Isolation of Complementary Deoxyribonucleic Acids Encoding Putative Secreted and Membrane-Bound Folate Binding Proteins from Endometrium of Swine1

Abstract

Two distinct forms of endometrial folate binding protein (FBP) cDNAs were isolated using reverse transcription-polymerase chain reaction and 3' and 5' rapid amplification of cDNA ends (RACE) procedures. On the basis of the absence or presence of an intact glycophosphatidylinositol linkage site in the C terminus of the predicted amino acid sequences, the two forms appear to encode secreted and membrane-bound forms of FBP. The cDNAs for the putative secreted and membrane forms encoded 252- and 249-amino acid proteins, respectively, that were 73% identical with each other and were 66-82% identical with other known FBPs. However, the nucleotide sequences within the 5' untranslated region and from codons 224 and 223 of the secreted and membrane forms, respectively, to the 3' ends of each RNA, were divergent. The divergence in the 3' ends of the two cDNAs was exploited to determine changes in concentrations of each mRNA in the endometrium during the estrous cycle and early pregnancy. Northern blots of endometrial total RNA probed with a putative secreted FBP specific probe indicated that mRNA concentrations do not change during early pregnancy. In contrast, blots probed with a putative membrane FBP specific probe indicated that mRNA concentrations increase dramatically from Day 15 to Day 24 of pregnancy. Finally, N-terminal amino acid sequencing of FBP purified from Day 15 pregnant uterine flushings matched the secreted form of FBP mRNA. These data are consistent with a role for putative secreted and membrane-bound forms of FBPs in the transport of folate to the developing swine conceptus during early pregnancy.