Fibrillins play a critical role in tissue/organ development and cardiopulmonary function. Here we showed that O-glucosylation of the fibrillins’ epidermal growth factor-like repeats by protein O-glucosyltransferase 2 (POGLUT2) and POGLUT3 was indispensable for in vivo function of mouse fibrillins. Loss of O-glucosylation of fibrillins caused neonatal death with cardiopulmonary, skeletal, and eye defects reminiscent of fibrillin/elastin mutations. Analyses of FBNs in Poglut2/3 double knockout (DKO) lung, and from DKO dermal fibroblast medium and matrix, provided overwhelming evidence that fibrillins were more susceptible than other POGLUT2/3 substrates to loss of O-glucose. In the DKO, defects in microfibril structure impaired elastic fiber formation, reduced TGF-β/pERK signaling, caused structural defects in late gestation lung blood vessels and terminal bronchioles, and impaired cell differentiation and primary septation in the saccules/alveoli. Collectively, these data support an essential role for POGLUT2/3-mediated O-glucosylation in fibrillin trafficking, microfibril assembly/stability, and function in the ECM environment during lung development.

Primary fibroblast cultures established from lung tissue of E18.5 mouse embryos were grown to confluency in 6 cm dishes. At confluency, medium was switched to DMEM high glucose media with no serum or antibiotics. After 3 days, medium was collected, cleared, and stored at -20 C for mass spectral analysis. BCA assays were used to determine protein concentration of each sample. Media volume equivalent to 10 μg was acetone precipitated in ice-cold acetone (1:4 media:acetone) overnight at -20 C. Samples were spun down at max speed, 10 minutes, 4 C. Supernatant was removed and pellet was processed for mass spectral analysis. BCA assays were used to determine protein concentration of each sample. Media volume equivalent to 10 μg was acetone precipitated in ice-cold acetone (1:4 media:acetone) overnight at -20 oC. Samples were spun down at max speed, 10 minutes, 4 C. Supernatant was removed and pellet was processed for mass spectral analysis. Media samples were denatured and reduced using 15 μL of reducing buffer containing 8 M Urea, 400 mM ammonium bicarbonate, and 10 mM TCEP at 50 C for 5 min. Alkylation of both media and extracellular matrix samples was performed at room temp by adding 100 mM iodoacetamide in 50 mM TrisHCl to a final concentration of approximately 33 mM iodoacetamide and incubating in the dark for 30 min to 1 h. Mass spectral grade water was added to each sample to dilute urea to approximately 1 M. 500 ng of trypsin (cleaves C-terminal to lysine and arginine, Thermo Scientific Pierce 90057) protease was added per sample. Samples were incubated in 37 C water bath for 3-4 h. Samples were acidified with 5% of formic acid and sonicated for 15 min. Samples were desalted with Millipore C18 Zip Tip Pipette Tips. After elution in 50% acetonitrile, 0.1% acetic acid, samples were diluted to an approximate concentration of 1 μg/μL, 15% acetonitrile, and 0.1% formic acid. Approximately 2-3 μg of each sample was injected on a Q-Exactive Plus Orbitrap mass spectrometer (Thermo Fisher) with an Easy nano-LC HPLC system with a C18 EasySpray PepMap RSLC C18 column (50 μm × 15 cm, Thermo Fisher Scientific). A 90 min binary gradient solvent system (Solvent A: 0.1% formic acid in water and Solvent B: 90% acetonitrile, 0.1% formic acid in water) with a constant flow of 300 nL/min was used. Positive polarity mode was used with an m/z range of 350-2,000 at a resolution of 35,000 and automatic gain control set to 1 x 106. Higher energy collisional dissociation-tandem mass spectrometry (HCD-MS/MS) was used on the top 10 precursor ions in each full scan (collision energy set to 27%, 2 x 105 gain control, isolation window m/z 3.0, dynamic exclusion enabled, and 17,500 fragment resolution.

Primary fibroblasts established from lung tissue of wild type and Poglut2 and 3 double knockout mouse embryos at embryonic day 18.5. All fibroblast cultures were no older than 3 passages.