Bartfeld N S, Law J H
Department of Biochemistry, University of Arizona, Tucson 85721.
J Biol Chem. 1990 Dec 15;265(35):21684-91.
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.
已从成年烟草天蛾（Manduca sexta）的血淋巴中分离出一种分子量为77,000的铁结合糖蛋白。由于该蛋白在体内和体外均能结合铁离子，并且通过圆二色光谱（CD光谱）判断其二级结构与人类血清转铁蛋白和人乳铁蛋白相似，我们决定克隆其cDNA，以确定它与脊椎动物转铁蛋白的关系。针对该蛋白产生的抗血清用于筛选幼虫脂肪体cDNA文库。分离出一个2.0千碱基的克隆，该克隆选择的mRNA在体外翻译时能产生一种可免疫沉淀的77 kDa蛋白。当用该2.0千碱基的克隆作为探针重新筛选文库时，分离出了三个全长克隆，并确定了一个2,183碱基对插入片段的完整核苷酸序列。推导的蛋白质序列包含一个18个氨基酸的信号序列和一个由663个氨基酸组成的成熟蛋白质序列，计算分子量为73,436。该序列用于搜索国家生物医学研究基金会（NBRF）蛋白质数据库，结果显示与脊椎动物转铁蛋白有显著相似性，脊椎动物转铁蛋白是一类80 kDa的糖蛋白，在血液和其他体液中运输和螯合铁。多重序列比对显示，相似性最大的区域位于两个铁结合位点周围，不过昆虫蛋白似乎只含有一个这样的功能位点。此外，与其他转铁蛋白相比，昆虫蛋白的24个半胱氨酸残基中有23个占据相同位置，表明其整体三级结构相似。对昆虫序列的两半进行比较表明，该蛋白可能是基因复制的结果。烟草天蛾序列与脊椎动物转铁蛋白的相似性可能为转铁蛋白的进化提供重要线索。
