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 Fc␣ 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.

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

“…Previous experiments showed that all the different bacterial IgA1 proteases cleaved recombinant wild-type human IgA1 to give fragments of mass consistent with cleavage in the hinge (22,27). However, recombinant human IgA2 was resistant to cleavage with these enzymes.…”

“…A series of mutants were produced based on a hybrid IgA2/IgA1 half hinge Ab in which a sequence representing half of the duplicated hinge region of IgA1 was incorporated into the equivalent position in IgA2 (22) (Fig. 1).…”

“…1). The IgA2-IgA1 half hinge expression vector pBS2 carried downstream of the mouse V NP gene, the ␣-chain gene of human IgA2m(1) with nucleotides coding for half of the hinge of human IgA1 inserted between the CH1 and CH2 domains (22). A BamHI and XhoI fragment (ϳ760 bp) comprising the 5Ј end of this ␣-chain construct was ligated into the multiple cloning site of pSP73 to generate pBS3.…”

Immunoglobulin gene analysis as a tool for investigating human immune responses

“…IgA is a mucosal antibody and can be secreted across barriers in the gut, breast, lungs, GU tract to block pathogens at mucosal surfaces. The major differences between IgA1 and IgA2 lies in the presence of the drastically extended hinge region of IgA1, thought to improve antigen recognition by increasing affinity with antigen epitopes that are spatially distant, but making it vulnerable to proteases 37, 38, 39. The IgE antibody has received an increasing amount of attention because of its role in hypersensivity responses and allergy in the developed world, although initially thought to have evolved to target parasites (eg, helminths and parasitic arthropods) that are too large to be phagocytosed 40, 41, 42…”

Are anti-HIV IgAs good guys or bad guys?

“…cleavage by proteases derived from bacterial pathogens, such as Haemophilus influenzae and Neisseria meningitidis [20][21][22]. It is currently not known whether SIgA1 and SIgA2 exhibit differential susceptibility to proteolytic cleavage by normal microbial flora in the various mucosal fluids.…”