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J. Biol. Chem., Vol. 282, Issue 23, 16969-16980, June 8, 2007
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1
234
From the
Department of Biochemistry and Molecular Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, United Kingdom and the Service d'Immunologie et d'Allergie, Centre Hospitalier Universitaire Vaudois, BH19-650, Rue du Bugnon, CH-1011 Lausanne, Switzerland
Received for publication, February 12, 2007 , and in revised form, March 9, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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SC consists of five immunoglobulin-like domains (D1–D5) and up to seven glycan chains. SC bound to pIgA protects the antibody against proteolytic digestion (6) and governs anchoring of SIgA at mucosal surfaces (7). Free SC is also found in secretions and is now recognized as an active antibacterial participant to protect mucosal surfaces against Helicobacter pylori (8), enteropathogenic Escherichia coli and Clostridium difficile toxin A (9), and Streptococcus pneumoniae choline binding Protein A (10, 11). Recombinant human SC produced from transfected Chinese hamster ovary (CHO) cells behaves identically, and its neutralizing properties rely on the presence of oligosaccharides on SC (12).
Despite the importance of SC and its glycosylation, only partial information is available on its structure. SC comprises the first 585 residues in pIgR (Fig. 1A) (13). Homology modeling of rabbit SC domains 1 and 2 (D1 and D2) indicates that both exhibit the typical features of Ig variable (V)-type superfamily members with seven 
-strands A–G and an extra two designated C' and C'' (14, 15). A similar topology was determined in the modeling of domains 2 and 3 (D2 and D3) of mouse SC (16). These were confirmed by the crystal structure of D1 of human SC, which revealed a V-type fold (17). So far, the three-dimensional domain arrangement of full-length SC has not been considered, even though this is essential to appreciate molecular mechanisms that are involved in mucosal immunity. Its high glycosylation and the presence of long interdomain linkers suggest that SC may be too flexible to be crystallized intact. In a situation such as this, x-ray and neutron scattering and analytical ultracentrifugation in combination with constrained modeling techniques will reveal the domain arrangement of SC (18). Here, we determine a solution structure for recombinant human SC produced in CHO cells. We show that SC forms a compact domain arrangement, and this structure rationalizes many of the biological roles of both free and bound SC for the first time.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For the x-ray scattering and ultracentrifugation experiments, samples were recovered in PBS. For neutron scattering, the buffer was Dulbecco's PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.5), made up in 100% 2H2O. The neutron samples were dialyzed at 6 °C into this 100% 2H2O buffer for 36 h with four buffer changes. The two buffer densities in H2O solvent were predicted to be 1.00485 g/ml and 1.00543 g/ml, respectively, from these compositions using SEDNTERP. The SC, D1–D3, and D4–D5 concentrations were determined using absorption coefficients of 12.3, 7.1, and 18.7 (1%, 280 nm, and 1-cm path length) that were calculated from its composition by assuming the presence of seven biantennary complex-type oligosaccharides Man3GlcNAc4Gal2NeuNAc2 (see Fig. 1) (19). The partial specific volume 
for SC, D1–D3, and D4–D5 was calculated to be 0.717, 0.717, and 0.718 ml/g, respectively, from their compositions (19). The calculated molecular masses for SC, D1–D3, and D4–D5 were 79.6, 45.5, and 34.1 kDa, respectively, their unhydrated volumes were 99.7, 56.9, and 42.7 nm3, respectively, and their hydrated volumes were 132.2, 75.5, and 56.7 nm3, respectively (assuming 0.3 g of H2O/g of glycoprotein).
X-ray and Neutron Scattering and Ultracentrifugation Data—X-ray scattering data were obtained in two sessions on the Beamline ID02 at the European Synchrotron Radiation Facility (Grenoble, France), operating at 6.03 GeV (20). To reduce the incident flux, the experiments were performed in 16-bunch and 4-bunch mode with storage ring currents ranging between 92 and 53 mA (SC) and 4-bunch mode with currents ranging between 41 and 23 mA (D1–D3 and D4–D5). The sample-to-detector distance of 3.0 m, yielded a Q range from 0.07 to 2.1 nm–1 (where Q = 4
sin 
/
; 2 = scattering angle; = wavelength). Samples were measured at concentrations between 0.62 and to 2.0 mg/ml (SC), 0.39 to 1.6 mg/ml (D1–D3), and 0.40 to 0.73 mg/ml (D4–D5). Data were acquired using five or ten time frames of 0.5, 1, 2, or 4 s to establish the absence of radiation damage. Neutron scattering data for SC were collected in one session in a Q range between 0.02 and 1.93 nm–1 on Instrument LOQ at the ISIS facility at the Rutherford Appleton laboratory, Didcot, UK (21). The proton beam current was 172–173 µA, and acquisition times were 1–2 h for concentrations between 0.62 and 2.0 mg/ml, measured at 15 °C. Other details, including calibrations and data reduction, the Guinier analyses to determine the radius of gyration RG and the radius of gyration of the cross-sectional structure RXS, and the calculation of the distance distribution function P(r) using GNOM for which Q ranged between 0.08 and 2.06 nm–1, are described previously (22, 23).
Analytical ultracentrifugation was performed at 20 °C on a Beckman XL-I instrument, equipped with an AnTi50 rotor. Sedimentation equilibrium data sets for SC between 0.20 and 2.50 mg/ml were acquired over 45 h with column heights of 2 mm at rotor speeds of 8,000, 11,000, 14,000, 18,000, and 23,000 rpm, until equilibrium had been reached at each speed as shown by the perfect overlay of runs measured at 5-h intervals. Sedimentation velocity data for SC, D1–D3, and D4–D5 were acquired over 16 h at rotor speeds of 20,000, 25,000, 30,000, and 35,000 rpm (and 42,000 rpm for D1–D3 and D4–D5) with column heights of 12 mm. SC was studied at five concentrations from 0.30 to 2.50 mg/ml. D1–D3 and D4–D5 were each studied at two concentrations of 0.39–1.6 mg/ml and 0.40–0.73 mg/ml respectively. DCDT+ g(s*) time-derivative analyses and SED-FIT Lamm equation fits based on the non-interacting species model were performed as previously described (23).
Constrained Modeling of SC—SC corresponds to residues 19–603 of the polymeric immunoglobulin receptor (SwissProt code P01833
[GenBank]
). The start and end of D2–D5 is defined as residues 1–104 in Fig. 1. The homology modeling of D2–D5 employed MODELLER v7 (24) and was based on a topological sequence alignment with the D1 crystal structure identified using BLASTP (PDB code 1xed) (17). The models were structurally validated using PROCHECK. Seven biantennary oligosaccharide chains Man3GlcNAc4Gal2NeuNAc2 were incorporated in extended conformations in the D1–D5 models (25, 23). The first SC residue Lys was added to the D1 crystal structure using MODELLER v7. To model the other interdomain linkers, residues 109–116, 219–221, 324–335, 440–446, and 543–585 (Fig. 1B) were created in extended -strand conformations using INSIGHT II 98.0 with BIOPOLYMER, DISCOVER, HOMOLOGY, and DELPHI modules (Accelrys, San Diego, CA), and 5000 randomized conformations for each were generated using DISCOVER3 (22). Linker length constraints of 95–100% were applied to generate sufficient conformational variability when needed. Full SC models were created by superimpositions of the domains and the linkers.
Each SC, D1–D3, and D4–D5 model was converted into Debye spheres to calculate the x-ray and neutron scattering curves (22, 23). A cube side length of 0.542 nm in combination 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 nm3, respectively, calculated from their compositions. Hence the optimal totals of unhydrated spheres were 626, 268, and 353 respectively, which were then hydrated. The number of spheres N in the unhydrated and hydrated models after grid transformation was used to assess steric overlap between the D1, D2, D3, D4, and D5 domains.
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were calculated directly from the hydrated SC, D1–D3, and D4–D5 sphere models using the GENDIA and HYDRO programs (22, 23). Control calculations of were performed using HYDROPRO shell modeling directly from the molecular models for SC, D1–D3, and D4–D5 (27). The ten best SC 
-carbon co-ordinate models were deposited in the Protein Data Bank with the accession code 2OCW. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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X-ray and Neutron Scattering—X-ray scattering identified the domain arrangements in SC, D1–D3, and D4–D5 at concentrations between 0.40 and 2.7 mg/ml (see "Materials and Methods"). Because no radiation damage was detectable, the ten time frames of each acquisition were averaged for analyses. SDS-PAGE analyses before and after runs showed that, although SC and D4–D5 generally remained intact, further cleavages occurred in D1–D3 to give single domain fragments as well as the three-domain fragment. All the SC and D4–D5 results below correspond to samples shown to be intact by SDS-PAGE before and after the experiment.
At low Q values, Guinier analyses of ln I(Q) against Q2 resulted in linear plots for SC and its two fragments, yielding the radius of gyration (RG) (Fig. 2, A, E, and F). At larger Q values, analyses of ln I(Q)Q against Q2 yielded the RG of the cross-section (RXS). For SC, a mean x-ray RG of 3.53 (± 0.03) nm and mean RXS of 1.76 (± 0.08) nm were recorded from seven runs (Table 1). Another SC sample gave a similar mean x-ray RG of 3.64 (± 0.17) nm and RXS of 1.63 (± 0.12) nm from six runs. A partially cleaved sample with 
33% SC, 33% D1–D3, and 33% D4–D5 showed a small increase in the mean RG to 3.77 (± 0.17) nm and a small decrease in the mean RXS to 1.60 (± 0.61) nm. From five runs, the partially cleaved sample of D1–D3 showed an apparent RG of 3.44 (± 0.27) nm and an apparent RXS of 1.07 (± 0.07) nm (supplemental Fig. S1). From five runs, D4–D5 was found to have a mean RG of 3.16 (± 0.04) nm and a mean RXS of 1.11 (± 0.08) nm (supplemental Fig. S1). The Guinier analyses were confirmed by the I(0)/c values, which are proportional to molecular masses, where c is the concentration (18). The observed I(0)/c values were in the ratio of 1:0.30:0.53, respectively, relative to Lupolen. The sequence-derived molecular mass ratios were 1:0.57:0.43 in that order. I(0)/c for D1–D3 was lower than expected, which is attributed to its cleavage, whereas that for D4–D5 was in fair agreement with the sequence.
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0 values, the lengths were calculated to be 11.9, 9.1, and 10.8 nm, respectively (Table 1). Despite the cleavage in the D1–D3 sample, the similarity of these three lengths is explained by postulating that SC is folded back upon itself. If the end-to-end length of the SC domains is taken to be 3.7 nm from the D1 crystal structure (17), SC, D1–D3, and D4–D5 are predicted to be 18.5, 11.1, and 7.4 nm long if they are fully extended but with no linkers. The disparity between the observed SC length of 10.6–11.9 nm and the 18.5 nm prediction shows that SC must be folded back. That D4–D5 (Table 1) is observed to be longer than the predicted value may result from the 42 C-terminal residues in SC (Fig. 1A). The observed lengths for D1–D3 are in fair agreement with the predicted length of 11.1 nm.
Neutron scattering data for SC confirm the x-ray RG values (Table 1). These were obtained as a control of radiation damage by x-rays, because these effects are absent in neutron scattering. The neutron data are complementary in that the hydration shell surrounding SC is not detectable (26). The high negative protein-solvent scattering contrast acts as a control of any scattering inhomogeneity effects caused by the high proportion of carbohydrate in SC. The mean of three RG and RXS values for SC were 3.63 (± 0.28) nm and 1.30 (± 0.10) nm, respectively (Fig. 2). The decrease in the neutron RXS compared with the x-ray RXS value is attributed to the absence of hydration effects on the cross-section of SC (25). The length of SC from the neutron RG and RXS was 11.7 nm and from I(0) and [I(Q)Q]Q0 was 13.1 nm, which is consistent with the x-ray determined lengths. The neutron data gives molecular masses from the expression I(0)/c x 9 x 105 (22). The observed I(0)/c of 0.087 (± 0.009) resulted in 78 (± 8) kDa, which is close to the sequence-derived value of 79.6 kDa.
The transformation of I(Q) into the distance distribution function, P(r) confirmed that SC has a folded back conformation in solution. The P(r) curves for SC (x-rays and neutrons) and for D1–D3 and D4–D5 (x-rays) were reproducible with single maxima (Fig. 3). The RG from these were 3.66 (± 0.19) nm and 3.71 (± 0.12) nm (two SC x-ray samples), 3.73 (± 0.17) nm (SC neutrons), 3.52 (± 0.27) nm (D1–D3), and 3.15 (± 0.18) nm (D4–D5), all of which were consistent with the Guinier analyses (Table 1). The maximum, M, corresponds to the most frequently occurring interatomic distance. These were 3.8 (± 0.2) nm and 3.2 nm, respectively, for the x-ray and neutron data for SC and were slightly smaller at 2.9 nm and 2.7 nm for D1–D3 and D4–D5, respectively. The length, L, is determined when P(r) becomes zero at large r values and is not dependent on shape assumptions. For SC, L was determined to be 13 (± 1) nm (x-rays) and 12 (± 1) nm (neutrons). For D1–D3 and D4–D5, L was determined to be 12 (± 1) nm and 10 (± 1) nm, respectively. Although the D1–D3 analysis may be affected by cleaved products, this analysis will reveal the maximum length of the intact protein as any cleavage products will be shorter in length. These lengths were consistent with the Guinier-derived analyses (Table 1).
Analytical Ultracentrifugation—The oligomerization of SC at concentrations between 0.2 and 2.5 mg/ml was determined by sedimentation equilibrium to show that this is monomeric. Assuming a single species was present, good curve fits with small random residuals were obtained (Fig. 4). SDS-PAGE analyses before and after runs confirmed that any SC cleavage was not detectable. A small decrease in the molecular mass of SC was observed with increasing rotor speed, which is attributable to sample polydispersity (30). This is most likely to result from heterogeneous occupation at the seven N-linked glycosylation sites, and/or any cleavage products (Fig. 1). Thus the presence of seven bi-antennary oligosaccharides would give a molecular mass of 79,600 Da, whereas seven tetra-antennary ones would give 88,800 Da. In addition, the molecular masses slightly increased on dilution (lower panels, Fig. 4). This was attributed to decreased interactions between SC molecules at lower concentrations, because any oligomerization would result in a decrease in molecular masses on dilution. Regressions resulted in molecular masses of 79,500 (± 2,900) Da (interference) and 81,600 (± 5,500) Da (absorbance) at zero concentration. These agree well with the sequence-derived molecular mass. The equilibrium data therefore showed that all seven putative glycosylation sites are filled.
The sedimentation coefficient, , monitors the macromolecular elongation, independently from RG. Sedimentation velocity was used to examine SC, D1–D3, and D4–D5, as well as SC that was cleaved into an equimolar mixture of the three forms, by incubating this at room temperature. The advantage of SEDFIT c(s) distribution plots is that these identify the values of any species present in each sample (Table 1), hence these values are independent of degradation effects. All the boundary fits showed good agreements (Fig. 5, A–D). The c(s) plots showed that SC sedimented as a single species at 4.2 S, whereas major species were seen at 3.1 S for D1–D3 and 2.4 S for D4–D5 (Fig. 5, E–G). This interpretation was confirmed by the three major peaks seen in the cleaved SC sample (Fig. 5H). The c(s) plot for D4–D5 showed a minor peak at 0.7 S (Fig. 5G). In contrast, the D1–D3 c(s) analysis showed peaks at 0.7 S and 2.2 S to indicate more extensive cleavage, in agreement with the x-ray scattering and SDS-PAGE analyses above. The c(M) analyses represent the c(s) peaks as molecular mass values. The three major peaks in Fig. 5H correspond to 24,500 (± 5,600), 44,700 (± 6,200), and 79,000 (± 7,200) Da. These agree well with the sequence-derived values of 34,100, 45,500, and 79,600 Da.
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values were determined to be 4.0 S and 3.9 S for SC (interference and absorbance optics, respectively) and 2.4 S and 2.3 S for D4–D5 (interference and absorbance optics, respectively (Table 1)). D1–D3 could not be analyzed by DCDT+.
The values lead to frictional ratios f/fo that confirm the folded back structure of SC, where fo is the frictional coefficient of the sphere of volume equal to that of the hydrated protein. Many globular proteins show f/fo of 1.2 to 1.3. For SC, D1–D3, and D4–D5, the f/fo ratios were determined to be 1.55, 1.42, and 1.48. These show that all three glycoproteins possess similar degrees of elongation. They are consistent with the RG/R0 ratios of 1.44–1.49, 1.69, and 1.70 in that order.
Constrained Modeling—The constrained modeling of the solution data utilized the D1 crystal structure, which has a -sheet sandwich formed from the DEBA and A'GFCC'C'' -strands (17). These -strands are defined in Fig. 1A. D1 showed sequence identities from 24% to 36% with D2–D5, where over 30% is considered significant (31). Cys22–Cys92 forms the internal disulfide bridge between -strands B and F and was conserved in D1–D5. Sequence gaps and insertions were minimal, except that -strand C'' is deleted in D5. The alignment using BLASTP thus defined the D1–D5 domains and their six flanking peptides. Homology models for the D2–D5 domains were constructed and validated, and oligosaccharides were attached ("Materials and Methods"). The four linkers were defined to run from the end of -strand G to -strand A between domain pairs (25). Linkers (boxed in Fig. 1A) were created from conformational libraries, from which random selections were used to generate full SC models in all orientations. Secondary structure analyses of the 42-residue C-terminal peptide predicted little secondary structure (32). BLASTP sequence searches of over 39,000 structures in the Protein Data Bank did not reveal any significant structural similarity for this C-terminal peptide (33). Its amino acid composition showed a high proportion of 12 negatively charged Asp/Glu residues and nine positively charged Arg/Lys residues, together with 14 small Gly/Ala/Ser residues, all of which promote random structures (27). Accordingly the C-terminal peptide was modeled as another unconstrained structure.
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Good D1–D3 curve fits were obtained by postulating that the observed x-ray scattering corresponded to a mixture of D1–D3 and single-domain fragments (cf.: the SDS-PAGE and c(s) analyses above). The D1–D3 modeling was achieved in five searches. The use of three unconstrained linkers yielded few good fits. Hence the linkers were constrained to be 95–100% extended, and this gave models that spanned a sufficiently wide range of RG, but with RXS that were too high. The final search was based on the randomization of only the D1–D2 linker. The D2–D3 linker proved too short to permit movement without steric overlap, and the C-terminal D3–D4 linker was held fixed in an extended conformation. Even with this, whereas 65 models showed compatible RG values, none showed a low enough RXS of 1.07 nm. All ten best-fit models exhibited linear domain arrangements. Alterations of the oligosaccharide conformations were also unable to account for the reduced RXS value. The final good D1–D3 curve fits were obtained only by computing composite scattering curves from the best-fit D1–D3 model with the lowest R-factor of 13.2% and a single domain D1 model. The sum of 74% D1–D3 and 26% D1 gave the best curve fit with an R-factor of 6.0%. The linear D1–D3 model gave a sedimentation coefficient of 3.02 S that agrees well with the observed value of 3.1 S. Other poor-fit D1–D3 models confirmed this interpretation. For example, combining a U-shaped D1–D3 model with the D1 model did not reduce the R-factor or RXS, even though it gave a good RG. The D1–D3 constrained modeling parameters and curve fits are shown in panels A–C in the supplemental Figs. S3 and S4.
Intact SC (for which the x-ray scattering curve was shown to be free of cleavage products) was modeled using either five unconstrained linkers or the best-fit D1–D3 and D4–D5 models. The unconstrained search is summarized by the filled cyan circles in Fig. 7 (A–F). Convex distributions of RXS as a function of RG were observed. Their maxima agreed well with the observed RG of 3.53 nm (x-rays) and 3.63 nm (neutrons) in Fig. 7(A and D), and their minima corresponded to the lowest R-factors in Fig. 7 (B and E). However, many models showed steric overlap; accordingly, this search was not pursued further. The second search varied only the D3–D4 linker between the best-fit D1–D3 and D4–D5 models. The unfilled circles in Fig. 7(A–F) showed that these followed the same trends as the cyan circles, but resulted in fewer sterically overlapping models with too low RG values. Filtering showed that 268 SC models agreed with the experimental x-ray RG of 3.53 nm, of which 70 agree with the experimental RXS of 1.76 nm (Table 2). The removal of partially overlapping SC models gave the 10 best-fit SC models (yellow in Fig. 7, A–C). All 10 best-fit SC models showed a folded-back arrangement in which D1–D3 is in proximity to D4–D5 in an overall J-shaped conformation. This arrangement was also found in the best-fit models from the unconstrained search, although their R-factors were slightly higher. The sedimentation coefficient of 4.06 S is close to the experimental values of 3.9–4.0 S. The neutron modeling in Fig. 7 (D–F) also resulted in the same J-shaped best-fit models and a good curve fit in Fig. 8D. A linear domain arrangement showed significantly worsened fits (blue in Figs. 7 (A–F), 8C, and 8F). When the extended oligosaccharide orientations in the best-fit model were replaced by more compact ones, slightly worsened curve fits were usually seen (green in Figs. 7 (A–F), 8B, and 8E). These results are summarized in the models of Fig. 9, in the form of the ten best-fit models (A), a stereo view of the best-fit model with oligosaccharide chains (B), an electrostatic map of the fragments D1–D3 and D4–D5 (C), and a view showing the availability of pIgA binding motifs (D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In view of the importance of glycosylation, and inter-domain flexibility to the SC structure, all the analyses were conducted in near-physiological solution conditions using complementary methods based on x-ray and neutron scattering and analytical ultracentrifugation. When analyzed with constrained modeling, the three-dimensional structure of free SC was found to be a compact, folded-back structure. The ten best-fit models of Fig. 9A are very similar to each other. As all adopted a J-shaped structure, this emphasized the robustness of the procedure. A stereo view of the best-fit model is presented in Fig. 9B. The J-shaped structure is confirmed by the lengths of Fig. 3, the sedimentation coefficients (Figs. 5 and 6) and the constrained modeling (Fig. 8). The point at which SC is folded back to form a compact J-shaped structure was located to the junction between the D3 and D4 domains in both modeling searches of all five SC domains. In support of this model, the length and sedimentation coefficient data obtained with the D1–D3 and D4–D5 fragments both fit perfectly with the SC structure (Figs. 3 and 5) and reinforce the accuracy of the SC model.
This compact, folded-back structure of SC was unexpected in the light of the extended domain arrangements of the structurally related seven Ig-like domains in carcinoembryonic antigen and four FnIII domains in anosmin-1 (25, 34). In fact, the linker between D3 and D4 comprises 10 amino acids and is unusually long (Fig. 1). This length provides the requisite flexibility needed to form the J-shaped conformation. It is noteworthy that the cleavage site between Arg336 and Ser337 is exposed on the surface in all ten best-fit structures (Fig. 9A), thus accounting for its susceptibility to protease(s) and justifying the appearance of the two D1–D3 and D4–D5 polypeptides. The Poisson-Boltzmann electrostatic maps of the carbohydrate-free surfaces in D1–D3 and D4–D5 (Fig. 9C) suggest that there are spatially well defined areas of negative and positive charges, respectively, on these that can attract each other to form the bent structure of SC. The short linker between D2–D3 may reduce flexibility between D2 and D3, hence facilitating the binding of D4 and D5 to these.
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The seven Asn residues that constitute the attachment points for carbohydrate residues mostly appear on the same side of SC, implying that the glycans are clustered within a restricted part of the SC structure (Fig. 9B). The curve fits showed a preference for modeling the seven oligosaccharide chains in conformations extending away from the protein surface (middle panels of Fig. 8). Although their precise spatial locations remain unassigned by constrained scattering modeling, a preferential distribution of oligosaccharides on one face of SC is not ruled out. If this is so, this can contribute to tether free and/or IgA-bound SC efficiently to antigenic structures found at the surface of microorganisms (12).
In the context of the interaction between SC and pIgA, the location of the oligosaccharides on one face of SC leave uncovered, and thus accessible, the three CDR-like motifs present in D1, as well as Cys502 in D5 (Fig. 9D). These constitute two critical elements for the interaction with pIgA (35, 36). Indeed, the oligosaccharides of SC are not involved in the interaction with pIgA but are nonetheless important for its resistance to proteases and its correct anchoring within the mucus laying on the epithelium (7). Studies comparing deglycosylated and partly glycosylated D1 revealed nearly equal binding affinities for pIgA (17). Recovery of the Fd fragment after digestion of pIgA with proteases from intestinal fluids indicates that the Fc portion is particularly sensitive in the absence of bound SC (6). The determined length of SC fits very well with its likely association with pIgA without affecting the F(ab')2 and the hinge region. This is supported by the observation that pIgA and SIgA of the same specificity show the same affinity for their cognate antigen (37). Additionally, the labile link between D3 and D4 of SC is likely to be concealed within pIgA, thus providing an explanation for the mutual protection previously observed between the two partners (6). This translates into the generation of a complex molecule optimally designed to operate at mucosal surfaces.
Although not covalent, the interaction between the C-terminal part of the J chain in pIgA and SC is a critical step for transport of the pIgR complex to mucosal surfaces (4). Pentameric IgM and pIgA are both transported by pIgR, but it is known that D1 can discriminate among Ig subclasses in favor of IgA (35), and that D1–D2 and D1–D3 bind pIgA, but not mIgA nor IgG (38), explaining the predominance of SIgA in secretions. As far as pIgA is concerned, the integrity of D2 and D3 is necessary for a covalent interaction to take place (28), yet without establishing close contact with the pIgA backbone (39, 28). This argues in favor of the "zipper effect" model initiated by the interaction of D1 with one C chain of pIgA, progression to the J chain, and establishment of the disulfide bond between Cys311 of the C2 domain of IgA and Cys502 of D5 (36). In the J-shaped structure model, D1 remains appropriately exposed to initiate this "zipper" binding interaction.
Several unsolved issues remain, such as the identification of the residues in SC that are involved in the interactions with the J chain and with the residues 402–410 of the C3 domain in the Fc fragment of pIgA (40). Likewise, even though the CDR-like motifs of D1 have been extensively described, their ligands on the pIgA molecule remain unidentified. In this respect, based on electron micrographs of human pIgA displaying a partly bent tail-to-tail arrangement (41), one can envisage that the Fc domain spacing is suitable for the binding of at least one SC molecule. Whether the compact conformation of SC opens up upon binding to pIgA is another issue. Moreover, structural differences between human IgA1 and IgA2 exist (22, 23) that are consistent with recent findings showing distinct binding modes of human SC to pIgA1 and pIgA2 (42). Future experiments aimed at unraveling the contribution of SC in terms of the architecture of the whole SIgA molecule are now being planned. Knowledge of the three-dimensional structure of SC represents a mandatory first step toward the resolution of the complete structure of SIgA. If we assume that the model of SC is not that different from the extracellular portion of pIgR, it might also be helpful per se to map binding site(s) to permit epithelial drug targeting through specific interaction with pIgR itself (43, 44, 45).
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FOOTNOTES |
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The atomic coordinates and structure factors (code 2OCW) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). 
The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.
1 Supported by the Biotechnology and Biological Sciences Research Council for financial support.
2 Supported by a grant from the Nestlé Ph.D. Programme.
3 Supported by the Swiss Science Research Foundation Grant 3200-109545.
4 To whom correspondence should be addressed. Tel.: 44-20-7679-7048; Fax: 44-20-7679-7193; E-mail: s.perkins{at}medsch.ucl.ac.uk.
5 The abbreviations used are: SIgA, secretory immunoglobulin A; SC, secretory component; Ig, immunoglobulin; pIgR, polymeric immunoglobulin receptor; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; V, variable type domain.
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ACKNOWLEDGMENTS |
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