ABSTRACT
The Kresge Hearing Research Institute-3 (KHRI-3) antibody binds to a guinea pig inner ear supporting cell antigen (IESCA) and causes hearing loss. To gain insight into the mechanism of antibody-induced hearing loss, we used antibody immunoaffinity purification to isolate the IESCA, which was then sequenced by mass spectroscopy, revealing 10 guinea pig peptides identical to sequences in human choline transporter-like protein 2 (CTL2). Full-length CTL2 cDNA sequenced from guinea pig inner ear has 85.9% identity with the human cDNA. Consistent with its expression on the surface of supporting cells in the inner ear, CTL2 contains 10 predicted membrane-spanning regions with multiple N-glycosylation sites. The 68 and 72 kDa molecular forms of inner ear CTL2 are distinguished by sialic acid modification of the carbohydrate. The KHRI-3 antibody binds to an N-linked carbohydrate on CTL2 and presumably damages the organ of Corti by blocking the transporter function of this molecule. CTL2 mRNA and protein are abundantly expressed in human inner ear. Sera from patients with autoimmune hearing loss bind to guinea pig inner ear with the same pattern as CTL2 antibodies. Thus, CTL2 is a possible target of autoimmune hearing loss in humans.
Subject(s)
Ear, Inner/metabolism , Glycoproteins/genetics , Glycoproteins/isolation & purification , Hearing Disorders/immunology , Labyrinth Supporting Cells/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/isolation & purification , Membrane Transport Proteins/genetics , Membrane Transport Proteins/isolation & purification , Animals , Antibodies/immunology , Antibodies/pharmacology , Carbohydrates/analysis , Cloning, Molecular , DNA, Complementary/genetics , Ear, Inner/immunology , Ear, Inner/pathology , Glycoproteins/biosynthesis , Glycosylation , Guinea Pigs , Hearing Disorders/chemically induced , Humans , Labyrinth Supporting Cells/immunology , Membrane Glycoproteins/biosynthesis , Membrane Transport Proteins/biosynthesis , Molecular Sequence Data , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
To study the mechanisms of immune responses and immune injuries in inner ears, labyrinthitis was induced by inoculation of keyhole limpet hemocyanin (KLH) into the scala tympani of systemically sensitized guinea pigs. Inner ears were then immunostained for KLH, immunoglobulin G (IgG), albumin, connexin26 (Cx26), and sodium-potassium adenosine triphosphate (Na,K-ATPase). Inflammatory cells containing KLH were observed in the scala tympani and in the collecting venule of the spiral modiolar vein (SMV). Spiral ligament, spiral limbus, and blood vessels including the SMV were diffusely positive for IgG and albumin. Immunoreactivity for Cx26 and Na,K-ATPase was decreased compared with the normal ears in the fibrocytes of the spiral ligament. These results suggest that inflammatory cells and blood constituents could extravasate into the cochlea from blood vessels and that fibrocyte damage in the spiral ligament could cause cochlear dysfunction.