Isolation, characterization, cDNA cloning and deduced amino acid sequence of transferrin from the tobacco hornworm, Manduca sexta.

Abstract

An iron-binding 77 kilodalton glycoprotein was isolated from hemolymph of the adult sphinx moth, Manduca sexta. This protein bound a single ferric ion both in vivo and in vitro and had a secondary structure similar to that of human serum transferrin and human lactoferrin, as judged by CD spectra. Antiserum generated against this protein was used to screen a fifth instar, day four, larval fat body cDNA library. A 2.0 kilobase clone was isolated and used to probe a northern blot of both total and poly(A)⁺ RNA from fat body, revealing a message of 2.3 kilobases. The message is expressed throughout the fourth instar, fifth instar, wandering, pupal and adult stages. The 2.0 kilobase clone selected an mRNA which, when translated in vitro, produced an immunoprecipitable 77 kDa protein. The 2.0 kb clone was used as a probe to further screen the cDNA library, resulting in the isolation of three full-length clones. The complete nucleotide sequence of one 2183 base pair cDNA insert was determined. The deduced amino acid sequence contained an 18 amino acid signal sequence which, when cleaved, resulted in a mature protein sequence of 663 amino acids with a calculated molecular weight of 73,436. The first 34 residues of the mature protein were identical to those determined by Edman degradation of the intact protein. The sequence contained four consensus N-linked glycosylation sites (Asn-X-Thr/Ser). The sequence was used to search the National Biomedical Research Foundation protein database. The proteins exhibiting the greatest similarity were human serum transferrin, chicken ovotransferrin, human lactoferrin and human melanotransferrin. When the five sequences were aligned using a multiple alignment program, the insect protein contained approximately 27% identical residues when compared to each of the other transferrins. The greatest areas of similarity were around the iron binding sites. Moreover, 23 of the 24 cysteine residues in the insect protein occupied identical positions as compared to the other transferrins, indicating a similar overall tertiary structure. The insect protein also exhibited some internal homology between the N-terminal and C-terminal halves of the molecule. Ligands capable of binding an iron atom were present in the N-terminal half, but most were lacking in the C-terminal half. Based upon sequence comparisons and other structural and functional data, we believe that we have isolated and sequenced an invertebrate transferrin, the first such molecule to have its entire sequence determined.