Proprotein convertases process and thereby inactivate formylglycine-generating enzyme

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Formylglycine-generating enzyme (FGE) is crucial for activating sulfatases by post-translationally modifying a cysteine residue to formylglycine (FGly).
• Sulfatases, activated by FGly, are essential for human development and homeostasis, with 17 nonredundant enzymes identified.
• FGE, normally an endoplasmic reticulum resident, is also secreted, with a significant portion being N-terminally truncated.

Purpose of the Study:

• To elucidate the mechanism and identify the protease responsible for the intracellular generation of N-terminally truncated FGE.
• To investigate the functional consequences of FGE processing on its catalytic activity.
• To understand the physiological role of FGE processing in regulating enzyme activity in higher eukaryotes.

Main Methods:

• Limited proteolysis assays to identify the processing site and responsible proteases.
• Site-directed mutagenesis to determine critical residues for FGE cleavage.
• Enzyme activity assays to compare the FGly-generating capacity of processed and unprocessed FGE.
• Cell-based assays using furin-deficient cells and transient furin expression.

Main Results:

• N-terminal truncation of secreted FGE (lacking residues 34-72) is mediated by proprotein convertases (PCs) via limited proteolysis at the RYSR(72)↓ motif.
• Furin is identified as the major protease responsible for FGE processing, as processing is abolished in furin-deficient cells and restored upon furin re-expression.
• Mutations at Arg-69 and Arg-72 abolish FGE processing, while Tyr-70 and Ser-71 contribute to the partial processing observed.
• Truncated, secreted FGE lacks FGly-generating activity, whereas the unprocessed form remains active.

Conclusions:

• Furin-mediated intracellular processing of FGE generates an inactive, truncated form.
• This processing occurs along the secretory pathway, primarily intracellularly but with some extracellular activity.
• The selective inactivation of FGE through furin processing represents a physiological mechanism for regulating sulfatase activity in higher eukaryotes upon enzyme secretion.