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Determining the Role of O‐fucose Modifications of the Stabilin‐2 Receptor Expression and Function
Author(s) -
Nour Aiah,
Harris Edward
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.01889
Subject(s) - ectodomain , fucosylation , chinese hamster ovary cell , fucosyltransferase , microbiology and biotechnology , transfection , biology , endoplasmic reticulum , brefeldin a , receptor , cell culture , fucose , golgi apparatus , glycoprotein , biochemistry , gene , genetics
Protein O‐fucosyltransferase‐1 (POFUT‐1) is a fucosyltransferase that hypothetically modifies over 100 human proteins. POFUT1 is enzymatically active to the precise EGF domains of proteins in the endoplasmic reticulum. The importance of O‐fucosylation is evident in its involvement in NOTCH receptor signaling. NOTCH is the only studied and proved substrate to be modified by POFUT‐1 with its many matching EGF domains. Preliminary studies suggest that 190HARE may be O‐fucosylated due to protein expression of stab‐2 in Lec1 and Lec13 modified Chinese Hamster Ovary Cells (CHO). In this study, we proposed a mechanism for O‐fucosylation of the Stabilin‐2 receptor because of identical consensus sequences on 6 of the 20 EGF domains required for POFUT‐1 recognition and modification. To demonstrate this, two cell lines were constructed: a control unmodified line and a line that has undergone CRISPR/CAS9 to inactivate the POFUT‐1 gene. Stab2 cDNA was transfected in each cell line and assessed for expression and function. We verified expression of transfected cells by SDS‐PAGE followed by western blot. We found that O‐fucosylation does not affect receptor expression or endocytosis function. Based on preliminary data, we will be purifying the ectodomain of Stab‐2 to determine precisely which amino acids are modified by POFUT‐1.