Biological inferences from IgM binding characteristics of recombinant human secretory component mutants.
The polymeric immunoglobulin receptor (pIgR) or membrane secretory component (SC) selectively transports polymeric IgA and IgM across secretory epithelial cells to mucosal surfaces. The ligand binding ectodomain consists of five homologous Ig-like domains with domain I being an absolute requirement for binding. The role of DII to V in IgM binding remains unknown. Here, using in vitro refolded non-glycosylated recombinant domain deletion mutants of human SC, we show by biological and biophysical binding assays that DII to V are required for high affinity binding to IgM. Competitive binding analysis, by whole cell ELISA, showed that DII-V significantly increase the affinity of recombinant SC for IgM (K(i)=2.42 nM) as opposed to recombinant DI only (K(i)=44.8 nM). Lastly, we provide qualitative data highlighting the complexity of measuring the IgM/SC interaction using surface plasmon resonance spectroscopy.
Prinsloo
Earl
E
Department of Biochemistry and Microbiology, Faculty of Science, Nelson Mandela Metropolitan University South Campus, Summerstrand, Port Elizabeth, South Africa. e.prinsloo@ru.ac.za
Oosthuizen
Vaughan
V
Van de Venter
Maryna
M
Naudé
Ryno J
RJ
D001693:Biological Transport, Active; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005818:Genetic Engineering; D006801:Humans; D007075:Immunoglobulin M / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics / Q000276:immunology*; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics* / Q000276:immunology; D019056:Receptors, Polymeric Immunoglobulin / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017384:Sequence Deletion
D007075:Immunoglobulin M; D019056:Receptors, Polymeric Immunoglobulin; D011994:Recombinant Proteins; C024242:polymeric IgM