Cloning and sequencing of the immunoglobulin A1 protease gene (iga) of Haemophilus influenzae serotype b.

Secretion of immunoglobulin A1 (IgA1) proteases is a characteristic of Haemophilus influenzae and several other bacterial pathogens causing infectious diseases, including meningitis. Indirect evidence suggests that the proteases are important virulence factors. In this study, we cloned the iga gene encoding immunoglobulin A1 (IgA1) protease from H. influenzae serotype b into Escherichia coli, in which the recombinant H. influenzae iga gene was expressed and the resulting protease was secreted. Sequencing a part of a 7.5-kilobase DNA fragment containing the iga gene revealed a large open reading frame with a strongly biased codon usage and having the potential of encoding a protein of 1,541 amino acids and a molecular mass of 169 kilodaltons. Putative promoter and terminator elements flanking the open reading frame were identified. Comparison of the deduced amino acid sequence of this H. influenzae IgA1 protease with that of a similar protease from Neisseria gonorrhoeae revealed several domains with a high degree of homology. Analogous to mechanisms known from the N. gonorrhoeae IgA protease secretion, we propose a scheme of posttranslational modifications of the H. influenzae IgA1 protease precursor, leading to a secreted protease with a molecular mass of 108 kilodaltons, which is close to the 100 kilodaltons reported for the mature IgA1 protease.     

Effect of mutations in the human immunoglobulin A1 (IgA1) hinge on its susceptibility to cleavage by diverse bacterial IgA1 proteases

Components of the human immunoglobulin A1 (IgA1) hinge governing sensitivity to cleavage by bacterial IgA1 proteases were investigated. Recombinant antibodies with distinct hinge mutations were constructed from a hybrid comprised of human IgA2 bearing half of the human IgA1 hinge region. This hybrid antibody and all the mutant antibodies derived from it were resistant to cleavage by the IgA1 proteases from Streptococcus oralis and Streptococcus mitis biovar 1 strains but were cleaved to various degrees by those of Streptococcus pneumoniae, some Streptococcus sanguis strains, and the type 1 and 2 IgA1 proteases of Haemophilus influenzae, Neisseria meningitidis, and Neisseria gonorrhoeae. Remarkably, those proteases that cleave a Pro-Ser peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies lacking a Pro-Ser peptide bond in the hinge, and those that cleave a Pro-Thr peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies devoid of a Pro-Thr peptide bond in the hinge. Thus, the enzymes can cleave alternatives to their preferred postproline peptide bond when such a bond is unavailable. Peptide sequence analysis of a representative antibody digestion product confirmed this conclusion. The presence of a cleavable peptide bond near the CH2 end of the hinge appeared to result in greater cleavage than if the scissile bond was at the CH1 end of the hinge. Proline-to-serine substitution at residue 230 in a hinge containing potentially cleavable Pro-Ser and Pro-Thr peptide bonds increased the resistance of the antibody to cleavage by many IgA1 proteases.     

Cleavage of a recombinant human immunoglobulin A2 (IgA2)-IgA1 hybrid antibody by certain bacterial IgA1 proteases

To understand more about the factors influencing the cleavage of immunoglobulin A1 (IgA1) by microbial IgA1 proteases, a recombinant human IgA2/IgA1 hybrid molecule was generated. In the hybrid, termed IgA2/A1 half hinge, a seven-amino-acid sequence corresponding to one half of the duplicated sequence making up the IgA1 hinge was incorporated into the equivalent site in IgA2. Insertion of the IgA1 half hinge into IgA2 did not affect antigen binding capacity or the functional activity of the hybrid molecule, as judged by its ability to bind to IgA Fcalpha receptors and trigger respiratory bursts in neutrophils. Although the IgA2/A1 hybrid contained only half of the IgA1 hinge, it was found to be cleaved by a variety of different bacterial IgA1 proteases, including representatives of those that cleave IgA1 in the different duplicated halves of the hinge, namely, those of Prevotella melaninogenica, Streptococcus pneumoniae, S. sanguis, Neisseria meningitidis types 1 and 2, N. gonorrhoeae types 1 and 2, and Haemophilus influenzae type 2. Thus, for these enzymes the recognition site for IgA1 cleavage is contained within half of the IgA1 hinge region; additional distal elements, if required, are provided by either an IgA1 or an IgA2 framework. In contrast, the IgA2/A1 hybrid appeared to be resistant to cleavage with S. oralis and some H. influenzae type 1 IgA1 proteases, suggesting these enzymes require additional determinants for efficient substrate recognition.     

New method that uses binding of immunoglobulin A to group A streptococcal immunoglobulin A Fc receptors for demonstration of microbial immunoglobulin A protease activity.

A new method is described for the detection of bacterial immunoglobulin A (IgA) protease which splits IgA into Fab and Fc fragments. The method takes advantage of a recent finding that receptors for IgA fragments occur commonly among type 4 group A streptococci. The bacterial preparation to be tested for protease activity was first incubated with radiolabeled purified IgA1 myeloma protein, and the proportion of radioactivity bound to a standard suspension of the streptococci was then measured. Since isolated Fab fragments do not bind to streptococcal IgA receptors, a decrease in the amount of radioactivity bound to the streptococci, as compared with the amount before digestion, indicates the presence of protease in the test preparation. Using this method, protease activity was detected in Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, and Streptococcus sanguis, but not in Escherichia coli or Branhamella catarrhalis.     

Cloning of the gene encoding streptococcal immunoglobulin A protease and its expression in Escherichia coli.

We have identified and cloned a 6-kilobase-pair segment of chromosomal DNA from Streptococcus sanguis ATCC 10556 that encodes immunoglobulin A (IgA) protease activity when cloned into Escherichia coli. The enzyme specified by the iga gene in plasmid pJG1 accumulates in the periplasm of E. coli MM294 cells and has a substrate specificity for human IgA1 identical to that of native S. sanguis protease. Hybridization experiments with probes from within the encoding DNA showed no detectable homology at the nucleotide sequence level with chromosomal DNA of gram-negative bacteria that excrete IgA protease. Moreover, the S. sanguis iga gene probes showed no detectable hybridization with chromosomal DNA of S. pneumoniae, although the IgA proteases of these two streptococcal species cleaved the identical peptide bond in the human IgA1 heavy-chain hinge region.     

Comparative analysis of immunoglobulin A1 protease activity among bacteria representing different genera, species, and strains.

Immunoglobulin A1 (IgA1) proteases cleaving human IgA1 in the hinge region are produced constitutively by a number of pathogens, including Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, and Streptococcus pneumoniae, as well as by some members of the resident oropharyngeal flora. Whereas IgA1 proteases have been shown to interfere with the functions of IgA antibodies in vitro, the exact role of these enzymes in the relationship of bacteria to a human host capable of responding with enzyme-neutralizing antibodies is not clear. Conceivably, the role of IgA1 proteases may depend on the quantity of IgA1 protease generated as well as on the balance between secreted and cell-associated forms of the enzyme. Therefore, we have compared levels of IgA1 protease activity in cultures of 38 bacterial strains representing different genera and species as well as strains of different pathogenic potential. Wide variation in activity generation rate was found overall and within some species. High activity was not an exclusive property of bacteria with documented pathogenicity. Almost all activity of H. influenzae, N. meningitidis, and N. gonorrhoeae strains was present in the supernatant. In contrast, large proportions of the activity in Streptococcus, Prevotella, and Capnocytophaga species was cell associated at early stationary phase, suggesting that the enzyme may play the role of a surface antigen. Partial release of cell-associated activity occurred during stationary phase. Within some taxa, the degree of activity variation correlated with degree of antigenic diversity of the enzyme as determined previously. This finding may indicate that the variation observed is of biological significance.     

Cleavage of the human immunoglobulin A1 (IgA1) hinge region by IgA1 proteases requires structures in the Fc region of IgA

Secretory immunoglobulin A (IgA) protects the mucosal surfaces against inhaled and ingested pathogens. Many pathogenic bacteria produce IgA1 proteases that cleave in the hinge of IgA1, thus separating the Fab region from the Fc region and making IgA ineffective. Here, we show that Haemophilus influenzae type 1 and Neisseria gonorrhoeae type 2 IgA1 proteases cleave the IgA1 hinge in the context of the constant region of IgA1 or IgA2m(1) but not in the context of IgG2. Both C(alpha)2 and C(alpha)3 but not C(alpha)1 are required for the cleavage of the IgA1 hinge by H. influenzae and N. gonorrhoeae proteases. While there was no difference in the cleavage kinetics between wild-type IgA1 and IgA1 containing only the first GalNAc residue of the O-linked glycans, the absence of N-linked glycans in the Fc increased the ability of the N. gonorrhoeae protease to cleave the IgA1 hinge. Taken together, these results suggest that, in addition to the IgA1 hinge, structures in the Fc region of IgA are required for the recognition and cleavage of IgA1 by the H. influenzae and N. gonorrhoeae proteases.     

Immunoglobulin A1 protease types of Neisseria gonorrhoeae and their relationship to auxotype and serovar.

Immunoglobulin A1 (IgA1) proteases are extracellular bacterial proteolytic enzymes which correlate with virulence in several species of human pathogens. We report that Neisseria gonorrhoeae produced two distinct types of IgA1 protease, each of which cleaved a different peptide bond in the hinge region of human IgA1. The type of IgA1 protease produced correlated with both nutritional auxotype and outer membrane protein I serovar in this organism. Gonococcal type 1 IgA1 protease was produced primarily by N. gonorrhoeae strains which require arginine, hypoxanthine, and uracil (AHU) and which belong to the protein IA-1 or IA-2 serovar. Most isolates of other auxotypes and serovars produced type 2 IgA1 protease. Although both the AHU auxotype and protein IA serogroup were found to be associated with disseminated gonococcal infection, there was no direct correlation of IgA1 protease type with disseminated or with uncomplicated gonorrhea.     

Characterization of the Streptococcus pneumoniae immunoglobulin A1 protease gene (iga) and its translation product.

Bacterial immunoglobulin A1 (IgA1) proteases constitute a very heterogenous group of extracellular endopeptidases which specifically cleave human IgA1 in the hinge region. Here we report that the IgA1 protease gene, iga, of Streptococcus pneumoniae is homologous to that of Streptococcus sanguis. By using the S. sanguis iga gene as hybridization probe, the corresponding gene from a clinical isolate of S. pneumoniae was isolated in an Escherichia coli lambda phage library. A lysate of E. coli infected with hybridization-positive recombinant phages possessed IgA1-cleaving activity. The complete sequence of the S. pneumoniae iga gene was determined. An open reading frame with a strongly biased codon usage and having the potential of encoding a protein of 1,927 amino acids with a molecular mass of 215,023 Da was preceded by a potential -10 promoter sequence and a putative Shine-Dalgarno sequence. A putative signal peptide was found in the N-terminal end of the protein. The amino acid sequence similarity to the S. sanguis IgA1 protease indicated that the pneumococcal IgA1 protease is a Zn-metalloproteinase. The primary structures of the two streptococcal IgA1 proteases were quite different in the N-terminal parts, and both proteins contained repeat structures in this region. Using a novel assay for IgA1 protease activity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we demonstrated that the secreted IgA1 protease was present in several different molecular forms ranging in size from approximately 135 to 220 kDa. In addition, interstrain differences in the sizes of the pneumococcal IgA1 proteases were detected. Southern blot analyses suggested that the S. pneumoniae iga gene is highly heterogenous within the species.     

Localization of the cleavage site specificity determinant of Haemophilus influenzae immunoglobulin A1 protease genes.

Immunoglobulin A1 (IgA1) proteases are produced by a number of different species of bacteria which cause infection at human mucosal surfaces. The sole substrate of these proteases is human IgA1. Cleavage is within the hinge region of IgA1, although there is variability in the exact peptide bond within the hinge region that is cut by a particular protease. The cleavage site of the Haemophilus influenzae type 1 protease is located four amino acids from the cleavage site of the type 2 enzyme. In this study, the region of the H. influenzae IgA1 protease gene (iga) that determines the cleavage site specificity was localized through the comparison of the type 1 and type 2 genes and the construction and analysis of type 1-type 2 hybrid genes. The hybrid genes were generated by in vivo and in vitro techniques which facilitated the selection and screening of randomly generated hybrids. The cleavage site determinant was found to be within a 370-base-pair region near the amino-terminal coding region, in one of two large areas of nonhomology between the two types of H. influenzae iga genes. DNA sequence analysis of the cleavage site determinant and surrounding regions did not reveal a simple mechanism whereby one enzyme type could be converted to the other type. Comparison of the type 2 gonococcal IgA1 protease gene to the two Haemophilus genes revealed a significant amount of homology around the cleavage site determinant, with the two type 2 genes showing greater homology.     

Loss of antibody activity in human immunoglobulin A exposed extracellular immunoglobulin A proteases of Neisseria gonorrhoeae and Streptococcus sanguis.

Immunoglobulin A (IgA) proteases are extracellular enzymes elaborated by Neisseria gonorrhoeae, N. meningitidis, and Streptococcus sanguis. These enzymes each cleave human IgA1 at a critically situated prolyl-threonyl peptide bond to yield Fab alpha and Fc alpha fragments. To study their effect on the antibody activity of human IgA, we enzymatically digested a group of five human IgA monoclonal immunoglobulins with high-titer rheumatoid factor or cold agglutinin activity and human serum macroamylase, an amylase-IgA complex. In contrast to four control IgM rheumatoid factor monoclonal proteins, whose activity was unaffected by enzyme, gonococcal and streptococcal IgA proteases caused prompt, major reductions of IgA antibody activity to negligible levels and converted macroamylase activity to amylase of normal size, as determined by molecular sieve chromatography. In addition, both enzymes promptly deagglutinated sensitized cells that had been aggregated by IgA rheumatoid factors, indicating that IgA bound to antigen is also susceptible to enzyme cleavage. Fab fragments of Iga protein Chr, a rheumatoid factor, showed essentially no antigen-binding activity despite the high titers observed with the parent protein. These studies emphasize the high degree of specificity of the microbial proteases for IgA and their potential for interfering with antibody activity in the IgA1 subclass.     

Characterization of interferons induced by bacteria and interferon-producing leukocytes in human peripheral blood.

Indirect evidence suggests that immunoglobulin A1 (IgA1) proteases may be factors in the pathogenesis of certain infectious diseases, including meningitis, gonorrhoea, and destructive periodontitis. Bacterial IgA1 proteases are therefore potential candidates as vaccines. In this study, IgA1 proteases from 166 clinical isolates and reference strains of Haemophilus influenzae and Haemophilus aegyptius were compared with regard to specific activity and pattern of enzyme inhibition by antisera raised against IgA1 protease from nine selected strains of H. influenzae. A total of 93% of H. influenzae strains and all H. aegyptius strains had detectable IgA1 protease activity. The majority of strains cleaved a prolyl-seryl or a prolyl-threonyl peptide bond in the alpha 1 hinge region, whereas occasional H. influenzae strains possessed two separate IgA1 proteases with these two specific activities. Of the 155 IgA1 protease-producing strains, all except 12 could be assigned to one of 14 IgA1 protease "inhibition types," each defined by a characteristic pattern of inhibition by the nine antisera. There was no correlation between IgA1 protease type and biotype of the strains. However, among 92 encapsulated H. influenzae strains, a close correlation between capsular serotype and IgA1 protease type was observed. With the exception of serotype f, strains of all capsular serotypes produced an exclusive antigenic type of IgA1 protease. All 38 strains of serotype b produced IgA1 protease of inhibition type 1, which was never demonstrated in non-encapsulated H. influenzae strains. These results facilitate the detection of an antibody response against specific IgA1 proteases and are of practical value for a possible future vaccine against H. influenzae serotype b infections.     

Molecular biology of haemophilus influenzae IgA1 proteases

IgA1 proteases of two distinct specificities were demonstrated among 95 isolates of Haemophilus influenzae and nine isolates of H. aegyptius. The two enzymes cleaved two different peptide bonds in the hinge region of the α chain of IgA1: a prolyl-seryl bond located at position 231–232 (type A cleavage) and a prolyl-threonyl peptide bond between residues 235 and 236 (type B cleavage). Each strain of H. influenzae produced either one or both of these types of enzymes, whereas all H. aegyptius strains produced type A enzyme only. The application of enzyme-neutralizing antibodies to the study of IgA1 proteases produced by the 104 strains of H. influenzae and H. aegyptius revealed at least 15 different types of protease activities based on inhibition patterns in nine selected antibody preparations. The types of IgA1 proteases closely correlated with the serotype of encapsulated strains of H. influenzae. The study suggests that H. influenzae strains produce at least two serologically different IgA1 proteases with distinct or identical enzymatic activities.     

Amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by streptococcal IgA1 proteases

The amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by IgA1 proteases of different species of Streptococcus were investigated. Recombinant IgA1 antibodies were generated with point mutations at proline 227 and threonine 228, the residues lying on either side of the peptide bond at which all streptococcal IgA1 proteases cleave wild-type human IgA1. The amino acid substitutions produced no major effect upon the structure of the mutant IgA1 antibodies or their functional ability to bind to Fcalpha receptors. However, the substitutions had a substantial effect upon sensitivity to cleavage with some streptococcal IgA1 proteases, with, in some cases, a single point mutation rendering the antibody resistant to a particular IgA1 protease. This effect was least marked with the IgA1 protease from Streptococcus pneumoniae, which showed no absolute requirement for either proline or threonine at residues 227 to 228. By contrast, the IgA1 proteases of Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis had an absolute requirement for proline at 227 but not for threonine at 228, which could be replaced by valine. There was evidence in S. mitis that proteases from different strains may have different amino acid requirements for cleavage. Remarkably, some streptococcal proteases appeared able to cleave the hinge at a distant alternative site if substitution prevented efficient cleavage of the original site. Hence, this study has identified key residues required for the recognition of the IgA1 hinge as a substrate by streptococcal IgA1 proteases, and it marks a preliminary step towards development of specific enzyme inhibitors.     

Antigenic relationships among immunoglobulin A1 proteases from Haemophilus, Neisseria, and Streptococcus species.

To investigate the antigenic variation and relationships of immunoglobulin A1 (IgA1) proteases among different species and genera, we examined a comprehensive collection of serine type and metallo-type IgA1 proteases and corresponding antisera in enzyme neutralization assays. Sharing of neutralizing epitopes of metallo-type IgA1 proteases from Streptococcus pneumoniae, Streptococcus sanguis, Streptococcus mitis, and Streptococcus oralis and of serine type IgA1 proteases from Haemophilus and pathogenic Neisseria species was extremely limited. A number of limited to strong cross-reactions in such epitopes were found among serine type IgA1 proteases released by members of the genera Haemophilus and Neisseria, reflecting the common origin of their iga gene. However, the relatively limited prevalence of shared "neutralizing" epitopes of IgA1 proteases from the two genera indicates that they rarely induce immunity to each other. In contrast, extensive sharing of neutralizing epitopes was found between N. meningitidis and N. gonorrhoeae IgA1 proteases, making them potentially attractive vaccine components. Among metallo-type IgA1 proteases, several pneumococcal proteases were found to induce neutralizing antibodies to IgA1 proteases of oral streptococci whereas the opposite was not the case.     

IgA protease production as a characteristic distinguishing pathogenic from harmless neisseriaceae.

IgA proteases are extracellular enzymes of bacteria that have human immunoglobulin A of the IgA1 subclass as their only known substrate. The identification of this enzyme in neisseria prompted us to determine whether IgA protease production correlates with pathogenicity within this genus. Multiple clinical isolates of Neisseria gonorrhoeae, N. meningitidis and eight species of non-pathogenic neisseria that commonly colonize the normal human nasopharynx were examined for IgA protease activity. All N. gonorrhoeae and N. meningitidis strains were enzyme positive; all non-pathogenic strains were negative. Among meningococci, the enzyme occurred in strains carried harmlessly in the nasopharynx as well as those isolated from systemic infections. Because mucosal immune defense is largely mediated by antibodies of the IgA isotype, the finding that IgA protease activity is linked specifically to the pathogenic neisseria suggests that the enzyme may be involved in the pathogenesis of neisserial infection.     

Analysis of the specificity of bacterial immunoglobulin A (IgA) proteases by a comparative study of ape serum IgAs as substrates.

Immunoglobulin A (IgA) proteases are bacterial enzymes with substrate specificity for human serum and secretory IgAs. To further define the basis of this specificity, we examined the ability of IgA proteases of Clostridium ramosum, Streptococcus pneumoniae (EC 3.4.24.13), Neisseria meningitidis (EC 3.4.21.72), and Haemophilus influenzae (EC 3.4.21.72) to cleave serum IgAs of gorillas, chimpanzees, and orangutans. All enzymes cleaved the IgAs of the three apes despite differences in ape IgA1 hinge sequence relative to the human prototype. To directly compare the ape and human hinge cleavage sites, the sites were identified in eight ape IgA digests. This analysis confirmed that ape proteins were all cleaved in the IgA hinge region, in all but one case after proline residues. The exception, C. ramosum protease, cleaved gorilla and chimpanzee IgAs at peptide bonds having no proline, but the scissile bonds were in the same hinge location as the Pro-221-Val-222 cleaved in human IgA1. These data indicate that proline is not an invariant substrate requirement for all IgA proteases and that the location of the scissile bond, in addition to its composition, is a critical determinant of cleavage specificity.     

Proteolysis of bacterial membrane proteins by Neisseria gonorrhoeae type 2 immunoglobulin A1 protease.

The immunoglobulin A1 (IgA1) proteases of Neisseria gonorrhoeae have been defined as having human IgA1 as their single permissive substrate. However, in recent years there have been reports of other proteins which are susceptible to the proteolytic activity of these enzymes. To examine the possibility that gonococcal membrane proteins are potential substrates for these enzymes, isolated outer and cytoplasmic membranes of N. gonorrhoeae were treated in vitro with exogenous pure IgA1 protease. Analysis of silver-stained sodium dodecyl sulfate-polyacrylamide gels of outer membranes indicated that there were two outer membrane proteins of 78 and 68 kDa which were cleaved by IgA1 protease in vitro in GCM 740 (a wild-type strain) and in two isogenic IgA1 protease-negative variants. Similar results were observed with a second gonococcal strain, F62, and its isogenic IgA1 protease-negative derivative. When GCM 740 cytoplasmic membranes were treated with protease, three minor proteins of 24.5, 23.5, and 21.5 kDa were cleaved. In addition, when outer membranes of Escherichia coli DH1 were treated with IgA1 protease, several proteins were hydrolyzed. While the identities of all of these proteolyzed proteins are unknown, the data presented indicate that there are several proteins found in the isolated membranes of gram-negative bacteria which are permissive in vitro substrates for gonococcal IgA1 protease.     

Synthetic peptide substrates for the immunoglobulin A1 protease from Neisseria gonorrhoeae (type 2).

Neisseria gonorrhoeae secretes protease which inactive human immunoglobulin A1 (IgA1) by cleavage of specific peptide bonds in the hinge region. The type 2 IgA1 protease (EC 3.4.24.13) is secreted as a 169-kDa precursor which undergoes autoproteolysis at three sites (A, B, and C) to release the 106-kDa active form of the enzyme (J. Pohlner, R. Halter, K. Beyreuther, and T. F. Meyer. Nature [London] 325:458-462, 1987). Synthetic decapeptides consisting of five residues on each side of the three autoproteolytic cleavage sites and their potential pentapeptide catabolites were prepared by solid-phase synthesis. Cleavage of the decapeptides by the type 2 IgA1 protease from N. gonorrhoeae was monitored by high-performance liquid chromatography. Peptides homologous with the amino acid sequences around the B and C sites are cleaved by the IgA1 protease. Amino acid analysis and Edman degradation show that the cleavage products have both the composition and amino acid sequence which would be expected from cleavage at the predicted sites. Km values of 1.35 mM and 3.43 mM and kcat values of 280 pmol/h/U and 439 pmol/h/U for the site B and site C peptides, respectively, were determined. The catalytic efficiency (kcat/Km) for the synthetic substrates is about 10% of that reported for intact IgA1. Cleavage of the peptides is inhibited by IgA1 protease inhibitors such as the tetrapeptide substrate analog inhibitor HRP-48, human colostrum, and a peptide-boronate transition state inhibitor. An extract from an N. gonorrhoeae construct lacking active IgA1 protease failed to cleave the synthetic substrate, while an extract from the control construct which secretes active enzyme completely hydrolyzed the synthetic peptide. Neither the site A peptide nor synthetic decapeptides encompassing cleavage sites in the hinge region of IgA1 are hydrolyzed by IgA1 protease. These are the first synthetic substrates to be reported for any IgA1 protease.     

Nucleotide sequence homology between the immunoglobulin A1 protease genes of Neisseria gonorrhoeae, Neisseria meningitidis, and Haemophilus influenzae.

Isolated DNA fragments encoding the immunoglobulin A1 (IgA1) protease of Neisseria gonorrhoeae were used as hybridization probes to search for homologous sequences in whole cell DNA from Neisseria meningitidis and Haemophilus influenzae. Significant homology was detected. That the detected homology represented IgA1 protease-specific sequences was confirmed by the cloning of these sequences in Escherichia coli HB101 and demonstrating the expression of IgA1 protease by these transformed cells. Molecular probing of commensal Neisseria and Haemophilus species, which do not elaborate IgA1 protease activity, revealed that they were devoid of sequence homology with the cloned IgA1 protease gene DNA.