Biophysical and Biochemical Characterization of Avian Secretory Component Provides Structural Insights into the Evolution of the Polymeric Ig Receptor

Abstract

The polymeric Ig receptor (pIgR) transports polymeric Abs across epithelia to the mucosa, where proteolytic cleavage releases the ectodomain (secretory component [SC]) as an integral component of secretory Abs, or as an unliganded protein that can mediate interactions with bacteria. SC is conserved among vertebrates, but domain organization is variable: mammalian SC has five domains (D1-D5), whereas avian, amphibian, and reptilian SC lack the D2 domain, and fish SC lacks domains D2-D4. In this study, we used double electron-electron resonance spectroscopy and surface plasmon resonance binding studies to characterize the structure, dynamics, and ligand binding properties of avian SC, avian SC domain variants, and a human SC (hSC) variant lacking the D2 domain. These experiments demonstrated that, unlike hSC, which adopts a compact or "closed" domain arrangement, unliganded avian SC is flexible and exists in both closed and open states, suggesting that the mammalian SC D2 domain stabilizes the closed conformation observed for hSC D1-D5. Experiments also demonstrated that avian and mammalian pIgR share related, but distinct, mechanisms of ligand binding. Together, our data reveal differences in the molecular recognition mechanisms associated with evolutionary changes in the pIgR protein.

Figure 1. pIgR domain organization and SEC-MALS data

(A) Schematic showing the domain organization of the pIgR protein from humans, birds, reptiles, amphibians, and teleost fish. Each mammalian domain (D1-D5) is indicated by color and domains from other species are colored according to their mammalian homolog. (B) SEC-MALS data for ggSC showing elution time versus refractive index units (RIU) and Molar Mass (Da).

Figure 2. Comparison of ggSC and hSC sequences

Sequence alignment of ggSC (top) and hSC (bottom) with domain numbering for ggSC (orange) and hSC (blue) indicated. The locations of regions implicated in mammalian SC-pIg interactions, including D1 CDR loops and D5 CDR1 and DE loops, are indicated above the ggSC sequence along with the position of Cys residues involved in potential covalent binding to pIgA (black asterisk), ggSC potential N-linked glycosylation sites (orange asterisk), and residues mutated to Cys for nitroxide labeling (R1). The hSC secondary structure elements, determined from the hSC crystal structure (15), are shown below the hSC sequence along with residues found to stabilize domain interfaces (colored according to the key below the sequence alignment).

Figure 3. DEER data

(A–D) Time domain data (left) and associated distance distribution, probability [P(r)] versus distance (Å), for the indicated mutants obtained after model-free fitting of the dipolar evolution function (right). The gray and orange bars indicate the upper limit of reliable distance and shape of the distribution (see Materials and Methods). Distances 70 Å are beyond detection limits (dashed traces).

Figure 4. ggSC and hSC binding to dIgA and pIgM

(A–C) Sensorgrams showing ggSC, ggSC D1, ggSC tCDR1/tDE binding human dIgA. The highest concentration for all variants binding to human dIgA was 1024nM. (D) Overlay of the normalized binding response for concentration-matched samples (32nM) from A–C and Supplemental Fig. 3A, 3B, (hSC and hSC D1-D3-D4-D5). (E–G) Sensorgrams showing ggSC, ggSC D1, ggSC tCDR1/tDE binding human pIgM. Highest concentrations of ggSC variant binding to human pIgM were: 32nM (ggD1), 1024nM (ggSC), 1024nM (ggSC tCDR1/tDE). (H) Overlay of the normalized binding response for concentration-matched samples (32nM) from E–G and Supplemental Fig. 3C, 3D, (hSC and hSC D1-D3-D4-D5). Sensorgrams shown are equivalent to those obtained from replicate, independent experiments (not shown).

Figure 5. Models of SC variants

(A) Model of a closed four-domain SC variant based on the hSC crystal structure with loop residues (which replace D2; black arrow) modeled in geometrically reasonable positions. Labels indicate mammalian SC domain identity (blue) and avian SC domain identity (orange, parentheses) and orange arrows indicate putative regions of flexibility. (B) Schematic summary of SC conformation throughout evolution. Fish SC has just two domains and exists in an open conformation; avian SC has four domains and exists in an equilibrium between open and closed states; hSC remains closed until encountering ligand.