Fibrillin-1 (FBN1) is an essential component of the extracellular matrix, forming microfibril bundles that are important for proper development of elastic tissues found in the aorta and lung, as well as non-elastic tissue found in the eyes and skeleton. Many missense mutations in the FBN1 gene are associated with Marfan syndrome (MFS), a common developmental disorder. FBN1 contains 47 Epidermal Growth Factor-Like (EGF) repeats, which are protein domains characterized by six cysteines and three ...disulfide bonds. Over half of these EGF repeats are modified with an O-glucose monosaccharide added by Protein O-glucosyltransferase 2 and/or 3 (POGLUT2/3). Previous studies showed that O-glucose modifies the serine within the putative consensus sequence between cysteines 3 and 4: C3-x-N-T-x-G-S-F/Y-x-C4. These residues are common among modified EGFs, but it is unknown if they are required for O-glucosylation. To address this, we used a glycoproteomic approach by analyzing O-glucosylation levels of individual EGF repeats from overexpressed N-terminal FBN1 variants in HEK293T cells. Surprisingly, only the serine (S) was required for O-glucosylation, leading to the revised consensus sequence, C3-x-x-x-x-x-S-x-x-C4. Using this open consensus in database searches, the possible number of POGLUT2/3 substrates in humans has doubled. While some variants displayed reduced O-glucose monosaccharide modification, other variants, including MFS variants, displayed elongation of the O-glucose monosaccharide by additional glycosyltransferases. MFS variants reduction or elongation of O-glucose warrants further investigation on their influence on FBN1 function, which could play a role in the molecular mechanism of the disease.[Show more]

Purified protein (1-2 μg) was acetone precipitated in lo-bind tubes (8:1 acetone:Ni-NTA eluate, -20 C overnight). Precipitated protein was centrifuged at 14,000 RPM in a microcentrifuge for 10 minutes, 4 oC. Acetone was removed, and the pellet was air dried at room temperature for 30 minutes. Proteins were denatured and reduced using 10 μl of reducing buffer containing 8 M Urea, 400 mM ammonium bicarbonate, and 10 mM TCEP at 50 oC for 5 min. Alkylation was performed at room temperature in the d...ark by adding 5 μl 100 mM iodoacetamide in 50 mM Tris-HCl (pH 8.0) for 30 min. 45 μl of mass spectral grade water was added to alkylated samples. Digestion was performed with either trypsin (500 ng, 37 oC for 4 hours) or chymotrypsin (500 ng, 37 oC for 1 hour). Formic acid (5%, 7 μl) was added to digested samples, which were then sonicated for 20 minutes. Samples were desalted with Millipore C18 Zip Tip Pipette Tips (Millipore). After elution in 50% acetonitrile and 0.1% formic acid, samples were either diluted to 20-25% acetonitrile, 0.1% formic acid to a concentration of 10 ng/μl, or dried down using a speed-vac system and resuspended in 0.1% formic acid without acetonitrile before injection. Approximately 10 ng 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 x 15 cm, Thermo Fisher Scientific). A 30 or 40 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 to 2000 or 200 to 2000 at a resolution of 35,000 and automatic gain control set to 1 x 10^6. Higher-energy collisional dissociation–tandem mass spectrometry (HCD-MS/MS) was used on the top ten precursor ions in each full scan (collision energy set to 27%, 2 x 10^5 gain control, isolation window m/z 3.0, dynamic exclusion enabled, and 17,500 fragment resolution).[Show more]

N-terminal fibrillin-1 (FBN1-N) over expressed in HEK293T adherent and suspension cell cultures (WT and glycosyltransferase KO cell lines). FBN1-N constructs were expressed for 72 hours post transfection in adherent cells, and 96 hours post transfection for suspension cells.