Abstract
Trophoblasts of ruminant species secrete multiple forms of a novel Type I interferon (IFN-tau) for a few days preceding implantation. These IFN-tau are structurally distinct from IFN-alpha, -beta, and -omega, but it remains unclear whether they have any distinguishing biological properties. It has been of interest, therefore, to study their interaction with Type I IFN receptors. However, a recombinant bovine IFN-tau (boIFN-tau 1) prepared for such use is rapidly inactivated during chemical iodination with 125I, presumably because a critical tyrosine becomes modified. Here we describe the synthesis of a novel IFN-tau 1 in which Leu169 and Leu171 in the carboxyl terminus have been replaced by 2 tyrosine residues. The recombinant product (rboIFN-tau 1Y2) can be readily iodinated without significant loss of antiviral and antiproliferative activities, provided that reaction time with 125I is minimized. Both rboIFN-tau 1Y2 and rboIFN-alpha 1 compete with each other for binding to bovine endometrial cell membranes and Madin-Darby bovine kidney cells. Dissociation constants of both IFN are quite similar (Kd = 3.7 x 10(-10) and 3.5 x 10(-10) M, respectively). Cross-linking studies reveal a single size class of receptor polypeptide on endometrium and Madin-Darby canine kidney cells whose molecular weight is reduced from approximately 130,000 to approximately 70,000 by treatment with N-glycosidase. These studies with a single recombinant form of boIFN-tau avoid the difficulties that arise from use of a mixture of naturally occurring isoforms. They provide further evidence for the presence of a receptor common to both IFN-tau and IFN-alpha in bovine tissues and no indication for a unique IFN-tau-binding polypeptide in endometrium.
Highlights
Trophoblasts of ruminant species secrete multiple IFN-T appears to reduce the pulsatile reloefasperostaglandin forms of a novel TypeI interferon (IFN-T)for a few days F2, a luteolytic hormone that in a nonpregnant animal causes preceding implantation
Second,IFN-T genexpression is poorly induced by virus and is trophoblast-specific [12,13,14]
Evidence is accumulating that the IFN-T may be considerably more potent in nated without significant loss of antiviral and antipro- their ability to extend inter-estrous intervals than IFN-a in liferative activities,provided that reaction time with 1' nonpregnant ewes
Summary
Muterials-Chemicals of special note were purchased from the following companies: NaIZK(I14.9 mCi/pg) from Amersham Corp.; IODOGEN (1,3,4,6-tetrachloro-3a,6a-diphenylglycourfirlo~m Pierce Chemical Cod.;isuccinimidyl suberate (DSS) from ICNBiomedicals, Inc.(Irvine, CA); Protein A-Sepharose, phenylmethylsulfonyl fluoride, deoxycholic acid, and Nonidet P-40 from Sigma; N-glycosidaseF from Boehringer Mannheim, and nucleic acid modifymg enzymes from Promega Biotech, Madison,WI (except where stated otherwise).Recombinant bovine interferon a1 (rboIFN-al) (2.5 mg/ml) and a rabbit anti-. Competitive Binding of 1251-rboIFN-rlY2to the Receptor onMDBK CellLine--Two ng of 1261-rboIFN-~1Yw2ere incubated with 7.5 x 10' cells (added last) in absence (total binding) or in presence of different amounts of unlabeled IFN from 0.1 ng to1pg (nonspecificbinding) in 500 pl of Dulbecco's modifiedEagle's mediumcontaining 5% fetal bovine serum at 4 "C for 12-14 h. Binding of lZ5I-IFNto Receptor onEndometrial Membranes-For the IFN competitive binding assay on endometrial membranes, 4 ng of 12'I-IFN (rboIFN-a1 or rboIFN-rlY2) were incubated with 250pg of endometrial membrane protein (added last) inthe absence (total binding) or in the presence of different amounts of unlabeled IFN from 0.1 ng to 1pg (nonspecificbinding) in 500 pl of an assay buffer (25 nm Tris-HC1 (pH 7.4), 1 nm MgCl,, 1 nm CaCl,, and 0.1% bovine serum albumin).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
