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We recently reported that the lectin surfactant protein D (SP-D) suppresses epidermal growth factor receptor (EGFR) signaling by interfering with ligand binding to EGFR through an interaction between the carbohydrate-recognition domain (CRD) of SP-D and N -glycans of EGFR. Here, we report that surfactant protein A (SP-A) also suppresses EGF signaling in A549 human lung adenocarcinoma cells and in CHOK1 cells stably expressing human EGFR and that SP-A inhibits the proliferation and motility of the A549 cells. Results with 125 I-EGF indicated that SP-A interferes with EGF binding to EGFR, and a ligand blot analysis suggested that SP-A binds EGFR in A549 cells. We also found that SP-A directly binds the recombinant extracellular domain of EGFR (soluble EGFR or sEGFR), and this binding, unlike that of SP-D, was not blocked by EDTA, excess mannose, or peptide: N -glycosidase F treatment. We prepared a collagenase-resistant fragment (CRF) of SP-A, consisting of CRD plus the neck domain of SP-A, and observed that CRF directly binds sEGFR but does not suppress EGF-induced phosphorylation of EGFR in or proliferation of A549 cells. These results indicated that SP-A binds EGFR and down-regulates EGF signaling by inhibiting ligand binding to EGFR as well as SP-D. However, unlike for SP-D, SP-A lectin activity and EGFR N -glycans were not involved in the interaction between SP-A and EGFR. Furthermore, our results suggested that oligomerization of SP-A is necessary to suppress the effects of SP-A on EGF signaling., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Heregulin signaling is involved in various tumor proliferations and invasions; thus, receptors of heregulin are targets for the cancer therapy. In this study we examined the suppressing effects of extracellular domains of ErbB2, ErbB3, and ErbB4 (soluble ErbB (sErbB)) on heregulin β signaling in human breast cancer cell line MCF7. It was found that sErbB3 suppresses ligand-induced activation of ErbB receptors, PI3K/Akt and Ras/Erk pathways most effectively; sErbB2 scarcely suppresses ligand-induced signaling, and sErbB4 suppresses receptor activation at ∼10% efficiency of sErbB3. It was revealed that sErbB3 does not decrease the effective ligands but decreases the effective receptors. By using small interfering RNA (siRNA) for ErbB receptors, we determined that sErbB3 suppresses the heregulin β signaling by interfering ErbB3-containing heterodimers including ErbB2/ErbB3. By introducing the mutation of N418Q to sErbB3, the signaling-inhibitory effects were increased by 2-3-fold. Moreover, the sErbB3 N418Q mutant enhanced anticancer effects of lapatinib more effectively than the wild type. We also determined the structures of N-glycan on Asn-418. Results suggested that the N-glycan-deleted mutant of sErbB3 suppresses heregulin signaling via ErbB3-containing heterodimers more effectively than the wild type. Thus, we demonstrated that the sErbB3 N418Q mutant is a potent inhibitor for heregulin β signaling.

Although more than 100 mutations have been identified in the copper/zinc superoxide dismutase (Cu/Zn-SOD) in familial amyotrophic lateral sclerosis (FALS), the mechanism responsible for FALS remains unclear. The finding of the present study shows that FALS-causing mutant Cu/Zn-SOD proteins (FALS mutant SODs), but not wild-type SOD, are barely detected by three monoclonal antibodies (mAbs) in Western blot analyses. The enzyme-linked immunosorbent assay for denatured FALS mutant SODs by dithiothreitol, SDS, or heat treatment also showed a lowered immunoreactivity against the mAbs compared with wild-type SOD. Because all the epitopes of these mAbs are mapped within the Greek key loop (residues 102-115 in human Cu/Zn-SOD), these data suggest that different conformational changes occur in the loop between wild-type and FALS mutant SODs during the unfolding process. Circular dichroism measurements revealed that the FALS mutant SODs are sensitive to denaturation by dithiothreitol, SDS, or heat treatment, but these results do not completely explain the different recognition by the mAbs between wild-type and FALS mutant SODs under the denatured conditions. The study on the conformational changes in local areas monitoring with mAbs may provide a new insight into the etiology of FALS.