Generation of 13C-labeled MUC5AC mucin oligosaccharides for

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Generation of C-labeled MUC5AC mucin oligosaccharides for stable isotope probing of host-associated microbial communities Clayton Evert, Tina Loesekann, Ganapati Bhat, Asif Shajahan, Roberto Sonon, Parastoo Azadi, and Ryan Hunter ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00296 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019

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ACS Infectious Diseases

Generation of 13C-labeled MUC5AC mucin oligosaccharides for stable isotope probing of host-associated microbial communities Clayton Evert1, Tina Loesekann1, Ganapati Bhat2, Asif Shajahan2, Roberto Sonon2, Parastoo Azadi2, Ryan C. Hunter1*

Department of Microbiology & Immunology, University of Minnesota, 689 23rd Ave SE, Minneapolis, MN 55455 USA

1

2 Complex

Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA 30602 USA

*Address correspondence to Ryan C. Hunter, [email protected]

1

ACS Paragon Plus Environment

ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Stable isotope probing (SIP) has emerged as a powerful tool to address key questions about microbiota structure and function. To date, diverse isotopically labeled substrates have been used to characterize in situ growth activity of specific bacterial taxa and have revealed the flux of bioavailable substrates through microbial communities associated with health and disease. A major limitation to the growth of the field is the dearth of biologically-relevant ‘heavy’ labeled substrates. Mucin glycoproteins, for example, comprise an abundant source of carbon in the gut, oral cavity, respiratory tract and other mucosal surfaces, but are not commercially available. Here we describe a method to incorporate a

13C-labeled

monosaccharide into MUC5AC, a predominant

mucin in both gastrointestinal and airway environments. Using the lung adenocarcinoma cell line, Calu-3, polarized cell cultures grown in 13C-labeled D-glucose resulted in liberal mucin production on the apical surface. Mucins were isolated by size-exclusion chromatography, and O-linked glycans were released by -elimination, permethylated, and analyzed by ESI-MS/MS and MALDITOF-MS techniques. We demonstrate a 98.7% incorporation of 13C in the heterogeneous O-linked oligosaccharides that make up >80% of mucin dry weight. These ‘heavy’ labeled glycoproteins represent a valuable tool for probing in vivo activity of host-associated bacterial communities and their interactions with the mucosal barrier. The continued expansion of labeled substrates for use in SIP will eventually allow bacterial taxa that degrade host compounds to be identified, with longterm potential for improved health and disease management.

Keywords: mucin, MUC5AC, Calu-3, stable isotope probing, microbial ecology

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Stable isotope probing (SIP) has become a popular and powerful tool in microbial ecology, linking taxonomic identity and function of microorganisms in their natural environments. SIPbased approaches have also facilitated culture-independent studies on the effect of environmental stimuli (e.g. pH, temperature, nutrient availability) on functionally active groups of microorganisms in complex ecosystems. In a typical SIP experiment, environmental samples are supplemented with “heavy” stable isotope (e.g. 2H, 13C, 15N, 18O) substrates. As these substrates are assimilated by active organisms, labeled atoms are incorporated into newly synthesized biomass. Downstream analyses of labeled macromolecules (e.g. DNA, RNA, proteins, polar lipid derived fatty acids (PLFA), and other metabolites) can then be carried out by isotope ratio mass spectrometry or molecular analyses to provide insight into the flux of specific growth substrates through microbial communities in situ

1-6.

SIP is now also regularly used in combination with

fluorescence in situ hybridization (FISH) coupled with single cell techniques such as nanoscale secondary ion mass spectrometry (nanoSIMS) and Raman microspectroscopy to provide spatial information about phylogeny and function at high resolution 7, 8. While early applications of SIP were used to study elemental cycling and bioremediation in soil and aquatic environments 1-3, there has been a surge in SIP-based studies characterizing the ecophysiology of host-associated bacterial communities (reviewed in 9). Most notably, diverse 13C

or 15N-labeled dietary substrates (e.g. glucose, inulin, galacto-oligosaccharides, bicarbonate,

potato starch) and host-derived compounds (e.g. sialic acid) have been used to probe nutrient utilization by intestinal microbiota both in vitro and in vivo 5, 10-16. For example, Berry et al. used a combination of SIP-NanoSIMS and

13C/15N

labeled threonine to show that Akkermansia

muciniphila and Bacteroides acidifaciens were the most abundant consumers of secreted host proteins in mouse ceca 5. In a follow-up Raman-SIP study, deuterated water (2H2O) was used as a measure of general growth activity and revealed distinct response patterns of both A. muciniphila and B. acidifaciens to amendments of simple and complex carbohydrates 17. Similar approaches have been used to probe the microbial activity in the oral cavity and sputum derived

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from cystic fibrosis patients 18-20, attesting to the power and versatility of SIP to reveal insights into the structure and function of host-associated bacterial communities in situ. Development and application of new biologically-relevant labeled substrates will be critical for the continued use of SIP to the study of human microbiota. Mucin glycoproteins, for example, comprise approximately half of the bioavailable carbon in the colon

21

and are among the most

abundant host-derived compounds used to support bacterial growth throughout the body 22. Yet, studies of their assimilation by microbiota are limited by the dearth of commercially available 13C or

15N-labeled

mucin substrates. As a step in this direction, we established a method to

incorporate a 13C-labeled isotope into MUC5AC – a prominent secreted mucin in airway, vaginal, eye, middle ear and gastrointestinal environments. Using a mucin-overproducing human adenocarcinoma cell line, Calu-3

23,

polarized cells grown in the presence of

13C

labeled D-

glucose resulted in abundant mucin production at the apical interface and 98.7% displacement of 12C

residues in O-linked oligosaccharides that make up ~80% of mucin weight. Because of their

broad applicability across several mucosal environments, heavy isotopically-labeled mucins represent a valuable tool for the study of mucin-microbe interactions in a range of health and disease contexts.

RESULTS AND DISCUSSION Labeled MUC5AC production by Calu-3 cells. Calu-3 cells are a well-characterized cell line derived from a pleural serous effusion from the proximal airways 23. They form well-differentiated, polarized, electrically resistant cell layers, and are regarded as a reliable model for studies of drug metabolism, ion transport, and bacterial colonization

24-26.

Calu-3 cells form a pseudostratified,

mixed-phenotype layer of ciliated and secretory cells, and are one of the few respiratory cell lines that form tight junctions 27. When grown at an air-liquid interface, macromolecular constituents of apical secretions are also similar to those derived from primary airway cultures they secrete a viscous mucus gel predominated by MUC5AC

4


29,

28.

Importantly,

a major secreted mucin

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glycoprotein found at several body sites 30-32. We sought to exploit this characteristic to generate a heavy labeled (13C) mucin substrate for downstream SIP studies. To do so, Calu-3 cells were continuously propagated in a light (12C) D-glucose medium (mEMEM), in which they grew rapidly and formed confluent monolayers within 5 days postseeding. Upon reaching confluence, cells were passaged to Transwell supports where they were grown in mEMEM containing heavy-labeled (13C) glucose for 3-7 days prior to the establishment of an air-interface culture (AIC) (Figure 1A). Here, cells progressively differentiated and formed a polarized monolayer resistant to media seepage from the basolateral well. After 10 days of polarization, both 12C- and 13C-grown cultures began showing a distinct sheen of secreted mucins on the apical surface that increased with culture time (Figure 1B). When processed by cryo-section and visualized by immunofluorescent staining, morphology of Calu-3 cell cultures grown in both light and heavy mEMEM was consistent with previous reports 33, 34. After 21-28 days of AIC growth, cultures consisted of a monolayer of cells of irregular depth, with only few instances of multilayering. Intracellular mucin granules were not observed by fluorescence microscopy as they were previously via transmission electron microscopy

29, 34,

though anti-MUC5AC immunostaining revealed a confluent mucus layer

covering the apical surface that varied in thickness between 2 and 10 m (Figure 1C). These mucins were harvested by dissolution in 6M GuHCl reduction buffer after 21-28 days of AIC culture for downstream purification and characterization.

Mucin isolation and purification by FPLC-Size Exclusion Chromatography. Following extraction, reduction and dialysis, mucin integrity was analyzed by FPLC sizeexclusion chromatography using a Sepharose CL-4B matrix. Consistent with previous results 36,

35,

chromatograms revealed an abundance of high-molecular weight components running in the

exclusion limit (void) of the column (Figure 2A), with fewer compounds of low molecular weight in the inclusion volume. These data suggest a relatively intact molecular size for purified mucins from Calu-3 cells. To confirm the presence of MUC5AC, 48 x 1mL fractions were collected and 5



analyzed

by

immunoblotting

using

a

monoclonal

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MUC5AC

antibody

(Figure

2B).

Immunoreactivity was highest in fractions 5 to 20, with little to no signal in fractions 25 through 50. Based on these data, fractions 5 through 20 were collected, ethanol precipitated, air-dried and stored at -80C for further analysis. In a typical mucin isolation experiment using either 13C-grown

12C-

or

cells, we were able to isolate 1.77 +/- 0.24 mg (dry weight) of mucin per 6-well

Transwell plate.

Mass spectrometry of purified MUC5AC. Mass spectrometry has emerged as an important approach for structural analyses of mucin glycoprotein structure. Among available methods, matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) and electrospray ionization (ESI) are the most commonly used soft ionization techniques to obtain mass spectra of intact macromolecules. MALDI-TOF enables rapid and sensitive analysis of large biomolecules, and has the advantages of relative simplicity, high sensitivity, high throughput, and no requirement for substrate labeling. However, MALDI-TOF analysis of native glycans is challenging due to structural complexity and low ionization efficiency. Further, only one degree of tandem MS is possible, limiting the amount of structural information that can be obtained. Thus, further structural details can be achieved by ESI-MS and tandem MS fragmentation, which enables characterization of branching and monosaccharide attachment patterns

37.

Here, we

combined both MALDI-TOF-MS and ESI-MS/MS to maximize advantages of each ionization method for qualitative analysis and relative quantitation of MUC5AC oligosaccharides isolated from Calu-3 cells. Prior to MS analysis, mucin O-linked glycans were released by -elimination followed by desalting and permethylation to increase sensitivity and ionization efficiency. During this process, reducing ends of the glycans are derivatized, and amine, hydroxyl, and carboxyl groups are replaced with methyl groups. Permethylation improves sample purification, the sensitivity for detection by increasing the ionization efficiency of glycans up to 20-fold, stabilization of

6


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carbohydrate residues, and simplified assignment of fragmentation patterns to glycan sequences with low false discovery rates

38-41.

Notably, permethylation offers the advantage (over native

glycans) of providing monosaccharide sequence information and the positions by which they are linked to one another. This is a result of non-methylation of linked positions, which creates a 14mass unit “scar” that can be localized by MS/MS42. Using this approach, we determined the structures and monosaccharide composition of O-linked glycans derived from Calu-3 cells. Figure 3A shows the MALDI-TOF-MS mass spectrum of glycan alditols isolated from mucins derived from 12C-grown cultures. Glycan composition and relative abundances can be deduced from these analyses and calculated based on peak integration. Here, nine oligosaccharides were observed, and Table 1 gives an interpretation of the spectrum. Glycans were 2-8 residues long, and most oligos recovered were Gal-GalNAc (18.42%), Neu5Ac-Gal-GalNAc (45.69%), 2Neu5Ac-Gal-GalNAc (14.53%) and Neu5Ac-GalGlcNAc-Gal-GalNAc (11.19%). Structures were confirmed by ESI MS/MS spectra (Figure 4)42, and isoforms were identified based on the CID MS2 fragmentation patterns (Figures S1-S14). These glycans are consistent with previous studies showing similar oligosaccharide composition in MUC5AC

43, 44,

yet they lack mono- and difucosylated sugars characteristic of human gastric

and respiratory mucins45. By comparing MALDI-TOF-MS spectra of

12C

glycans to those derived from

13C-grown

cultures (Figure 3B), we determined the degree of incorporation of the heavy isotope into MUC5AC oligosaccharides. Here, we identified a strong increase in the level of expression of glycans at m/z 548.33 (Gal-GalNAc, 23.53%), 801.53 (GlcNAc-Gal-GalNAc, 1.74%), 920.61 (Neu5Ac-Gal-GalNAc, 58.88%), 1011.68 (GlcNAc-2Gal-GalNAc), 1292.76 (2Neu5Ac-GalGalNac, 5.54%) and 1381.84 (Neu5Ac-Gal-GlcNAc-Gal-GalNAc, 4.96%) corresponding, respectively, to native glycans at m/z 534.31 (not detected in 13C-grown culture), 779.47 (0.63%), 895.53 (0.59%), 983.58 (0.06%), 1256.72 (nd), and 1344.78 (nd) (see Table 1). By comparing

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Table 1. Relative quantification of 12C and 13C-labeled mucin glycans determined by MALDI-TOF-MS and ESI-MS (spectra shown in Figure 3). -GalNAc, -Gal, -GlcNac, -Neu5Ac (sialic acid). m/z = monoisotopic.

Glycan structure

12C m/z* measured

12C relative abundance (%)

13C m/z measured

13C relative abundance (%)

 Mass

534.31

18.42

548.33

23.53

14.02

779.47

2.48

779.47 801.53

0.63 1.74

22.06

73.4

895.52 920.61

0.59 58.88

25.09

99.0

983.58 1011.68

0.06 2.35

28.09

97.5

895.52

983.59

45.69

5.91

1256.72

14.53

1292.77

5.54

36.05

1344.78

11.19

1381.85

4.95

37.07

1705.97

1.49

1749.04

1.67

43.07

1794.02

0.19

-

-

-

2155.0

0.07

-

-

-

8


%13C

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isotope intensities between glycans isolated from “light” and “heavy” culture conditions, we determined 98.7% incorporation rate (i.e. displacement of

12C)

of the heavy label into mucin

glycans when Calu-3 cells are grown in 13C D-glucose (Table 1). The approach described herein builds upon the growing number of metabolic labeling approaches used for the study of O-linked glycans. Radiolabeling methods have long been used to study mucin biology and biochemistry of its metabolism and have demonstrated the utility of in vitro cell culture to study changes in mucin regulation and ultrastructure during disease progression 46-48. In 2002, stable isotope labeling by amino acids in cell culture (SILAC) was first introduced, in which cell cultures are grown in media lacking a standard amino acid but supplemented with an isotopically labeled form (e.g. deuterated leucine, Leu-D3)49. Leu-D3 incorporation into newly synthesized biomass allows for straightforward quantitative proteomic studies of cell proteins (including mucins)50. Others have modified this approach through the use of a GalNAc analog (N-azidoacetylgalactosamine, GalNAz), which is metabolized by host cells, incorporated into surface glycans, and the azide-containing GalNAz serves as a selective handle that facilitates detailed studies of mucin structure 51. Most recently, Orlando et al. were the first to describe an isotope-tagging approach for comparative glycan analysis

52.

Termed IDAWG

(isotopic detection of aminosugars with glutamine), culture of mouse embryonic stem cells in the presence of amide-15N-Gln resulted in near complete incorporation of

15N

into GlcNAc, GalNAc

and sialic acids found in both N-linked and O-linked glycans. Importantly, here we report a near complete incorporation rate (98.7%) of

13C

D-glucose, establishing our method as an additional

tool for the study of O-linked glycan biology. The success of glycan labeling opens up exciting opportunities for studying the dynamics of bacterial mucin degradation. As with other SIP approaches, 13C-mucin-derived metabolites can be analyzed using MS-based metabolomics analyses to track their flux throughout bacterial communities in situ. Other potential powerful approaches include Raman microspectroscopy and nanoSIMS; two high-resolution imaging techniques which allow for the examination of single cells

9



for isotopic enrichment 7. When coupled with fluorescence in situ hybridization, these tools will allow us to link the structure, function and taxonomic identities of individual cells within host microbial communities. We envision future in situ studies in which labeled mucins are directly added to clinical samples derived from patients with cystic fibrosis, colitis, chronic sinusitis and other conditions where mucin degradation has been linked to disease progression 53, 54. While heavy labeled MUC5AC is relevant to the lung, cervix, eye, GI tract and middle ear, we acknowledge that Calu-3-derived mucins have their limitations. Most notably, Calu-3 cells are an adenocarcinoma cell line, which are known to express altered glycosylation patterns and compromised biosynthetic regulation relative to normal tissues55, and may not reflect mucin glycan composition that varies with body site or disease state43, 56, 57. However, given that crude preparations of MUC5AC derived from porcine gastric mucosa or bovine maxillary glands are the commonly used and currently accepted commercially available substrates for in vitro studies of mucin-microbe interactions53, 58-61, Calu-3-derived mucins described here can be regarded as a rational model for SIP-based studies of mucosal-associated microbiota. Future studies will be aimed and generalizing the approach to other immortalized cell lines or even primary cell culture, which will likely enable the isolation of other secreted and tethered mucins. For example, MUC1 and MUC2 are known to be overproduced by LS174T or Caco2 cell lines52. By expanding the availability of labeled mucin substrates, detailed insights into mucin-microbe dynamics applicable to health and disease at several body sites will become attainable.

Conclusion. We have demonstrated that continuous culture of the mucin-overproducing Calu-3 cell line in the presence of

13C

D-glucose leads to the accumulation of labeled MUC5AC

glycoproteins in the cells and culture medium. Subsequent isolation and purification of these mucins expands the repertoire of isotopically labeled substrates available for stable isotope probing of the human microbiota. SIP methods are now seeing widespread use in microbial ecological studies, and we predict that their continued development will generate critical insights

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into bacterial communities associated with mucosal surfaces throughout the human body. The generation of heavy labeled mucins is an important step in that direction; tracking the degradation and the fate of the heavy isotope through clinical samples will help provide insight into specific disease processes and may help identify targets for novel therapeutics. METHODS Preparation of

13C-labeled

Culture Media. A modified Eagle’s minimum essential medium

(mEMEM) was prepared using the following components (per L): 10 mL 100X non-essential amino acids (Sigma M7145), 20 mL 50X essential amino acids (Sigma 5550), 10 mL 100X vitamin mix (Thermo Fisher 1112052), 10 mL 100X sodium pyruvate (Sigma 5280), 10 mL 100X phenol red sodium salt (Sigma 3532), 292 mg L-glutamine, 200 mg CaCl2, 97 mg MgSO4, 400 mg KCl, 1.5 g NaHCO3, 140 mg NaH2PO4H2O, and 6.8 g NaCl.

12C

D-glucose (Sigma) or 13C D-glucose

(Cambridge Isotope Labs, CLM-1396-5) was added to a final concentration of 1.0g/L or 1.33 g/L, respectively. The medium was then vacuum-filtered through a 0.22-m filter, followed by the addition of 10% FBS (Thermo), penicillin G (100 U/mL) and streptomycin (100 g/mL), prior to storage at 4C. Cell culture. Calu-3 cells

23

were obtained from the ATCC (Rockville, MD) and maintained in

unlabeled (12C) mEMEM in a humidified atmosphere with 5% CO2 at 37C. Cells were plated in 75 cm2 cell culture flasks (Corning) and growth medium was replaced every 48h. Upon reaching ~80% confluence, cells were passaged using a 0.25% trypsin solution in EDTA (Sigma T4049). Cells from passages 11-18 were then used to generate MUC5AC at the air-liquid interface of polarized cell cultures. To do so, cells were collected and resuspended in 13C-labeled mEMEM at a concentration of 1x106 cells/mL. 1.5 mL aliquots were then seeded into the apical chamber of each 24mm insert of a Transwell microtiter plate (Corning 3450), and 2.5 mL of 13C mEMEM was added to the basolateral chamber. Cells reached ~90% confluency between 3-7 days, at which point the apical medium was removed to establish an air-liquid interface. Apical medium was

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removed daily until seepage from the basolateral side had stopped. Polarized cells were maintained for additional 21-28 days in 13C mEMEM with medium replacement every 48h prior to mucin collection. Unlabeled control cultures were grown in parallel using

12C

mEMEM at all

stages. Immunofluorescent imaging of Calu-3 cells. Transwell supports were removed from the microtiter dish and chemically fixed with 100 L of 4% paraformaldehyde in PBS for 4h at 4C, followed by two gentle washes in PBS. Following fixation, polycarbonate filters supporting adherent monolayers were covered with a small aliquot of OCT embedding medium and separated from the Transwell inserts using a 6mm dermal tissue biopsy punch. Once removed, the membrane was gently placed onto the surface of an embedding mold partially filled with OCT. Additional OCT was then placed over the exposed side of the membrane (containing the cell monolayer), which was then frozen on a metal stage cooled with liquid nitrogen. Just prior to solidification, the embedding mold was removed and placed on dry ice. Cryo-sections (10 m) of the monolayer/filter samples were obtained using a CM1950 cryostat with a Cryojane tape transfer system (Leica) to facilitate sample adherence to glass slides. Slides were stored at -80C prior to staining. Upon warming to room temperature, slides were rehydrated in PBS for 10 minutes, followed by blocking in 1% goat serum in PBS for 30 minutes at room temperature. Samples were then treated with a human anti-MUC5AC primary antibody (1:250 dilution) in an incubation buffer consisting of 1% bovine serum albumin, 1% goat serum and 0.1% triton X-100 in PBS. Slides were incubated overnight at 4C, followed by washing three times in PBS, and incubation in a goat-anti-human secondary antibody (alexafluor 488, 1:250) in incubation buffer for 1. Following a final wash, slides were counterstained using DAPI (300 nM) and mounted using Vectashield anti-fade mounting medium. Samples were imaged using an Olympus IX83 microscope and images were processed using CellSens software. Isolation of MUC5AC. Secreted mucins were collected as described by Corfield et al. 62. Briefly, Transwells were first cleared of any residual medium before solubilization with a reduction buffer 12


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consisting of 6M guanidine hydrochloride, 0.1 M Tris-HCl buffer, and 5mM EDTA (pH 8). Immediately prior to solubilization, DTT was added to a final concentration of 10mM, in addition to cOmplete Mini protease inhibitor (one tablet per 400 mL, Roche) to minimize MUC5AC degradation. Buffer (1 mL) was added to each apical chamber and incubated at room temperature for 5 minutes. Upon detachment, cell suspensions were gently agitated by pipetting to dislodge remaining biomass, and each of the six Transwell suspensions per plate were pooled into a single aliquot. Reduction buffer (4mL) was then used to rinse residual cell debris from the Transwell membranes and added to pooled aliquots for a final volume of 10mL. This crude mixture was incubated for 5h at 37C, followed by addition of 25 mM iodoacetamide and incubation overnight at room temperature. Mucin purification and immunoblotting. Mucin suspensions were dialyzed (1000 kDa MWCO) against 1L of a 4 M GuHCl buffer containing 2.25 mM NaH2PO4H20 and 76.8mM Na2HPO4. Dialysis proceeded for 36h with buffer exchanges every 12h. The dialyzed sample was then processed by FPLC-size exclusion chromatography using an AKTA pure chromatography system (GE) and a 10/200 mm Tricorn column packed with Sepharose CL-4B base matrix. The column was pre-washed with 4M GuHCl buffer, and samples were run in 5mL aliquots at a flow rate of 0.2 mL/min. Eluate was analyzed using a UV monitor and collected in 20 x 1mL aliquots. FPLC-SEC fractions were screened for MUC5AC by dot-blot analysis. From each fraction, 5L was spotted onto nitrocellulose film, dried and blocked using Tris-buffered saline containing 0.05% Tween 20 (TBS-T) and non-fat powdered dry milk (5% w/v) for 30 minutes at room temperature. The film was then incubated overnight at 4C in TBS-T + milk with 1:500 mouse anti-MUC5AC primary antibody (Life Technologies 182261). The film was then washed three times with TBS-T, followed by incubation in 1:100 goat anti-mouse horseradish peroxidase conjugate secondary antibody (Life G21040) in TBS-T + milk. Samples were washed three more times and the film was developed for 5 minutes using SuperSignal West Femto Substrate Kit

13



(Thermo) according the manufacturer’s instructions. Imaging was performed on an Odyssey Imaging System (LI-COR). Fractions containing MUC5AC (typically 5 thru 20) were pooled and ethanol precipitated. Briefly, samples were mixed with -20C anhydrous ethanol in a 1:9 ratio and left at -20C overnight. Samples were then centrifuged at 15,000 x g for 30 minutes at 4C. Supernatants were discarded, followed by resuspension of pellets in cold ethanol and centrifugation at 15,000 x g for an additional 15 minutes. Pellets were allowed to air dry in a laminar flow hood followed by resuspension in PBS. Final suspensions were verified a second time using dot blot analysis (described above) and quantified using Qubit fluorometry. Reductive -elimination and permethylation of 12C and 13C mucin O-glycans. Isolated mucins were sent to the Complex Carbohydrate Research Center (Athens, GA) for analysis. Mucin samples were further dialyzed (3 kDa MWCO; G-Biosciences, 786611) and freeze-dried before treatment with 1.0 M sodium borohydride (NaBH4 ; ACROS,USA, 20005-1000) in a 50 mM NaOH solution (JT Baker, 3727-01). Samples were then heated to 45C for 18 h to release O-glycans, followed by neutralization by 10% acetic acid (JT Baker, 9508-03), passed through a hydrogen foam resin column (AG 50w-x2 H+ resin, Bio-Rad, 142-1241) and lyophilized. Excess borate crystals were removed under a stream of nitrogen. Dried glycans were derivatized (permethylated) for structural characterization by mass spectrometry as described previously

40, 42, 63.

Briefly, reduced glycans were dissolved in 0.2 mL

dimethyl sulfoxide (DMSO; Thermo Fisher, D128-4) and methylated using 0.1 mL methyl iodide (Sigma- Aldrich, 289566) under alkaline conditions in DMSO/NaOH (50 % vol/vol). After vigorous mixing for 10 min, the reaction was quenched with water, extracted with 2 mL dichloromethane (DCM; Sigma-Aldrich, 650463) and excess methyl iodide was removed by sparging with nitrogen. Permethylated glycans were dissolved in 20 L of methanol (Sigma-Aldrich, 900688) and stored at -20C until further use.

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MALDI-TOF-MS and ESI-MS analysis of O-linked glycans. To characterize MUC5AC O-linked oligosaccharides, permethylated glycans were dissolved in methanol (20 L). A small aliquot (1 L) was spotted onto a MALDI plate (Opti-TOF-384 well insert, Applied Biosystems) and crystallized with 1 L of 2,5-dihydroxybenzoic acid (DHB matrix, 10 mg/mL; Sigma-Aldrich, 149357) in 50% methanol/water. Data were obtained from an Applied Biosystems SCIEX MALDI TOF/TOF 5800 mass spectrometer in reflector positive-ion mode 42, 63, 64. To confirm glycan structure by ESI-MSn, a small aliquot (2 L) of permethylated O-glycans was dissolved in ESI-MS direct infusion buffer (33% H2O, 33% acetonitrile, and 33% 1 mM NaOHmethanol (1:1 vol/vol)). Further, glycans were infused on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific) through a nanospray ionization (NSI) probe. The MSn spectra (collision-induced dissociation, CID and higher energy collisional dissociation, HCD) of the glycans were acquired at high resolution by total ion mapping (TIM) program and detected on Orbitrap / Ion trap 42, 65. Data analysis was performed using Data Explorer V4.5, and the assignment of glycan structures was based on the primary m/z coupled with MS/MS fragmentation patterns using the Expasy tool (https://web.expasy.org/glycomod/) and GlycoWorkbench 1.1 software.

ACKNOWELDGEMENT This research was supported in part by the NIH grants 1S10OD018530 and P41GM10349010 to PA and the CCRC, and 1HL136919 to RCH. We acknowledge Jennifer McCurtain and Scott O’Grady (University of Minnesota) for their technical assistance and members of the Hunter lab for their critical review of the manuscript.

SUPPORTING INFORMATION Collision-induced dissociation (CID) MS2 spectra of permethylated O-glycans derived from Calu3 cells grown in 12C-D-glucose and 13C-D-glucose media. 15



CORRESPONDING AUTHOR INFORMATION Correspondence should be addressed to Ryan C. Hunter ([email protected])

ABBREVIATIONS SIP, stable isotope probing; FISH, fluorescence in situ hybridization; PLFA, polar lipid fatty acid; mEMEM, modified Eagle’s Minimal Essential Medium; AIC, air-interface culture; IDAWG, isotopic detection of aminosugars with glutamine.

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Figure 1. (A) Schematic of mucin hyper-producing Calu-3 cells grown on Transwell permeable supports. Basolateral culture medium containing 12C D-glucose is replaced with 13C D-glucose EMEM to stimulate incorporation of heavy label into MUC5AC oligosaccharides. (B) Mucins (arrow) are produced after 10 days of culture and increase with culture time. (C) Cryo-sectioning and immunofluorescent detection of MUC5AC (green) reveals a robust mucus layer on the apical surface of Calu-3 cells (blue, DAPI) after 21 days. Scale bar = 10 μm.



Figure 2. (A) Purified mucins were analyzed by Sepharose CL-4B size exclusion chromatography. Abundant, high molecular weight compounds ran in the void fraction of the column. (B) Collected fractions were analyzed via slot blot and probed with anti-MUC5AC monoclonal antibody. Fraction number is indicated for each image. Fractions 5-20 were collected for mass spectrometry characterization.


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Figure 3. Structural analysis of Isotopically labeled MUC5AC glycans. Full MALDI-TOF-MS analysis of permethylated O-linked oligosaccharides released by β-elimination from Calu-3 cells grown in either (A) 12C glucose or (B) 13C glucose. Monosaccharide notations are presented as insets, and heavy isotopically labeled residues are denoted by blue brackets. Abundance of each glycan isoform is denoted as a percentage of the total glycan pool. Proposed masses are indicated by red text (m/z, z=1).



Figure 4. ESI-MS full mass of O-glycans from (A) 12C mucin and (B) 13C mucin from Calu-3 cells. Monosaccharide notations are presented as an inset.


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For Table of Contents Use Only


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