A comparison of the binding of secretory component to immunoglobulin A (IgA) in human colostral S-IgA1 and S-IgA2

Abstract

A detailed investigation of the binding of secretory component to immunoglobulin A (IgA) in human secretory IgA2 (S-IgA2) was made possible by the development of a new method of purifying S-IgA1, S-IgA2 and free secretory component from human colostrum using thiophilic gel chromatography and chromatography on Jacalin-agarose. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of unreduced pure S-IgA2 revealed that, unlike in S-IgA1, a significant proportion of the secretory component was bound non-covalently in S-IgA2. When S-IgA1 was incubated with a protease purified from Proteus mirabilis the secretory component, but not the alpha-chain, was cleaved. This is in contrast to serum IgA1, in which the alpha-chain was cleaved under the same conditions - direct evidence that secretory component does protect the alpha-chain from proteolytic cleavage in S-IgA. Comparisons between the products of cleavage with P. mirabilis protease of free secretory component and bound secretory component in S-IgA1 and S-IgA2 also indicated that, contrary to the general assumption, the binding of secretory component to IgA is different in S-IgA2 from that in S-IgA1.

Figure 1

Coomassie-Blue-stained 10% SDS–PAGE of clarified human colostrum and fractions from chromatography of colostrum on thiophilic resin Gels were run under (a) non-reducing conditions or (b) reducing conditions. Lane 1, protein markers; lane 2 clarified colostrum starting material; lanes 3–9, every second fraction eluted from thiophilic resin; lane 10 column run-through.

Figure 3

Western blot (10% SDS–PAGE run under non-reducing conditions) of dimeric S-IgA2 peak fractions from Sephacryl S-300 column probed with rabbit anti-SC antibody (SC), goat anti-human-light chains (λ and κ) antibody (LC) or 5% SDS–PAGE blotted with monoclonal anti-α2 (α2) Protein markers are shown in lane 1.

Figure 2

Coomassie-Blue-stained, 10% SDS–PAGE of fractions of purified S-IgA2 (lanes 1–3, 4–6) and S-IgA1 (lanes 7, 8) run under non-reducing (lanes 1–3, 7) or reducing (lanes 4–6, 8) conditions. Lane M, protein markers. The 45 000 MW protein band marked by the arrow (lanes 1, 2) is a light-chain dimer.

Figure 4

(a) Coomassie-Blue-stained 10% SDS–PAGE run under non-reducing conditions of fractions (lanes 1–5) from the third peak eluted from an S300 column loaded with proteins from the Jacalin–agarose column run-through. Lane 3 is the peak fraction. (b,c) FSC fractions from the S300 column pooled and chromatographed on a pIgA1-Sepharose column. Material eluted from pIgA1-Sepharose column is shown stained with Coomassie Blue (b) or after Western blot with rabbit anti-human SC antibody (c). (b) Lane 1 contains molecular weight markers; lane 2, material loaded onto column; lanes 3–5, material eluted from column; lane 6 is column run-through. (c) Lanes 1 and 2 are eluted fractions.

Figure 5

Coomassie-blue-stained 10% SDS–PAGE under reducing conditions of S-IgA1 incubated at 37° with either buffer alone for 40 hr (lane 1) or with P. mirabilis purified protease S-IgA1 for 8 hr (lane 2), 16 hr (lane 3) and 40 hr (lane 4). The 76 500 MW fragment derived from secretory component (SC) is indicated by the arrow.

Figure 6

Western blot of 10% SDS–PAGE run under reducing conditions showing structural features of bound and of untreated (control – C) free secretory component, SC-IgA2 and SC-IgA1 and these after cleavage with P. mirabilis protease at 37° for 24, 48 and 72 hr and probed with rabbit anti-human-SC antibody (detecting antibody is a goat anti-rabbit IgG conjugated with alkaline phosphatase). Cleavage of FSC and the SC in S-IgA2 was complete within 24 hr but that of the SC in S-IgA1 remained uncleaved after 72 hr.