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.
Highlights
SAS1B, is a cortical granule and oocyte surface-associated zinc matrix metallo-proteinase (MMP, EC 3.4.24.21) with reported roles in sperm-egg interaction [1, 2] and in the block to polyspermy [3] during eutherian fertilization
In humans, where mono-ovular menstrual cycles occur, the relative abundance of SAS1B mRNAs isolated from whole ovaries is predicted to be low, since the pool of growing oocytes in secondary follicle stages and beyond is small and many secondary follicles undergo atresia [20]
RT-PCR of a panel of normal human tissues identified ASTL amplicons only in the ovary (Figure 1B). Using both a rabbit polyclonal antibody generated to recombinant human SAS1B (IM) (Figure 1C, IM) and a commercial ASTL pro-peptide antibody (Figure 1D), SAS1B was immunolocalized in human ovarian sections within oocytes in a early primary to secondary transition follicle (Figure 1C3 and inset, black arrow, where perinuclear staining was first detected), secondary follicles and subsequent stages but not in the ovarian reserve of primordial follicles, in primary follicles (C2), or in any other ovarian cell type
Summary
SAS1B (sperm acrosomal SLLP1 binding protein, a.k.a ovastacin, astacin-like or ASTL, GenBank ID NM_001002036.3), is a cortical granule and oocyte surface-associated zinc matrix metallo-proteinase (MMP, EC 3.4.24.21) with reported roles in sperm-egg interaction [1, 2] and in the block to polyspermy [3] during eutherian fertilization. Beginning at its N-terminus this ~46 kDa metalloproteinase consists of a signal peptide, pro-peptide motif, proteinase domain containing an active hex-box [HEXXHXXGXXH) catalytic site motif, and a unique C terminus [2]. The pattern of SAS1B protein expression within the ovary is conserved in several mammalian groups including non-human primates, canines, felines, artiodactyls, and rodents where SAS1B protein is restricted to the pool of growing oocytes beginning within primary-secondary transition follicles and persisting through ovulation [2]. Interrogation of the NCBI human EST database did reveal a single EST in human uterus [5], but detailed examination of this GENBANK deposit revealed that the cDNA sequence was from an un-classified human uterine tumor Based on this information, the current study was undertaken to investigate the frequency of ASTL transcription and translation in an exploratory cohort of uterine tumors and to evaluate if SAS1B might offer opportunities as a target for a novel immunotherapeutic approach
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