Annexin A5 is the Most Abundant Membrane-Associated Protein in Stereocilia but is Dispensable for Hair-Bundle Development and Function

Abstract

The phospholipid- and Ca2+-binding protein annexin A5 (ANXA5) is the most abundant membrane-associated protein of ~P23 mouse vestibular hair bundles, the inner ear’s sensory organelle. Using quantitative mass spectrometry, we estimated that ANXA5 accounts for ~15,000 copies per stereocilium, or ~2% of the total protein there. Although seven other annexin genes are expressed in mouse utricles, mass spectrometry showed that none were present at levels near ANXA5 in bundles and none were upregulated in stereocilia of Anxa5−/− mice. Annexins have been proposed to mediate Ca2+-dependent repair of membrane lesions, which could be part of the repair mechanism in hair cells after noise damage. Nevertheless, mature Anxa5−/− mice not only have normal hearing and balance function, but following noise exposure, they are identical to wild-type mice in their temporary or permanent changes in hearing sensitivity. We suggest that despite the unusually high levels of ANXA5 in bundles, it does not play a role in the bundle’s key function, mechanotransduction, at least until after two months of age in the cochlea and six months of age in the vestibular system. These results reinforce the lack of correlation between abundance of a protein in a specific compartment or cellular structure and its functional significance.

Detection of BDNF-Related Proteins in Human Perilymph in Patients With Hearing Loss

“…Plexins are members of a family of transmembrane proteins that control axonal growth and have been demonstrated in the developing spiral ganglion (Lu et al, 2011). Annexin A5 has been described as being present in high concentration in hair cell stereocilia (Krey et al, 2016). Within this group, the patients with profound hearing loss expressed a significantly reduced proteome, when considering proteins regulated by BDNF (Figure 2).…”

Analysis of level of apoptosis markers in patients with coronary heart disease

“…Одним из ведущих направлений для изучения выраженности апоптоза у пациентов с различными заболеваниями является определение концентрации в плазме и сыворотке крови биомаркеров апоптоза. Среди наиболее широко изучаемых в настоящее время биомаркеров апоптоза особое значение исследователи придают AnxА5, который продуцируется за счет апоптотических и разрушенных клеток, и Bcl-2, который является антиапоптическим белком [8,9]. Изменение уровня данных маркеров к настоящему времени изучено при широком ряде сердечно-сосудистых заболеваний, в том числе и у пациентов с ФП [6,10].…”

Analysis of the Proteome of Hair-Cell Stereocilia by Mass Spectrometry

“…We have used both shotgun (Francis, Krey, Krystofiak, Cui, Nanda, Xu et al, 2015; Krey et al, 2015; Wilmarth et al, 2015; Ebrahim, Avenarius, Grati, Krey, Windsor, Sousa et al, 2016; Krey, Drummond, Foster, Porsov, Vijayakumar, Choi et al, 2016) and targeted mass spectrometry (Krey et al, 2016; Ebrahim et al, 2016) to characterize mouse utricle proteins; similar methods are used with chick and rat hair bundles (Fig. 1B–G).…”

“…Details of sample loading, chromatography, electrospray ionization, and mass spectrometry are described elsewhere (Shin et al, 2013; Krey, Wilmarth, Shin, Klimek, Sherman, Jeffery et al, 2014; Francis et al, 2015; Wilmarth et al, 2015; Krey et al, 2015; Krey et al, 2016; Ebrahim et al, 2016). Data from these ThermoFisher instruments are saved as RAW files, which are then analyzed using the PAW and MaxQuant pathways described below.…”

“…In the absence of calibrated heavy standards, relative protein abundance can be estimated by comparing two or more conditions, for example hair bundles from wild-type and knockout mice (Krey et al, 2016). Again, results are improved by normalizing the signal in each sample to actin or another protein that is not expected to change between the samples.…”