Hemoglobin Switching in Sheep and Goats

Abstract

Abstract Initiation of sheep globin synthesis was studied to determine if a unique initiation mechanism might be a control point for hemoglobin A to C switching. Polysomes from the reticulocytes of anemic adult sheep direct cell-free synthesis of sheep α-, βb-, and/or βc-globin chains. The first two amino acids on the NH2 terminus of these three globins are valine-leucine, methionine-leucine, and proline-asparagine, respectively. The initiator tRNA, methionyl-tRNAf, donates [35S]methionine to the βb chain but not to the α or βc. When methionine on the initiator tRNA is formylated, formyl[35S]methionine is donated to all three globins. α-, βb-, and βc-Globins with an NH2-terminal formylmethionine were isolated from the cell-free reaction by carboxymethylcellulose chromatography and subjected to deformylation and proteolytic digestion. [35S]Methionine on formylmethionine α-globin is released as free methionine, methionine-valine, or methionine-valine-leucine (as determined by electrophoresis and chromatography). Similarly, the [35S]methionine on formylmethionine βb-globin is released as methionine-leucine. Conditions were not found which would cleave a dipeptide from the formylmethionine βc chain, so the globin was labeled with [3H]proline, [3H]asparagine, or [3H]lysine in addition to [35S]methionine and subjected to automated Edman degradation. The data show that the initial sequence of formylmethionine βc is methionine-proline-asparaginelysine. We conclude that methionyl-tRNAf initiates all three globin chains but that this methionine is subsequently cleaved from the α and βc chains. We infer that βa globin is initiated in a manner similar to βb because their NH2-terminal sequences are identical. Thus, the mechanism of sheep globin initiation is similar to that of rabbit and human globin initiation; this conclusion suggests a pretranslational control for hemoglobin switching in the sheep.