Secreted ferritin subunits are of two kinds in insects - Molecular cloningof cDNAs encoding two major subunits of secreted ferritin from Calpodes ethlius

In insects, holoferritin is easily visible in the vacuolar system of tissue s that filter the hemolymph and, at least in Lepidoptera, is abundant in th e hemolymph. Sequences reported for insect secreted ferritins from Lepidopt era and Diptera have high sequence diversity. We examined the nature of thi s diversity for the first time by analyzing sequences of cDNAs encoding two ferritin subunits from one species, Calpodes ethlius (Lepidoptera, Hesperi idae). We found that insect secreted ferritin subunits are of two types wit h little resemblance to each other. Ferritin was isolated from iron loaded hemolymph of C. ethlius fifth instar larvae by differential centrifugation. The N-terminal amino acid sequences for the nonglycosylated subunit with M r 24,000 (S) and the largest glycosylated subunit with Mr 31,000 (G) were d etermined. The N-termini of the two subunits were different and were used t o construct degenerate PCR primers. The same cDNA products were amplified f rom cDNA libraries from the midgut which secretes holoferritin and from the fat body which secretes iron-poor apoferritin. The G subunit most closely resembles the glycosylated ferritin subunit from Manduca sexta and the S su bunit resembles the Drosophila small subunit. The S and G subunits from Cal podes were dissimilar and distinct from the cytosolic ferritins of vertebra tes and invertebrates. Additional sequences were obtained by 5' and 3' RACE from separate fat body and midgut RACE libraries. cDNAs encoding both subu nits had a consensus iron responsive element (IRE) in a conserved cap-dista l location of their 5' UTR. An integrin-binding RGD motif found in the G su bunit and conserved in Manduca may facilitate iron uptake through a calreti culin (mobilferrin)/integrin pathway. Calpodes and other insect ferritins h ave conserved cysteine residues to which fatty acids can be linked. Dynamic acylation of ferritin may slow but not prevent its passage out of the ER. (C) 1999 Published by Elsevier Science Ltd. All rights reserved.