Functions of unique middle loop and C-terminal tail in GnT-III activity and secretion

• Published in: Biochimica et biophysica acta. General subjects Vol. 1869; no. 1; p. 130734
• Main Authors: Bao, WanXue, Yamasaki, Takahiro, Nakano, Miyako, Nagae, Masamichi, Kizuka, Yasuhiko
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2025
• Subjects: Animals; Disordered segment; Glycosylation; Glycosyltransferase; GnT-III (MGAT3); HEK293 Cells; Humans; N-Acetylglucosaminyltransferases - genetics; N-Acetylglucosaminyltransferases - metabolism; N-glycan; Polysaccharides - metabolism; Protein Domains; SSA; GnT-III (MGAT3); CBB; KO; Glycosyltransferase; Disordered segment; LC-ESI MS; PHA; DAPI; Glycosylation; GnT; ER; DMEM; RCA; CHX; FBS; N-glycan; WT
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2024.130734

N-Glycan branching modulates the diversity of protein functions. β1,4-N-acetylglucosaminyltransferase III (GnT-III or MGAT3) produces a unique GlcNAc branch, “bisecting GlcNAc”, in N-glycans, and is involved in Alzheimer's disease and cancer. However, the 3D structure and catalytic mechanism of GnT-III are unclear. According to AlphaFold-based structure prediction, GnT-III likely contains two putative disordered segments, a long middle loop (Loop) and a C-terminal tail (Tail). We hypothesized that these segments play important roles in regulating the activity or intracellular behaviors of GnT-III. We expressed wild-type GnT-III (GnT-III-WT), GnT-III-Loop- and -Tail-deletion mutants in cells. Their in vitro catalytic activity and glycan biosynthesis in cells were examined using high-performance liquid chromatography, UDP-Glo glycosyltransferase assays, and glycomic analysis. Subcellular localization of WT and GnT-III mutants was investigated by immunostaining, and degradation rate and secretion were also examined. The Loop-deletion mutant had higher in vitro and in cellulo activity than GnT-III-WT, indicating that Loop suppresses catalytic activity. In contrast, the Tail-deletion mutant showed weaker activity, increased ER localization, and faster degradation than GnT-III-WT, indicating that Tail is required for proper folding. In addition, deletion of Loop led to aberrant shedding of GnT-III, indicating that Loop contains the cleavage site or regulates GnT-III shedding. Loop and Tail of GnT-III play important roles in catalytic activity, folding and shedding. Our results provide further understanding of the catalysis and shedding mechanisms of GnT-III and can help in the development of methods for modifying the levels of bisecting GlcNAc on glycoproteins and in cells. [Display omitted] •GnT-III biosynthesizes a unique N-glycan branch, bisecting GlcNAc.•GnT-III contains two unique disordered segments in structure.•The long middle loop suppresses activity of GnT-III.•The long middle loop is important for shedding of GnT-III.•The C-terminal tail is essential for proper folding of GnT-III.