Bernard W. Senior

Purification and characterisation of human immunoglobulin IgA1 and IgA2 isotypes from serum

A method is described for the simultaneous purification of IgA1 and IgA2 from human serum. Ammonium sulphate precipitation, gel filtration and ion-exchange chromatography on DEAE-Sephacel yielded a partially purified IgA preparation which was separated quantitatively into IgA1 and IgA2 by affinity chromatography on jacalin-Sepharose. The IgA1 which bound to the jacalin was eluted with 0.8 M D-galactose. The IgA1 preparation was apparently homogeneous by SDS-PAGE but contained a trace of C1-inhibitor and a second protein detected by immunoelectrophoresis. The IgA2 which did not bind to the jacalin was purified to apparent homogeneity by chromatography on columns of Protein G-Sepharose, Fastflow-S Sepharose and Superose 6. Typical yields were 95% and 58% for IgA1 and IgA2 respectively or 253 mg and 24 mg per 100 ml serum. The IgA1 and IgA2 were characterised by their reactivity with isotype specific monoclonal antibodies and sensitivity to bacterial proteinases. The IgA2 preparation apparently contained both allotypes, IgA2m(1) and IgA2m(2).

Amino Acid Sequence Requirements in the Hinge of Human Immunoglobulin A1 (IgA1) for Cleavage by Streptococcal IgA1 Proteases

ABSTRACT 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 Fcα 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.

The Influences of Hinge Length and Composition on the Susceptibility of Human IgA to Cleavage by Diverse Bacterial IgA1 Proteases

The influences of IgA hinge length and composition on its susceptibility to cleavage by bacterial IgA1 proteases were examined using a panel of IgA hinge mutants. The IgA1 proteases of Streptococcus pneumoniae, Streptococcus sanguis strains SK4 and SK49, Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae cleaved IgA2-IgA1 half hinge, an Ab featuring half of the IgA1 hinge incorporated into the equivalent site in IgA1 protease-resistant IgA2, whereas those of Streptococcus mitis, Streptococcus oralis, and S. sanguis strain SK1 did not. Hinge length reduction by removal of two of the four C-terminal proline residues rendered IgA2-IgA1 half hinge resistant to all streptococcal IgA1 metalloproteinases but it remained sensitive to cleavage by the serine-type IgA1 proteases of Neisseria and Haemophilus spp. The four C-terminal proline residues could be substituted by alanine residues or transferred to the N-terminal extremity of the hinge without affect on the susceptibility of the Ab to cleavage by serine-type IgA1 proteases. However, their removal rendered the Ab resistant to cleavage by all the IgA1 proteases. We conclude that the serine-type IgA1 proteases of Neisseria and Haemophilus require the Fab and Fc regions to be separated by at least ten (or in the case of N. gonorrhoeae type I protease, nine) amino acids between Val222 and Cys241 (IgA1 numbering) for efficient access and cleavage. By contrast, the streptococcal IgA1 metalloproteinases require 12 or more appropriate amino acids between the Fab and Fc to maintain a minimum critical distance between the scissile bond and the start of the Fc.

Effect of Mutations in the Human Immunoglobulin A1 (IgA1) Hinge on Its Susceptibility to Cleavage by Diverse Bacterial IgA1 Proteases

ABSTRACT 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.

ABSTRACT 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 Fcα 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.

Structural and Functional Consequences of Cleavage of Human Secretory and Human Serum Immunoglobulin A1 by Proteinases from Proteus mirabilis and Neisseria meningitidis

ABSTRACT The cleavage of human serum monomeric immunoglobulin A1 (IgA1) and human secretory IgA1 (S-IgA1) by IgA1 proteinase of Neisseriameningitidis and cleavage by the proteinase from Proteusmirabilis have been compared. For serum IgA1, both proteinases cleaved only the α chain. N. meningitidis proteinase cleaved only in the hinge. P. mirabilis proteinase sequentially removed the tailpiece, the CH3 domain, and the CH2 domain. The cleavage of S-IgA1 by N. meningitidis proteinase occurred only in the hinge and was as rapid as that of serum IgA1. P. mirabilis proteinase predominantly cleaved the secretory component (SC) of S-IgA1. The SC of S-IgA1, whether cleaved or not, appeared to protect the α1 chain. Purified Fc fragment derived from the cleavage of serum IgA1 by N. meningitidis proteinase stimulated a respiratory burst in neutrophils through Fcα receptors, whereas the (Fcα1)2-SC fragment from digested S-IgA1 did not. The loss of the tailpiece from serum IgA1 treated with P. mirabilis proteinase had little effect, but the loss of the CH3 domain was concurrent with a rapid loss in the ability to bind to Fcα receptors. S-IgA1 treated with P. mirabilis proteinase under the same conditions retained the ability to bind to Fcα receptors. The results are consistent with the Fcα receptor binding site being at the CH2-CH3 interface. These data shed further light on the structure of S-IgA1 and indicate that the binding site for the Fcα receptor in S-IgA is protected by SC, thus prolonging its ability to activate phagocytic cells at the mucosal surface.

A comparison of the binding of secretory component to immunoglobulin A (IgA) in human colostral S-IgA1 and S-IgA2.

A detailed investigation of the binding of secretory component to immunoglobulin A (IgA) in human secretory IgA2 (S‐IgA2) was made possible by the development of a new method of purifying S‐IgA1, S‐IgA2 and free secretory component from human colostrum using thiophilic gel chromatography and chromatography on Jacalin‐agarose. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis of unreduced pure S‐IgA2 revealed that, unlike in S‐IgA1, a significant proportion of the secretory component was bound non‐covalently in S‐IgA2. When S‐IgA1 was incubated with a protease purified from Proteus mirabilis the secretory component, but not the α‐chain, was cleaved. This is in contrast to serum IgA1, in which the α‐chain was cleaved under the same conditions – direct evidence that secretory component does protect the α‐chain from proteolytic cleavage in S‐IgA. Comparisons between the products of cleavage with P. mirabilis protease of free secretory component and bound secretory component in S‐IgA1 and S‐IgA2 also indicated that, contrary to the general assumption, the binding of secretory component to IgA is different in S‐IgA2 from that in S‐IgA1.

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

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 α1α2γ3, 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 Cα3 domain motif Pro440-Phe443 into the corresponding position in the Cγ3 domain of α1α2γ3 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 HuBovα3 in which the Cα3 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.

Cleavage of the Hormone Human Chorionic Gonadotropin, by the Type 1 IgA1 Protease of Neisseria gonorrhoeae and Its Implications

The hormone human chorionic gonadotropin (hCG) serves to maintain the fetus during early pregnancy and regulate the onset of labor in late pregnancy. hCG also prevents Neisseria gonorrhoeae from developing invasive characteristics. Part of the beta subunit of hCG has an amino acid sequence similar to that of the hinge of human IgA1, which is the site of action of IgA1 proteases. This study examined the sensitivity of hCG to gonococcal IgA1 proteases, by means of autoradiography, immunoblotting, and RIA. hCG was cleaved in the beta subunit by the type 1 but not the type 2 IgA1 proteases of N. gonorrhoeae. hCG cleavage by gonococcal IgA1 proteases in vivo may increase the invasiveness of the pathogen and destroy its natural biologic activity, with major consequences for the fetus and the pregnancy.

Decision Analysis of Histamine H2-Receptor Antagonist Maintenance Therapy Versus Helicobacter pylori Eradication Therapy: A Randomised Controlled Trial in Patients with Continuing Pain After Duodenal Ulcer

AbstractBackground: Much has been published on the efficacy and cost effectiveness of Helicobacter pylori eradication treatment as an alternative to histamine H2-receptor antagonist maintenance treatment in peptic ulcer disease. However, most studies have analysed and emphasised H. pylori eradication rates rather than management/control of symptoms and the associated cost savings. Although H. pylori eradication therapy is very successful in clearing the infection, dyspeptic symptoms may persist and management of these can be expensive. Objective: The aim of this studywas to assess the cost implications in controlling symptoms using either H2-receptor antagonist maintenance therapy or H. pylori eradication therapy in patients with duodenal ulcer disease. Design: This was a non-blind, prospective, randomised, parallel-group study comparing maintenance H2-receptor antagonist treatment using ranitidine with H. pylori eradication therapy, with a 1-year follow-up. Setting: This was a study of outpatients from general practices in Dundee, Scotland, or the Ninewells Hospital, Dundee, gastroenterology clinic. Patients and participants: 119 patientswith confirmed duodenal ulcer, free from active ulceration at study entry but positive for H. pylori infection, who were receiving maintenance H2-receptor antagonist therapy. Interventions: Patients were randomised to receive either continuing maintenance therapy with ranitidine (initially 150mg daily; 58 patients) or H. pylori eradication therapy using an omeprazole/amoxicillin/metronidazole regimen (or omeprazole/clarithromycin if allergic to penicillin). Main outcome measures and results: Overall, H. pylori eradication rates were 100% per protocol and 95.1% intention-to-treat. At completion of 1 year of follow-up, 12 of the 61 (19.7%) patients successfully eradicated of H. pylori were still dependent on acid suppression for symptom relief. H. pylori eradication treatment was the least-cost strategy in managing/controlling symptoms at 1 year (£168 vs £210 per patient; 1996 values). However, over time, post-eradication treatment costs were greater than H2-receptor antagonist therapy costs. Any potential savings were directly related to the proportion of patients needing further treatment post-eradication, the cost of endoscopy and the urea breath test. Conclusions: If dyspepsia persists long term, H. pylori eradication treatment may not be the least-cost option for patients with duodenal ulcer.