster from mouse LMTK- cells than wild-type ETA, suggesting that the insertion impaired the ability of ETA-60EF61 to interact with exotoxin A receptors. The location of the insert is within a major concavity on the surface of domain I of the exotoxin A molecule, suggesting that this concavity is important for toxin-receptor interaction.     

Proteases from human immunodeficiency virus and avian myeloblastosis virus show distinct specificities in hydrolysis of multidomain protein substrates.

The virally encoded proteases from human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) have been compared relative to their ability to hydrolyze a variant of the three-domain Pseudomonas exotoxin, PE66. This exotoxin derivative, missing domain I and referred to as LysPE40, is made up of a 13-kilodalton NH2-terminal translocation domain II connected by a segment of 40 amino acids to enzyme domain III of the toxin, a 23-kilodalton ADP-ribosyltransferase. HIV protease hydrolyzes two peptide bonds in LysPE40, a Leu-Leu bond in the interdomain region and a Leu-Ala bond in a nonstructured region three residues in from the NH2-terminus. Neither of these sites is cleaved by the AMV enzyme; hydrolysis occurs, instead, at an Asp-Val bond in another part of the interdomain segment and at a Leu-Thr bond in the NH2-terminal region of domain II. Synthetic peptides corresponding to these cleavage sites are hydrolyzed by the individual proteases with the same specificity displayed toward the protein substrate. Peptide substrates for one protease are neither substrates nor competitive inhibitors for the other. A potent inhibitor of HIV type 1 protease was more than 3 orders of magnitude less active toward the AMV enzyme. These results suggest that although the crystallographic models of Rous sarcoma virus protease (an enzyme nearly identical to the AMV enzyme) and HIV type 1 protease show a high degree of similarity, there exist structural differences between these retroviral proteases that are clearly reflected by their kinetic properties.     

Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine

Glutamic acid-148, an active-site residue of diphtheria toxin identified by photoaffinity labeling with NAD, was replaced with aspartic acid, glutamine, or serine by directed mutagenesis of the F2 fragment of the toxin gene. Wild-type and mutant F2 proteins were synthesized in Escherichia coli, and the corresponding enzymic fragment A moieties (DTA) were derived, purified, and characterized. The Glu----Asp (E148D), Glu----Gln (E148Q), and Glu----Ser (E148S) mutations caused reductions in NAD:EF-2 ADP-ribosyltransferase activity of ca. 100-, 250-, and 300-fold, respectively, while causing only minimal changes in substrate affinity. The effects of the mutations on NAD-glycohydrolase activity were considerably different; only a 10-fold reduction in activity was observed for E148S, and the E148D and E148Q mutants actually exhibited a small but reproducible increase in NAD-glycohydrolytic activity. Photolabeling by nicotinamide-radiolabeled NAD was diminished ca. 8-fold in the E148D mutant and was undetectable in the other mutants. The results confirm that Glu-148 plays a crucial role in the ADP-ribosylation of EF-2 and imply an important function for the side-chain carboxyl group in catalysis. The carboxyl group is also important for photochemical labeling by NAD but not for NAD-glycohydrolase activity. The pH dependence of the catalytic parameters for the ADP-ribosyltransferase reaction revealed a group in DTA-wt that titrates with an apparent pKa of 6.2-6.3 and is in the protonated state in the rate-determining step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of a region of the S1 subunit of pertussis toxin required for efficient ADP-ribosyltransferase activity

Purified recombinant S1 subunit of pertussis toxin (rS1) possessed similar NAD glycohydrolase and ADP-ribosyltransferase activities as S1 subunit purified from pertussis toxin. Purified rS1 and C180 peptide, a deletion peptide which contains amino acids 1-180 of rS1, had Km values for NAD of 24 and 13 microM and kcat values of 22 and 24 h-1, respectively, in the NAD glycohydrolase reaction. In contrast, under linear velocity conditions, the C180 peptide possessed less than 1% of the ADP-ribosyltransferase activity of rS1 using transducin as target. Radiolabeled tryptic peptides of transducin that had been ADP-ribosylated by either rS1 or C180 peptide were identical which suggested that both rS1 and C180 peptide ADP-ribosylated the same amino acid within transducin. To extend the functional primary amino acid map of the S1 subunit, two carboxyl-terminal deletions were constructed. One deletion, C195, removed the 40 carboxyl-terminal amino acids and the other, C219, removed the 16 carboxyl-terminal amino acids of the S1 subunit. Both C195 and C219 migrated in reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular masses of 22,000 and 27,500 Da, respectively. Relative to the C180 peptide C195 possessed 10-20-fold increase and C219 possessed 100-150-fold increase in ADP-ribosyltransferase activities. In addition, C219 appeared to have the same ADP-ribosyltransferase activity as rS1. These studies indicate that (i) rS1, purified from Escherichia coli, possesses biochemical properties similar to S1 subunit purified from pertussis toxin, (ii) amino acids 1-180 of the S1 subunit contain residues required for NAD binding, N-glycosidic cleavage, and transfer of ADP-ribose to transducin, and (iii) residues between 181 and 219 of the S1 subunit are required for efficient ADP-ribosyltransferase activity.     

Mutational analysis of domain I of Pseudomonas exotoxin. Mutations in domain I of Pseudomonas exotoxin which reduce cell binding and animal toxicity

Pseudomonas exotoxin (PE) is a single polypeptide chain that contains 613 amino acids and is arranged into three structural domains. Domain I is responsible for cell recognition, II for translocation of PE across membranes and III for ADP ribosylation of elongation factor 2. Treatment of PE with reagents that react with lysine residues has been shown to lead to a reduction in cytotoxic activity apparently due to a modification of domain I (Pirker, R., FitzGerald, D. J. P., Hamilton, T. C., Ozols, R. F., Willingham, M. C., and Pastan, S. (1985) Cancer Res. 45, 751-757). To determine which lysine residues are important in cell recognition, all 12 lysines in domain I were converted to glutamates by site-directed mutagenesis. Also, two deletion mutants encompassing almost all of domain I (amino acids 4-252) or most of domain I (amino acids 4-224) were studied. The mutant proteins were produced in Escherichia coli, purified, and tested for their cytotoxic activity against Swiss 3T3 cells and in mice. The data indicate that conversion of lysine 57 to glutamate reduces cytotoxic activity towards 3T3 cells 50-100-fold and in mice about 5-fold. Deletion of amino acids 4-224 causes a similar reduction in toxicity towards cells and mice. Deletion of most of the rest of domain I (amino acids 4-252) causes a further reduction in toxicity toward cells and mice indicating this second region between amino acids 225 and 252 of domain I is also important in the toxicity of PE. Competition assays indicated that the ability of PEGlu57 to bind to 3T3 cells was greatly diminished, accounting for its diminished cytotoxic activity.     

Use of synthetic peptides and site-specific antibodies to localize a diphtheria toxin sequence associated with ADP-ribosyltransferase activity.

Diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A have the same molecular mechanism of toxicity; both toxins ADP-ribosylate a modified histidine residue in elongation factor 2. To help identify amino acids involved in this reaction, sequences in DT that share homology with P. aeruginosa exotoxin A were synthesized and examined for a role in the ADP-ribosyltransferase reaction. By using this approach, residues 32 to 54 of DT were found to define an epitope associated with antibody-mediated inhibition of DT enzyme activity. This lends further support to the notion that residues in this region of DT are involved in the enzymatic reaction.     

Crystal structure of the C3bot-RalA complex reveals a novel type of action of a bacterial exoenzyme

C3 exoenzymes from bacterial pathogens ADP-ribosylate and inactivate low-molecular-mass GTPases of the Rho subfamily. Ral, a Ras subfamily GTPase, binds the C3 exoenzymes from Clostridium botulinum and C. limosum with high affinity without being a substrate for ADP ribosylation. In the complex, the ADP-ribosyltransferase activity of C3 is blocked, while binding of NAD and NAD-glycohydrolase activity remain. Here we report the crystal structure of C3 from C. botulinum in a complex with GDP-bound RalA at 1.8 A resolution. C3 binds RalA with a helix-loop-helix motif that is adjacent to the active site. A quaternary complex with NAD suggests a mode for ADP-ribosyltransferase inhibition. Interaction of C3 with RalA occurs at a unique interface formed by the switch-II region, helix alpha3 and the P loop of the GTPase. C3-binding stabilizes the GDP-bound conformation of RalA and blocks nucleotide release. Our data indicate that C. botulinum exoenzyme C3 is a single-domain toxin with bifunctional properties targeting Rho GTPases by ADP ribosylation and Ral by a guanine nucleotide dissociation inhibitor-like effect, which blocks nucleotide exchange.     

Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02

Two fibrinolytic enzymes (QK-1 and QK-2) purified from the supernatant of Bacillus subtilis QK02 culture broth had molecular masses of 42,000 Da and 28,000 Da, respectively. The first 20 amino acids of the N-terminal sequence are AQSVPYGISQ IKAPALHSQG. The deduced protein sequence and its restriction enzyme map of the enzyme QK-2 are different from those of other proteases. The enzyme QK-2 digested not only fibrin but also a subtilisin substrate, and PMSF inhibited its fibrinolytic and amidolytic activities completely; while QK-1 hydrolyzed fibrin and a plasmin substrate, and PMSF as well as aprotinin inhibited its fibrinolytic activity. These results indicated QK-1 was a plasmin-like serine protease and QK-2 a subtilisin family serine protease. Therefore, these enzymes were designated subtilisin QK. The sequence of a DNA fragment encoding subtilisin QK contained an open reading frame of 1149 base pairs encoding 106 amino acids for signal peptide and 257 amino acids for subtilisin QK, which is highly similar with that of a fibrinolytic enzyme, subtilisin NAT (identities 96.8%). Asp32, His64 and Ser221 in the amino acid sequence deduced from the QK gene are identical to the active site of nattokinase (NK) produced by B. subtilis natto.     

A Pseudomonas aeruginosa alginate-exotoxin A conjugate that elicits anti-alginate and exotoxin A-neutralizing antibodies

Pseudomonas aeruginosa alginate was covalently coupled to exotoxin A by reductive amination using adipic acid dihydrazide as spacer. The conjugate was composed of 25% alginate and 75% exotoxin A and possessed an average molecular mass higher than 700 kDa as determined by polyacrylamide gel electrophoresis. The conjugate had virtually no ADP-ribosyltransferase activity and a reduced cytotoxicity for TSA8 murine cells, derived from Friend erythroleukemia cells, as indicated by a greater than 50-fold increased LD50. Anti-conjugate antibodies recognized exotoxin A and alginate. A booster injection resulted in markedly increased antibody ELISA titers to both exotoxin A and alginate. The antibodies neutralized the exotoxin A toxicity.