Gene expression during estivation in spadefoot toads, Scaphiopus couchii: upregulation of riboflavin binding protein in liver.

A cDNA library constructed from liver of 2-month estivating female spadefoot toads, Scaphiopus couchii, was differentially screened to reveal genes that were induced or upregulated during estivation. After two rounds of screening a clone was isolated that showed 60% higher expression in liver of estivating, versus control, toads. The clone possessed a 1.0 kb insert which annealed to a single 0.7 kb band on Northern blots. Sequencing revealed a 1053 nucleotide full-length cDNA; the largest potential open reading frame was 708 nucleotides which encoded a protein of 235 amino acids. A homology search in Genbank indicated that the protein was a riboflavin binding protein (RfBP), a monomeric phosphoglycoprotein produced by the liver of female birds, reptiles, and mammals that functions to bind plasma riboflavin and load the vitamin into eggs or fetus. To our knowledge, this is the first demonstration that RfBP is also present in amphibians. Toad RfBP showed 50% of residues identical with the chicken or turtle liver proteins and many essential structural features were conserved in the toad protein including 18 cysteine residues, two asparagine glycosylation sites, and 6 tryptophan residues. However, a region with eight phosphoserines in the chicken or turtle proteins that functions in RfBP binding to the oocyte membrane contained only three serine residues in toad RfBP, suggesting that recognition and binding to oocyte receptors must be different in toads. Northern hybridization showed that toad RfBP was largely liver-specific; no mRNA transcripts were detected in brain, gut, heart, or kidney but low message levels occurred in hind leg skeletal muscle of estivating, but not control, toads. Upregulation of RfBP in liver of estivating toads may be linked with maturation of eggs in preparation for the explosive breeding that occurs immediately upon emergence from estivation but might also have a role for the adult in "caching" riboflavin to maintain an endogenous vitamin pool over the 9-10 months of each year that toads are dormant.