Abstract
Secretory component (SC) in association with polymeric IgA (pIgA) forms secretory IgA, the major antibody active at mucosal surfaces. SC also exists in the free form, with innate-like neutralizing properties against pathogens. Free SC consists of five glycosylated variable (V)-type Ig domains (D1-D5), whose structure was determined by x-ray and neutron scattering, ultracentrifugation, and modeling. With a radius of gyration of 3.53-3.63 nm, a length of 12.5 nm, and a sedimentation coefficient of 4.0 S, SC possesses an unexpected compact structure. Constrained scattering modeling based on up to 13,000 trial models shows that SC adopts a J-shaped structure in which D4 and D5 are folded back against D2 and D3. The seven glycosylation sites are located on one side of SC, leaving known IgA-binding motifs free to interact with pIgA. This work represents the first analysis of the three-dimensional structure of full-length free SC and paves the way to a better understanding of the association between SC and its potential ligands, i.e. pIgA and pathogenic-associated motifs.
Highlights
The D1–D3 constrained modeling parameters and curve fits are shown in panels A–C in the supplemental Figs
Intact SC was modeled using either five unconstrained linkers or the best-fit D1–D3 and D4 –D5 models
The second search varied only the D3–D4 linker between the ditions using complementary methods based on x-ray and best-fit D1–D3 and D4 –D5 models
Summary
Recombinant Secretory Component and Its Fragments— CHO cells producing human SC [7] were cultured in suspension in CHO-S-SFM II medium (Invitrogen) using CELLine 350 bioreactors (Integra-Biosciences). 6), and anti-D5 (lanes 7 and 8) identify full-length SC at 75 kDa, D1–D3 as a major band at 45 kDa, and D4 –D5 at 30 kDa. with a cutoff of 4 atoms consistently produced sphere models within 95% of the total unhydrated volume of 99.7, 56.9, and 43.8 nm, respectively, calculated from their compositions. A flat background correction of 2.7% of I(0) was applied to the final neutron scattering curve fits in Fig. 8 to allow for a uniform incoherent scattering of residual protons in the sample. Other details, including those of calibration studies used to validate this approach, are given elsewhere [22, 26]. The ten best SC ␣-carbon co-ordinate models were deposited in the Protein Data Bank with the accession code 2OCW
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