Analyzing the Stabilizing Effects of O‐Fucose Glycans on Thrombospondin Type 1 Repeats
Authors: Berardinelli Steven John , Haltiwanger Robert S. 
Published: June 2021

Peters Plus Syndrome (PPS) is an autosomal recessive disorder that results in debilitating quality of life phenotypes such as Peters Anomaly of the eye (anterior eye chamber defect), short stature, brachydactyly, developmental delay and others. PPS is caused by inactivating mutations in a gene encoding β1,3‐glucosyltransferase (B3GLCT) and is classified as a Congenital Disorder of Glycosylation. B3GLCT is highly specific for adding glucose to O‐linked fucose on a Thrombospondin Type 1 Repeat (TSR) resulting in the formation of a Glucose‐β1,3‐Fucose disaccharide. TSRs are small protein motifs defined by six cysteines forming three disulfide bonds and are found in over 65 cell‐surface or secreted proteins. TSR‐containing proteins function in a variety of cellular processes that are essential for proper cell‐cell interactions and formation and processing of the Extracellular matrix (ECM). The O‐linked fucose is added to TSRs containing the consensus sequence C1XX(S/T)C2 by Protein O‐fucosyltransferase (POFUT2), and the consensus is found in 49 TSR‐containing proteins. Elimination of Pofut2 in mice results in embryonic lethality with defects in gastrulation, suggesting that loss of the entire Glucose‐β1,3‐Fucose disaccharide from TSRs has more severe effects than loss of the Glucose alone. Both POFUT2 and B3GLCT are localized to the endoplasmic reticulum (ER), and all proteins that they target move through the secretory pathway to the cell surface or to be secreted from cells. Interestingly, POFUT2 will only modify properly folded TSRs. The addition of Fucose and sometimes the Glucose‐Fucose disaccharide are essential for adequate secretion of POFUT2 target proteins, offering a potential explanation for the difference in severity of phenotypes observed in the Pofut2 null mice and PPS patients. Based on this knowledge we hypothesize that both POFUT2 and B3GLCT are part of a non‐canonical ER quality control mechanism in which the sugar modifications stabilize the folded state of a TSR by interacting with certain underlying amino acids that are in close proximity to the sugars. To test this hypothesis, preliminary NMR data has given insight to potential amino acids that favorably interact with the sugars. A series of MD simulations will be performed to evaluate stabilizing interactions between sugars and amino acids. Site directed mutagenesis of these amino acids will be performed in a single TSR, and HPLC unfolding assays of the unmodified TSR, or TSR modified with Fucose or Glucose‐Fucose disaccharide, will be used to test our hypothesis. Understanding how POFUT2 and B3GLCT act as a quality control mechanism for TSR folding inside the ER will provide molecular insight into how sugar modifications affects the stability of TSRs and how TSR‐containing proteins are inadequately secreted to the ECM in PPS patients.Support or Funding InformationThis research is supported by NIH grant R01HD090156.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.