Abstract
The amino acid sequence of an insect apolipoprotein, apolipophorin-III from Locusta migratoria, has been deduced from the sequence of its cloned cDNA. The mature hemolymph protein consists of 161 amino acids. Optimized alignments of this protein with apolipophorin-III from the tobacco hornworm, Manduca sexta, disclosed an overall sequence identity of only 29%, even though the two proteins are functionally equivalent. The L. migratoria sequence is composed of 12 repeating peptides that are variable in length. Six amphipathic helical segments of varying length were identified in each protein using a newly described algorithm for detecting such secondary structures. The degree of sequence identity between the two insect apoproteins is considerably less than that observed among orthologous mammalian apolipoproteins. However, calculation of the rates of synonymous and nonsynonymous nucleotide substitutions indicates that the insect genes may be evolving at rates similar to the mammalian apolipoprotein genes. Further comparative analyses of insect and mammalian apolipoproteins should provide insights about the limits of sequence diversity tolerated by their predicted amphipathic helical domains.
Highlights
From the $Department of Biochemistry, University of Arizona, Tucson, Arizona 85721, the §Department of Biology, Queen’s University, Kingston, Ontario K7L 3N6, Canada, and the qDepartment of Pathology and IlDepartment of Biological Chemistry, Washington University School ofMedicine, St
The in a larger, less dense lipoprotein particle which contains a maturehemolymphproteinconsists of 161 amino third apoprotein, apolipophorin-I11(apoLp-111, M, 20,000)
Six (Shapiro and Law,1983;Van der Horst et al, 1984; Goldsworthy et al, 1985; Chino et al, 1986).apoLp-I11exists free in the hemolymph in resting animals and associates with lipophorin during lipid loading until each particle contains16 molecules ofapoLp-111 in M. sexta (Wells et al, 1987) and amphipathic helical segments of varying length were nine molecules (Chino and Yazawa, 1986) or14 molecules identified in each protein using a newly describeadl- (Van der Horst et al, 1988) of apoLp-I11in L. rnigratoria
Summary
Animals-African migratory locusts ( hcusta migratoria migratorioides) were reared a t Queen’s University as previously described (Chen et al, 1978). Fractions which contained apoLp-111 (detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were combined, dialyzed against 0.1 M acetic acid, and lyophilized, yielding 40 mg of protein. Part of this preparation (3.5 mg)was further purified by reversed phase high performance liquid chromatogtaphy (pBondapak C-18, Waters, 25 X 0.5 cm). ApoLp-I11 prepared by this method contained a single band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Kanost et al, 1987).This preparation of apoLp-I11was used as an antigen for production of antibodies in rabbits and for analysis of amino acid composition and NHz-terminal sequence. Computer-assisted Analysis of Sequencing Data-All calculations were carried out on a MicroVAX I1 (Digital Equipment Corp.) running the VMS operating system (version 4.4).Program AMPHI for the prediction of amphipathic helical segments was adhpted from the published FORTRAN source code (see Appendix to Margalit et al, 1986).Program LWL85 for estimating synonymous and nonsynonymous rates of nucleotide substitution(Li et al, 1985) was kindly provided by Dr Wen-Hsiung Li (University of Texas Health Science Center at Houston)
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