Structural Studies on Lipophorin, an Insect Lipoprotein*

An insect high density lipoprotein, lipophorin, can be rapidly isolated from larval Munduca sextu (tobacco hornworm) hemolymph by single vertical spin density gradient ultracentrifugation. The two apolipoproteins (M, = 245,000 and 78,000; designated apoLp-I and apoLp-11, respectively) were readily dissociated and separated in 6 M guanidine HC1 by gel permeation chromatography. ApoLp-I and apoLp-I1 showed no im- munological cross-reactivity on electrophoretic blots of sodium dodecyl sulfate-polyacrylamide gels. ApoLp-I and apoLp-I1 from lipophorin of adult M. sextu be- haved identically to their larval counterparts. Amino acid compositions of larval apoLp-I and apoLp-I1 were similar except with respect to tryptophan and cysteine; apoLp-I contained 32 residues/mol of tryptophan (1.5 mol %) and 22 residues/mol (1.1 mol %) of cysteine; apoLp-I1 contained 2 residues/mol of tryptophan (0.2 mol %) and 14 residues/mol of cysteine (2.1 mol %). In double immunodiffusion tests, antiserum against apoLp-I or whole lipophorin strongly precipitated lipophorin, while antiserum against apoLp-I1 caused only minor precipitation. This indicates relatively greater exposure of apoLp-I to the aqueous environ- ment.

3 Present address, Monsanto Agricultural Products Co., 800 North Lindbergh Boulevard, St. Louis, MO 63167. § To whom correspondence should be addressed.
ified cholesterol (Chapman, 1980). Furthermore, lipophorins contain two relatively large apoproteins (Mr = 250,000 and -80,000). In contrast, mammalian lipoproteins contain little diacylglycerol and a much higher proportion of nonpolar lipid (primarily triacylglycerol and cholesterol esters). Most mammalian lipoproteins also contain numerous small (M, = <40,000) apoproteins, with the exception of the low density lipoproteins. These compositions suggest a difference in structural organization between mammalian and insectan lipoproteins.
Little is known about the structure of native lipophorin. Improved methods of lipophorin and apoprotein isolation from larval M. sexta reported here permit structural studies using immunological probes. We also report complete amino acid compositions of the two apoproteins, since cysteine and tryptophan contents were not previously reported (Pattnaik et al., 1979). The marked similarity in amino acid content of the apoproteins indicated possible homology, and a recent report on locust lipophorin (Gellissen and Wyatt, 1981) suggested that a large apoprotein, not consistently present in lipophorin preparations, represented an aggregate of small apoproteins. We have, therefore, compared the apoproteins of M. sexta lipophorin, which we designate apoLp-1"' ( M , = 245,000) and apoLp-I1 ( M , = 78,0001, and demonstrate unequivocally that they are not homologous.
We have used Roman numerals to designate these insect apolipoproteins to avoid premature comparison to the lettered mammalian apolipoproteins.
Portions of this paper (including "Experimental Procedures," Figs. 1-3, and   through the side of the tube with a hypodermic syringe (see Fig. 4, lanes 1 and 6 ) . Recentrifugation of isolated lipophorin and comparison with a density gradient reference tube yielded a buoyant density of 1.13 g/ml (Fig. 1). Gel permeation HPLC demonstrated a single major peak, with a small amount (<lo%) appearing at the void volume as aggregated material.

SDS-Polyacrylamide Gel Electrophoresis: Molecular Weight Determination and Carbohydrate Content-Earlier molecular weight determinations on
SDS-polyacrylamide gels were made by comparison to cross-linked standards (Pattnaik et al., 1979). Our more recent experience with these standards has been less satisfactory than with non-cross-linked standards. We, therefore, slightly revised our molecular weight estimates, using non-cross-linked standards (Bio-Rad high molecular weight standards, plus ferritin (220,000) and thyroglobulin (330,000), not shown) on 3-8% SDS-polyacrylamide gradient slab gels (Fig. 4). Molecular weights of apoLp-I and apoLp-I1 were calculated as 245,000 & 7,000 and 78,000 & 3,000 (MI f S.D., n = 6).
Scanning densitometry of stained apoproteins yielded an approximate apoLp-1:apoLp-I1 ratio of 3:l by weight, corresponding to the 1:l molar ratio previously observed (Pattnaik et al., 1979). The stoichiometric ratio was further confirmed by separation of the two apoproteins by preparative SDS-gel electrophoresis. The fractions containing the two apoproteins were identified by analytical SDS-slab gel electrophoresis and the amounts of protein present in pooled fractions of each apoprotein were determined quantitatively by the Folinphenol method of Peterson (1983), using SDS solutions of the pure apoproteins as standards. In two runs, apoLp-I/apoLp-I1 molar ratios of 1:13 and 0.87 were determined.
After separation on slab gels, both apoLp-I and apoLp-I1 stained with periodate-Schiff reagent, and both bound fluorescein-labeled concanavalin A.
Amino Acid Composition and UVAbsorbance-Apoproteins were analyzed for content of amino acids, including tryptophan and cysteine (Table 11). Spectrophotometric determination of tryptophan yielded values of 29 and 2 residues/mol of protein in apoLp-I and apoLp-11, respectively, compared to 32 and 2 residues/mol when determined by mild hydrolysis and chromatographic analysis. Specific absorbances for apoLp-I and apoLp-I1 a t 280 nm in 6 M guanidine HCl were 0.716 mg" ml-' and 0.300 mg" ml-', respectively. A visible wavelength scan of native lipophorin in PBS revealed a maximum absorbance a t 455 nm (due to the presence of carotenoids) and profile identical to that observed in Locusta migratoria lipophorin (Peled and Tietz, 1975).
Immunological Comparison of Apoproteins-Immunological reaction of nitrocellulose-bound proteins was used to determine whether the apoproteins have common structural features. Lipophorin was subjected to electrophoresis on SDSpolyacrylamide slab gels, transferred electrophoretically to nitrocellulose, and reacted with antisera specific to whole lipophorin or to either apoprotein. IgG-bound proteins were located by reacting blotted proteins with Staphylococcus lz5Iprotein A followed by autoradiography. Clearly, the antilipophorin showed immunoreactivity to both apoLp-I and apoLp-I1 (Fig. 4). However, anti-apoLp-I did not bind to apoLp-11, and anti-apoLp-I1 did not bind to apoLp-I. ApoLp-I and apoLp-I1 were the only proteins in fifth instar larval hemolymph that reacted with anti-lipophorin. Lipophorin was isolated from adult M. sexta hemolymph and analyzed in the same manner, using antisera against larval lipophorin and apoproteins (Fig. 5). ApoLp-I and apoLp-I1 from adult lipophorin migrated with the larval apoproteins and reacted identically with antisera against larval apoproteins, again showing no cross-reactivity between apoproteins.
Exposure of Proteins in the Lipophorin Particle-In double immunodiffusion tests, antiserum against lipophorin or against apoLp-I reacted effectively against lipophorin or hemolymph (Fig. 6, A and B ) . However, weak precipitin lines resulted when lipophorin was reacted with antiserum against apoLp-I1 (Fig. 6C). A stronger reaction was observed in crude hemolymph. Therefore, proteins from the subnatant of the KBr density gradient were run on SDS-slab gels and blotted against anti-apoLp-11. Immunologically cross-reactive material was present at the position of apoLp-11, indicating that some of this apoprotein was free in the hemolymph.

DISCUSSION
Insect lipophorin is thought to be a lipid shuttle, reutilized many times without new synthesis or degradation (Chino and Kitazawa, 1981). In the adult locust, the lipophorin particle can absorb additional diacylglycerol from the fat body under the influence of the decapeptide adipokinetic hormone (Mwangi and Goldsworthy, 1981). Lipophorin thereupon increases in size and decreases in density to the low density lipoprotein class. These changes are reversed as diacylglycerol is deposited for use as fuel in the flight muscles. Thus, unlike mammalian lipoproteins, which may change in density but not fluctuate, insect lipophorin fluctuates between lipid density classes in the course of its transport function. Diacylglycerol transport is apparently facilitated by association with a smalI protein, designated C protein (Mwangi and Goldsworthy, 1981;van der Horst et al., 1981;Wheeler andGoldsworthy 1983a, 1983b), though the protein and nature of its association with lipophorin have not been characterized. While not present in larvae, a similar protein has recently been isolated from adult M. ~e x t a .~ We propose to call this small apoprotein apoLp-111, in accordance with our proposed terminology and to avoid premature comparison with mammalian apolipoproteins C.
In order to understand how insect lipophorin functions during lipid transport, we need to know more about the structure of the particle and of its apoprotein components. We have developed a gentle and rapid density gradient procedure for isolating the larval lipophorin from the hemolymph of M. sextu and an efficient gel permeation chromatography procedure for separating the apoproteins. The larval lipophorin is homogeneous with respect to molecular weight and density, while lipophorin from adult M. sextu is polydisperse and less dense than the larval form and thus resembles the diacylglycerol-loaded lipophorin of the adult locust (Shapiro and Law, 1983).
We have substantially improved separation of the apoproteins by gel permeation chromatography through use of guanidine hydrochloride as a chaotrope rather than SDS, which was used previously (Pattnaik et al., 1979). We performed complete amino acid analysis on the isolated apoproteins and found that the cysteine and tryptophan contents, not previously determined, were of particular interest. ApoLp-I contained 29-32 residues/mol (1.5 mol %) of tryptophan, compared to only 2 residues/mol (0.2 mol %) for apoLp-11. This observation accounts for the low absorbance of apoLp-I1 at 280 nm seen during chromatography in 6 M guanidine hydrochloride (Fig. 3). Cysteine content of the apoproteins also differed; apoLp-I contained 22 residues/mol(1.1 mol %), while apoLp-I1 contained 14 residues/mol (2.1 mol %). Assuming that most cysteine residues are involved in disulfide linkages, the higher percentage of cysteine in apoLp-I1 may contribute to a more compact structure.
Some studies on the lipophorin of Locustu rnigratoriu have demonstrated a single apoprotein 'that is comparable in molecular weight to apoLp-I1 of Manduca lipophorin (Gellissen and Emmerich, 1980;Gellissen and Wyatt, 198l), while others have found both apoproteins corresponding to those of M. sexta (Chino et al., 1981b;Chino and Kitazawa, 1981). Chino and Kitazawa (1981) found a definite difference in the apoproteins of locust lipophorin; apoLp-I stained for carbohydrate in SDS-polyacrylamide gels, while apoLp-I1 did not. We have isolated two distinct apoproteins from Manduca lipophorin, both of which are stained by the periodate-Schiff reagent and both of which bind fluorescein labeled concanavalin A, indicating that they are both high mannose glycoproteins. We have clearly demonstrated through immunological methods that they are not homologous (Fig. 4). This effectively rules out aggregation of apoLp-I1 as the origin of apoLp-I and indicates that apoLp-I1 is not a result of fragmentation of genes for apoLp-I or a processing product of an apoLp-I transcript. It is possible, however, that the two apoproteins are discrete products of a single large gene or messenger, as are the apoproteins of some vitellogenins (Harnish et al., 1982). We also showed that two apoproteins found in adult lipophorin are immunologically identical to those in larval lipophorin (Fig. 5).

A B C
FIG. 6. Double immunodiffusion of lipophorin and hemolymph against antisera to apoproteins or whole lipophorin. 10 pl of antiserum against ( A ) whole lipophorin (5-fold dilution), ( B ) apoLp-I (2-fold dilution), or ( C ) apoLp-I1 (undiluted) was placed into center wells. Peripheral wells in A and B contained 10 pl of: lipophorin (10 pg, well 1 ; 2 pg, well 2; 1 pg, well 3), whole hemolymph (2-fold dilution, well 4 ; 5-fold dilution, well 5 ; 10-fold dilution, well 6 ) . Peripheral wells in C contained 10 pl of: apoLp-I1 in PBS (>lo pg, well I ; >5 pg, well 2), whole hemolymph (2-fold dilution, well 3). or lipophorin (20 pg, well 4 ; 10 pg, well 5; 5 pg, well 6 ) . Samples were allowed to diffuse for 3 days at room temperature and 3 days at 4 "C. Plates were dried and stained as described under "Experimental Procedures." tions and hydrophobic core interactions (Shen et al., 1977). lipophorin may demand a greater role for apoproteins in The former are contributed by polar apoprotein residues and formation of an apolar core. This concept is supported by our lipid groups, the latter by apolar lipids. The small proportion experiments on relative accessibility of the two apoproteins of apolar hydrocarbons, triacylglycerols, and sterol esters in to proteases, immunoglobulins, and radiolabeling reagents.
An earlier report found apoLp-I to be much more susceptible to trypsin digestion than apoLp-I1 (Pattnaik et al., 1979) and radioiodination . Here we have demonstrated that apoLp-I in the intact particle is more susceptible than apoLp-I1 to reaction with antibodies. Low accessibility of apoLp-I1 to bulky probes suggests that this polypeptide is sheltered from the aqueous environment and may lie partly within the particle, perhaps constituting a part of the core.
Though lipophorins readily exchange lipid (diacylglycerol and cholesterol) with tissue both in vitro (Chino and Gilbert, 1965;1971) and in vivo (van der Horst et al., 1981), no exchange mechanism has yet been proposed (Chino and Kitazawa, 1981). However, the apoproteins, especially apoLp-I, may participate in exchange by reversible association with plasma membranes. In mammalian systems, polar lipids exchange from lipoproteins more readily than the apolar triacylglycerols and cholesterol esters found in the lipoprotein core (Morton and Zilversmit, 1982). Several soluble plasma proteins that facilitate triacylglycerol and cholesteryl ester exchange have been isolated (Rajaram et al., 1980;Zilversmit et al., 1975;Pattnaik et al., 1978Ihm et al., 1980, 1982Chajek and Fielding, 1978). Since insect lipophorins contain a larger proportion of polar lipid than mammalian lipoproteins, a larger proportion of lipid should be freely exchangeable. This does not exclude the presence of hemolymph proteins that facilitate lipid transfer, especially in life stages requiring rapid turnover of polar lipid. In adult M. sexta a new apoprotein, apoLp-111, is found in the hemolymph. ApoLp-111, which can reversibly associate with lipophorin, may serve a lipid transfer function during the adult stage, a period of great demand for lipid, both as flight fuel in many insects (Bailey, 1975) and as a constituent of egg yolk. Furthermore, apoLp-I11 might serve as a recognition signal to promote interaction between lipophorin and muscle cell membranes, or it might serve to activate lipolytic enzymes at the muscle to aid in assimilation of transported diacylglycerol, much as apolipoprotein C-I1 in human very low density lipoprotein serves to activate lipoprotein lipase in peripheral tissue (Smith et al., 1978).