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Cupulin is a zona pellucida-like domain protein and major component of the cupula from the inner ear.

Dernedde J, Weise C, Müller EC, Hagiwara A, Bachmann S, Suzuki M, Reutter W, Tauber R, Scherer H - PLoS ONE (2014)

Bottom Line: The extracellular membranes of the inner ear are essential constituents to maintain sensory functions, the cupula for sensing torsional movements of the head, the otoconial membrane for sensing linear movements and accelerations like gravity, and the tectorial membrane in the cochlea for hearing.So far a number of structural proteins have been described, but for the gelatinous cupula precise data are missing.Here, we describe for the first time a major proteinogenic component of the cupula structure with an apparent molecular mass of 45 kDa from salmon.

View Article: PubMed Central - PubMed

Affiliation: Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité -Universitätsmedizin Berlin, Berlin, Germany.

ABSTRACT
The extracellular membranes of the inner ear are essential constituents to maintain sensory functions, the cupula for sensing torsional movements of the head, the otoconial membrane for sensing linear movements and accelerations like gravity, and the tectorial membrane in the cochlea for hearing. So far a number of structural proteins have been described, but for the gelatinous cupula precise data are missing. Here, we describe for the first time a major proteinogenic component of the cupula structure with an apparent molecular mass of 45 kDa from salmon. Analyses of respective peptides revealed highly conserved amino-acid sequences with identity to zona pellucida-like domain proteins. Immunohistochemistry studies localized the protein in the ampulla of the inner ear from salmon and according to its anatomical appearance we identified this glycoprotein as Cupulin. Future research on structure and function of zona pellucida-like domain proteins will enhance our knowledge of inner ear diseases, like sudden loss of vestibular function and other disturbances.

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Related in: MedlinePlus

Zona pellucida-like domain protein homology and structure.A, protein sequences from UniProt database: salmon: C0H9B6; chicken: E1C8E6; human: Q8TCW7 were aligned by applying the ClustalW2 program. Asteriks (★) marked below the sequence highlight conserved amino-acids (∼70%) between the three organisms. Conserved cysteine residues 1–8 that constitute the zona pellucida-like domain (blue box) are shown in red letters. Arrows mark the mature protein after cleavage of the N-terminal signal sequence (SP, green box) predicted by the SignalP algorithm and the C-terminal furin cleavage site (CFCS, black and bold letters). IHP and EHP (red boxes) show the potential internal and external hydrophobic patches of the zona pellucida-like-domain (ZLPD). The transmembrane domain (TMD, orange box) was predicted by the TMHMM 2.0 software. In pink and bold are highlighted the peptides 1–7, identified by mass spectrometry. Individual peptides 1, 2 and 3 are highlighted with blue lines. Underlined peptide sequences were used for immunization. Grey diamonds indicate the asparagine residue of potential N-glycosylation sites (NXS/T) determined with the program NetNGlyc 1.0. B, scheme of zona pellucida-like domain protein structure.
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pone-0111917-g003: Zona pellucida-like domain protein homology and structure.A, protein sequences from UniProt database: salmon: C0H9B6; chicken: E1C8E6; human: Q8TCW7 were aligned by applying the ClustalW2 program. Asteriks (★) marked below the sequence highlight conserved amino-acids (∼70%) between the three organisms. Conserved cysteine residues 1–8 that constitute the zona pellucida-like domain (blue box) are shown in red letters. Arrows mark the mature protein after cleavage of the N-terminal signal sequence (SP, green box) predicted by the SignalP algorithm and the C-terminal furin cleavage site (CFCS, black and bold letters). IHP and EHP (red boxes) show the potential internal and external hydrophobic patches of the zona pellucida-like-domain (ZLPD). The transmembrane domain (TMD, orange box) was predicted by the TMHMM 2.0 software. In pink and bold are highlighted the peptides 1–7, identified by mass spectrometry. Individual peptides 1, 2 and 3 are highlighted with blue lines. Underlined peptide sequences were used for immunization. Grey diamonds indicate the asparagine residue of potential N-glycosylation sites (NXS/T) determined with the program NetNGlyc 1.0. B, scheme of zona pellucida-like domain protein structure.

Mentions: To identify specific peptide sequences from the salmon protein, gel electrophoresis was performed, the 45 kDa band was cut-out, and the protein was trypsinized and further analyzed by mass spectrometry. The peptide mass fingerprint analysis with annotated peptide sequences is shown in Fig. S1 and exemplarily a detailed MS/MS spectrum for one peptide is presented in Fig. S2. Database searches revealed identity to several predicted open reading frames of zona pellucida-like domain proteins. Here, to the best of our knowledge we identified for the first time corresponding peptide sequences from a zona pellucida-like domain protein (Table 1). The overall match of seven identified peptides with the predicted sequences from salmon, chicken, and human origin is convincing (Fig. 3A) and covers about 26% of the mature extracellular protein ranging from amino-acids 21 to 319 (Fig. 3) With peptide 1 we most probably identified the N-terminus of the secreted zona pellucida-like domain protein. Cleavage of the signal peptide between amino-acids A19 and Q20 is predicted by the SignalP algorithm (data not shown). Furthermore the conversion of glutamine to pyroglumate (Table 1) argues for the N-terminal position, where spontaneous intramolecular cyclization can occur. In addition it is interesting to note, that we observed one difference to the published sequence (C0H9B6) from the salmon zona pellucida-like domain protein. Instead of the uncharged asparagine (N22) we identified the acidic aspartic acid residue (D22) in peptides 1 and 1a. Further amino-acid changes might be explained by the interindividual variations due to our randomly pooled material from wild and farm-raised salmon from diverse origin. In detail, in addition to F30 present in peptide 1 a substitution to Y30 was identified in peptide 1a. Further in addition to the correctly matching sequence from peptide 5, two variations were identified compared to the database entry within peptide 5a, where V229 was changed to I229 and A239 to P239. In each case the amino-acid substitution was conservative and hydrophobic apolar amino-acids were used.


Cupulin is a zona pellucida-like domain protein and major component of the cupula from the inner ear.

Dernedde J, Weise C, Müller EC, Hagiwara A, Bachmann S, Suzuki M, Reutter W, Tauber R, Scherer H - PLoS ONE (2014)

Zona pellucida-like domain protein homology and structure.A, protein sequences from UniProt database: salmon: C0H9B6; chicken: E1C8E6; human: Q8TCW7 were aligned by applying the ClustalW2 program. Asteriks (★) marked below the sequence highlight conserved amino-acids (∼70%) between the three organisms. Conserved cysteine residues 1–8 that constitute the zona pellucida-like domain (blue box) are shown in red letters. Arrows mark the mature protein after cleavage of the N-terminal signal sequence (SP, green box) predicted by the SignalP algorithm and the C-terminal furin cleavage site (CFCS, black and bold letters). IHP and EHP (red boxes) show the potential internal and external hydrophobic patches of the zona pellucida-like-domain (ZLPD). The transmembrane domain (TMD, orange box) was predicted by the TMHMM 2.0 software. In pink and bold are highlighted the peptides 1–7, identified by mass spectrometry. Individual peptides 1, 2 and 3 are highlighted with blue lines. Underlined peptide sequences were used for immunization. Grey diamonds indicate the asparagine residue of potential N-glycosylation sites (NXS/T) determined with the program NetNGlyc 1.0. B, scheme of zona pellucida-like domain protein structure.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219815&req=5

pone-0111917-g003: Zona pellucida-like domain protein homology and structure.A, protein sequences from UniProt database: salmon: C0H9B6; chicken: E1C8E6; human: Q8TCW7 were aligned by applying the ClustalW2 program. Asteriks (★) marked below the sequence highlight conserved amino-acids (∼70%) between the three organisms. Conserved cysteine residues 1–8 that constitute the zona pellucida-like domain (blue box) are shown in red letters. Arrows mark the mature protein after cleavage of the N-terminal signal sequence (SP, green box) predicted by the SignalP algorithm and the C-terminal furin cleavage site (CFCS, black and bold letters). IHP and EHP (red boxes) show the potential internal and external hydrophobic patches of the zona pellucida-like-domain (ZLPD). The transmembrane domain (TMD, orange box) was predicted by the TMHMM 2.0 software. In pink and bold are highlighted the peptides 1–7, identified by mass spectrometry. Individual peptides 1, 2 and 3 are highlighted with blue lines. Underlined peptide sequences were used for immunization. Grey diamonds indicate the asparagine residue of potential N-glycosylation sites (NXS/T) determined with the program NetNGlyc 1.0. B, scheme of zona pellucida-like domain protein structure.
Mentions: To identify specific peptide sequences from the salmon protein, gel electrophoresis was performed, the 45 kDa band was cut-out, and the protein was trypsinized and further analyzed by mass spectrometry. The peptide mass fingerprint analysis with annotated peptide sequences is shown in Fig. S1 and exemplarily a detailed MS/MS spectrum for one peptide is presented in Fig. S2. Database searches revealed identity to several predicted open reading frames of zona pellucida-like domain proteins. Here, to the best of our knowledge we identified for the first time corresponding peptide sequences from a zona pellucida-like domain protein (Table 1). The overall match of seven identified peptides with the predicted sequences from salmon, chicken, and human origin is convincing (Fig. 3A) and covers about 26% of the mature extracellular protein ranging from amino-acids 21 to 319 (Fig. 3) With peptide 1 we most probably identified the N-terminus of the secreted zona pellucida-like domain protein. Cleavage of the signal peptide between amino-acids A19 and Q20 is predicted by the SignalP algorithm (data not shown). Furthermore the conversion of glutamine to pyroglumate (Table 1) argues for the N-terminal position, where spontaneous intramolecular cyclization can occur. In addition it is interesting to note, that we observed one difference to the published sequence (C0H9B6) from the salmon zona pellucida-like domain protein. Instead of the uncharged asparagine (N22) we identified the acidic aspartic acid residue (D22) in peptides 1 and 1a. Further amino-acid changes might be explained by the interindividual variations due to our randomly pooled material from wild and farm-raised salmon from diverse origin. In detail, in addition to F30 present in peptide 1 a substitution to Y30 was identified in peptide 1a. Further in addition to the correctly matching sequence from peptide 5, two variations were identified compared to the database entry within peptide 5a, where V229 was changed to I229 and A239 to P239. In each case the amino-acid substitution was conservative and hydrophobic apolar amino-acids were used.

Bottom Line: The extracellular membranes of the inner ear are essential constituents to maintain sensory functions, the cupula for sensing torsional movements of the head, the otoconial membrane for sensing linear movements and accelerations like gravity, and the tectorial membrane in the cochlea for hearing.So far a number of structural proteins have been described, but for the gelatinous cupula precise data are missing.Here, we describe for the first time a major proteinogenic component of the cupula structure with an apparent molecular mass of 45 kDa from salmon.

View Article: PubMed Central - PubMed

Affiliation: Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité -Universitätsmedizin Berlin, Berlin, Germany.

ABSTRACT
The extracellular membranes of the inner ear are essential constituents to maintain sensory functions, the cupula for sensing torsional movements of the head, the otoconial membrane for sensing linear movements and accelerations like gravity, and the tectorial membrane in the cochlea for hearing. So far a number of structural proteins have been described, but for the gelatinous cupula precise data are missing. Here, we describe for the first time a major proteinogenic component of the cupula structure with an apparent molecular mass of 45 kDa from salmon. Analyses of respective peptides revealed highly conserved amino-acid sequences with identity to zona pellucida-like domain proteins. Immunohistochemistry studies localized the protein in the ampulla of the inner ear from salmon and according to its anatomical appearance we identified this glycoprotein as Cupulin. Future research on structure and function of zona pellucida-like domain proteins will enhance our knowledge of inner ear diseases, like sudden loss of vestibular function and other disturbances.

Show MeSH
Related in: MedlinePlus