Cochlin Isoforms and Their Interaction with CTL2 (SLC44A2) in the Inner Ear

Abstract

Choline transporter-like protein 2 (CTL2) is a multitransmembrane protein expressed on inner ear supporting cells that was discovered as a target of antibody-induced hearing loss. Its function is unknown. A 64 kDa band that consistently co-precipitates with CTL2 from inner ear extracts was identified by mass spectroscopy as cochlin. Cochlin is an abundant inner ear protein expressed as multiple isoforms. Its function is also unknown, but it is suspected to be an extracellular matrix component. Cochlin is mutated in individuals with DFNA9 hearing loss. To investigate the CTL2-cochlin interaction, antibodies were raised to a cochlin-specific peptide. The antibodies identify several cochlin polypeptides on western blots and are specific for cochlin. We show that the heterogeneity of the cochlin isoforms is caused, in part, by in vivo posttranslational modification by N-glycosylation and, in part, caused by alternative splicing. We verified that antibody to CTL2 co-immunoprecipitates cochlin from the inner ear and antibody to cochlin coimmunoprecipitates CTL2. Using cochlear crosssections, we show that CTL2 is more widely distributed than previously described, and its prominent expression on cells facing the scala media suggests a possible role in homeostasis. A prominent but previously unreported ribbon-like pattern of cochlin in the basilar membrane was demonstrated, suggesting an important role for cochlin in the structure of the basilar membrane. CTL2 and cochlin are expressed in close proximity in the inner sulcus, the spiral prominence, vessels, limbus, and spiral ligament. The possible functional significance of CTL2-cochlin interactions remains unknown.

Choline Transporter-Like Protein-2

“…13 Here, we first asked the question whether recombinant CTL-2 is able to interact with immobilized cochlin. To answer this question, we generated transfected human embryonic kidney cells stably expressing CTL-2.…”

“…Activated VWF regulates neutrophil recruitment toward endothelial surfaces and supports neutrophil extravasation. 15 Kommareddi et al's 13 observation prompted us to investigate whether CTL-2 can also interact with plasmatic VWF and whether neutrophil responses toward anti-HNA-3a alloantibodies observed in in vitro diagnostic testing and in the in vivo TRALI model are related to the presence of VWF; furthermore, we speculated that signaling via CD11b/CD18 may be involved in anti-HNA-3a alloantibodies-induced neutrophil activation.…”

“…Important information on how alloantibodies against CTL-2 might affect neutrophil activation came from studies performed by Kommareddi et al 13 Investigating the expression of CTL-2 in the inner ear, they reported an interaction between CTL-2 and cochlin. Cochlin carries 2 von Willebrand factor (VWF) A-domains, a motif identified in numerous proteins, but initially described as a major adhesion site of VWF, a central protein in hemostasis which also plays an important role in inflammation.…”

Cochlin-Tomoprotein: A Novel Perilymph-Specific Protein and a Potential Marker for the Diagnosis of Perilymphatic Fistula

“…Alternatively, CTP may be directly coded from a unique COCH gene splice variant or from a COCH homologue [Kommareddi et al, 2007]. The COCH gene was initially isolated by subtractive hybridization and Northern blot.…”

Expression of Cochlin mRNA Splice Variants in the Inner Ear

“…Cochlin isoforms are classified into 4 groups, p63, p44 and p40 in the inner ear, and a short isoform, cochlin-tomoprotein (CTP), in the perilymph [Ikezono et al, 2001[Ikezono et al, , 2004. The origin of cochlin isoforms has been attributed to a variety of mechanisms, including proteolysis at peptide bonds, chemical modification such as glycosylation, phosphorylation and/or deamination, and multiple transcripts [Robertson et al, 1997[Robertson et al, , 2001[Robertson et al, , 2003Grabski et al, 2003;Ikezono et al, 2001;Kommareddi et al, 2007].…”