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The Journal of Immunology, 2006, 177: 3913-3919.
Copyright © 2006 by The American Association of Immunologists, Inc.

Sites in the CH3 Domain of Human IgA1 That Influence Sensitivity to Bacterial IgA1 Proteases1

Bernard W. Senior and Jenny M. Woof2

Division of Pathology and Neuroscience, University of Dundee Medical School, Ninewells Hospital, Dundee, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The influence of regions, other than the hinge, on the susceptibility of human IgA1 to cleavage by diverse bacterial IgA1 proteases, was examined using IgA1 mutants bearing amino acid deletions, substitutions, and domain swaps. IgA1 lacking the tailpiece retained its susceptibility to cleavage by all of the IgA1 proteases. The domain swap molecule {alpha}1{alpha}2{gamma}3, in which the CH3 domain of IgA1 was exchanged for that of human IgG1, was resistant to cleavage with the type 1 and 2 serine IgA1 proteases of Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae, but remained sensitive to cleavage with the metallo-IgA1 proteases of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis. Substitution of the IgA1 C{alpha}3 domain motif Pro440-Phe443 into the corresponding position in the C{gamma}3 domain of {alpha}1{alpha}2{gamma}3 resulted now in sensitivity to the type 2 IgA1 protease of N. meningitidis, indicating the possible requirement of these amino acids for sensitivity to this protease. For the H. influenzae type 2 protease, resistance of an IgA1 mutant in which the CH3 domain residues 399–409 were exchanged with those from IgG1, but sensitivity of mutant HuBov{alpha}3 in which the C{alpha}3 domain of bovine IgA replaces that of human IgA1, suggests that CH3 domain residues Glu403, Gln406, and Thr409 influence sensitivity to this enzyme. Hence, unlike the situation with the metallo-IgA1 proteases of Streptococcus spp., the sensitivity of human IgA1 to cleavage with the serine IgA1 proteases of Neisseria and Haemophilus involves their binding to different sites specifically in the CH3 domain.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunoglobulin A1 proteases are enzymes that specifically cleave human IgA1 (reviewed in Refs. 1, 2, 3). Most are produced by major bacterial pathogens that instigate infection at mucosal surfaces of the respiratory tract, such as Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis, or the genital tract such as Neisseria gonorrhoeae. IgA1 proteases represent a group of structurally diverse enzymes—metalloproteases, serine-type proteases, and cysteine-type proteases—with different enzymatic mechanisms. Evidence of in vivo production of IgA1 proteases in human infections (4, 5, 6), the association of recovery from infection with the development of Abs to IgA1 proteases (7, 8, 9), and the failure of related but nonpathogenic species of bacteria to produce IgA1 proteases (10, 11) all point to IgA1 proteases being important virulence factors of the pathogens that produce them. Despite these persuasive indirect observations, the in vivo roles of IgA1 proteases in virulence are difficult to determine, because the substrate is restricted almost exclusively (12, 13, 14, 15) to IgA1 of humans, gorillas, and chimpanzees, and IgA of orangutans (16), and therefore convenient animal models are not available. However, IgA1 protease has been shown to compromise IgA-mediated killing of S. pneumoniae both in vitro and in vivo (17). S. pneumoniae-specific IgA1, when cleaved by IgA1 protease, loses its protective effects, and the resultant IgA fragments appear to enhance adherence of pneumococci to host cells (18).

The IgA1 hinge, an extended region that separates the Fab and Fc regions of the Ab and incorporates a duplication of an 8-aa sequence rich in proline, serine, and threonine, is the site of cleavage by all IgA1 proteases. IgA2 lacks this region and is thereby naturally resistant to IgA1 protease action. Although IgA1 proteases belong to very different families of enzymes, they are all postproline endopeptidases. They each cleave a specific peptide bond (either a Pro-Ser bond for type 1 enzymes or a Pro-Thr bond for type 2 enzymes) in only one of the duplicated halves of the IgA1 hinge.

The consequence of IgA1 cleavage is the generation of Fab and Fc fragments and thus the decoupling of recognition of Ags from mechanisms for their elimination. Moreover, the Fab formed may mask relevant epitopes from the immune system and thus prevent the binding of intact Abs of other isotypes (19, 20), activation of complement, and complement-mediated lysis. Some IgA1 proteases may have a role in virulence by mechanisms additional to or distinct from those arising through IgA1 cleavage (13, 15, 21).

It is becoming increasingly apparent that the sensitivity of human IgA1 to cleavage by IgA1 proteases is dependent not only on the presence of the hinge but also on structures elsewhere in IgA1. For example, it has been reported that structures, as yet undefined, in the IgA1 Fc region are required for cleavage by the type 1 IgA1 protease of H. influenzae and the type 2 IgA1 protease of N. gonorrhoeae (22). To understand more about the features of human IgA influencing its sensitivity to cleavage by bacterial IgA1 proteases, ultimately with a view to preparing IgA1 protease inhibitors, this study sought to localize structures in IgA1 outside the hinge that influence the sensitivity of human IgA1 to cleavage by IgA1 proteases. In anticipation of distinct structural requirements in terms of substrate recognition for different types of IgA1 protease, we tested a range of metallo-type and serine-type IgA1 proteases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Primers

Primer BOVA3'SAL (5'-CCGGCCGTCGACGGATCCACTAGT-3') contained a SalI restriction site (in italics) and bases complementary to the multiple cloning site (BamHI site underlined) of pBluescript SK vector. Primer HUMBOVDS1 (5'-TCTCAAAATCCGGGAACACGTTCC-3') incorporated bases from the 3' end of the CH2 exon of the human {alpha}1 gene (underlined) and bases from the 5' end of the CH3 exon of the bovine {alpha} gene (in italics). Primer HUMBOVDS2 was its complement.

Primer 399–409GAMS (5'-GTACCTGACTTGGCCACCCGTGCTGGATTCCGACGGTACCTTCGCTGTGACCAGC-3') corresponded to the sequence of bases 1401–1437 and (underlined) 1447–1464 of the C{alpha}3 domain of human IgA1, with bases in bold being changed to those coding for the corresponding amino acids in human IgG1. Primer 399–409GAMAS was its complement.

Primer AGS (5'-CCCTCAACTCCACCTACCCCATCTCCCTCAACTCCACCTACCCCATCTCCCTCATGCCCACCGTGCCCAGGTAAGC-3') corresponded to the nucleotide sequence of the human IgA1 hinge (underlined) and part of the IgG1 {gamma} H chain gene (nt 921–942). Primer AGAS (5'-TGAGGGAGATGGGGTAGGTGGAGTTGAGGGAGATGGGGTAGGTGGAGTTGAGGGAACTTTCTTGTCCACCT-3') corresponded to the complement of the nucleotide sequence of the human IgA1 hinge (underlined) and nt 486–502 of the C{gamma}1 exon of human IgG1. Primer AG3' (5'-GCGCGCGCGAATTCGCTTTATTTCCATGCTG-3') contained an EcoRI restriction site (underlined) and annealed ~100 nt downstream of the stop codon of human IgG1. Primer DS3 (5'-AGGACTCTACTCCCTCAGCAGC-3') annealed 5' of a unique BstEII site in the C{gamma}1 exon.

Construction of mutant Ab expression vectors

PCR overlap extension mutagenesis (23) was used to construct various H chain expression vectors. For the domain swap HuBov{alpha}3 (with C regions C{alpha}1, hinge, and C{alpha}2 from human IgA1 and C{alpha}3 from bovine IgA) (see Figs. 1 and 2), a previously described 5' flanking primer that annealed 5' of a unique XhoI site in the human IgA1 {alpha}-chain vector (24), and the 3' flanking primer BOVA3'SAL, were used. Sequence corresponding to part of the C{alpha}2 domain of human IgA1 was amplified with the 5' flanking primer and HUMBOVDS2 using a human IgA1 {alpha}-chain-containing plasmid as template. The C{alpha}3 exon of bovine IgA was amplified from a pBluescript SK plasmid bearing the {alpha}-chain gene of bovine IgA (25) using primers HUMBOVDS1 and BOVA3'SAL. A second round of PCR overlap extension produced a fragment of ~2 kb. After cleavage with SalI and XhoI, it was ligated into an IgA1 H chain expression vector, replacing the wild-type sequence in this region.


Figure 1
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  		FIGURE 1. Schematic diagram of the H chain C regions of the key mutants constructed and tested. Each region (CH1, hinge, CH2, CH3, and tailpiece) is indicated by a rectangular box (not to scale) filled to indicate its origins as following: white, human IgA1; dark gray, human IgG1; light gray, bovine IgA. Mutants {alpha}1{alpha}2{gamma}3PLAF and 399–409GAM feature single segment substitutions within their CH3 domains. wt, wild type.

 

Figure 2
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  		FIGURE 2. Alignment of CH3 domain sequences of Igs used in this study. Deletions are indicated by dots. Residue number (IgA1 Bur numbering) is shown above the sequences. The Pro440-Phe443 loop substituted in {alpha}1{alpha}2{gamma}3PLAF is boxed. The human IgG1-derived region substituted in 399–409GAM is highlighted in gray. Bovine IgA amino acids differing from the corresponding amino acids in human IgA1 are underlined in HuBov{alpha}3. Residues Pro440-Phe443 may be important for activity of the N. gonorrhoeae type 2 IgA1 protease. The Ala399-Thr409 region, and amino acids Glu403, Gln406, and Thr409, in particular, may be important for the activity of the type 2 IgA1 protease of H. influenzae.

 
In the IgA1 mutant 399–409GAM, amino acids Ala399 to Thr409 inclusive of the human IgA1 H chain were replaced by the corresponding amino acids from human IgG1 (Figs. 1 and 2). To generate the expression vector, internal mismatch primers 399–409GAMS and 399–409GAMAS, and the same 5' and 3' flanking primers as previously described (24) were used in PCR overlap extension, with a wild-type IgA1 {alpha}-chain-containing plasmid serving as template. The mutated PCR overlap product was ligated, as above, into unique XhoI and SalI restriction sites in the expression vector, replacing the wild-type sequence in this region.

In the TAHG-1 mutant, the hinge of human IgA1 (residues Pro223 to Ser240) was introduced into human IgG1, replacing the upper hinge region (residues Glu216 to Thr225 inclusive (Eu numbering) (26)) but retaining the inter-H chain disulfide bridges (Fig. 1). To generate the expression vector, PCR overlap extension was performed using internal primers AGS and AGAS, flanking primers DS3 and AG3', and a human IgG1 {gamma}-chain-containing plasmid as template (25). The mutant overlap fragment generated was inserted as a BstEII-EcoRI fragment into an anti-NIP (3-nitro-4-hydroxy-5-iodophenylacetate) IgG1 H chain expression vector, replacing the wild-type sequence in this region (25).

In each case, the derived plasmids were sequenced to confirm that the desired mutations had been incorporated and that no other base changes had occurred during the construction of the plasmids.

Expression and purification of the recombinant Igs

CHO-K1 cells stably transfected with an appropriate mouse {lambda} L chain (27) were transfected with the constructed H chain expression vectors, positive transfectants were selected, and high level expressors were identified by Ag ELISA as described previously (27). Abs were purified from transfectant supernatants by affinity chromatography on NIP-Sepharose as described previously (27), and supplemented with 0.1% sodium azide before storage in small aliquots at –20°C.

We have described previously the preparation of the following mutant human IgA1 Abs: PTerm455, an IgA1 in which the 18-aa C-terminal tailpiece is deleted (24) (Fig. 1); {alpha}1{alpha}2{gamma}3, a domain-swap Ab in which the CH3 domain of human IgA1 is exchanged for the CH3 domain of human IgG1 (25) (Figs. 1 and 2); {alpha}1{alpha}2{gamma}3PLAF, in which residues His433, Asn434, His435, and Tyr436 (Eu numbering) of the C{gamma}3 domain of the parent {alpha}1{alpha}2{gamma}3 construct are replaced by the Pro-Leu-Ala-Phe (PLAF) motif present at the equivalent position in the C{alpha}3 domain of human IgA1 (25) (Figs. 1 and 2); E254A, D255V, G259R, and C311S featuring single amino acid substitutions in the C{alpha}2 domain (25, 28); and E437A and A442R with point mutations in the C{alpha}3 domain (25, 28).

Microbial IgA1 proteases

The bacterial strains used as sources of IgA1 proteases were as described previously (29), with the addition of H. influenzae strain R 16, a producer of a type 1 enzyme. The culture conditions and preparation of the IgA1 proteases have been described previously (29).

Digestion of recombinant Igs with IgA1 proteases and immunoblotting

The digestion conditions, SDS-PAGE, and immunoblotting procedures were as described previously (29). All of the IgA1 protease preparations were initially calibrated with regard to activity on wild-type IgA1, and the optimal concentration of each enzyme was determined. Thereafter, for each enzyme, identical, optimal amounts were added to all digestion reactions. Following transfer to nitrocellulose, blots were blocked in 5% nonfat dried milk powder in PBS containing 0.1% Tween 20 (PBST), and then incubated in a 1/1000 dilution of HRP-labeled goat Ab to either human IgA ({alpha}-Fab specific) (Kirkegaard & Perry Laboratories) or human IgG ({gamma}-chain specific) (Sigma-Aldrich) in PBST for 2 h. After thorough washing, the membranes were developed in 10 ml of 50 mM Tris-HCl (pH 7.6) buffer containing 0.3 mg/ml nickel chloride, 10 mg diaminobenzidine, and 60 µl of 30% hydrogen peroxide. Some IgA1 molecules were heterogeneously glycosylated resulting in multiple H chain bands in immunoblots. Hence, some lanes contain Fab of more than one size, representing different glycoforms of the same cleavage product. The different IgA1 proteases cleave at a variety of previously defined sites in the hinge region. Those that cleave close to the CH1 domain generate Fab that run markedly more quickly in SDS gels than those generated by IgA1 proteases that cleave closer to the Fc region, presumably in part as a result of the O-linked glycans that are attached to the intervening sequence. In addition, some Fab appear to be more readily detected by the anti-IgA Ab than others, possibly due to epitope loss upon protease treatment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
When mutant PTerm455, which lacked the tailpiece, was incubated with the different bacterial IgA1 proteases, it was cleaved by all of them in a manner similar to that of wild-type IgA1 (see Fig. 3 and Table I). However, when mutant Ab TAHG-1, in which the hinge region of human IgA1 had been inserted into the corresponding position in human IgG1, was incubated with the different bacterial IgA1 proteases, it was found to be resistant to cleavage by all of them (Table I). Thus, the sensitivity of IgA1 to cleavage by the diverse bacterial IgA1 proteases was not dependent on the presence of the tailpiece in IgA1, but it was dependent on the presence of other elements in IgA1 (in addition to the hinge) that were not presented by IgG1.


Figure 3
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  		FIGURE 3. Western blot analysis of the action of different IgA1 proteases on PTerm455. After separation under reducing conditions, digests were probed with an anti-human {alpha}-chain-specific-HRP conjugate that bound to epitopes in the Fab region. PTerm455 was untreated (lane 1) or treated with the IgA1 protease of S. pneumoniae SK 690 (lane 2), H. influenzae R 16 (type 1) (lane 3), H. influenzae H 15 (type 2) (lane 4), N. meningitidis HF13 (type 2) (lane 5), N. gonorrhoeae 6092 (type 1) (lane 6), N. meningitidis 3564 (type 1) (lane 7), and N. gonorrhoeae 5489 (type 2) (lane 8). PTerm455 was cleaved by all the different IgA1 proteases.

 

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  		Table I. Sensitivity of different mutant Abs to bacterial IgA1 proteasesa

 
The cleavage of {alpha}1{alpha}2{gamma}3, in which the C{alpha}3 domain of human IgA had been replaced with the C{gamma}3 domain of human IgG1, with the different bacterial IgA1 proteases was dependent on the type of protease used (see Table I). {alpha}1{alpha}2{gamma}3 was resistant to cleavage with the type 1 and type 2 IgA1 proteases of N. gonorrhoeae, N. meningitidis, and H. influenzae, all of which cleave wild-type IgA1 at different positions in the C-terminal duplicated half of the hinge. By contrast, {alpha}1{alpha}2{gamma}3 was cleaved by the IgA1 proteases of all of the strains of S. pneumoniae, Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis, which cleave wild-type IgA1 in the N-terminal half of the hinge (Fig. 4 and Table I).


Figure 4
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  		FIGURE 4. Western blot analysis of the action of different IgA1 proteases on {alpha}1{alpha}2{gamma}3 and {alpha}1{alpha}2{gamma}3PLAF. After separation under reducing conditions, digests were probed with an anti-human {alpha}-chain-specific-HRP conjugate that bound to epitopes in the Fab region. {alpha}1{alpha}2{gamma}3 (lanes 1–5) and {alpha}1{alpha}2{gamma}3PLAF (lanes 6–10) were either untreated (lanes 1 and 6) or treated with the IgA1 protease of S. pneumoniae SK 690 (lanes 2 and 7), H. influenzae R 16 (type 1) (lanes 3 and 8), N. meningitidis HF13 (type 2) (lanes 4 and 9), or N. gonorrhoeae 6092 (type 1) (lanes 5 and 10). Both {alpha}1{alpha}2{gamma}3 and {alpha}1{alpha}2{gamma}3PLAF were cleaved by the IgA1 protease of S. pneumoniae but not by those of H. influenzae type 1or N. gonorrhoeae type 1. {alpha}1{alpha}2{gamma}3 was not cleaved by the type 2 IgA1 protease of N. meningitidis, whereas {alpha}1{alpha}2{gamma}3PLAF was cleaved partially by this enzyme.

 
The mutant {alpha}1{alpha}2{gamma}3PLAF, in which four amino acids (residues His433-Tyr436) (Eu numbering) in an interdomain loop of the C{gamma}3 domain of {alpha}1{alpha}2{gamma}3 had been replaced with the corresponding residues from the C{alpha}3 domain of human IgA1 (Pro440-Phe443) (Bur numbering), was found in general to be similar to {alpha}1{alpha}2{gamma}3 in its sensitivity to cleavage with different IgA1 proteases (Fig. 4 and Table I). Thus, it was sensitive to cleavage with the IgA1 proteases of all the strains of S. pneumoniae, S. oralis, S. sanguis, and S. mitis (Fig. 4 and Table I), and resistant to cleavage with the type 1 and 2 IgA1 proteases of N. gonorrhoeae and H. influenzae, and the type 1 IgA1 protease of N. meningitidis (Fig. 4 and Table I). However, the type 2 IgA1 protease of N. meningitidis appeared to give partial cleavage of {alpha}1{alpha}2{gamma}3PLAF but no cleavage of {alpha}1{alpha}2{gamma}3 (Fig. 4).

These findings indicated that the sensitivity of human IgA1 to the type 1 and 2 IgA1 proteases of N. gonorrhoeae and H. influenzae and the type 1 IgA1 protease of N. meningitidis required not only the presence of the IgA1-specific hinge, but also sequences in the CH3 domain of IgA1 that could not be substituted by the CH3 domain of human IgG1. Furthermore, the sensitivity of IgA1 to cleavage by the type 2 IgA1 protease of N. meningitidis, unlike that of the other serine-type IgA1 proteases of Neisseria and Haemophilus, may be dependent on the presence of one or more of the amino acids in the Pro440-Phe443 (PLAF) interdomain loop of the C{alpha}3 domain of human IgA1. In contrast, the IgA1 proteases of streptococcal species showed no requirement for the presence in human IgA1 of the C{alpha}3 domain for cleavage to occur.

Analysis of the IgA1 mutants E254A, D255V, G259R, C311S, each with a single amino acid change in the C{alpha}2 domain, and E437A and A442R, each with a point substitution in the C{alpha}3 domain, showed that all remained sensitive to cleavage with the type 1 and 2 IgA1 proteases of N. meningitidis, N. gonorrhoeae, and H. influenzae (Table II). With the exception of the C311S mutation, all of these point substitutions lie at or near the Fc interdomain region, and hence are close to or, in the case of A442R, within the PLAF loop. Because this latter substitution produces a substantial change in the size and nature of the residue at position 442, it appears that the sensitivity of IgA1 to cleavage by the N. meningitidis type 2 protease depends on elements within the PLAF loop other than residue Ala442.


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  		Table II. Sensitivity of IgA1 point mutants to cleavage by bacterial IgA1 proteasesa

 
Domain-swap molecule HuBov{alpha}3, in which the C{alpha}3 domain of human IgA1 was replaced by the C{alpha}3 of bovine IgA, was cleaved by all the IgA1 proteases except for that of the type 1 enzyme of N. gonorrhoeae (Fig. 5 and Table I). This suggests that one or more of the 17 aa in the C{alpha}3 domain of IgA1 that differ from those of the bovine IgA C{alpha}3 (see Fig. 2) may be required for this enzyme to cleave IgA1, and that this element(s) may not be required for cleavage by the other IgA1 proteases.


Figure 5
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  		FIGURE 5. Western blot analysis of the action of IgA1 proteases on HuBov{alpha}3. After separation under reducing conditions, digests were probed with an anti-human {alpha}-chain-specific-HRP conjugate that bound to epitopes in the Fab region. HuBov{alpha}3 was either untreated (lane 1) or treated with the IgA1 proteases of S. oralis SK 10 (lane 2), N. meningitidis 3564 type 1 (lane 3), N. meningitidis HF 13 type 2 (lane 4), N. gonorrhoeae 6092 type 1 (lane 5), N. gonorrhoeae 5489 type 2 (lane 6), S. sanguis SK1 (lane 7), S. sanguis SK4 (lane 8), S. mitis SK 564 (lane 9), and S. mitis SK599 (lane 10). HuBov{alpha}3 was cleaved by all the IgA1 proteases except that of N. gonorrhoeae 6092 type 1.

 
Mutant 399–409GAM, in which aa 399–409 of the C{alpha}3 domain of human IgA1 were replaced with the equivalent region of the C{gamma}3 domain of human IgG1 (see Fig. 1), was cleaved by all the IgA1 proteases except for the type 2 enzyme of H. influenzae (Fig. 6 and Table I). This may indicate that one or more of the amino acids Ala399-Thr409 (see Fig. 2) in the CH3 domain of IgA1 are required for the activity of this enzyme but not for that of the other IgA1 proteases.


Figure 6
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  		FIGURE 6. Western blot analysis of the action of IgA1 proteases on IgA1, {alpha}1{alpha}2{gamma}3, and 399–409GAM. After separation under reducing conditions, digests were probed with an anti-human {alpha}-chain-specific-HRP conjugate that bound to epitopes in the Fab region. Wild-type IgA1 (lanes 1, 4, and 7), {alpha}1{alpha}2{gamma}3 (lanes 2, 5, and 8) and 399–409GAM (lanes 3, 6, and 9) were either untreated (lanes 1–3) or treated with the IgA1 protease of N. meningitidis 3564 type 1 (lanes 4–6) or H. influenzae H15 type 2 (lanes 7–9). Molecular mass markers are shown on the left. Although the type 1 IgA1 protease of N. meningitidis cleaved 399–409GAM but not {alpha}1{alpha}2{gamma}3, the type 2 enzyme of H. influenzae was unable to cleave either 399–409GAM or {alpha}1{alpha}2{gamma}3.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Despite recent advances (22, 29, 30, 31), not all the factors determining the sensitivity of human IgA1 to cleavage by bacterial IgA1 proteases are completely understood. The resistance of synthetic decapeptides with sequences mirroring parts of the human IgA1 hinge to cleavage by the type 2 IgA1 protease of N. gonorrhoeae (32) may be a consequence of the size of the artificial hinge being too small (29). Alternatively, or in addition, this resistance to cleavage may indicate that, as well as a requirement for a specific amino acid sequence of adequate size in the hinge, the presence of appropriate structural features in the IgA substrate outside the hinge are also necessary for cleavage by IgA1 proteases. Furthermore, sequential incubation of IgA1 with IgA1 proteases of different cleavage specificities revealed that remaining potential cleavage sites in the Fc fragments, but not those in Fab, could be further cleaved by a second IgA1 protease (33). This suggests that structural features in human IgA1 C-terminal to the cleavable peptide bond are particularly important in determining susceptibility to IgA1 protease activity. The purpose of the present study was to pinpoint regions within the Fc portion of the Ab capable of influencing the sensitivity of human IgA1 to cleavage by bacterial IgA1 proteases. Given the structural and mechanistic diversity of the IgA1 proteases and therefore the likely distinctions in substrate recognition, we chose to investigate this for a range of different IgA1 proteases.

The sensitivity of PTerm455, an IgA1 mutant devoid of the tailpiece, to cleavage with all of the IgA1 proteases indicated clearly that there was no requirement for the presence of the tailpiece in IgA1 for susceptibility to any of the IgA1 proteases. This was not unexpected because of the tailpiece’s location distal to the hinge and its relatively small size compared with the CH2 and CH3 domains.

An earlier study reported that a human IgA1-IgG2 domain swap Ab, featuring the C{alpha}1, hinge, and C{alpha}2 domain of IgA1 and the C{gamma}3 domain of IgG2, was resistant to cleavage by the type 1 IgA1 protease of H. influenzae and the type 2 IgA1 protease of N. gonorrhoeae (22). In the present study, the observed resistance to cleavage by these enzymes of {alpha}1{alpha}2{gamma}3, a human IgA1-IgG1 domain swap, was consistent with this finding. In addition, we demonstrated that this molecule was also resistant to the type 1 IgA1 proteases of N. gonorrhoeae and N. meningitidis and the type 2 enzymes of H. influenzae and N. meningitidis. All of these IgA1 proteases cleave human IgA1 in the C-terminal half of the hinge. It would appear therefore that the ability of the serine IgA1 proteases of Neisseria and Haemophilus spp. to cleave IgA1 is dependent on the presence of particular amino acids or structures present in the CH3 domain of human IgA1 but not in the CH3 domain of human IgG. By contrast, all the metallo-IgA1 proteases of the different streptococci, which cleave wild-type IgA1 at the Pro227-Thr228 peptide bond in the N-terminal half of the IgA1 hinge, were able to cleave {alpha}1{alpha}2{gamma}3. Thus, either elements within the CH3 domain of human IgA1 are not required for cleavage of human IgA1 by the streptococcal IgA1 proteases, or structures contained with the C{gamma}3 domain can provide an adequate substitute for any requirements of this sort.

The reason for the resistance of {alpha}1{alpha}2{gamma}3 to cleavage by the IgA1 proteases of Neisseria and Haemophilus spp. was examined further by determining their activity on the {alpha}1{alpha}2{gamma}3PLAF mutant in which the PLAF loop from the C{alpha}3 domain replaced residues His433-Asn434-His435-Tyr436 at the equivalent position in the C{gamma}3 domain of {alpha}1{alpha}2{gamma}3 (25). With one exception, all of the IgA1 proteases that failed to cleave {alpha}1{alpha}2{gamma}3 also failed to cleave {alpha}1{alpha}2{gamma}3PLAF (Table I). Thus, the PLAF loop at the interface of the C{alpha}2 and C{alpha}3 domains of IgA1 is not involved in interaction of the serine IgA1 proteases of Neisseria and Haemophilus spp. with IgA1. The exception was the type 2 IgA1 protease of N. meningitidis, which appeared to partially cleave {alpha}1{alpha}2{gamma}3PLAF. This finding suggests that, for this particular protease, the PLAF residues contribute in some way toward optimal binding of the enzyme to IgA1 substrates, thereby facilitating appropriate orientation or position to permit cleavage in the hinge region. The lack of impact of the A442R point mutation on IgA1 protease susceptibility indicates that residues Pro440, Leu441, and Phe443 probably play the major role in this respect. The PLAF loop, lying at the domain interface (Fig. 7), forms an important interaction surface on IgA1 Fc, having been previously shown to be critical for binding of not only Fc{alpha}RI (CD89) (25, 28, 34, 35) but also streptococcal IgA-binding proteins (36) and staphylococcal toxin SSL7 (37). Its equivalent region on IgG is also involved in interaction with a variety of molecules and has been recognized as one of a limited number of regions on the Ig surface that is particularly suited to protein-protein interaction (38, 39). The loop is readily accessible and Fig. 7 illustrates how it might form an "anchor point" for the N. meningitidis type 2 IgA1 protease, assisting correct orientation to allow cleavage of the hinge above.


Figure 7
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  		FIGURE 7. Views of a molecular model of human IgA1 (coordinates from PBD accession 1iga). H chains are shown in blue, and L chains are shown in yellow. The H chain tailpieces (residues Pro455 onward) are not shown. Residues Pro440-Phe443 (green spheres) may modulate sensitivity to the N. meningitidis type 2 IgA1 protease, whereas the Ala399-Thr409 loop (white spheres) appears to influence sensitivity to the H. influenzae type 2 enzyme. In A, the peptide bond lying between Pro235 and Thr236 (red spheres), which is cleaved in wild-type IgA1 by both these enzymes, is indicated on one H chain by an arrow. A, Face-on view; B, side view, in which the Fab regions (H chains up to Pro219 and L chains) are not shown.

 
In an attempt to further define regions in the human IgA1 C{alpha}3 domain critical for substrate recognition and cleavage by one or more of the IgA1 proteases of N. meningitidis, N. gonorrhoeae, and H. influenzae, we generated mutant 399–409GAM. This mutant is identical with human IgA1 except that residues 399–409 in the C{alpha}3 domain have been replaced with the corresponding residues from human IgG1. The finding that the N. gonorrhoeae type 1 enzyme could partially cleave 399–409GAM but not HuBov{alpha}3 suggests that this enzyme requires elements in the human C{alpha}3 domain outside the 399–409 region that are present in human IgA1 but lacking in bovine IgA (see Fig. 2). Further experimentation will be required to define these elements more closely.

The finding that the type 2 IgA1 protease of H. influenzae failed to cleave 399–409GAM suggests that residues 399–409 (sequence Ala399-Ser400-Arg401-Gln402-Glu403-Pro404-Ser405-Gln406-Gly407-Thr408-Thr409) are necessary to allow cleavage by this enzyme. The Ab HuBov{alpha}3 was cleaved by this protease and the only amino acids common to wild-type IgA1 and HuBov{alpha}3 in this loop are Glu403, Gln406, and Thr409 (Fig. 2). These findings together may indicate that one or more of these amino acids or the entire 399–409 segment is required for the type 2 protease of H. influenzae to act. The 399–409 region lies within the upper central part of the C{alpha}3 domain (Fig. 7), and in a side view of the Fc (Fig. 7B) is seen partially to protrude out from the surface of the domain. It is tempting to speculate that the H. influenzae type 2 protease may use this accessible protrusion as a means to stabilize its interaction with IgA1 and facilitate correct orientation for optimal cleavage of the Pro235-Thr236 peptide bond lying above it (see Fig. 7B). Interestingly, the same region on the C{alpha}3 domain has been implicated in binding to the polymeric Ig receptor (40, 41). Taken together with the PLAF loop finding above, it appears that certain IgA1 proteases may take advantage of regions on the Fc region of IgA1 suited to protein-protein interaction that have been conserved because they form key interaction surfaces for host receptors.

In regard to the above conclusions, some possible caveats should be borne in mind. First, it is feasible that differential glycosylation profiles might impact on the susceptibility of substrates to certain IgA1 proteases. For example, it has been shown that hinge glycosylation can influence susceptibility to cleavage by the streptococcal metallo-enzyme IgA1 proteases (30, 42). However, we are not aware presently of evidence for the influence of hinge glycosylation on susceptibility to enzymes of Neisseria and Haemophilus spp., so considerations of hinge glycosylation patterns seem unlikely to significantly sway conclusions regarding sites in the CH3 domain that influence substrate susceptibility to such enzymes. Second, given the signs of significant intra- and interspecies homologous recombination revealed by sequence comparisons of serine-type IgA1 proteases, it is possible that, in certain cases, IgA1 proteases from different strains of the same species may have subtly different recognition requirements, and therefore that our conclusions may relate to IgA1 proteases from specific strains only. Although we cannot formally rule out this possibility, we do not consider it to be very likely, because our preliminary experiments on cleavage of wild-type IgA1 by IgA1 proteases from different strains have never revealed any interstrain differences.

An exciting aspect of our findings is that they point toward new possibilities for the design of both IgA1 protease inhibitors and therapeutic Abs. Before this study, peptide inhibitors modeled on the hinge itself would have been considered as the only possibilities for inhibitor development. Our results indicate, for the first time, that inhibitors designed to mimic aspects of the CH3 loops implicated in IgA1 recognition (Pro440-Phe443 and Ala399-Thr409) could also be used as starting points for inhibitor design. In addition, we have generated the first IgA1 molecules resistant to an IgA1 protease by virtue of mutations outside the hinge region. Thus, we have revealed the possibility of engineering IgA1 molecules with specific mutations within the Ala399-Thr409 loop that would be resistant to cleavage by the H. influenzae type 2 enzyme, without the necessity to mutate the hinge region. Retention of the normal hinge could be advantageous because the extended hinge region in IgA1 may preferentially allow bivalent Ag engagement leading to higher avidity interaction (43). Because H. influenzae is a major pathogen, such Abs may have potential as novel therapeutics.

In summary, through analysis of the effects of different directed Fc mutations, we have localized regions in the C{alpha}3 domain of IgA1 that impact on recognition and cleavage by certain IgA1 proteases. This information adds to the growing understanding of the features of human IgA1 influencing proteolytic cleavage by this group of enzymes.


   Acknowledgments
 
We are grateful to Professor Mogens Kilian for kindly supplying some of the bacterial strains, and to Drs. Richard Pleass, James Dunlop, and Catherine Anderson for preparation of some of the IgA1 mutant proteins.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Wellcome Trust. Back

2 Address correspondence and reprint requests to Dr. Jenny M. Woof, Division of Pathology and Neuroscience, University of Dundee Medical School, Ninewells Hospital, Dundee DD1 9SY, U.K. E-mail address: j.m.woof{at}dundee.ac.uk Back

Received for publication February 7, 2006. Accepted for publication June 20, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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