Purification of insect vitellogenin and vitellin by gel-immobilized ferric chelate

PROTEIN

EXPRESSION

AND

PURIFICATION

2, 24-28 (1991)

Purification of Insect Vitellogenin and Vitellin by Gel-Immobilized Ferric Chelate Miranda

C. van Heusden,’ Susan Fogarty, Jerker Porath, and John H. Law Department of Biochemistry, Biological Sciences West, University of Arizona, Tucson, Arizona 85721

Received September

17,1990, and in revised form January

3,199l

Vitellogenin and vitellin of Manduca sexta and some other insect species were purified by immobilized metal ion affinity chromatography. Ferric ion was chosen as the immobilized metal ion. Agarose-bound carboxymethylpicolylamine was used as the chelating adsorbent for the ferric ion. Vitellogenin and vitellin, both phosphorylated lipoproteins, were shown to bind specifically to the iron. The general applicability of immobilized ferric ion affinity chromatography for the purification of insect vitellogenin and vitellin is suggested. d 1991

Academic

Press,

Inc.

As with other egg-laying animals, insect embryonic development is totally dependent on the egg yolk content. Insect egg yolk is formed by endocytotic uptake of hemolymph proteins synthesized elsewhere in the body and transported to the ovary. Although yolk contains several different proteins, its principal component is a large glycophospholipoprotein, vitellin, which is derived from a hemolymph precursor, vitellogenin (1). Vitellogenin is synthesized in the fat body, secreted into the hemolymph, and finally sequestered by the developing oocyte. Vitellogenin and vitellin of the tobacco hornworm, Manduca sexta, are lipoproteins composed of two subunits, apoVg-I or apoVt-I (180 kDa) and apoVg-II or apoVt-II (45 kDa) (2). Studies of the structure, metabolism, cell biology, and control of gene expression of vitellogenin require efficient isolation of the protein. Problems have frequently been encountered because of the susceptibility of vitellogenin and vitellin to proteolytic degradation. Thus, a rapid and specific separation method for these lipoproteins is desirable. W e have explored immobilized metal ion affinity chromatography (IMAC; (3)) as a rapid method for the purification of vitellogenin. Since phosphoproteins have 1 To whom correspondence

should be addressed.

been described to adsorb specifically to immobilized ferric ion by means of their phosphate groups (4), we chose to use ferric ion, immobilized to a new IMAC adsorbent, carboxymethylpicolylamine (CMPA). In this paper we report the adsorption and specific displacement of insect vitellin and vitellogenin using this IMAC method. MATERIALS

AND

METHODS

Synthesis of CMPA-Agarose Swelled Sepharose 6B (100 g) was suspended in 25 ml distilled water, 33 ml 4 M NaOH, and 7 ml epichlorohydrin in a l-liter round-bottom flask provided with a stirrer and two funnels, one with 33 ml 4 M NaOH, the other with 33 ml epichlorohydrin. Sodium borohydride (0.3 g) was added to the suspension. The suspension was stirred for 2 h at room temperature. Stirring was continued for 2 h with slow addition of the reagents from the funnels. The reaction was allowed to proceed overnight (18 h total). The gel was collected on a sintered glass funnel (No. 2 or 3) and washed with water until the washings showed a neutral pH. The gel was washed with 0.4 M sodium carbonate buffer, pH 10.0, and suspended in 100 ml of the same buffer containing 4 ml of picolylamine. The reaction was allowed to proceed for 24 h at room temperature. The gel was washed with 1 M acetic acid followed by distilled water until a neutral pH was observed. Bromoacetate solution was prepared by dissolving 15 g of bromoacetic acid in 30 ml 3 M NaOH, adjusting the pH to 10.0 with solid NaOH, and mixing with 35 ml of 1 M sodium carbonate buffer, pH 10.0. The gel was suspended in the bromoacetate solution and carboxymethylation was allowed to proceed at room temperature overnight (17 h). The gel was washed with distilled water and stored until use. A sample was tested for its uptake of Cu2+ and was found to contain 2.07% Cu per gram of dried gel.

24 Copyright All

rights

0 1991 of reproduction

1046-5928/91 $3.00 by Academic Press, Inc. in any form reserved.

INSECT

VITELLOGENIN

2.0 -

Animals A4. sexta (tobacco hornworm, a sphinxmoth, Lepidoptera) was reared as described by Prasad et al. (5). Hyalophora cecropia (silkmoth, Lepidoptera) was reared as described by Kulakosky and Telfer (6). Diceroprocta semicinta (cicada, Homoptera) and Triatoma rubida (kissing bug, Hemiptera) were collected in the field (near Tucson, AZ). The hard tick Dermacentor variabilis (SAY, Ixodidae) was reared as described by Rosell-Davis and Coons (7), and adult mated, ovipositing females were used. Preparation

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PURIFICATION

z w u ;

1.5 0.6 II NaCl 0.1 N PO,

l.O-

2 m b 9

0.5 -

of Samples

Preparation of all samples was performed on ice. Hemolymph was isolated from adult female sphinxmoth M. sexta (1-2 days old) by the flushing out method (8) using Pipes buffer (0.05 M Pipes, 0.1 M NaCl, pH 6.5), containing 1 mM diisopropylfluorophosphate (DFP) and 20 mM glutathione, as bleeding solution. EDTA, a usual component of insect bleeding solutions, was omitted because of its interference during IMAC. Hemolymph was centrifuged (12,OOOg, 5 min) to remove hemocytes. In order to remove free metal ions which could interfere in IMAC, metal-free TED gel (tris[carboxymethyllethylenediamine, synthesized as described by Porath and Olin (9)) was added to the hemolymph (1 g/10 ml hemolymph) and mixed for 5 min. After the gel settled, hemolymph was removed and subjected to chromatography. Ovaries were dissected from 5- to 6-day-old female M. sexta and mature follicles were removed. Follicles were homogenized in Pipes buffer, pH 6.5, containing 1 InM DFP, 20 mM glutathione, and a cocktail of protease inhibitors (as described by Prasad et al. (lo)), by breaking the follicles with a micropestle in a microfuge tube. The homogenate was subjected to centrifugation (12,OOOg, 10 min) and subsequently the supernatant was mixed with metal-free TED in order to remove free metal ions (as described above for the preparation of a hemolymph sample). Pupal hemolymph from the silkmoth (H. cecropia), obtained by puncturing the cuticula, was diluted 1:l with Pipes buffer, pH 6.5, containing 10 mM glutathione. Subsequently the hemolymph was subjected to centrifugation (12,OOOg, 5 min) and the supernatant was used for chromatography. Samples of cicada (D. semicinta) and hard tick (D. uariabilis) ovaries were prepared similarly to the M. sexta follicles, except that in these cases total ovaries were used. With the kissing bug (T. rubida), mature (laid) eggs were used. Chromatography All chromatography steps were performed at 4°C. CMPA-Agarose was packed in a 1 X 6-cm column and

0.0 Jl0

10

20

30 fraction

40

50

60

FIG. 1. Chromatography of M. se&a hemolymph on FeWI)CMPA-agarose. The column was equilibrated in Pipes buffer, pH 6.5, and the hemolymph was loaded in the same buffer. Nonspecifically bound protein was eluted with Pipes buffer, pH 7.0, containing 0.6 M NaCl, after which the specifically adsorbed protein was eluted with a phosphate gradient (0.0-0.1 M in Pipes buffer, pH 7.0), as indicated by the arrows.

loaded with ferric ions by elution with 2 column volumes of 0.1 M FeCl, in distilled water. Excess metal was removed by extensive rinsing with distilled water followed by 2 M acetic acid. After being washed with acetic acid the iron remaining on the gel is very strongly bound. The column was equilibrated with Pipes buffer, pH 6.5. Samples were loaded and elution of proteins was monitored by absorbance measurements at 280 nm. Elution procedures were as described under Results. Following chromatography, the eluted proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (11). Gels were stained with 0.05% amido black 10B. After each experiment the bound iron is eluted with 0.2 M EDTA. Removal of the metal ion from some other chelating gels can be more difficult or sometimes impossible. However, CMPA-agarose can be rinsed and stored in distilled water containing 0.02% azide at 4°C. The gel can be recharged with ferric ion and used again. RESULTS

Isolation

of Vitellogenin

from M. sexta Hemolymph

Fractionation of M. sexta hemolymph on Fe(III)CMPA-agarose is shown in Fig. 1. The major portion of the hemolymph proteins did not adsorb to the column and appeared in the flowthrough fraction. A pH increase to 7.0 and inclusion of 0.6 M NaCl in the Pipes buffer resulted in the elution of small amounts of nonspecifically bound protein. Elution with a phosphate gradient (0.0 to 0.1 M phosphate in Pipes buffer, pH 7.0)

26

VAN

a

ApoLp-I

*

b

c

HEUSDEN

kD

4 ZDD

.116 -97

ApoLp-II

)

466

ApoVg-II >

422

ET

AL.

for the same binding sites and that vitellogenin places lipophorin. Isolation

of Vitellin

dis-

from M. sexta Follicles

Fractionation of soluble follicle proteins on Fe(III)CMPA-agarose (Fig. 3) results in an elution pattern similar to that of hemolymph proteins. Like hemolymph vitellogenin, vitellin appeared to adsorb to the gel and to be displaced by phosphate (see Fig. 4). Again, high salt concentration (0.6 M NaCl) did not displace the vitellin, indicating that the adsorption is not due to electrostatic interaction. In addition to vitellin, several other egg proteins present in smaller amounts appeared to adsorb to the gel. However, these proteins were more weakly bound and eluted at the leading edge of the vitellin peak in a phosphate gradient.

* 14

FIG. 2. SDS-PAGE analysis (4-15% slab gel) of M. sexto hemolymph fractionated on Fe(III)-CMPA-agarose. (a) Total hemolymph; (b) nonbound protein; and (c) adsorbed protein eluted with phosphate. ApoLp-I and -11, apolipophorin I and II; apoVg-I and -11, apovitellogenin I and II. Molecular weight markers (in kilodaltons) are as indicated.

resulted in the elution of a protein peak at approx 0.025 phosphate that contained vitellogenin as shown by SDS-PAGE analysis (Fig. 2). This indicates that vitellogenin was specifically adsorbed to and specifically eluted from Fe(III)-CMPA-agarose. The double band for apoVg-I after SDS-PAGE analysis (see Fig. 2) has been described previously by Imboden and Law (2). In addition to vitellogenin, in some experiments a trace amount of lipophorin (the major insect hemolymph lipoprotein involved in general lipid transport, see Kanost et al. (12)) was adsorbed to the Fe(II1) gel and coeluted with vitellogenin in the phosphate gradient. Since lipophorin from M. sexta follicles did not adsorb to the column (see below), the amount of phospholipid in the lipophorin particle (which is lower in the egg form of lipophorin than in the hemolymph form, Kawooya et al. (13)) might influence the affinity of lipophorin for immobilized iron. This, together with the high lipophorin concentration in hemolymph, could explain the adsorption of some lipophorin. Some vitellogenin is present in the flowthrough fraction (Fig. 2), which can be explained by overloading of the column. When the flowthrough fraction was reloaded on a regenerated column, all vitellogenin was removed from the sample and bound to the column (result not shown). When this vitellogenin was eluted from the column with 0.1 M phosphate, the fraction contained lipophorin as well. The higher ratio of lipophorin to vitellogenin in this fraction (compared to the phosphate-eluted vitellogenin fraction after the first adsorption of hemolymph to the column) suggests that vitellogenin and lipophorin compete M

Other Species

Vitellogenin of the silkmoth (H. cecropiu) has an apoprotein pattern similar to that of M. sexta vitellogenin, a larger apoprotein of 180 kDa and a smaller apoprotein of 42 kDa (14). Hemolymph from pupal H. cecropia was applied to the Fe(III)-CMPA-agarose column in Pipes buffer, pH 6.5. After elution of the nonbound protein, nonspecifically bound proteins were removed with 0.6 M NaCl in Pipes buffer, pH 7.0. Subsequently, vitellogenin was eluted with 0.1 M phosphate in Pipes buffer, pH 7.0. In addition to vitellogenin, trace amounts of lipophorin and of the extremely abundant storage proteins were adsorbed to the column (see Figs. 5a and 5b). A sample of cicada (D. semicintu) ovary extract was applied to the column. After removal of nonbound and nonspecifically bound protein, electrophoretically pure

2.01

e 1.5. c

0.6 II NaCl

z phosphate 6radicnt

0.0 0

lo

20

30

40

50

60

fraction

FIG. 3. Chromatography of M. sextu follicle extract on Fe(III)CMPA-agarose. Elution was as described in the legend to Fig. 1.

INSECT

ApoLp-I

VITELLOGENIN

w

ApcNt -I), ApoLp-II

m

ApoVt -II,,

FIG. 4. SDS-PAGE analysis (4-15% slab gel) of M. s&a follicle extract fractionated on Fe(III)-CMPA-agarose. (a) Total follicle extract; (b) nonbound protein; and (c) adsorbed protein eluted with phosphate. ApoVt-I and -11, apovitellin I and II; Other abbreviations are as in the legend to Fig. 2. Molecular weight markers (in kilodaltons) are as indicated.

vitellin was eluted with 0.1 M phosphate (see Figs. 5c and 5d). A sample of kissing bug (T. rubida) egg extract was applied to the column. The vitellogenin appeared to bind specifically and was eluted in a phosphate gradient at approx 0.05 M phosphate (result not shown). Affinity of vitellin from a non-insect invertebrate species, the hard tick D. variabilis, was investigated. Similar to the insect vitellogenins and vitellins, the tick vitellin was adsorbed to the immobilized iron and the electrophoretically pure protein was eluted with 0.1 M phosphate (result not shown).

27

PURIFICATION

immobilized ferric ion was shown to act as an ion exchanger, enabling selective pH-dependent elution of proteins according to their phosphate content (9). Ongoing projects have as one of their objectives the study of the behavior of iron-loaded gels with different chelating groups in order to find the optimal condition for selective isolation of phosphoproteins. CMPA-agarose is one of the ligands in the series being tested. Like vitellogenins in general, insect vitellogenins and vitellins are phosphorylated, although their degree of phosphorylation (O.l-0.3%, Ref. (1)) is low compared to that of some vertebrate species (16,17). In M. sexta, vitellogenin seems to be the only hemolymph protein which is significantly phosphorylated (18). Therefore we expected to achieve a good purification of insect vitellogenin using immobilized ferric ion affinity chromatography. To investigate the interaction between vitellogenin and immobilized ferric ions, we used CMPAagarose, which has not been characterized previously, as chelating adsorbent. Vitellogenin from M. sextu hemolymph appears to have a high affinity for the gel-immobilized ferric ions. Since vitellogenin is eluted with phosphate, but is not displaced by high salt concentrations, the affinity is due to specific interaction of the phosphate side groups of the lipoprotein with the immobilized ferric ions. Vitellogenin and vitellin of M. sexta are displaced by the same phosphate concentration (0,025 M phosphate), indicating that their phosphate content is similar. H. cecropia and M. sexta are two closely related insect species, and their respective vitellogenins are structurally very similar, also reflected by immunological cross-reactivity (18). Nevertheless, because of slight differences in the molecules (e.g., isoelec-

LD

DISCUSSION

Immobilized metal ion affinity chromatography is a rather new and promising method for protein purification, with application possibilities for an extremely large variety of biomolecules. The method is based on the specific affinity of proteins and other biomolecules for certain metal ions, which are attached to an insoluble matrix by means of chelating ligands (3,15). The differential metal ion affinity of biomolecules gives this method an extremely wide range of applications. Although characterization of the interaction between proteins and metal ions is still a developing field, some specific protein-metal ion interactions have been investigated and described in more detail. The specific adsorption of phosphoproteins to ferric ions, immobilized to chelating gels, was described by Muszynska et al. (4). They showed that the strength of binding depends on the phosphate content of the protein. In addition, the

FIG. 5. SDS-PAGE analysis (4-15% slab gel) of (a) silkmoth total hemolymph; (b) protein adsorbed to Fe(III)-CMPA-agarose and eluted with 0.1 M phosphate; (c) cicada total ovary extract; and (d) protein adsorbed to Fe(III)-CMPA-agarose and eluted with 0.1 M phosphate. Abbreviations are as in the legend to Fig. 2. Molecular weight markers (in kilodaltons) are as indicated.

28

VAN

HEUSDEN

tric point) different purification protocols were developed when more conventional methods were used (6,18). However, our results show that both vitellogenins adsorb to immobilized ferric ion, due to their phosphate content. Since vitellogenins generally are phosphorylated proteins, our results suggest the general applicability of this method for vitellogenin purification. This is supported by the Fe affinity of vitellogenin from two other insect species (cicada and kissing bug) belonging to unrelated orders (Homoptera and Hemiptera), as well as vitellogenin from a hard tick species. Therefore immobilized ferric ion affinity chromatography appears to be a fast and extremely efficient technique for the purification of insect and other invertebrate vitellogenins. In addition to the affinity of insect vitellogenin and vitellin for immobilized iron, we also observed that a large number of other M. se&a hemolymph proteins have affinity for different metal ions such as Zn, Cu, or Ni (M. C. van Heusden and J. Porath, unpublished observations) and therefore IMAC will be a very useful technique for the purification especially of less abundant (and less studied) proteins and peptides in insect hemolymph. ACKNOWLEDGMENTS The authors thank Dr. W. Telfer for supplying the H. cecropia hemolymph, Dr. F. Noriega for the D. semicintu ovary sample and the T. rubida eggs, and Dr. R. Rose11 for the D. uariabilis ovary sample. This work was supported by National Institutes of Health Grant GM 29238.

REFERENCES 1. Kunkel, J. G., and Nordin, J. H. (1985) Yolk proteins, in “Comprehensive Insect Physiology, Biochemistry and Pharmacology” (Kerkut, G. A., and Gilbert, L. I., Eds.), Vol. I, pp. 83-111, Pergamon, Elmsford, NY. 2. Imboden, H., and Law, J. H. (1983) Heterogeneity of vitellins and vitellogenins of the tobacco hornworm, Manduca sextu L. Time course of vitellogenin appearance in the haemolymph of the adult female. Insect Biochem. 13, 151-162. 3. Porath, J. (1989) High-performance immobilized-metal-ion affinity chromatography of peptides and proteins. J. Chromatogr.

443,3-11.

ET AL.

4. Muszynska,

G., Andersson, L., and Porath, J. (1986) Selective adsorption of phosphoproteins on gel-immobilized ferric chelate. Biochemistry 25,6850-6853.

5. Prasad, S. V., Ryan, R. O., Law, J. H., and Wells, M. A. (1986) Changes in lipoprotein composition during larval-pupal metamorphosis of an insect, Man&co sexta. J. Biol. Chem. 261,558-

562. 6. Kulakosky,

P. C., and Telfer, W. H. (1987) Selective endocytosis, in uitro, by ovarian follicles from Hyalophora cecropiu. Insect Biothem. 17,845-858.

7. Rosell-Davis, R., and Coons, L. B. (1989) The relationship between feeding, mating, vitellogenin production and vitellogenesis in the tick, Dermacentor variabilis. Exp. Appl. Acarol. 7,95-105.

8. Chino, H., Hirayama,

Y., Kiyomoto, Y., Downer, R. G. H., and Takahashi, K. (1987) Spontaneous aggregation of locust lipophorin during hemolymph collection. Insect Biochem. 17, 89-97.

9. Porath, J., and Olin, B. (1983) Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions. Biochemistry 22, 1621-1630. 10. Prasad, S. V., Fernando-Warnakulasurya, G. J. P., Sumida, M., Law, J. H., and Wells, M. A. (1986) Lipoprotein biosynthesis in the larvae of the tobacco hornworm, Munduca sexta. J. Biol. Chem. 261,17,174-17,176. 11. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T,. Nature 227,680-685. 12. Kanost, M. R., Kawooya, J. K., Law, J. H., Ryan, R. O., Van Heusden, M. C., and Ziegler, R. (1990) Insect haemolymph proteins, in “Advances in Insect Physiology” (Evans, P. D., and Wigglesworth, V. B., Eds.), Vol. 22, pp. 299-396, Academic Press, San Diego. 13. Kawooya, J. K., Osir, E. O., and Law, J. H. (1988) Uptake of the major hemolymph lipoprotein and its transformation in the insect egg. J. Biol. Chem. 263,8740-8747. 14. Telfer, W. H., and Pan, M. C. (1988) Adsorptive endocytosis of vitellogenin, lipophorin, and microvitellogenin during yolk formation in Hyalophora. Arch. Insect Biochem. Physiol. 9.339-355. 15. Porath, J., Carlsson, J., Olsson, J., and Beltrage, G. (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258, 598-599. 16. Redshaw, M. R., and Follett, B. K. (1971) The crystalline yolk platelet proteins and their soluble plasma precursor in an amphibian, Xenopus laevis. Biochem. J. 124, 759-766. 17. Christmann, J. L., Grayson, M. J., and Huang, R. C. C. (1977) Comparative study of hen yolk phosvitin and plasma vitellogenin. Biochemistry 16,3250-3256. 18. Osir, E. O., Wells, M. A., and Law, J. H. (1986) Studies on vitellogenin from tobacco hornworm, Manduca sexta. Arch. Insect Biothem. Physiol. 3.217-233.