Uterine Estrogen Response At Ulf-250 Protein Identification

In previous study,we characterized an estrogen-responsive protein with an estimated molecular weight of 250kD from ULF obtained from estradiol-treated ovariectomized rats. The immunohistochemical analysis was used to determine the 250 kDa in uteri during the estrous cycle.Positive immunostaining of the 250 kDa protein occurred in the epithelial cells of the uterus at all phases of the estrous cycle.The 250 kDa protein is produced by the glandular and luminal epithelial cells of the uterine endometrium and fluctuates with the phases of the estrous cycle.The preliminary study showed that anti-ULF-250 antibody reacted with some unknown protein in duct carcinoma and lobular carcinoma of mammary glands,but did not react with the normal tissues around the diseased tissues.Objectives of the present study are:to characterize the ULF-250 further,and to investigate the correlation between the ULF-250 and breast cancer.Methods:1,The ULF was collected from the estradiol-treated ovariectomized rats,and protein components of the ULF were separated by SDS-PAGE and two-dimensional electrophoresis (2-DE).After separation,the protein bands or spots were recognised by polyclonal antiserum against ULF-250 using Western blotting and 2D-Western.The targeted protein was analyzed by Mass Spectrometry and compared with database.The 2D-Western analysis showed that anti-ULF-250 antibody specifically recognised the protein spots at the molecular weight of 250 kD.The spots were excised from Coomassie blue-stained gel,and in-gel digestion with trypsin was performed.Tryptic peptides were analyzed by MALDI-TOF-MS.The Peptide Mass Fingerprinting(PMF) and sequences obtained were matched to SWISSPROT protein database using the Mascot search algorithm (www.matrixscience.com).2,Relationship between the ULF-250 and breast cancer.The protein samples of breast cancer were separated by SDS-PAGE and two-dimensional electrophoresis(2-DE).After separation,the protein bands or spots were recognized by polyclonal antiserum against ULF-250 using Western blotting and 2D-Western.The targeted protein was analyzed by Mass Spectrometry and compared with database.The spots were excised from Coomassie blue-stained gel,and in-gel digestion with trypsin was performed. Tryptic peptides were analyzed by MALDI-TOF-MS.The Peptide Mass Fingerprinting (PMF) and sequences obtained were matched to SWISSPROT protein database using the Mascot search algorithm(www.matrixscience.com).Results:1,The result indicates that the ULF-250 is ebnerin/DMBT1.Additionally,the 250 kD protein band excised from SDS-PAGE gel was subjected to the HPLC-ESI-MS/MS analysis.2,Several protein bands at about 160kD and 55kD were specifically recognized by rabbit anti-ULF-250 polyclonal antibody from breast cancer tissue extract by using Western blot analysis.Furthermore,the result showed that quantity,quality and Molecular Weight of the recognized protein bands varied from patient to patient.3,2DE-Western blot and Peptide mass fingerprinting combined with Internet searching of protein database revealed that three protein spots are identical to fibrinogen gamma,transferrin,fibrin beta.Conclusions:1,The comparison of two proteins indicates that both ULF-250 and ebnerin/DMBT1 are identical on localization and regulation.Therefore,we conclude that the ULF-250 is ebnerin/DMBT1 expressed in the uterus.The unknown estrogen-responsive protein ULF-250 is characterized and validated as ebnerin/DMBT1 by using 2-DE combined with MS as well as data searching.In addition,our results suggest that ebnerin/DMBT1 is not only expressed in epithelium of uterus but also secreted into the luminal fluid.The function of the protein needs further investigation.2,The cross reactions of the anti-ULF-250 antibody with human breast cancer were examined by Western blot,and the common antigen varied form patient to patient.For this reason,we could not investigate the correlation between ULF-250 and breast cancer.3,We must enlarge the breast cancer samples to determine whether ULF-250 can be a tumor biomarker.