Hexameric Storage Protein Research Articles

A role for storage proteins in autogenous reproduction in Aedes atropalpus

In the autogenous mosquito, Aedes atropalpus, storage proteins accumulated during the larval stage may serve as an amino acid reserve for oogenesis, in addition to metamorphosis. Hexameric storage proteins accumulate during larval development and include subunits of three different masses: 62.5, 66, 72.5 kDa. All three types of subunits are found in the female but only the larger two are in males. In females, storage proteins are only partially depleted by the time of eclosion. The remaining protein amounts to about 40% of the original store. Males, in contrast, exhaust their supply of stored protein during metamorphosis. In the female, the storage proteins disappear over the first days after eclosion, and are depleted before vitellogenin/vitellin levels reach their maximum. This suggests that the amino acids held in storage proteins are transferred to vitellogenesis, enabling autogenous egg development. The fact that these amino acids are not available for egg development until after eclosion, later than in many other insects, probably reflects a relatively recent evolution from blood-feeding ancestors.

Purification and characterization of two storage proteins from Locusta migratoria showing distinct developmental and hormonal regulation

Two major hemolymph proteins, belonging to the insect hexameric storage protein family, have been purified from Locusta migratoria. Larval storage protein 1 (LSP1) has M r = 410,000, s = 16.1 S, 10% hexose content and is composed of up to five different 75 kDa subunits. Isoelectric focusing of native LSP1 shows a pI of about pH 6.0 with some microheterogeneity. Persistent storage protein (PSP) has a M r = 460,000, s = 16.4 S, 7% hexose and contains major 74 kDa and minor 77 kDa subunits. Native PSP has a pI of pH 5.6 also with some microheterogeneity. PSP dissociates reversibly into monomers at alkaline pH. The N-terminal sequence was determined for the PSP 74 kDa subunit. A previously identified cyanoprotein (CP), with M r = 435,000 and subunits of 69 kDa, was also purified. A second larval-specific storage protein, LSP2, has been identified but not purified. LSP1 and PSP exhibit distinct developmental patterns and hormonal regulation. LSP1 is larval-specific, increasing to a high concentration in the late fifth instar and disappearing in the early adult. PSP, however, remains abundant through the last larval instar and persists at a lower concentration in the adult. In fifth instar larvae, treatment with the juvenile hormone (JH) analog, pyriproxyfen, totally repressed production of LSP1, and significantly lowered the hemolymph level of PSP. In contrast, JH analog treatment is found to elevate the level of PSP in adults. Storage proteins which persist in the adult stage, under hormonal regulation that is switched during metamorphosis, may be characteristic of some hemimetabolous insects.

Hexameric haemolymph protein related to adult diapause in the red firebug, Pyrrhocoris apterus (L.) (Heteroptera)

A hexameric storage protein was electrophoretically isolated from the haemolymph of diapausing adults of the red firebug, Pyrrhocoris apterus. This protein ( M rmr ~ 480,000, glycoprotein) was found to be a monohexamer. During the penultimate and last larval instar (both in diapausing and non-diapausing bugs), its concentration increased from about 1 mg/ml up to 8 and 13 mg/ml, respectively, and declined to almost zero value during ecdysis. In diapausing adults, the titre of the protein rose steeply from day 3 and reached a level of about 21 mg/ml in females and 24 mg/ml in males. In non-diapausing adult females there was an initial rise in the protein titre during the first 6 days but subsequently it stabilized to a level of about 3 mg/ml. In males the hexameric protein content reached only 1 mg/ml after imaginal ecdysis; an increase on day 21 and 30 was observed. In 14-day-old diapausing and non-diapausing bugs the hexameric protein constitutes about 15 and 4% of all haemolymph proteins, respectively. An amino acid analysis showed that the hexameric protein is neither an arylphorin nor a methionine-rich protein.

Binding of riboflavin to lipophorin and a hexameric protein in the hemolymph of Heliothis virescens

The hemolymph of late final instar Heliothis virescens larvae was analyzed for riboflavin and its metabolic derivatives. Riboflavin levels in the hemolymph were found to be relatively high at this developmental stage ( ca 25 μg/ml hemolymph) and all of the flavin was associated with high molecular weight proteins. Based on the recovery of riboflavin with purified hemolymph protein preparations, approx. 15% of the total was bound to lipophorin and the remainder to a hexameric storage protein comprised of 82 kDa subunits, designated p82. The recovery of p82-bound riboflavin was not quantitative, a result attributable to the effects of sample dilution during purification. The p82 hexamer (but not lipophorin, arylphorin or other hemolymph proteins) was bound by a flavinyl-agarose affinity matrix, which provided independent confirmation of its identity as a flavin-binding protein. In addition to the large hemolymph pool of protein-bound riboflavin, H. virescens larvae also contained substantial amounts of free riboflavin in malpighian tubules ( ca 19μg/g tissue) and in testes ( ca 25 μg/g tissue). These findings suggest a complex pattern of riboflavin binding and transport mediated by specific pathways of flavin metabolism, storage and excretion depending upon the target tissue.

Storage proteins of the larval root weevil Diaprepes abbreviatus (Coleoptera: Curculionidae): riboflavin binding and subunit isolation.

Proteins present at high concentrations in hemolymph of the larval weevil Diaprepes abbreviatus were previously shown to bind a synthetic coumarin, 7-amino-3-phenyl coumarin (coumarin-10). One of the two native proteins previously identified (protein I) is now shown to separate into two distinct bands (proteins Ia and Ib) using native gradient pore-limiting electrophoresis. The high concentration of proteins Ia, Ib, and II in larval hemolymph, their disappearance from hemolymph upon pupation, and an apparent hexameric structure shown by chemical crosslinking identify them as hexameric storage proteins (hexamerins). At least one chromatographic form of Ib isolated by anion exchange HPLC is now shown to bind riboflavin (Rb). Binding was also demonstrated by quenching of Rb fluorescence by a partially isolated mixture of the storage proteins. Lipophorin did not quench Rb fluorescence. Rb was heat-extracted from whole hemolymph and identified by its fluorescence spectra and by reverse phase HPLC with fluorescence detection. The two subunits shared by the three holoproteins have been isolated by sequential density gradient ultracentrifugation, gel permeation HPLC, and reverse phase HPLC. All three holoproteins shared the alpha subunit (M(r) 75,000), while the beta subunit (M(r) 71,000) was lacking from one of the three. Repeated passage through an anion exchange column yielded two of the three proteins (Ib and II) in homogeneous form. Chemical crosslinking with dimethylsuberimidate indicated a hexameric structure for the holoproteins. All subunits and holoproteins stained as high mannose glycoproteins when probed with biotinylated concanavalin A on PVDF membranes. The alpha subunit was high in Met, His, and Thr, and the beta subunit was high in Lys. Both were high in Pro and had approximately 16% Phe+Tyr. Sequences of the 20 N-terminal amino acid residues of each subunit showed 45-60% homology between subunits. These coleopteran proteins also showed some sequential homology but no immunological cross-reactivity with storage proteins from the lepidopterans Galleria mellonella and Heliothis virescens.

Purification and properties of a storage protein from the hemolymph of Rhodnius prolixus

A non-arylphorin hexameric storage protein (SPR) has been isolated from the larval hemolymph of the bug, Rhodnius prolixus. The purified protein contained 0.69% lipid and 1.04% carbohydrate. SPR had a mol. wt of 470,000, with a subunit mol. wt of 78,000. In electron micrographs, SPR molecules appeared very similar to calliphorin. After the metamorphic molt, SPR persisted at lower concentrations in the hemolymph of adult insects, and it disappeared only in fasting bugs.