TY - JOUR
T1 - Generation of 13C-Labeled MUC5AC Mucin Oligosaccharides for Stable Isotope Probing of Host-Associated Microbial Communities
AU - Evert, Clayton
AU - Loesekann, Tina
AU - Bhat, Ganapati
AU - Shajahan, Asif
AU - Sonon, Roberto
AU - Azadi, Parastoo
AU - Hunter, Ryan C.
N1 - Publisher Copyright:
© 2019 American Chemical Society.
PY - 2019/3/8
Y1 - 2019/3/8
N2 - 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 β-elimination, permethylated, and analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-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 long-term potential for improved health and disease management.
AB - 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 β-elimination, permethylated, and analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-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 long-term potential for improved health and disease management.
KW - Calu-3
KW - MUC5AC
KW - microbial ecology
KW - mucin
KW - stable isotope probing
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U2 - 10.1021/acsinfecdis.8b00296
DO - 10.1021/acsinfecdis.8b00296
M3 - Article
C2 - 30623643
AN - SCOPUS:85062596260
SN - 2373-8227
VL - 5
SP - 385
EP - 393
JO - ACS Infectious Diseases
JF - ACS Infectious Diseases
IS - 3
ER -