ABSTRACT
The metalloproteinase SAS1B [ovastacin, ASTL, astacin-like] was immunolocalized on the oolemma of ovulated human oocytes and in normal ovaries within the pool of growing oocytes where SAS1B protein was restricted to follicular stages spanning the primary-secondary follicle transition through ovulation. Gene-specific PCR and immunohistochemical studies revealed ASTL messages and SAS1B protein in both endometrioid [74%] and malignant mixed Mullerian tumors (MMMT) [87%] of the uterus. A MMMT-derived cell line, SNU539, expressed cell surface SAS1B that, after binding polyclonal antibodies, internalized into EEA1/LAMP1-positive early and late endosomes. Treatment of SNU539 cells with anti-SAS1B polyclonal antibodies caused growth arrest in the presence of active complement. A saporin-immunotoxin directed to SAS1B induced growth arrest and cell death. The oocyte restricted expression pattern of SAS1B among adult organs, cell-surface accessibility, internalization into the endocytic pathway, and tumor cell growth arrest induced by antibody-toxin conjugates suggest therapeutic approaches that would selectively target tumors while limiting adverse drug effects in healthy cells. The SAS1B metalloproteinase is proposed as a prototype cancer-oocyte tumor surface neoantigen for development of targeted immunotherapeutics with limited on-target/off tumor effects predicted to be restricted to the population of growing oocytes.
Subject(s)
Antibodies/pharmacology , Antigens, Neoplasm , Immunoconjugates/pharmacology , Immunotherapy/methods , Metalloproteases/antagonists & inhibitors , Mixed Tumor, Mullerian/drug therapy , Ribosome Inactivating Proteins, Type 1/pharmacology , Uterine Neoplasms/drug therapy , Amino Acid Sequence , Antibodies/metabolism , Antibodies/toxicity , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Antigens, Neoplasm/metabolism , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Endocytosis , Female , Humans , Immunoconjugates/metabolism , Immunoconjugates/toxicity , Immunotherapy/adverse effects , Metalloproteases/genetics , Metalloproteases/immunology , Metalloproteases/metabolism , Mixed Tumor, Mullerian/enzymology , Mixed Tumor, Mullerian/genetics , Mixed Tumor, Mullerian/immunology , Mixed Tumor, Mullerian/pathology , Molecular Sequence Data , Molecular Targeted Therapy , Oocytes/drug effects , Oocytes/enzymology , Ribosome Inactivating Proteins, Type 1/metabolism , Ribosome Inactivating Proteins, Type 1/toxicity , Saporins , Signal Transduction/drug effects , Time Factors , Uterine Neoplasms/enzymology , Uterine Neoplasms/genetics , Uterine Neoplasms/immunology , Uterine Neoplasms/pathologyABSTRACT
BACKGROUND: Sperm Acrosomal SLLP1 Binding (SAS1B) protein (ovastacin) is an oolemmal binding partner for the intra-acrosomal sperm protein SLLP1. RESULTS: Immunohistochemical localization revealed that SAS1B translation is restricted among adult tissues to the ovary and oocytes, SAS1B appearing first in follicles at the primary-secondary transition. Quiescent oocytes within primordial follicles and primary follicles did not stain for SAS1B. Examination of neonatal rat ovaries revealed SAS1B expression first as faint signals in postnatal day 3 oocytes, with SAS1B protein staining intensifying with oocyte growth. Irrespective of animal age or estrus stage, SAS1B was seen only in oocytes of follicles that initiated a second granulosa cell layer. The precise temporal and spatial onset of SAS1B expression was conserved in adult ovaries in seven eutherian species, including nonhuman primates. Immunoelectron micrographs localized SAS1B within cortical granules in MII oocytes. A population of SAS1B localized on the oolemma predominantly in the microvillar region anti-podal to the nucleus in ovulated MII rat oocytes and on the oolemma in macaque GV oocytes. CONCLUSIONS: The restricted expression of SAS1B protein in growing oocytes, absence in the ovarian reserve, and localization on the oolemma suggest this zinc metalloprotease deserves consideration as a candidate target for reversible female contraceptive strategies.
