Pigment Epithelium-derived Factor Behaves Like a Noninhibitory Serpin

Abstract

Pigment epithelium-derived factor (PEDF), a neuritepromoting factor, has an amino acid primary structure that is related to members of the serine protease inhibitor (serpin) family. Controlled proteolysis of native PEDF (50 kDa) with either trypsin, chymotrypsin, elastase, or subtilisin yields in each case one major limited product of 46 kDa as analyzed by SDS-polyacrylamide gel electrophoresis. N-terminal sequence analysis of the isolated 46-kDa products indicates a favored cleavage region located toward the C-terminal end of PEDF. A proteolyzed PEDF protein reaction mixture reveals two overlapping sequences: that of the N terminus of intact PEDF and that of an internal region, consistent with cleavage of PEDF about position 382. These data indicate that PEDF protein has a globular conformation with one protease-sensitive exposed loop that contains the homologous serpin-reactive site. Cleavage within the reactive-site loop of PEDF does not cause a conformational change in the molecules (the stressed (S) 3 relaxed (R) transition) and results in heat denaturation identical to its native counterpart. This lack of conformational change is also seen upon cleavage within the reactive-site loop of the noninhibitory serpin ovalbumin. Furthermore, the PEDF neurite-promoting function is not lost with cleavage of the exposed loop. Recombinant PEDF polypeptide fragments with larger truncations from the C-terminal end show neurotrophic activity. Our results clearly indicate that integrity of the PEDF homologous serpin reactive center is dispensable for neurotrophic activity. Thus, the PEDF induction of neurites must be mediated by a mechanism other than serine protease inhibition. Altogether our data indicate that PEDF belongs to the subgroup of noninhibitory serpins and that its N-terminal region confers a neuritepromoting activity to the protein. The neurotrophic active site of PEDF is separated from the serpin reactivesite loop, not only in the primary structure, but also in the folded protein structure. PEDF1 was first described as a neurite-promoting factor that is released by human fetal retinal pigment epithelial (RPE) cells. It was reported that PEDF isolated from medium conditioned by human fetal RPE primary cultures promotes neurite outgrowth in cultured human retinoblastoma Y-79 cells (1). Information about the PEDF peptide sequence has permitted the isolation and cloning of a human PEDF cDNA (2). From cDNA clones, expression vectors were constructed (3, 4) and, in turn, specific antisera to PEDF were developed from the recombinant PEDF proteins (4, 5). Antiserum Ab-rPEDF has been instrumental in the identification of PEDF protein in physiological sources. PEDF is present in bovine eyes in the interphotoreceptor matrix (IPM), i.e. the extracellular matrix located between RPE and the neural retina, and is the sole IPM component responsible for the IPM neurite-promoting activity (5). In addition to the effect on retinoblastoma cells, PEDF has the capacity of promoting neuronal survival of primary cerebellar granule...

Binding of Pigment Epithelium-derived Factor (PEDF) to Retinoblastoma Cells and Cerebellar Granule Neurons

“…However, PEDF has no known protease target for inhibition, and furthermore, its neurotrophic activity is determined by sequences away from its serpin-exposed loop, the structural determinant of serpin protease inhibition (25). The present study indicates that PEDF can interact with cells derived from the retina and the central nervous system.…”

“…Given these studies, we had previously attempted to determine the inhibitory capacity of PEDF against serine proteases. First, an inhibitory activity against proteases, among others thrombin, could not be demonstrated for PEDF (24,25). Second, PEDF, like the noninhibitory serpins ovalbumin, angiotensinogen, and maspin, lacks the serpin S 3 R conformational change upon cleavage of its serpin-exposed loop (25,26).…”

“…First, an inhibitory activity against proteases, among others thrombin, could not be demonstrated for PEDF (24,25). Second, PEDF, like the noninhibitory serpins ovalbumin, angiotensinogen, and maspin, lacks the serpin S 3 R conformational change upon cleavage of its serpin-exposed loop (25,26). Third, structure-function studies demonstrated that the serpin reactive loop located toward the carboxyl end of the polypeptide is dispensable, whereas a region toward the amino end (BA, amino acid positions 44 -121) confers the neurotrophic activity to the PEDF polypeptide (26).…”

Pigment Epithelium-derived Factor (PEDF) Prevents Retinal Cell Death via PEDF Receptor (PEDF-R)

“…PEDF is a 50-kDa glycoprotein and a member of the serpin superfamily without a demonstrable inhibitory activity against serine proteases (23). It has been shown that the biological activities of PEDF depend on binding to cell surfaces of target cells rather than inhibiting serine protease.…”

Identification of the antivasopermeability effect of pigment epithelium-derived factor and its active site

“…Pigment epithelium-derived factor (PEDF), a 418-aa 50-kDa glycoprotein, is a member of the serine protease inhibitor (serpin) family (10,11). Although PEDF has a putative proteasesensitive loop, unlike classical serpins such as ␣1-antichymotrypsin (ACT), PEDF lacks protease inhibitory activity.…”