Date of Award
2008
Degree Type
Thesis
Degree Name
Master of Science
Program
Physiology and Pharmacology
Supervisor
Dr. Graham Wagner
Second Advisor
Dr. Douglas Jones
Third Advisor
Dr. David Freeman
Abstract
Stanniocalcin-I (STC-1) is a 50 kDa homodimeric glycoprotein hormone which was originally identified in an endocrine gland in fish called the corpuscles of Stannius. Fish STC-1 consists of two identical 25 kDa subunits, with 256 amino acids and a carbohydrate moiety of 5 kDa. It regulates serum calcium. Alternatively, more highly glycosylated forms of fish STC-1 are expressed in other tissues such as ovary, where they are believed to function locally. Based on DNA and recombinant STC-1, the mammalian STC-1 is structurally similar to fish STC- 1 with 6-10 kDa carbohydrate moiety and STC-1 gene is also widely expressed like the fish hormone. However, in mammals the ovaries have the highest levels of STC-1 gene expression where the 50 kDa dimer form has never been reported. The ovarian STC-1 is not a regulator of serum calcium and does not normally circulate in the blood. Instead, it has been targeted to lipid storage droplet cells and implicated in physiological process such as neural differentiation, reproduction and cancer. The ovaries are the only tissue that releases STC-1 into the bloodstream during pregnancy and lactation. These data suggest that the ovarian STC-1 might be perhaps structurally unique. At present, little is known about the structure of native STC-1 in mammals. In this study, we provide the first characterization of native STC-1 from bovine ovary. We hypothesized that the ovarian hormone was structurally unique. Ovarian tissue was extracted and STC-1 was partially purified by immunoaffinity chromatography and HPLC. Western blotting was conducted to observe STC-1 immunoreactive bands (STCir), and electroelution was used to confirm the origin of the STCir observed in western blotting. An in situ ligandbinding assay was used to determine the bioactivity of STC-1, and the carbohydrate moiety was sized by PNGase digestion. The western blot of immunoaffinity bound STC-1 revealed a single band of ~ 23 kDa under reducing conditions in ovary and even in kidney. Interestingly, a similar size band was evident in nonreducing conditions. Electroelution confirmed πi that the 23 kDa band under reducing condition was derived from the similar sized band under nonreducing condition, it was also revealed that there are no dimer or polymer forms and monomer was the only isoform of ovarian STC-1. In situ ligand binding revealed that the immunoaffinity bound fraction contained bioactive STC-1. PNGase also revealed a carbohydrate moiety of 6-10 kDa of native ovarian STC-1. The ovarian STC-1 was not adequately purified by HPLC to be identified by mass spectrometry. The findings suggest that native ovarian STC-1 is approximately the same size as recombinant STC-1, but unlike recombinant STC-1, it exists as a monomer. The presence of this monomer in bovine kidney and other species further suggest that this isoform might be common in mammals. Further investigation is needed to identify the precise structure of this monomeric form of the hormone.
Recommended Citation
Talebian, Asghar, "Purification of Stanniocalcin-I from bovine ovary" (2008). Digitized Theses. 4747.
https://ir.lib.uwo.ca/digitizedtheses/4747