Role of POGLUT2 and POGLUT3 in regulating microfibril structure and function

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Protein O-glucosyltransferases 2 and 3 (POGLUT2 and POGLUT3) add an O-linked glucose to a serine residue in Epidermal Growth factor-like (EGF) repeats containing the putative consensus sequence C3xNTxGS(Y/F)xC4, where C3 and C4 are the third and fourth conserved cysteine in the EGF repeat. Database searches reveal that the fibrillins (FBNs) and Latent TGFβ Binding Proteins (LTBPs) have numerous EGF repeats with the consensus, while other proteins only have one or two. FBN1 and FBN2 are the major structural proteins in the 10-12 nm microfibrils in extracellular matrix, where they serve as the scaffold for elastin in elastic tissues, providing elasticity and recoil essential for function of tissues such as lung. Microfibrils also bind a number of other molecules, including LTBPs, that regulate tissue homeostasis. Mutations in FBN1 cause Marfan Syndrome (MFS) that can result in aortic aneurism/dissection and also cause lung, bone-growth, and eye defects. Elimination of Fbn1, Ltbp1, or Ltbp4 in mice results in perinatal lethality, recapitulating several of the phenotypes seen in MFS patients. Nothing is known about the impact of EGF O-glucosylation on FBN or LTBP function. Using our glycoproteomic mass spectral methods, we demonstrated that 27 of the 47 EGF repeats in FBN1 immunopurified from human dermal fibroblast cultures are modified with O-glucose. Similar levels of O-glucose were found in recombinant FBN2, LTPB1, and LTBP4 expressed in HEK293T cells. Secretion of an N-terminal fragment (EGF1-26) of FBN1 was significantly reduced when POGLUT2 and POGLUT3 were knocked out in HEK293T cells, suggesting that modification by these enzymes, both localized in the endoplasmic reticulum, are required for efficient folding and secretion of these substrates. The importance of this O-glucose modification is underscored by our recent observation that the majority of our Poglut2/Poglut3 double knockout (DKO) mice die perinatally. Survivors have abnormalities similar to Fbn and Ltbp KOs including lung defects, small size, and syndactyly. Based on these observations, we hypothesize that O-glucosylation of FBNs and LTPBs is essential for a functional microfibril network. We predict that loss of O-glucosylation will reduce secretion of these proteins and/or disrupt binding to microfibril associated proteins, leading to structural and/or signaling defects in tissues. We will test this hypothesis in three Aims. Aim 1 addresses how loss of O-glucose affects POGLUT2/3 substrate proteins using cell-based secretion assays, ultrastructural analysis of microfibrils, and analysis of FBN1-ligand interactions. Aim 2 tests whether conserved amino acids in the putative POGLUT2/3 consensus are required for O-glucosylation, and whether MFS mutations in these conserved residues mediate their effects on FBN1 by loss of O-glucose. Aim 3 examines whether loss of POGLUT2/3 impairs the microfibril network in developing lungs using mouse models, histological approaches, and genetic interaction studies. Combined, these aims will provide molecular mechanisms to explain how O-glucose affects the structure and function of the microfibril network.