Multiple cleavage sites for polymeric immunoglobulin receptor

Abstract

Human polymeric immunoglobulin receptor (pIgR) was expressed in baby hamster kidney (BHK) cells using a recombinant vaccinia virus transfection system. Cleavage of pIgR on the cell surface was partially inhibited by the proteinase inhibitor, leupeptin. We addressed the question whether some particular regions of pIgR could affect the efficient cleavage of this molecule, with the following results: (1) a mutant lacking the entire cytoplasmic region resulted in release of secretory component (SC) into the culture supernatant much faster than wild-type; (2) a pIgR mutant lacking the entire extracellular domain 6, the region containing the susceptible cleavage sites, could be cleaved and released as a mutant SC. The transport kinetics of this mutant between endoplasmic reticulum (ER) and Golgi or Golgi and the cell surface was equivalent to wild-type pIgR. Our results indicate that although the main cleavage site is in domain 6, at least one other cleavage site may exist.

Figure 1

pIgR cleavage on the surface of BHK cells is leupeptin-dependent. The pIgR transfectants were metabolically labelled with [³⁵S]-cysteine. Cells were then further cultured with or without 10 µg/ml of leupeptin for 16 h at 37°. The cell lysates and culture supernatants were prepared and immunoprecipitataed with polyclonal rabbit antihuman SC antibody, followed by protein G-Sepharose. In the presence of leupeptin, 70% of SC release was inhibited. Reciprocally, 70% more pIgR was detected in cell lysates.

Figure 2

Schematic diagram of the deletion mutants of pIgR. The bold region (69 amino acids) represents the extracellular Domain 6 and all the amino acids in this domain were deleted in ΔCL mutant. The conserved susceptible cleavage site within this area is depicted as ▪. The thick region (22 amino acids) highlighted by the vertical lines represents the transmembrane region and the following sequence (103 amino acids) corresponds to the intracellular region. In the ΔTMCP mutant, both the transmembrane and intracellular regions were deleted. For ΔCP mutant, only the intracellular region was deleted. GSRDVSLAKADAAPDEKVLDSGFREIENKAIQDPRLFAEEKAVADTRDQADGSRASVDSGSSEEQGGSSLVSTLVPLGLVLAVGAVAVGVARARHRKNVDRVSIRSYRTDISNSREFGANDNMGASSITQETSLGGKEEFVATTESTTETKEPKKAKRSSKEEAEMAYKDFLLQSSTVAAEAQDGPQEA.

Figure 3

Expression of wild-type and mutants of pIgR. The wild-type, ΔCP, ΔTMCP and ΔCL mutants were transfected into BHK cells. 5·5 hr after transfection, cell lysates were prepared and loaded onto 8% SDS–PAGE gels. Western blotting was performed using polyclonal rabbit anti-human SC antibody followed by HRP-conjugated goat anti-rabbit IgG (H + I) antibody. The expression levels of the mutant proteins were equivalent to wild-type protein.

Figure 4

Release of free SC from wild-type- and mutant-transfectants. The wild-type- and mutant-transfectants were metabolically labelled as in Fig. 1 and further cultured with 10% FCS-DMEM for 16 hr. The cell lysates and culture supernatants were then immunoprecipitated with polyclonal rabbit anti-human SC and protein G-Sepharose. The samples were loaded onto 8% SDS–PAGE gels. In cell lysates of ΔCP and ΔTMCP mutants, no labelled protein was detected. In the culture supernatant of the ΔCL mutant, two different molecular weight bands were detected.

Figure 5

(a) The ΔCL mutant has the same ER–Golgi transport rate as wild-type pIgR. The wild-type and ΔCL mutant were transfected into BHK cells and metabolically labelled with [³⁵S]-cysteine for 15 min. The cell lysates were prepared at the indicated times and subjected to immunoprecipitation. The samples were loaded onto 8% SDS–PAGE gels. The molecular maturation kinetics were identical in both wild-type and ΔCL mutant. At 0 min of chase, only single bands were observed. At 30 min of chase, both ER and Golgi forms were detected and at 1 hr of chase all labelled protein were matured to the Golgi form.(b) Detection of cell surface-expressed wild-type and ΔCL mutant molecules. Wild-type or ΔCL mutant were transfected into BHK cells. Detection of the cell surface-expressed pIgR molecules was performed as described in Materials and Methods. The transport of the wild-type and ΔCL mutant to the cell surface was kinetically equivalent.