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.

FIG. 1.

Amino acid sequences of the hinge region of wild-type human IgA1 and IgA2m(1) and of the mutant recombinant IgA antibodies constructed. The wild-type IgA1 hinge contains two identical halves, one underlined by a solid line, the other underlined by a dashed line. The half-hinge insert is shown highlighted in grey. Mutations are shown boxed. The sites of cleavage of bacterial IgA1 proteases in the wild-type IgA1 hinge are indicated above.

FIG. 2.

Western blot analysis under reducing conditions of antibody IgA2-IgA1 half hinge (hh) (lanes 1 and 2) and other antibodies as indicated (lanes 3 to 10) treated with (+) S. pneumoniae IgA1 protease (lanes 2, 4, 6, 8, and 10) or untreated (−) (lanes 1, 3, 5, 7, and 9) and probed with a horseradish peroxidase-conjugated antibody detecting the Fab part of human IgA1. The positions of molecular mass markers in kilodaltons are indicated on the left. The IgA1 protease of S. pneumoniae, which cleaves a Pro-Thr peptide bond in wild-type IgA1, cleaved all the antibodies, including hhP227S, which lacked a Pro-Thr peptide bond in the hinge.

FIG. 3.

Partial view of mass spectrometric spectrum of digestion product of hhS224/230P following incubation with H. influenzae type 1 IgA1 protease, expanded in the region encompassing the 2203 mass peak. The inset shows the amino acid sequence obtained for the 2203 peak by LC MS-MS.

FIG. 4.

Western blot analysis under reducing conditions of antibody IgA2-IgA1 half hinge (hh) (lanes 1 and 2) and other antibodies as indicated (lanes 3 to 10) treated with (+) the type 1 IgA1 protease of N. gonorrhoeae (lanes 2, 4, 6, 8, and 10) or untreated (−) (lanes 1, 3, 5, 7, and 9) and probed with a horseradish peroxidase-conjugated antibody detecting the Fab part of human IgA1. The type 1 IgA1 protease of N. gonorrhoeae, which cleaves a Pro-Ser peptide bond in wild-type IgA1, cleaved all the antibodies, including hhS224/230P, which lacks a Pro-Ser peptide bond in the hinge.

FIG. 5.

Western blot analysis under reducing conditions of antibodies IgA2-IgA1 half hinge (hh) (lanes 1, 2, 7, and 8) and other antibodies as indicated (lanes 3 to 6 and 9 to 12) untreated (lanes 1 and 7) or treated with the type 1 IgA1 protease of H. influenzae (lanes 2 to 6) or N. meningitidis (lanes 8 to 12) and probed with an alkaline phosphatase-conjugated antibody detecting the Fc part of human IgA1. The positions of molecular mass markers in kilodaltons are indicated on the left. The position of the cleavable Pro-Ser peptide bond in the hinge influences sensitivity to cleavage. The type 1 IgA1 proteases of H. influenzae and N. meningitidis more readily cleaved antibody hhS224P, whose Pro-Ser peptide bond was at the CH2 end of the hinge, than antibody hhS230P, whose Pro-Ser peptide bond was at the CH1 end of the hinge.

FIG. 6.

Western blot analysis under reducing conditions of antibodies IgA2-IgA1 half hinge (hh) (lanes 1 and 2) and other antibodies as indicated (lanes 3 to 10) treated with (+) the type 2 IgA1 protease of H. influenzae (lanes 2, 4, 6, 8, and 10) or untreated (−) (lanes 1, 3, 5, 7, and 9) and probed with a horseradish peroxidase-conjugated antibody detecting the Fab part of human IgA1. The type 2 IgA1 protease of H. influenzae, which cleaves a Pro-Thr peptide bond in wild-type IgA1, cleaved all the antibodies, including hhP227S, which lacked a Pro-Thr peptide bond in the hinge.