Phosphatidylethanolamine binding protein 4 (PEBP4) is a secreted protein and has multiple functions

Abstract

Phosphatidylethanolamine binding proteins (PEBP) represent a superfamily of proteins that are conserved from bacteria to humans. In mammals, four members have been identified, PEBP1-4. To determine the functional differences among PEBP1-4 and the underlying mechanism for their actions, we performed a sequence alignment and found that PEBP4 contains a signal peptide and potential glycosylation sites, whereas PEBP1-3 are intracellular proteins. To test if PEBP4 is secreted, we made constructs with Myc epitope at the amino (N) terminus or carboxyl (C) terminus to mask the signal sequence or keep it free, respectively. Our data revealed that both mouse and human PEBP4 were secreted when the epitope was tagged at their C-terminus. To our surprise, secretion was dependent upon the C-terminal conserved domain in addition to the N-terminal signal sequence. When the epitope was placed to the N-terminus, the recombinant protein failed to secrete and instead, was retained in the cytoplasm. Mass spectrometry detected asparagine (N)-glycosylation on the secreted PEBP4. Although overexpression of N-terminal tagged PEBP4 resulted in an inhibition of ERK activation by EGF, that with a C-terminal epitope tag did not have such an effect. Likewise, transfection of PEBP4 shRNA did not appear to affect ERK activation, suggesting that PEBP4 does not participate in the regulation of this pathway. In contrast, PEBP4 siRNA suppressed phosphorylation of Act at S473. Therefore, our results suggest that PEBP4 is a multifunctional protein and can be secreted. It will be important to investigate the mechanism by which PEBP4 is secreted and regulates cellular events.

Keywords: Act; ERK; Glycosylation; PEBP4; Secretion; Signal peptide.

Figure 1. Sequence alignment of PEPB1–4

Access numbers are P70296 for mouse PEBP1 (mPEBP1), AF307146 for mouse PEBP2 (mPEBP2), AAB32786 for rat PEBP3 (rPEBP3), and NP_082802 for mouse PEBP4 (mPEBP4). Multiple sequence alignment was performed using ClusterW2 software. The degree of similarity is designated (*>:>.). PEBP1 and PEBP2 show 79.7% identity and 93.6% similarity; mouse PEBP1 and rat PEBP3 show 85.9% identity and 95.1% similarity; PEBP1 and PEBP4 show 26.7% identical and 38.2% similarity.

Figure 2. Sequence alignment of mouse and human PEBP4

Sequence alignment shows 45.2% identify and 59.5% similarity between mouse and human PEBP4 and predicts signal peptide highlighted yellow and asparagine glycosylation sites (marked red N).

Figure 3. Effects of epitope tagging to different ends of PEBP4

PEBP4 was tagged with GFP (A, B) or Myc epitope to at its N-terminus or C-terminus and transiently expressed in HEK293T cells. The cells were examined under confocal microscopy (A, B) or extracts and culture media subjected to western blot analysis using an anti-Myc antibody (C). The images in A and B were observed at 63×. Scale bar: 10 microns.

Figure 4. Secretion of PEBP4 variants to culture media

PEBP4 full length, point mutation or truncation mutations were made at different sites and tagged with Myc or MycHis₆, as indicated in (A). The recombinant proteins were transiently expressed in HEK293T cells and blotted with anti-Myc antibody in cell extracts or culture media (B).

Figure 5. N-glycosylation of PEBP4

A. Mouse and human PEBP4 tagged with MycHis₆ at the C-terminus was expressed in HEK293T cells, respectively, purified by NTA affinity chromatography and resolved on SDS-PAGE. B. The PEBP4 bands in A were excised and digested with PNGaseF. The released N-glycans were analyzed and the compositions are listed C. Proteomics analysis (only the data with human PEBP4 were presented). The PEBP4 band was deglycosylated in-gel, digested with trypsin and the resulting peptides analyzed using LC-MS/MS analysis as described in the Materials and Methods section. Data interpretation is described in the text.

Figure 6. The effect of PEBP4 on ERK activation

A. shRNA for PEBP4 or empty vector was transfected into HEK293T cells on 12 well plates, which were the treated with EGF (10 ng/ml) for 10 min. A. The cells were transfected with PEBP4 tagged at the N-terminus (N-Myc PEBP4) or C-terminus (C-Myc PEBP4) with Myc epitope at different doses. C. The experiments were conducted as B, except that the cells were treated with EGF. Cell extracts were analyzed by western blot with antibodies as indicated.

Figure 7. The effect of PEBP4 on Akt activation

A. HEK293T cells were transfected with PEBP4 shRNA or empty vector and treated as described in Figure 6A. B. The cells were transfected with C-Myc PEBP4 or N-Myc PEBP4. Western blot analysis was performed as indicated.