Carcinoembryonic antigen-related cell adhesion molecule 16 interacts with alpha-tectorin and is mutated in autosomal dominant hearing loss (DFNA4)

Abstract

We report on a secreted protein found in mammalian cochlear outer hair cells (OHC) that is a member of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family of adhesion proteins. Ceacam16 mRNA is expressed in OHC, and its protein product localizes to the tips of the tallest stereocilia and the tectorial membrane (TM). This specific localization suggests a role in maintaining the integrity of the TM as well as in the connection between the OHC stereocilia and TM, a linkage essential for mechanical amplification. In agreement with this role, CEACAM16 colocalizes and coimmunoprecipitates with the TM protein α-tectorin. In addition, we show that mutation of CEACAM16 leads to autosomal dominant nonsyndromic deafness (ADNSHL) at the autosomal dominant hearing loss (DFNA4) locus. In aggregate, these data identify CEACAM16 as an α-tectorin-interacting protein that concentrates at the point of attachment of the TM to the stereocilia and, when mutated, results in ADNSHL at the DFNA4 locus.

Fig. 1.

(A) CEACAM16 amino acid sequence and structural domains. Yellow indicates IgV-like domains; green indicates IgC-like domains. Predicted N-glycosylation sites are in red. Asterisks indicate cysteines; arrow shows the proteolytic cleavage site. Amino acids used to generate anti-CEACAM16 are underlined. (B) CEACAM16 forms oligomers in the cell pellet and is degraded by subtilisin. (C) CEACAM16 oligomers also are found in the cell-culture medium collected from Ceacam16-transfected HEK293T cells (CEACAM16) but not from untransfected cells (control). DTT reduces CEACAM16 oligomers to monomers.

Fig. 2.

Ceacam16 mRNA is present in OHC. (A) In situ hybridization of cochlear tissue derived from P42 129/C57BL6 mice hybridized with a negative RNA probe shows no staining. (B) Brown staining was observed in OHC only when tissue was hybridized with an anti-sense Ceacam16 RNA probe. (C) CEACAM16 (anti-CEACAM16, green) is localized at the tip of OHC but not IHC stereocilia. Red staining for actin helps identify the stereocilia core.

Fig. 3.

(A–G) CEACAM16 protein in adult cochleae is degraded by subtilisin. (A–C) Immunofluorescent images of an adult mouse cochlea stained with anti-CEACAM16 (A, green) and Texas Red-X phalloidin (B, red). Merged image is shown in C. (D–F) Similar to A–C, but samples were treated with subtilisin. (G) The CEACAM16 band also is degraded by subtilisin. Cochlear samples were separated on NEXT-PAGE and blotted with anti-CEACAM16. DTT reduced CEACAM16 oligomers to monomers. (H–I) Immunofluorescent images show the location of CEACAM16 in the organ of Corti. (H) Cross-section of the negative-control mouse cochlea. Anti-CEACAM16 is replaced with rabbit Ig control (2.5 μg/mL). (I) Immunofluorescent image shows that CEACAM16 (green) is present in the TM. The organ of Corti (indicated by circles) is visualized by actin staining.

Fig. 4.

(A–C) Immunofluorescent images show colocalization of CEACAM16 (A, green, polyclonal anti-V5) with α-tectorin (B, red, anti-myc) in OK cells cotransfected with myc-Tecta+V5-Ceacam16. Images are merged in C. (Scale bar: 24 μm.) (D–H) Coimmunoprecipitation experiments. Cell lysates (input) and eluted proteins (output) were separated on 4–20% gradient gel. Anti-V5, anti-myc, anti-oncomodulin, anti-Flag, and anti-GFP were used to identify CEACAM16, α-tectorin, oncomodulin, CEACAM1, and prestin. (D and E) A plasmid encoding CEACAM16 was cotransfected into OK cells with GFP-prestin (CEACAM16+GFP-prestin) and Tecta (CEACAM16+Tecta) plasmids, respectively. CEACAM16 is pulled down with 2 μg of anti-V5/protein A Sepharose. α-Tectorin coimmunoprecipitates with CEACAM16, but prestin does not. (F and G) Plasmids encoding α-tectorin were cotransfected into HEK293T cells with oncomodulin (α-tectorin+oncomodulin) and CEACAM16 (α-tectorin +CEACAM16). Cell lysates were subjected to coimmunoprecipitation by using 2 μg of anti-myc protein A Sepharose (pulling down α-tectorin). CEACAM16 coimmunoprecipitates with α-tectorin but not with oncomodulin. (H) Plasmid encoding α-tectorin was cotransfected into OK cells with CEACAM1 (α-tectorin +CEACAM1). Cell lysates were subjected to coimmunoprecipitation by using 2 μg of anti-Flag/protein G Sepharose (pulling down CEACAM1). CEACAM1 did not coimmunoprecipitate with α-tectorin.

Fig. 5.

T140P mutation of CEACAM16 is found in DFNA4. (A and B) Sequence results (A) and pedigree (B) showing that the heterozygous T140P mutation segregates with the dominant deafness in family 1070. (C) Diagram showing CEACAM16 structural domains. Potential N-glycosylation sites are marked by lollipops. [Modified from Zebhauser et al. (24), copyright (2005), with permission from Elsevier]. The T140P mutant is marked by a red asterisk. (D) Sequence alignment showing conservation of T140 (in bold) in mammalian species. (E) Western blot of WT and P142-CEACAM16. Proteins were separated on 10% NEXT-PAGE gels and blotted with anti-V5. P142-CEACAM16 appears to have a smaller monomer band than WT CEACAM16.

Fig. 6.

CEACAM16 with the T142P change is still associated with α-tectorin. (A–C) Immunofluorescent images show colocalization of the T142P mutant (green, polyclonal anti-V5) with α-tectorin (red, monoclonal anti-myc) in OK cells cotransfected with myc-Tecta+V5-T142P-Ceacam16. Images are merged in C. (Scale bar: 11.9 μm.) (D–G) HEK293T cell lysates from V5-CEACAM16+myc-α-tectorin–expressing cells (WT+α-tectorin), V5-prestin+myc-α-tectorin–expressing cells (prestin+α-tectorin), and V5-P142-Ceacam16+myc-α-tectorin–expressing cells (T142P+α-tectorin) were subjected to coimmunoprecipitation by using 2 μg of anti-V5 and protein A Sepharose. The eluted proteins were separated on 7.5% NEXT-PAGE. α-Tectorin was visualized by anti-myc. Prestin and CEACAM16 were visualized by anti-V5. (D) α-Tectorin precipitated with WT CEACAM16 and P142-CEACAM16, but not with prestin. (E) Cell lysates (collected before coimmunoprecipitation) show that α-tectorin was detected in all three samples. (F and G) CEACAM16 (WT and P142 forms) and prestin proteins were found in samples eluted from protein A beads as expected (F) and cell lysates (G).