Molecular Cloning and Immunologic Characterization of a Novel cDNA Coding for Progesterone-Induced Blocking Factor

Abstract

Previous studies from our laboratory showed that the immunomodulatory effects of progesterone are mediated by a 34-kDa protein, named the progesterone-induced blocking factor (PIBF). Lymphocytes of women with threatened abortion fail to produce this factor. Via inducing a Th2 biased cytokine production and blocking of NK activity, PIBF prevents induced pregnancy loss in mice, suggesting that substitution therapy with PIBF could be useful as an alternative treatment of certain forms of recurrent spontaneous abortions. Our study was aimed at mapping the sequence and structure of PIBF coding cDNA and characterizing the encoded protein product. Screening of a human liver cDNA library revealed a 2765-bp clone with a 2271-bp open reading frame. The PIBF1 cDNA encodes a protein of 757 amino acid residues with an 89-kDa predicted molecular mass, which shows no significant amino acid sequence homology with any known protein. PIBF produced via recombinant technique is recognized by the Ab specific for the secreted lymphocyte PIBF Ab, and possesses the biological activities of the secreted lymphocyte PIBF. The full-length PIBF is associated with the nucleus, whereas secretion of shorter forms, such a 34-kDa protein is induced by activation of the cell. The 48-kDa N-terminal part of PIBF is biologically active, and the part of the molecule, responsible for modulating NK activity is encoded by exons 2–4. These data provide an initial step for exploiting the possible diagnostic and therapeutic potential of this immunomodulatory molecule.

Urinary Progesterone-Induced Blocking Factor Concentration Is Related to Pregnancy Outcome1

“…Based on our discovery that PIBF is overexpressed by certain human primary tumors and transformed cell lines and, moreover, is secreted by MCF-7 human breast cancer cells [12,20], we hypothesized that high PIBF levels in body fluids can be indicative for the presence of malignant tissues. Indeed, this assay described here detected elevated PIBF levels in cancer patients that were correlated with tumor mass and corrected by the removal of the tumor (unpublished results).…”

“…In resting cells, the full length PIBF has perinuclear localization. Following activation, several smaller molecular weight forms are secreted [12]; thus, PIBF might appear in the sera and, considering the small molecular mass of certain isoforms, also in the urine. Moreover, this heterogeneity can be the result of degradation or processing of nascent PIBF protein, as well as the reflection of the complex alternative mRNA splicing pattern detected in various cells and tissues [20].…”

“…Polyclonal antibodies were generated in our laboratory by immunizing rabbits with a secreted 34-kDa lymphocyte PIBF and the 48-kDa N-terminal part of the human recombinant PIBF [12]. The antibody titers were determined by ELISA using the recombinant human PIBF protein as the antigen and the IgG from immune sera was affinity purified on protein-A or protein-G columns (AP Hungary Ltd., Budapest, Hungary).…”

“…Because PIBF is a secreted molecule, it might be present in biological fluids. PIBF has been cloned and sequenced, and now the recombinant human PIBF is available [12].…”

Complex humoral immune response against a benign tumor: Frequent antibody response against specific antigens as diagnostic targets

“…Recently, overexpression of this gene was associated with breast cancer and highly proliferating cells. The PIBF1 protein is associated with the centrosome (28,29). Notably, loss of progesterone receptor in meningioma patients is correlated with tumor grade and a poor prognosis (30,31).…”