Discovery of a novel splice variant of Fcar (CD89) unravels sequence segments necessary for efficient secretion: A story of bad signal peptides and good ones that nevertheless do not make it

Abstract

The IgA receptor, Fcar (CD89) consists of 5 sequence segments: 2 segments (S1, S2) forming the potential signal peptide, 2 extracellular EC domains that include the IgA binding site, and the transmembrane and cytoplasmic tail (TM/C) region. Numerous Fcar splice variants have been reported with various combinations of the sequence segments mentioned above. Here, we report a novel splice variant termed variant APD isolated from a healthy volunteer that lacks only the IgA-binding EC1 domain. Despite possessing the complete signal peptide S1+S2, the variant APD is only found in the intracellular space whereas the wild-type variant 1 is efficiently secreted and variant 4 leaks to the extracellular space. Further mutational experiments involving signal peptide replacements, cleavage site modifications, and studies on alternative isoforms demonstrate that despite the completeness of the signal peptide motif, the presence of the EC1 domain is essential for efficient extracellular export.

Role of FcαR EC2 region in extracellular membrane localization

“…Like the EC1 region, EC2 also plays a significant role in extracellular membrane localization for FcaR. A deletion of EC2 in variants 3 and 5 gave similar findings to variants that were lacking EC1 in our previous study [4]. It is indeed surprising to find a role in membrane localization for both EC1 and EC2 domains, especially given the more distal location of EC2 from the signal peptide.…”

“…Carrying on our investigation after our previous publication [4], we investigated the role of EC2 in the extracellular membrane localization by studying two additional FcaR variants that lacked EC2: FcaR variants 3 (lacking only EC2 [3], Accession No; NM_133271.3) and 5 (lacking both S2 and EC2, Accession No; NM_133273.3). Following the same methodology [4], we transiently transfected variants 3 and 5, followed by confocal microscopy and fluorescent activated cell sorting (FACS) analysis. For confocal microscopy, HeLa cells (chosen for their non-overlapping monolayer characteristics) were seeded at 1 £ 10 4 cell/well on glass coverslips.…”

Perspective: The promises of a holistic view of proteins—impact on antibody engineering and drug discovery

“…The natural variant of this receptor molecule contains a full signal peptide and extracellular (EC) domains that bind to IgA antibody. Lua et al [44] discovered that when a natural variant of the receptor lacking only the EC1 domain responsible for binding the IgA molecule [44] but having the full signal peptide was studied, the variant was found spatially constrained intracellularly rather than extracellularly. Attempts to ‘force’ EC localization, using other secretory signal peptides and mutations at the signal peptide cleavage sites, yielded no success [44].…”

“…Lua et al [44] discovered that when a natural variant of the receptor lacking only the EC1 domain responsible for binding the IgA molecule [44] but having the full signal peptide was studied, the variant was found spatially constrained intracellularly rather than extracellularly. Attempts to ‘force’ EC localization, using other secretory signal peptides and mutations at the signal peptide cleavage sites, yielded no success [44]. Further studying other variants (in the presence of the EC1 domain and the complete signal peptide) showed that the lack of the other EC domain, EC2 located more distantly from the signal peptide than EC1, also prevented the EC localization [45].…”

“…We found the residue positions W13 and F17 on the FcεRIα to be potential mutation targets (Figure 3B,C). In the process of making both the mutants and the wild-type control using site-directed mutagenesis and transient transfection methods [15,36,44,67], the W13A mutant could not be produced at detectable amounts and therefore could not be subjected to subsequent experiments. Results of bio-layer interferometry (using nickel-NTA biosensors to capture the purified FcεRIα proteins followed by interacting with IgE at various concentrations from 200 to 12.5 nM) shown in Figure 3D demonstrated that the F17A mutation experimentally reduced the IgE-FcεRIα responses.…”