A Proteomic Analysis of Human Cilia

Cilia play an essential role in protecting the respiratory tract by providing the force necessary for mucociliary clearance. Although the major structural components of human cilia have been described, a complete understanding of cilia function and regulation will require identification and characterization of all ciliary components. Estimates from studies of Chlamydomonas flagella predict that an axoneme contains ≥ 250 proteins. To identify all the components of human cilia, we have begun a comprehensive proteomic analysis of isolated ciliary axonemes. Analysis by two-dimensional (2-D) PAGE resulted in a highly reproducible 2-D map consisting of over 240 well resolved components. Individual protein spots were digested with trypsin and sequenced using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Peptide matches were obtained to 38 potential ciliary proteins by this approach. To identify ciliary components not resolved by 2-D PAGE, axonemal proteins were separated on a one-dimensional gel. The gel lane was divided into 45 individual slices, each of which was analyzed by LC/MS/MS. This experiment resulted in peptide matches to an additional 110 proteins. In a third approach, preparations of isolated axonemes were digested with Lys-C, and the resulting peptides were analyzed directly by LC/MS/MS or by multidimensional LC/MS/MS, leading to the identification of a further 66 proteins. Each of the four approaches resulted in the identification of a subset of the proteins present. In total, sequence data were obtained on over 1400 peptides, and over 200 potential axonemal proteins were identified. Peptide matches were also obtained to over 200 human expressed sequence tags. As an approach to validate the mass spectrometry results, additional studies examined the expression of several identified proteins (annexin I, sperm protein Sp17, retinitis pigmentosa protein RP1) in cilia or ciliated cells. These studies represent the first proteomic analysis of the human ciliary axoneme and have identified many potentially novel components of this complex organelle. Cilia play an essential role in protecting the respiratory tract by providing the force necessary for mucociliary clearance. Although the major structural components of human cilia have been described, a complete understanding of cilia function and regulation will require identification and characterization of all ciliary components. Estimates from studies of Chlamydomonas flagella predict that an axoneme contains ≥ 250 proteins. To identify all the components of human cilia, we have begun a comprehensive proteomic analysis of isolated ciliary axonemes. Analysis by two-dimensional (2-D) PAGE resulted in a highly reproducible 2-D map consisting of over 240 well resolved components. Individual protein spots were digested with trypsin and sequenced using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Peptide matches were obtained to 38 potential ciliary proteins by this approach. To identify ciliary components not resolved by 2-D PAGE, axonemal proteins were separated on a one-dimensional gel. The gel lane was divided into 45 individual slices, each of which was analyzed by LC/MS/MS. This experiment resulted in peptide matches to an additional 110 proteins. In a third approach, preparations of isolated axonemes were digested with Lys-C, and the resulting peptides were analyzed directly by LC/MS/MS or by multidimensional LC/MS/MS, leading to the identification of a further 66 proteins. Each of the four approaches resulted in the identification of a subset of the proteins present. In total, sequence data were obtained on over 1400 peptides, and over 200 potential axonemal proteins were identified. Peptide matches were also obtained to over 200 human expressed sequence tags. As an approach to validate the mass spectrometry results, additional studies examined the expression of several identified proteins (annexin I, sperm protein Sp17, retinitis pigmentosa protein RP1) in cilia or ciliated cells. These studies represent the first proteomic analysis of the human ciliary axoneme and have identified many potentially novel components of this complex organelle. Mucociliary clearance is an essential mechanism for defending the airways against exposure to inhaled toxins and pathological organisms (1.Wanner A. Salathe M. O’Riordan T.G. Mucociliary clearance in the airways.Am. J. Respir. Crit. Care Med. 1996; 154: 1868-1902Google Scholar). The continuous, coordinated beating of the cilia transports mucus and foreign material out of the airways to help maintain a sterile environment. The importance of this process is clearly illustrated by the disease primary ciliary dyskinesia (PCD), 1The abbreviations used are: PCD, primary ciliary dyskinesia; HBE, human bronchial epithelial; DHC, dynein heavy chain; LC/MS/MS, liquid chromatography-tandem mass spectrometry; LC/LC/MS/MS, two-dimensional liquid chromatography-tandem mass spectrometry; EST, expressed sequence tag; 2-D, two-dimensional; 1-D, one-dimensional. in which genetic defects cause dysfunctional cilia and result in chronic otitis media, sinusitus, and bronchitis (2.Meeks M. Bush A. Primary ciliary dyskinesia (PCD).Pediatr. Pulmonol. 2000; 29: 307-316Google Scholar). In addition to their role in mucociliary clearance, cilia and other axonemal structures are clearly essential to many other specialized cell biological functions. Nodal cilia have been shown to be important for the determination of situs (3.Wagner M.K. Yost H.J. Left-right development: the roles of nodal cilia.Curr. Biol. 2000; 10: R149-R151Google Scholar), and recently the primary cilium of renal epithelial cells has been demonstrated to be responsive to flow (4.Praetorius H.A. Spring K.R. Bending the MDCK cell primary cilium increases intracellular calcium.J. Membr. Biol. 2001; 184: 71-79Google Scholar). Motile cilia are also present in the oviduct, the epididymis, and the eppendymal cells of the brain. Sperm flagella possess a 9 + 2 axoneme, and the photoreceptor cells of the retina contain a modified cilium. Although these diverse axonemes may share many common structural features, it is clear that each is specialized for a particular function. The structure of respiratory tract cilia has been the subject of many previous microscopic studies. These studies have demonstrated the 9 + 2 arrangement of microtubules, the inner and outer dynein arms, the radial spokes, and other features common to all motile cilia. However, much of our understanding of cilia structure and function has been derived by analogy to model systems, such as Chlamydomonas and Paramecium. Although these models have provided a wealth of information, to understand the structure and regulation of human cilia it is clear that complementary studies of human material are required. An important step toward a complete understanding of ciliary function is the identification of all the protein components that comprise the ciliary axoneme (a ciliary proteome). Early studies of Chlamydomonas suggested that an axoneme may be composed of over 250 individual proteins (5, 6), and a number of these have been identified and studied. However, whereas the sequencing of the human genome has allowed the identification of homologs of these Chlamydomonas proteins, biochemical evidence is still necessary to demonstrate their axonemal nature in human cilia. Further, it is likely that human cilia will contain important novel proteins not found in simpler model systems. Although the difficulty of obtaining human cilia has limited previous studies, recent advances in cell culture techniques allow ciliated cell differentiation of human airway epithelial cells to occur in vitro (7.Gray T.E. Guzman K. Davis C.W. Abdullah L.H. Nettesheim P. Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells.Am. J. Respir. Cell Mol. Biol. 1996; 14: 104-112Google Scholar, 8.Bernacki S.H. Nelson A.L. Abdullah L. Sheehan J.K. Harris A. William Davis C. Randell S.H. Mucin gene expression during differentiation of human airway epithelia in vitro. Muc4 and muc5b are strongly induced.Am. J. Respir. Cell Mol. Biol. 1999; 20: 595-604Google Scholar), providing a reliable source of material. Coupled with the improvements in sensitivity and throughput of mass spectrometry, the identification of the ciliary proteome is now feasible. Because it is not yet clear which separation technique, is we and two-dimensional gel as well as multidimensional liquid J. analysis of protein using mass 1999; Scholar), in this characterization of the ciliary bronchial epithelial cells were obtained from by the of cells were obtained from normal and were cilia from the normal and cells. or 2 cells were an using (7.Gray T.E. Guzman K. Davis C.W. Abdullah L.H. Nettesheim P. Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells.Am. J. Respir. Cell Mol. Biol. 1996; 14: 104-112Google Scholar, 8.Bernacki S.H. Nelson A.L. Abdullah L. Sheehan J.K. Harris A. William Davis C. Randell S.H. Mucin gene expression during differentiation of human airway epithelia in vitro. Muc4 and muc5b are strongly induced.Am. J. Respir. Cell Mol. Biol. 1999; 20: 595-604Google with cells were on culture with was from the and were from the ciliated cells of the culture was Cilia were isolated as Randell S.H. K. of heavy as an inner of human cilia that is not in a of primary ciliary Biol. Scholar, P. L. of an axonemal dynein heavy expressed in airway epithelial J. Respir. Cell Mol. Biol. 2000; Scholar, of cilia from and of dynein Scholar). ciliated were with to mucus and cell of cilia from and of dynein was to the and the culture was for The was and the was The were and the ciliary axonemes were by and the of these studies, an additional step was in preparations to further of the axonemes. were in and of was to the and the axonemes were for on by were using the was out using on an to the were directly in and for and for and for in the was on a gel using a were and analyzed using 2-D mass spectrometry, axonemes were of axonemes were and the gel was with with of the first Individual spots were digested with trypsin as A. M. M. sequencing of proteins 1996; Scholar), and analyzed by LC/MS/MS ciliary axonemes were separated on a gel and with The gel was into individual from were and was as analysis by LC/MS/MS, a of ciliary axonemes was using The axonemes were a and digested in with in Scholar). analysis by LC/LC/MS/MS, a was using of and to the The was to 2 with a and digested in with was used for it in the and of ciliary axonemal proteins were analyzed by LC/MS/MS using an and to a mass as K. A. The of the Analysis of the of proteins Biol. 2001; Scholar). In addition to the LC/MS/MS a multidimensional approach to that by and J. analysis of protein using mass 1999; was using in the first and in the were from the inner with material a using a or 200 from each were analyzed by LC/MS/MS data from each step were using the of to peptide of was used in to to This into the of the of the mass for the and mass data from each step were against an protein using the protein identification by sequence using mass spectrometry 1999; 20: Scholar). with matches from the protein were against a human expressed sequence used for all were as allow to peptide mass 2 mass analyzed from or 2-D the were used in the analyzed from or in was used as a peptide matches by with were examined to the of the that were used to matches of the of an or features such as as well as biological a particular protein that toward in cilia. In many a protein be identified on the of a peptide was as Randell S.H. K. of heavy as an inner of human cilia that is not in a of primary ciliary Biol. with the was used was a from M. and was used a of were with in and was with to the of were with as Randell S.H. K. of heavy as an inner of human cilia that is not in a of primary ciliary Biol. using a of of Sp17, of well cells were in in and in for were with or with an of of by were with an of normal was using and a was isolated from of cells using an and was using the to the used were and for an for of for for of for for by a cells were on an these cells to a ciliated axonemes were isolated from these using a in the of As these preparations are highly for ciliary axonemes and are well for biochemical As a first step toward a ciliary axonemes isolated from several were separated by 2-D PAGE, and analyzed using analysis In the of proteins was highly reproducible gel was as a gel. four other to the an gel was This gel of over 240 protein spots in that in of the analyzed of To identification of these proteins by mass spectrometry, axonemal proteins were resolved by 2-D PAGE, the of protein was and the gel was with The of proteins was to that obtained with the was individual spots of were isolated from the digested with and analyzed by LC/MS/MS. was obtained on of the and over 200 individual peptides were the studies peptide matches to in the have been by to the number have been and it is clear that number for the the have been of all the matches identified for each is in I, and the spots are in The number of peptides obtained for each protein is also in of the proteins were identified by these may represent axonemal components and have been spots and several of the spots proteins were identified in that the protein was modified or in peptides in spots and an sperm by LC/MS/MS of human axonemal proteins separated by 2-D of protein human protein protein protein to protein to to to to to protein to protein sperm to protein to to sperm in a this 38 proteins were identified. contains a of all proteins identified by each of the approaches used in this The data from the analysis of the 2-D gel the first 38 in this of these 38 matches identified that were to be or components of cilia, on the or the in the These proteins are in which also or likely axonemal components found in each of the other approaches used in this In addition to the major and other ciliary components identified by 2-D analysis from ciliary primary structure as from the sequence and with Cell Scholar), proteins and homologs are with in cilia and the of Cell Scholar, in cilia and for protein 1999; Scholar), and H.A. and in Chlamydomonas 2001; Scholar). proteins were potentially novel components of human cilia, the sperm a a protein found in and an from a human of the proteins are likely and the the protein is to are with the ciliary axoneme or are proteins that with the of four proteomic of human ciliary sperm to protein protein protein to human protein protein of protein ciliary dynein heavy sequence protein to of several to radial to radial dynein intracellular protein dynein dynein protein heavy protein cell protein protein protein protein I, protein pigmentosa complex protein sperm protein cell to dynein dynein dynein heavy heavy dynein heavy heavy to protein protein protein protein protein protein protein to protein protein dynein heavy protein protein protein protein protein dynein heavy protein ciliary by dynein outer protein protein protein protein protein protein to radial protein protein protein heavy protein protein in a identified by that are or axonemal sperm protein protein of ciliary dynein heavy to radial to radial dynein dynein dynein protein protein protein protein protein protein I, protein pigmentosa sperm protein dynein dynein dynein heavy heavy dynein heavy heavy protein dynein heavy protein dynein heavy protein ciliary by dynein outer protein protein protein to radial heavy in a Although 2-D PAGE much important a protein it will not all of the proteins to be present in an dynein heavy which are to be a major axonemal are not resolved on 2-D of their mass To a complete of the proteins the human a of axonemes was separated on a gel The gel were into individual of a Each of the was to trypsin and the peptides were sequenced by LC/MS/MS. This approach sequence data on over peptides that protein and and several resulted in a of axonemal proteins ciliary proteins were that were not found in the 2-D PAGE whereas peptides were identified from in the 2-D over peptides were found to in the were found to dynein and as potentially novel ciliary components were also identified by this approach, a a sperm and the retinitis pigmentosa of the matches were to the axonemal whereas were proteins as potential identified axonemal proteins. of these matches were to or proteins and represent novel ciliary components. peptides that were also identified contains a of all identified in this and these may also represent novel axonemal components. Although the of and 2-D gel with LC/MS/MS the proteins that the ciliary axoneme, techniques have As 2-D gel not all proteins, and the and 2-D approach are As an approach to all the proteins in the ciliary axonemal preparations were and the resulting peptides were analyzed the gel In axonemal proteins were digested with Lys-C, and peptides were separated by and sequenced by Although this experiment a limited of sequence data the the of several of the proteins found in the analysis of the and 2-D peptides in the not identified in the and 2-D a peptide that a disease gene to be present in cilia Chlamydomonas and disease gene are for of cilia and Cell Biol. 2000; Scholar, P. The C. of the disease gene in a and is in 2001; Scholar). were also identified by this approach in To the of sequence obtained from a axonemal proteins were digested with were to an additional separation step analysis by Peptide were to a and with a of The peptides were further resolved on an and sequenced by This approach sequence data on an additional 250 were obtained to in the of which were also found in the studies, these the were to the axonemal protein whereas were likely As in the studies, the of the matches are to novel or proteins, for which is present evidence to are axonemal proteins. the data from the four in peptide matches to over proteins As matches were obtained to many components of the axoneme, and the and heavy of axonemal and the radial proteins are likely that with the and proteins. are the many proteins that have not been as of the ciliary To the identified potentially novel components of human cilia, were to for the expression of proteins identified by in cilia or ciliated cells. In the analysis of ciliary proteins gel many peptides were obtained that the proteins as This is not be by the of used during the cilia and of I, and in J. 1996; Scholar), and this of cilia was not to ciliary To was present in the cilia preparations used for isolated axonemes were separated on an gel and to for the with a to resulted in a of the clearly that was present in preparations of isolated cilia cells were with the to the of the Cilia were clearly by the whereas with the of ciliated cells also for of has been in the and of I, and in J. 1996; Scholar). that the retinitis pigmentosa protein in a gene a photoreceptor protein cause retinitis 1999; Scholar, J. A. M. S.H. in a novel gene cause retinitis 1999; Scholar), were also found in the proteomic of ciliary axonemes. photoreceptor cells contain a modified and an retinitis an disease resulting in and PCD, an disease of ciliary has been C. J. with and cilia in Med. the expression of was examined in of cells. were to was used to the expression of the isolated from cells of differentiation was using to be for ciliated cells have not yet a for was and ciliated cells in these a much for was This of expression has been for other axonemal proteins, Randell S.H. K. of heavy as an inner of human cilia that is not in a of primary ciliary Biol. Scholar, P. L. of an axonemal dynein heavy expressed in airway epithelial J. Respir. Cell Mol. Biol. 2000; Scholar, Nettesheim P. of axonemal dynein heavy expression during ciliated cell Biol. 1996; 71-79Google Scholar). in the of whereas with demonstrated the of the These demonstrate that is expressed by cells and that an in expression may be with in airway epithelial cells. of the peptides sequenced proteins identified as or Because sperm flagella contain the highly 9 + 2 axonemal structure found in cilia, it is likely that proteins identified as may also be components of airway cilia. is a well sperm protein that is present in the and of the sperm flagella and may also function in sperm to the of the of a protein that J. 2001; Scholar, and sequencing of the human sperm 1996; Scholar). of cell from with an demonstrated the of an protein that with the as human 2 and whereas in this was in from lane To was to the cilia of airway epithelial isolated ciliary axonemes were by lane of the was as well as many These may be the result of human is to or may be to other axonemal components. of cells were with the Although the the strongly the cilia and These clearly demonstrate that is a of human airway cilia, the result obtained by mass Although the axonemes of cilia and flagella have been the subject of many studies using model such as Chlamydomonas and recently have studies of human material feasible. In this we have begun to a complete proteome of the ciliary axoneme from human airway cells. the of ciliary components will be for a complete understanding of the and regulation of ciliary complete proteome also allow for identification of proteins that are modified in to that function or in such as primary ciliary in which defects in ciliary structure are is clear from the studies and that the of 2-D gel by mass spectrometry to identify individual protein spots is from several clear from this is the of 2-D to the dynein heavy that are essential components of the ciliary Although of the with 2-D this approach is also In gel proteins, and proteins of may be to identify to a such as In a of isolated axonemes in many of the and in gel Further, the of multidimensional liquid to and the resulting peptide for the identification of the proteins present in the However, each of the four approaches in the identified peptides not identified by of the other of the proteins were identified by approach. This is not previous studies demonstrated that of the complex protein by LC/MS/MS resulted in peptides sequenced in each K. A. The of the Analysis of the of proteins Biol. 2001; Scholar). of the in the present are also of However, it that a approach will be to all the proteins present in a complex such as an it is to the obtained by the also be as complementary approaches that be to The of these approaches has resulted in toward obtaining a complete proteome of human ciliary axonemes. The of the number of proteins in an axoneme is from studies of isolated flagella using 2-D PAGE, and analysis of proteins from and of Chlamydomonas A. Scholar, and biochemical of the Cell Biol. that an axoneme of proteins. This number may represent a it is clear that 2-D PAGE not all proteins and proteins may be present in a the number of individual axonemal proteins may be the protein may in and of the proteins are that with the As a first step toward obtaining a human axonemal we have a 2-D map of isolated cilia that of over 240 well resolved components. This number is to the of 250 proteins in a Chlamydomonas Although this map not all axonemal components this map will be for the identification of axonemal for proteins from the cilia of of individual spots by LC/MS/MS allowed to identify 38 proteins present this which will for by of additional proteomic peptide to other potential axonemal proteins were of axonemal proteins on a gel allowed to identify proteins not resolved on the 2-D axonemes directly in we were to the gel and analysis these by LC/MS/MS or LC/LC/MS/MS, additional data that proteins and common to each of the other In total, peptide were obtained to over potential axonemal proteins. an axoneme is to be of proteins, this data represent of the axonemal proteins. In we obtained peptide matches to over 200 human Although many of these may be it is likely that of represent additional novel axonemal proteins. characterization of these will the of axonemal proteins identified. of the matches have been as or likely axonemal components on or that are with the protein a potential axonemal These the of many proteins that are to be components of cilia, and radial proteins, and and heavy of many peptides were obtained that the sequence of human dynein heavy This is not this outer has been sequenced from In peptides were obtained that the sequence for to several from other Because are highly axonemal with additional studies will be to identify the of human axonemal present in cilia. These data also demonstrate that highly of proteins, which may have a peptide sequence may not proteins were identified that were not to be components of airway cilia. of the these proteins that are axonemal proteins. several or proteins were an sperm protein A. of proteins that with proteins in a to the of Biol. 2001; Scholar), and to the Chlamydomonas protein which to the M. and characterization of a human sperm with to the of the Chlamydomonas 1999; Scholar), whereas has been to the and the sperm from other that contain axonemal structures were also the retinitis pigmentosa and a These data demonstrate the of axonemal diverse In the a proteomic of axonemal structures will be to not identify proteins to identify the proteins that are for the specialized function of each particular The proteins, which the of proteins are proteins for which is to are axonemal components or Although the used in a highly of ciliary it is clear that other structures were with the axonemes during it is not that peptides were obtained from proteins to be components of the and However, the number of proteins identified in these studies to be is that the of the proteins identified may be axonemal components. to that these in resulted in the identification of novel axonemal we several to the proteins identified in the proteomic are with cilia or ciliated cells. In the of proteins for which are of isolated axonemes was used to demonstrate the of these proteins in the ciliary was also used to demonstrate that and to the cilia in of cells. proteins or proteins for which are not be used to the for the protein is expressed in ciliated and also the expression of the with the expression of retinitis pigmentosa protein was demonstrated clearly in ciliated and the of expression to with ciliated cell proteins, identified as components of ciliary axonemes by proteomic were by biochemical studies. Although each of the potentially novel axonemal components identified in this will require these data approaches to validate the of the proteomic In we have used proteomic techniques to identify many potentially novel components of human cilia. studies will on of these techniques to identify additional axonemal proteins. In will be to the ciliary and the components. The of these studies will to a understanding of cilia structure and regulation and will a for in the ciliary proteome in to complete ciliary proteome will also be for cilia from normal and and for proteins that are axonemes from airway cilia and sperm M. for providing to Sp17, Randell and the Cell for of K. and for K. for and the many this be with