Identification and characterization of an estrogen-inducible glycoprotein (uterine secretory protein-1) synthesized and secreted by rat uterine epithelial cells.
An estrogen-inducible 97-kilodalton (kDa) secretory glycoprotein, designated as uterine secretory protein (USP)-1, synthesized by rat uterine epithelial cells was identified and characterized. Uterine luminal fluid (ULF) proteins were labeled by direct administration of [35S]methionine into uterine lumen of rats. Incorporation of [35S]methionine into ULF proteins was negligible in ovariectomized rats. However, when 17 beta-estradiol (E2) was administered in the ovariectomized rat as sc paraffin pellets, a marked increase of [35S]methionine incorporation was noted after 2 days of treatment, showing that the de novo synthesis and secretion of ULF proteins were induced by E2. Six estrogen-inducible polypeptides (130, 110, 97, 65, 42, and 39 kDa) were identified in the analysis of the labeled ULF proteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography. Four of these polypeptides (110, 65, 42, and 39 kDa) were adsorbed by the immobilized antibody against rat serum proteins, indicating that these polypeptides are antigenically similar to serum proteins. Two polypeptides (130 and 97 kDa) not adsorbed by the antibody column were suggested to be uterine-specific secretory proteins. The 97-kDa protein (USP-1) was further purified by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis and electroelution. Reactivity of purified USP-1 to various lectins and carbohydrate composition analysis suggested that USP-1 possesses biantennary N-linked complex-type carbohydrate chain with fucose. Rabbit polyclonal antibody which can specifically immunoprecipitate [35S]methionine-labeled USP-1 was developed. Dot blot enzyme immunoassay showed that 4-day E2-treated ULF contains 78.3 +/- 24.1 (+/- SE) micrograms USP-1/mg protein. Immunohistochemical staining of rat uterine tissue showed that this protein localized only in the epithelial cells treated with estrogen (E2 and diethylstilbestrol). Testosterone, progesterone, and dexamethasone failed to induce synthesis and secretion of USP-1 as assessed by dot blot enzyme immunoassay, immunoprecipitation of [35S]methionine-labeled ULF proteins, and immunohistochemical staining of uterine tissue. The present result, for the first time, revealed that estrogen can induce synthesis and secretion of specific secretory protein which could be the useful marker to analyze the molecular mechanism of estrogen action in rat uterine epithelial cells.
Takeda
A
A
Aichi Cancer Center Research Institute, Nagoya, Japan.
Takahashi
N
N
Shimizu
S
S
D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D002241:Carbohydrates / Q000032:analysis; D004848:Epithelium / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D006023:Glycoproteins / Q000096:biosynthesis*; D007124:Immunoenzyme Techniques; D008715:Methionine / Q000378:metabolism; D010052:Ovariectomy; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D015843:Serpins; D014599:Uterus / Q000187:drug effects / Q000378:metabolism*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D002241:Carbohydrates; D006023:Glycoproteins; D011506:Proteins; D015843:Serpins; C029708:uterine luminal fluid proteins; D004958:Estradiol; D008715:Methionine