ABSTRACT
PURPOSE: Protease nexin-1 (PN-1), a serpin encoded by the SERPINE2 gene, has serine protease inhibitory activity and neurotrophic properties in the brain. PN-1 inhibits retinal angiogenesis; however, PN-1's neurotrophic capacities in the retina have not yet been evaluated. Pigment epithelium-derived factor (PEDF) is a serpin that exhibits neurotrophic and antiangiogenic activities but lacks protease inhibitory properties. The aim of this study is to compare PN-1 and PEDF. METHODS: Sequence comparisons were performed using computer bioinformatics programs. Mouse and bovine eyes, human retina tissue, and ARPE-19 cells were used to prepare RNA and protein samples. Interphotoreceptor matrix lavage was obtained from bovine eyes. Gene expression and protein levels were evaluated with reverse-transcription PCR (RT-PCR) and western blotting, respectively. Recombinant human PN-1, a version of PN-1 referred to as PN-1[R346A] lacking serine protease inhibitory activity, and PEDF proteins were used, as well as synthetic peptides designed from PEDF and PN-1 sequences. Survival activity in serum-starved, rat-derived retinal precursor (R28) cells was assessed with terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) cell death assays. Bcl2 levels were measured with RT-PCR. RESULTS: PN-1 is analogous in primary and tertiary structure to PEDF. A region in PN-1 shares homology with the neurotrophic active region of PEDF, a 17-residue region within alpha helix C. The native human retina, ARPE-19 cells, and murine RPE and retina expressed the gene for PN-1 (SERPINE2 and Serpine2 mRNA). The retina, ARPE-19 cell lysates, and bovine interphotoreceptor matrix contained PN-1 protein. The addition of PN-1, PN-1[R346A], or the 17mer peptide of PN-1 to serum-starved retina cells decreased the number of TUNEL-positive nuclei relative to the untreated cells, such as PEDF. PN-1, PN-1[R346A], and PN-1-17mer treatments increased the Bcl2 transcript levels in serum-starved cells, as seen with PEDF. CONCLUSIONS: PN-1 and PEDF share structural and functional features, and expression patterns in the retina. These serpins' mechanisms of action as cell survival factors are independent of serine protease inhibition. We have identified PN-1 as a novel factor for the retina that may play a neuroprotective role in vivo, and small peptides as relevant candidates for preventing retinal degeneration.
Subject(s)
Eye Proteins/chemistry , Eye Proteins/pharmacology , Nerve Growth Factors/chemistry , Nerve Growth Factors/pharmacology , Serpin E2/chemistry , Serpin E2/pharmacology , Serpins/chemistry , Serpins/pharmacology , Amino Acid Sequence , Animals , Apoptosis/drug effects , Cattle , Cell Line , Cell Survival , Cytoprotection/drug effects , Eye/metabolism , Humans , Mice, Inbred C57BL , Nerve Growth Factors/genetics , Nerve Growth Factors/metabolism , Protein Structure, Tertiary , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Serpin E2/genetics , Serpin E2/metabolism , Structural Homology, ProteinABSTRACT
Clinical immunity to pregnancy associated-malaria (PAM) in multigravida women has been attributed to antibodies that recognize VAR2CSA on the infected erythrocyte (IE) surface. The size and complexity of VAR2CSA have focused efforts on selecting one or more of its six Duffy binding-like (DBL) domains for vaccine development. Presently, however, there is no consensus as to which DBL domain(s) would be most effective in eliciting immunity. This is because antibodies to a number of the DBL domains have been found to block the adhesion of VAR2CSA-expressing erythrocytes to chondroitin sulfate A (CSA)-a major criterion for evaluating vaccine candidacy. Opsonization of IEs by cytophilic antibodies that recognize VAR2CSA represents an important yet understudied effector mechanism in acquired immunity to PAM. To date, no studies have sought to determine the targets of those antibodies. In this study, we found that IgGs from multigravida Malian women showed (i) higher reactivity to recombinant DBL domains by enzyme-linked immunosorbent assay (ELISA), (ii) more binding to VAR2CSA-expressing IEs, and (iii) greater opsonization of these IEs by human monocytic cells than IgGs from malaria-exposed Malian men and malaria-naive American adults. Preincubation of IgGs from multigravida women with recombinant DBL2χ, DBL3χ, or DBL5ε domains significantly diminished opsonization of VAR2CSA-expressing IEs by human monocytes. These data identify the DBL2χ, DBL3χ, and DBL5ε domains as the primary targets of opsonizing IgGs for the first time. Our study introduces a new approach to determining the antigenic targets of opsonizing IgGs in phagocytosis assays.
