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

Biochim Biophys Acta Gen Subj

Institute for Glyco-core Research (iGCORE), Gifu University, Gifu 501-1193, Japan. Electronic address:

Published: January 2025

Background: 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.

Methods: 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.

Results: 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.

Conclusions: Loop and Tail of GnT-III play important roles in catalytic activity, folding and shedding.

General Significance: 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.

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Source
http://dx.doi.org/10.1016/j.bbagen.2024.130734DOI Listing

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