Isolation and molecular cloning of transferrin from the tobacco hornworm, Manduca sexta. Sequence similarity to the vertebrate transferrins.
An iron-binding glycoprotein of Mr = 77,000 has been isolated from hemolymph of the adult sphinx moth Manduca sexta. Since this protein binds ferric ion both in vivo and in vitro and has a secondary structure similar to that of human serum transferrin and human lactoferrin as judged by CD spectra, we decided to clone its cDNA in order to determine its relationship to the vertebrate transferrins. Antiserum generated against this protein was used to screen a larval fat body cDNA library. A 2.0 kilobase clone was isolated that selects an mRNA which, when translated in vitro, produces an immunoprecipitable 77-kDa protein. When the library was rescreened using the 2.0-kilobase clone as a probe, three full-length clones were isolated, and the complete nucleotide sequence of one 2,183-base pair insert was determined. The deduced protein sequence contains an 18-amino acid signal sequence and a mature protein sequence of 663 amino acids with a calculated Mr of 73,436. The sequence was used to search the National Biomedical Research Foundation (NBRF) protein database, revealing significant similarity to the vertebrate transferrins, a family of 80-kDa glycoproteins which transport and sequester iron in the blood and other body fluids. A multiple sequence alignament shows the greatest areas of similarity to be around the two iron binding sites, although the insect protein seems to contain only one such functional site. Moreover, 23 of the 24 cysteine residues in the insect protein occupy identical positions as compared with the other transferrins, indicating a similar overall tertiary structure. Comparison of the two halves of the insect sequence indicates that the protein may have arisen as a result of gene duplication. The similarity of the M. sexta sequence to the vertebrate transferrins may provide important clues to transferrin evolution.
Bartfeld
N S
NS
Department of Biochemistry, University of Arizona, Tucson 85721.
Law
J H
JH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D007501:Iron / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths / Q000235:genetics*; D015183:Restriction Mapping; D014168:Transferrin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D000596:Amino Acids; D014168:Transferrin; D004247:DNA; D007501:Iron