Quantitative Proteomic Analysis of Human Airway Cilia Identifies Previously Uncharacterized Proteins of High Abundance

Abstract

Cilia are essential to many diverse cellular processes. Although many major axonemal components have been identified and studied, how they interact to form a functional axoneme is not completely understood. To further our understanding of the protein composition of human airway cilia, we performed a semi-quantitative analysis of ciliary axonemes using label-free LC/MSE which identified over 400 proteins with high confidence. Tubulins were the most abundant proteins identified, with evidence for 20 different isoforms obtained. Twelve different isoforms of axonemal dynein heavy chain were also identified. Absolute quantification of the non-tubulin components demonstrated a greater than 75-fold range of protein abundance (RSPH9;1850 fmol vs CCDC103;24 fmol), adding another level of complexity to axonemal structure. Of the identified proteins, approximately 70% are known axonemal proteins. In addition, many previously uncharacterized proteins were identified. Unexpectedly, several of these, including ERICH3, C1orf87, and CCDC181, were present at high relative abundance in the cilia. RT-PCR analysis and immunoblotting confirmed cilia specific expression for eight uncharacterized proteins, and fluorescent microscopy demonstrated unique axonemal localizations. These studies have provided the first quantitative analysis of the ciliary proteome and have identified and characterized several previously unknown proteins as major constituents of human airway cilia.

FAP57/WDR65 targets assembly of a subset of inner arm dyneins and connects to regulatory hubs in cilia

“…FAP57/WDR65 is also highly conserved throughout vertebrate species, and in humans, FAP57 is especially abundant in testes, respiratory tissue, and fallopian tubes (Fagerberg et al, 2014;Blackburn et al, 2017). We suggest that FAP57/WDR65 should be screened as a candidate gene for those patients with PCD whose mutations have not been identified in the more commonly known PCD loci.…”

“…Although several studies have identified FAP57/WDR65 as a conserved component of motile cilia and flagella (Broadhead et al, 2006;McClintock et al, 2008;Arnaiz et al, 2010;Sigg et al, 2017;Blackburn et al, 2017), functional studies on fap57/wdr65 mutations are very limited. One report identified a missense mutation in WDR65 in a patient with Van der Woude syndrome, a cleft palate disorder, but this work did not establish a clear connection between the mutation and the cleft palate defects (Rorick et al, 2011).…”

The outer dynein arm assembly factor CCDC103 forms molecular scaffolds through multiple self‐interaction sites

“…Further analysis using circular dichroism spectroscopy revealed that this protein has a melting temperature above 85 C and can refold to its native state even after heating to 100 C. Biochemical and immunofluorescence studies in Chlamydomonas identified CCDC103 in both the cytoplasm and the cilium where it appeared to be arrayed along the entire axonemal length (King & Patel-King, 2015). CCDC103 has also been identified by mass spectrometry in human airway cilia (Blackburn, Bustamante-Marin, Yin, Goshe, & Ostrowski, 2017). Axonemal CCDC103 was found to be stable to high salt treatment but could be readily extracted with 0.3% Sarkosyl as were the outer dynein arms.…”

“…CCDC103 exhibits unusual biophysical properties including the formation of dimers and higher order structures that are stable to, or reform after, heating in the presence of detergent (King & Patel-King, 2015;Panizzi et al, 2012). CCDC103 has also been identified by mass spectrometry in human airway cilia (Blackburn, Bustamante-Marin, Yin, Goshe, & Ostrowski, 2017). CCDC103 has also been identified by mass spectrometry in human airway cilia (Blackburn, Bustamante-Marin, Yin, Goshe, & Ostrowski, 2017).…”

The transcriptional signature associated with human motile cilia

“…Moreover, in conjunction with other transcriptional regulators, such as HNF1B and SOX5, further ciliary diversity is introduced for mechanosensory renal cilia and bronchiolar cilia, respectively (Gresh et al, 2004, Kiselak et al, 2010). Proteomic profiling studies have sought to define the components of motile cilia by dysregulating such transcriptional regulators and analysing the proteome of isolated cilia preparations using mass spectrometry (Choksi et al, 2014a, Blackburn et al, 2017, Ostrowski et al, 2002, El Zein et al, 2009, Campbell et al, 2016). Each of these studies has produced a list of cilia-associated proteins and accordingly a number of databases have been established.…”