Secretion of pigment epithelium-derived factor. Mutagenic study.
Pigment epithelium-derived factor (PEDF), a neurotrophic and antiangiogenic protein, is an extracellular component of the retinal interphotoreceptor matrix which has been shown to be secreted by human fetal retinal pigment epithelial cells. It belongs to the serpin superfamily and contains the typical exposed reactive center loop. The function of this loop is still unknown. In this study we used site-directed mutagenesis of the cDNA encoding PEDF to show that (a) truncation of the C-terminal tail (Pro415-Pro418) of PEDF, (b) deletion of the Pro373-Ala380 segment that resides within the reactive center loop of the protein, and (c) alanine substitution of amino-acid residues Asn391-Thr403 located within its hydrophobic core inhibit PEDF secretion, but not its transcription, by cells transfected with the various PEDF cDNAs. On the basis of the crystal structure of PEDF, these mutations are presumed to alter the protein conformation, suggesting that conservation of the 3D structure of PEDF is essential for its secretion. In addition, we show that replacement of Gly376 and Leu377 with alanine prevents PEDF secretion. As these two residues are located within the highly exposed segment of the reactive center loop, we propose a novel function for this loop in PEDF. Our results imply that the reactive center loop, specifically Gly376 and Leu377, is involved in the interaction of PEDF with components of the quality control system in the endoplasmic reticulum, thus ensuring its efficient secretion.
Shao
Hanshuang
H
Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel.
Schvartz
Iris
I
Shaltiel
Shmuel
S
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002454:Cell Differentiation; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary; D005136:Eye Proteins; D005998:Glycine / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015843:Serpins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000099174:Pigment Epithelium-Derived Factor
D017931:DNA Primers; D018076:DNA, Complementary; D005136:Eye Proteins; D005998:Glycine; D007930:Leucine; D009414:Nerve Growth Factors; D011506:Proteins; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor