Marfan syndrome variation of the POGLUT2 and POGLUT3 consensus sequence can produce aberrant fibrillin-1...

Fibrillin-1 (FBN1) is an essential component of the extracellular matrix, forming microfibril bundles that are important for the proper development of elastic tissues found in the aorta and lung, as well as nonelastic 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 (C) 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 C three and four: 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.