Files
Abstract
O-glycosylation of Epidermal Growth Factor-like (EGF) repeats plays crucial roles in protein folding, trafficking and function. The Notch extracellular domain has been used as a model to study these mechanisms due to its many O-glycosylated EGF repeats. Three enzymes were previously known to O-glycosylate Notch EGF repeats: Protein O-Glucosyltransferase 1 (POGLUT1), Protein O-Fucosyltransferase 1 (POFUT1), and EGF Domain Specific O-Linked N-Acetylglucosamine Transferase (EOGT). All of these modifications affect Notch activity. Recently, we identified POGLUT2 and POGLUT3 as two novel O-glucosyltransferases that modify a few Notch EGF repeats at sites distinct from those modified by POGLUT1. Comparison of these modification sites revealed a putative consensus sequence which predicted modification of many extracellular matrix (ECM) proteins including fibrillins (FBNs) and Latent TGF-beta-binding proteins (LTBPs). These proteins are essential for formation of microfibrils and elastic fibers in the ECM, which provides structural support to respective tissues and regulates TGF-beta signaling. Glycoproteomic analysis revealed that approximately half of the 47 EGF repeats in FBN1 and FBN2, and half of the 18 EGF repeats in LTBP1, are modified by POGLUT2 and/or POGLUT3. Cellular assays showed that loss of modifications by POGLUT2 and/or POGLUT3 significantly reduces secretion of a FBN1 fragment, suggesting a possible mechanism for the effects of POGLUT2/3-mediated O-glucosylation. Alternatively, it is possible that these O-glucose modifications added by POGLUT2/3 affect protein-protein interactions, as has been demonstrated by research on POGLUT1 and POFUT1 modifications on Notch. Mouse knockouts of either Poglut1 or Pofut1 lead to embryonic lethality with Notch phenotypes. Deletion of both Poglut2 and Poglut3 leads to perinatal lethality in mice with developmental defects affecting mouse limbs and lung development similar to previously described Fbn and Ltbp mouse models. These results highlight the physiological importance of POGLUT2 and POGLUT3, and strongly suggests their role in development and proper function of the ECM. Here we discuss the identification and function of POGLUT2 and POGLUT3 and the ongoing research that continues to elucidate the biological significance of these novel enzymes.