The major progesterone-modulated proteins secreted into the sheep uterus are members of the serpin superfamily of serine protease inhibitors.
The uterine milk proteins (UTMP) are a pair of structurally related basic glycoproteins that when newly synthesized carry phosphorylated mannosyl residues on their carbohydrate chains. They are the major proteins secreted by ovine endometrium under the influence of progesterone. RNA from a late pregnant ewe endometrium was isolated for use in in vitro translation assays and for constructing cDNA libraries. Translation experiments, initially with total cellular RNA and subsequently with RNA selected by hybridization with a specific cDNA, demonstrated the production of two polypeptides (Mr = 47,000 and 55,000) that were precipitated with antiserum to the UTMP. With microsomal membranes in the translation assay, there was increased production of an Mr = 57,000 form that was protected from protease digestion. Antibody screening of a cDNA library in lambda gt11 identified a short clone representing the 3' terminus of the mRNA that was shown by epitope selection experiments to be UTMP specific. This clone was then used to screen a lambda gt10 library. A longer clone (1.3 kilobases) was isolated and sequenced but lacked the 5' terminus to the mRNA. The latter sequence was obtained directly from the mRNA. Interesting features of the UTMP mRNA sequence, which was 1,352 bases long and contained a 1,287-base open reading frame, were two strong start codons, two potential sites for N-glycosylation and a repeat of 21 bases, six bases apart, that resulted in a repeat of seven amino acids. The inferred amino acid sequence agreed closely with the NH2-terminal amino acid sequence obtained directly from the UTMP and clearly placed the UTMP in the serpin superfamily of protease inhibitors. However, we have been unable to demonstrate inhibitory activity toward any serine protease so far tested.
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Department of Biochemistry and Molecular Biology, University of Florida, Gainesville 32610.
Roberts
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RM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004717:Endometrium / Q000032:analysis; D005260:Female; D006023:Glycoproteins; D006031:Glycosylation; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011247:Pregnancy; D011480:Protease Inhibitors; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics; D011506:Proteins / Q000235:genetics*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D015843:Serpins; D012756:Sheep
D006023:Glycoproteins; D011480:Protease Inhibitors; D011498:Protein Precursors; D011506:Proteins; D012333:RNA, Messenger; D015843:Serpins; C029708:uterine luminal fluid proteins; D012313:RNA