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 spadefoo t toads, Scaphiopus couchii, was differentially screened to reveal genes th at were induced or upregulated during estivation. After two rounds of scree ning a clone was isolated that showed 60% higher expression in liver of est ivating, versus central, toads. The clone possessed a 1.0 kb insert which a nnealed to a single 0.7 kb band on Northern blots. Sequencing revealed a 10 53 nucleotide full-length cDNA; the largest potential open reading frame wa s 708 nucleotides which encoded a protein of 235 amino acids. A homology se arch in Genbank indicated that the protein was a riboflavin binding protein (RfBP), a monomeric phosphoglycoprotein produced by the liver of female bi rds, reptiles, and mammals that functions to bind plasma riboflavin and loa d the vitamin into eggs or fetus. To our knowledge, this is the first demon stration that RfBP is also present in amphibians. Toad RfBP showed 50% of r esidues identical with the chicken or turtle liver proteins and many essent ial structural features were conserved in the toad protein including 18 cys teine residues, two asparagine glycosylation sites, and 6 tr;tryptophan res idues. However, a region with eight phosphoserines in the chicken or turtle proteins that functions in RfBP binding to the oocyte membrane contained o nly three serine residues in toad RfBP, suggesting that recognition and bin ding 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 i n hind leg skeletal muscle of estivating, but not control, toads. Upregulat ion of RfBP in liver of estivating toads may be linked with maturation of e ggs in preparation for the explosive breeding that occurs immediately upon emergence from estivation but might also have a role for the adult in "cach ing" riboflavin to maintain an endogenous vitamin pool over the 9-10 months of each year that toads are dormant. (C) 1999 Wiley-Liss,Inc.