The immunoglobulin D-binding part of the outer membrane protein MID from Moraxella catarrhalis comprises 238 amino acids and a tetrameric structure.
Moraxella catarrhalis IgD-binding protein (MID), a 200-kDa outer membrane protein comprising 2,139 amino acids, has recently been isolated and shown to display a unique and specific affinity for human IgD. To identify the IgD-binding region, MID was digested with proteases. In addition, a series of truncated fragments of MID were manufactured and expressed in Escherichia coli followed by analysis for IgD binding in Western and dot blots. The smallest fragment with essentially preserved IgD binding was comprised of 238 amino acid residues (MID(962-1200)). Shorter recombinant proteins gradually lost IgD-binding capacity, and the shortest IgD-binding fragment comprising 157 amino acids (MID(985-1142)) displayed a 1,000-fold reduced IgD binding compared with the full-length molecule. The truncated MID(962-1200) was efficiently attracted to a standard IgD serum and to purified myeloma IgD(kappa) and IgD(lambda) sera but not to IgG, IgM, or IgA myeloma sera. Furthermore, the fragment specifically bound to peripheral blood B lymphocytes, and the binding was inhibited by preincubation with anti-IgD-Fab polyclonal antibodies. Results obtained by introducing five amino acids randomly into MID(962-1200) using transposons suggested that alpha-helix structures were important for IgD binding. Ultracentrifugation experiments and gel electrophoresis revealed that native MID(962-1200) was a tetramer. Interestingly, tetrameric MID(962-1200) attracted IgD more than 20-fold more efficiently than the monomeric form. Thus, a tetrameric structure of MID(962-1200) is crucial for optimal IgD-binding capacity.
Department of Medical Microbiology, Lund University, University Hospital Malmö, S-205 02 Malmö, Sweden.
Therése
T
Nordström
Arne
A
Forsgren
Kristian
K
Riesbeck
D018829:Adhesins, Bacterial; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007072:Immunoglobulin D / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D014461:Ultracentrifugation
D018829:Adhesins, Bacterial; D002352:Carrier Proteins; C439616:IgD-binding protein MID, Moraxella catarrhalis; D007072:Immunoglobulin D