0031-3998/10/6803-0231 Vol. 68, No. 3, 2010 PEDIATRIC RESEARCH Printed in U.S.A. Copyright © 2010 International Pediatric Research Foundation, Inc. MUC5B Is the Predominant Mucin Glycoprotein in Chronic Otitis Media Fluid DIEGO PRECIADO, SAMITA GOYAL, MICHAEL RAHIMI, ALAN M. WATSON, KRISTY J. BROWN, YETRIB HATHOUT, AND MARY C. ROSE Department of Pediatric Otolaryngology-Head and Neck Surgery [D.P.], Center for Genetic Medicine Research [D.P., S.G., M.R., A.M.W., K.J.B., Y.H., M.C.R.], Children’s National Medical Center, Washington, District of Columbia 20001 ABSTRACT: Chronic otitis media (COM), e.g. “glue” ear is char- placement is the most common pediatric surgical procedure acterized by middle ear effusion and conductive hearing loss. Al- requiring anesthesia in the United States (11). though mucous glycoproteins (mucins), which contribute to in- Mucins are heavily glycosylated proteins that are consid- creased effusion viscosity, have been analyzed in ear tissue ered primarily responsible for the gel-like characteristics of specimens, no studies have been reported that characterize the mo- mucoid middle ear fluids (12,13). They are comprised by a lecular identity of secreted mucin proteins present in actual middle heavy carbohydrate content on a core protein backbone con- ear fluid. For this study, effusions from children with COM under- sisting of numerous tandem repeats, whose primary amino going myringotomy at Children’s National Medical Center, Wash- acid sequence is unique to each mucin (14,15). These tandem ington, DC were collected. These were solubilized and gel fraction- repeat regions contain proline and are high in serine and/or ated, and the protein content was identified using a liquid threonine residues, the sites of O-glycosylation (16). Mucins chromatography tandem mass spectrometry (LC-MS/MS) proteom- ics approach. Western blot analyses with mucin specific antibodies are broadly classified as either cell membrane-bound or se- and densitometry were performed to validate the mass spectrometry creted (14,16,17). To date, 18 human mucin genes coding for findings. LC-MS/MS results identified mucin MUC5B by Ͼ26 mucin glycoproteins have been identified. Of these, MUC2, unique peptides in six of six middle ear effusion samples, whereas MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, and mucin MUC5AC was only identified in one of six middle ear MUC19 are secreted. From studies of human airway secre- effusions. These findings were validated by Western blot performed tions, it is clear that MUC5AC and MUC5B, and to a much on the same six and on an additional 11 separate samples where lesser extent, MUC2, are key determinants of airway mucus densitometry revealed on average a 6.4-fold increased signal in gel properties (16,18). MUC5B when compared with MUC5AC (p ϭ 0.0009). In summary, Past studies have examined mRNA and protein expression although both MUC5AC and MUC5B mucins are detected in middle levels of mucin gene products in human middle ear tissues by ear effusions, MUC5B seems to be predominant mucin present in various approaches (Table 1). However, probably because of COM secretions. (Pediatr Res 68: 231–236, 2010) lack of readily available mucin specific antibodies, no study has successfully identified the specific mucin protein compo- titis media (OM) is a ubiquitous condition of early child- sition of pathologic middle ear effusion samples. O hood accounting for an enormous amount of public Emerging techniques in proteomics have significantly im- health costs (1–3). Chronic otitis media (COM) is character- proved the ability to globally analyze the proteins within ized by the persistence of middle ear effusion, which is most biologic samples (19). Currently, the use of mass spectrometry often highly viscous (4). This thick, viscous middle ear effu- (MS) and database searching is becoming a routine method to identify thousands of proteins in a single sample. One sion has been classically described as “glue ear” (5) and is approach relies on one-dimensional gel electrophoresis associated with conductive hearing loss, effusion nonclear- (SDS-PAGE) separation of proteins followed by gel digestion ance, and increased likelihood of requiring surgical tympanos- of specific bands and liquid chromatography tandem mass tomy tube placement (6–10). In turn, tympanostomy tube spectrometry (LC-MS/MS) analysis of the resulting peptides. Previous studies have successfully used proteomics ap- proaches to characterize the nature of gel-forming mucins in Received February 24, 2010; accepted May 17, 2010. biospecimens such as pancreatic cyst fluid and saliva (20–24). Correspondence: Diego Preciado, M.D., Ph.D., Division of Pediatric Otolaryngology- We hypothesized that by using an unbiased global protein Head and Neck Surgery, Children’s National Medical Center, 111 Michigan Avenue detection proteomics MS approach, the secreted mucin glyco- Northwest, Washington, DC 20010; e-mail: [email protected] Supported by NIH National Heart, Lung and Blood Institute, K12 Genomics of Lung proteins present in human middle ear effusions could be Grant, HL090020-01. Intellectual and Developmental Disability Research Centers Core definitively identified. Grants 1P30HD40677 and 5R24HD050846 to the Center for Genetic Medicine at Children’s National Medical Center, and an Avery Scholar Award from Children’s National Medical Center [D.P.]. Abbreviations: COM, Chronic otitis media; LC-MS/MS, liquid chromatog- Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the raphy tandem mass spectrometry; MS, mass spectrometry; MS/MS, tandem journal’s Web site (www.pedresearch.org). mass spectrometry; MUC, mucin; OM, otitis media 231 232 PRECIADO ET AL. Table 1. Summary of published studies attempting to characterize middle ear mucins Reference MUC5AC mRNA MUC5AC protein MUC5B mRNA MUC5B protein Kawano et al. (43) Tissue (ϩ) ISH, Northern blot Tissue (ϩ) IHC Tissue (ϩ) ISH, Northern blot Tissue (ϩ) IHC Chung et al. (4) NE Tissue (Ϫ) IHC NE Tissue (ϩ) IHC Lin et al. (27,28) Tissue (Ϫ) ISH, Northern blot Tissue (Ϫ) IHC Tissue (ϩ) ISH, Northern blot Tissue (ϩ) IHC Takeuchi et al. (31) Tissue (ϩ) RT-PCR NE NE NE Elsheikh and Mahfouz (35) 16% Effusion samples (ϩ) RT-PCR NE 96 % Effusion samples (ϩ) RT-PCR NE Kerschner (12) Cell explants (ϩ) RT-PCR NE Cell explants (ϩ) RT-PCR NE Previous studies of MUC5AC and MUC5B characterization in the human middle ear. ISH, in situ hybridization; IHC, immunohistochemistry; NE, not examined. ϭ ϭ ϭ ϭ ϭ METHODS 1.9 for z 1, Xcorr 2.2 for z 2, and Xcorr 2.5 for z 3), peptide probability based score with a p Ͻ 0.005. Sample collection and preparation. Effusions from children with COM Western blot analysis. Western blot analysis on middle ear secretions was undergoing myringotomy with tube placement at Children’s National Medical performed using protocols adapted from Berger et al. (25) as previously Center irrespective of race, ethnicity, or gender were collected under institu- described. Briefly, samples containing 40 ␮g total proteins were electropho- tional review board approval and parental consent. Patients included subjects resed in 1.0% agarose gels. After electrophoresis, samples were transferred to presenting to the operating room in a longitudinal fashion between January polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA). The 2008 and June 2008. Exclusion criteria included purulent effusion more MUC5B and MUC5AC positive biologic controls were human saliva to consistent with acute OM, cleft palate or other craniofacial dysmorphic which protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO) was syndromes, immunosuppressive state or condition, or early history of skull added at a 1:60 dilution on collection and Calu-3 lung carcinoma cell lysates, base radiation therapy or skull base malignancy. Bilateral effusions were respectively. Affinity-purified specific anti-MUC5:TR3A polyclonal antibod- combined into one sample per patient. Collected middle ear effusions were ies generated in our laboratory (25) were used. MG2 anti-MUC5B antibodies frozen at Ϫ80°C and freeze dried by lyophilization for 24 h. Six effusion were procured by Dr. Robert Troxler (26). Primary antibodies were diluted samples were collected longitudinally for proteomics and Western blot anal- 1:2000. Secondary antibody used was horse-peroxidase goat anti-rabbit anti- ysis. Eleven other separate effusions were collected for further subsequent body in 25 mL of 5% milk at a dilution of 1:5000. Blots were developed using confirmatory Western blot analysis. the horseradish flouro-illuminescence detection protocol using SuperSignal Because some of the effusion samples had blood contamination, serum West Dura Extended Duration Substrate (Pierce, Rockford, IL) for 5 min. The from a child with OM was collected as a control. This was done to demon- developed blots were exposed and visualized with a charge-coupled device strate as expected that blood does not contain mucin proteins and the camera equipped Gel Doc 2000 chemiluminescent imaging documentation contamination does not account for the potential source of the identified station (Bio-Rad, Hercules, CA). Exposure time was 1 s and equal for all mucins in the middle ear effusions. blots. The respective intensities of each MUC5AC or MUC5B positive band SDS-PAGE electrophoresis. Effusion samples containing 150 ␮g of pro- relative to the positive control in the resulting images were densitometrically teins were dissolved in Laemili buffer containing 0.1 mM DTT and subjected semi-quantified with Quantity One 4.3.1 image processing software (BioRad, to one-dimensional SDS gel electrophoresis fractionation at 200 V for 50 min. Hercules, CA) using equal-sized box-shaped markers. The gel was fixed with methanol and stained with Coomassie for protein visualization. Each gel lane was sliced into 32 segments, and each slice was digested with trypsin as follows. Briefly, the gel cuts were placed in 100 ␮L RESULTS of water and then subjected to two washes with a 1:1 by volume solution of water and acetonitrile.