Secretion of pigment epithelium-derived factor. Mutagenic study

Area of Science:

• Molecular Biology
• Protein Biochemistry
• Ophthalmology

Background:

• Pigment epithelium-derived factor (PEDF) is a secreted protein with neurotrophic and antiangiogenic properties.
• PEDF is found in the retinal interphotoreceptor matrix and is secreted by retinal pigment epithelial cells.
• PEDF belongs to the serpin superfamily and possesses a reactive center loop whose function is largely unknown.

Purpose of the Study:

• To investigate the role of PEDF structure and its reactive center loop in protein secretion.
• To identify specific regions and residues essential for the efficient secretion of PEDF.

Main Methods:

• Site-directed mutagenesis of PEDF cDNA was employed to create various mutant forms.
• Mutated PEDF constructs were transfected into cells to assess their transcription and secretion.
• Analysis was based on the known crystal structure of PEDF.

Main Results:

• Mutations affecting the C-terminal tail, reactive center loop, or hydrophobic core inhibited PEDF secretion but not transcription.
• Specific mutations, including alanine substitution of Gly376 and Leu377 within the reactive center loop, abolished PEDF secretion.
• These findings suggest that the 3D conformation of PEDF is critical for its secretion.

Conclusions:

• The conserved 3D structure of PEDF is essential for its secretion.
• The reactive center loop, particularly residues Gly376 and Leu377, plays a novel role in PEDF secretion.
• PEDF's reactive center loop likely interacts with the endoplasmic reticulum quality control system to facilitate secretion.