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.

Secreted and Placental Membrane Forms of Folate-Binding Protein Occur Sequentially During Pregnancy in Swine1

“…While folate binding in the uterine flushings increased on Days 13 and 15 of the estrous cycle and pregnancy in this study and in previous studies [14,15], endometrial expression of sFBP mRNA did not change between Days 10 and 15 [16]. Increased intrauterine content of sFBP was hypothesized to be due either to the release of stored sFBP from the endometrium or increased translation of sFBP mRNA [16].…”

“…Finally, two pig membrane-bound folate receptors are known [16,27]. One was cloned from pig endometrium [16] and the other from pig liver [27]. It is possible that the binding sites reported here are the result of expression of more than one folate receptor gene and that the relationship between the expression of the different folate receptor genes changes during late pregnancy.…”

“…Alternatively, changes in the plasma membrane lipid environment could also affect the affinity of the folate receptor. Finally, two pig membrane-bound folate receptors are known [16,27]. One was cloned from pig endometrium [16] and the other from pig liver [27].…”

“…However, the secretion of FBP beyond Day 15 of pregnancy has not been investigated. In addition to secreted FBP (sFBP), a cDNA and gene corresponding to a placental membrane form of FBP (mFBP) have been cloned and sequenced [16,17]. Both forms of FBP are expressed in the intrauterine environment during pregnancy in swine [16] and thus may be important for folate transport to the conceptus during pregnancy.…”

“…In addition to secreted FBP (sFBP), a cDNA and gene corresponding to a placental membrane form of FBP (mFBP) have been cloned and sequenced [16,17]. Both forms of FBP are expressed in the intrauterine environment during pregnancy in swine [16] and thus may be important for folate transport to the conceptus during pregnancy. The objectives of this study were to 1) compare specific folate binding during the estrous cycle and early pregnancy in uterine flushings and in allantoic fluid and by placental microsomal membranes during later pregnancy and 2) determine the localization of sFBP in the intrauterine environment throughout pregnancy.…”