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Purification and characterization of a very high density chromolipoprotein from the hemolymph of Trichoplusia ni (H├╝bner).

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Archives of insect Biochemistry and Physiology 7:l-11 (1988)
Purification and Characterization of a Very
High Density Chromolipoprotein From the
Hemolymph of Trichoplusia ni (Hubner)
Grace Jones, Robert 0. Ryan, Norbert H. Haunerland, and John H. Law
Department of Entomology, University of Kentucky, Lexington, Kentucky (G.J.); Departments of
Biochemistry (R.O.R., J.H.L.) and Entomology (N.H.H.), University of Arizona, Tucson
A biliverdin-carrying protein was purified t o homogeneity from the larval
hemolymph of Trichoplusia ni. The native protein (density = 1.26 g/ml)
contains both lipid and covalently bound carbohydrate, as well as 150,000 M,
apolipoproteins. The protein is immunologically related to a similar protein
from an insect belonging t o the same family but is not related to known
proteins from insects of other families. Also, the protein is not
immunologically related to any of the other abundant hemolymph proteins
found in larval Trichoplusia ni.
Key words: biliverdin, very high density lipoprotein, chromolipoprotein, Heliofhis zea
INTRODUCTION
Chromoproteins are common components of insect hemolymph. Blue
chromoproteins have been isolated and characterized from four species,
Manducu sextu (L.) [l-41, Pieris brussicue (L.) [5],Heliothis zeu (Boddie) [6], and
Locustu rnigrutoriu (L.)
In each of these cases the chromophore is biliverdin
IX. Major differences, however, exist in their respective apoprotein components. Manducu sextu insecticyanin and P. brussicue bilin-binding protein exist
as tetramers of M, = 21,000 nonglycosylated subunits, each with a noncovalently bound biliverdin [2,4,8]. Heliothis zeu chromoprotein is a VHDL* (den-
[v.
*Abbreviations: FlTC = fluorescein isothiocyanate; PBS = phosphate buffered saline; SDSPAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis; VHDL = very high
density lipoprotein.
Acknowledgments: This study was supported, in part, by NIH grant DK 39197 (GJ). Published
with the approval of the Director of the Kentucky Agricultural Experiment Station (87-7-189).
Robert 0. Ryan’s present address is: The Lipid and Lipoprotein Research Group, Department
of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2E1.
Received August 21,1987; accepted December 21,1987
Address reprint requests to Dr. Grace Jones, Dept. of Entomology, University of Kentucky,
Lexington, KY 40546.
0 1988 Alan R.
Liss, Inc.
2
Jonesetal.
sity = 1.26 glml) and is a tetramer of M, = 150,000 apoproteins, which
possess covalently bound mannose-containing oligosaccaride residues [6].
Locustu rnigrutoriu cyanoprotein is a M, 350,000 tetrameric protein with a
subunit M, of 83,000. While this protein lacks lipid, the apoproteins are
glycosylated. Furthermore, the stoichiometry of biliverdin:apoprotein was
2:l.
Munducu sextu insecticyanin and L. rnigrutoriu cyanoprotein are present in
hemolymph throughout the life cycle, whereas H. zeu chromoprotein is
specific for the ultimate larval instar. Its pattern of appearance and disappearance from hemolymph, together with its lipid content, have led to the
suggestion that this chromoprotein may have a role in lipid metabolism or
function as a storage protein [9] during development of H. zed [6]. Thus, the
previously ascribed function of chromoproteins as camouflage proteins [lo]
may have to be expanded to include other roles as well.
We have isolated a blue chromoprotein from hemolymph of the cabbage
looper, Trichoplusiu ni, and report here its chemical and immunological
properties.
MATERIALS AND METHODS
Animal Rearing
Trichoplusiu ni (Hubner) (Lepidoptera), was reared as described previously
Ell]. Heliothis zeu were reared and hemolymph was collected as described by
Haunerland and Bowers [12]. Mundum sextu (L.) eggs were a gift from Dr. J.
Bucker, USDA, Fargo, North Dakota, and larvae were reared as previously
described [2]. PupiZio polyxenes (Stroll) were collected and reared as described
elsewhere [13]. Eumorphu sp. were obtained from Dr. J.O. Schmidt, USDA,
Tucson, Arizona.
Purification of Chromoprotein
Hemolymph was collected by piercing the prolegs of day 2 fifth instar
larvae of Trichoplusiu ni. The chromoprotein was separated by KBr density
gradient ultracentrifugation essentially as described by Haunerland et al.
[14]. Potassium bromide was added to 0.7 ml of collected hemolymph, and
the mixture was then solubilized in PBS (0.15 M NaC1, 0.10 M sodium
phosphate, pH 7.0) containing 1mM diisopropylphosphorofluoridate and 5
mM glutathione at a final concentration of 1.11g KBrl2.5 ml of solution. The
2.5 ml of solution was then transferred into a 5.1-ml Beckman (Fullerton,
CA) Quick-Seal tube and overlayed with 22% KBr (wlv) in PBS. The sample
was then spun at 65,000 rpm for 4.5 h in a Beckman VTi 65.2 rotor. The blue
protein fraction was removed by puncturing the side of the ultracentrifuge
tube and withdrawing the contents with a syringe. KBr was then added to
the blue fraction to achieve a final concentration of 44% KBr (wlv) in 2.5 ml
PBS (density = 1.31 glml). After transfer to a Quick-Seal tube the solution
was overlayed with 33% KBr (wlv) in PBS and centrifuged at 65,000 rpm for
4.5 h. Following centrifugation, the blue protein fraction was collected and
dialyzed extensively against water to remove KBr.
Chromolipoprotein From Hemolymph of Trichoplusiani (Hiibner)
3
Munducu sextu insecticyanin was prepared from fifth instar larval animals
as described previously [2]. Pupilio polyxenes hemolymph was processed as
described by Ryan et al. 1131 with collection and use of the blue fractions
following the gel permeation chromatography step. Eurnorphu sp. and H. zeu
hernolymph was collected and used for electrophoretic procedures without
prior processing.
Purity and Molecular Size Determination
The purity of the T. ni chromoprotein preparation was assessed by SDSPAGE as described by Laemmli [15]. A 412% acrylamide gradient slab to
which samples had been loaded was subjected to electrophoresis at 30 mA
for 3.5 h. Molecular weight standards were obtained from Bio-Rad (Richmond, CA). The native molecular weight of the protein was determined by
gel filtration chromatography. The purified protein was loaded onto a 1.5 x
170 cm AcA 22 (LKB, Gaithersburg, MD) gel filtration column equilibrated in
PBS and eluted at a flow rate of 10 mllh with collection of 2.8-ml fractions.
The concentration of the protein eluate was determined spectrophotometrically.
Chemical Crosslinking
Chemical crosslinking was performed according to the procedure described by Davies and Stark 1161. The purified chromoprotein was dissolved
in 0.2 M triethanolamine buffer, pH 8.3, to a final concentration of 1mglml.
One hundred micrograms was then incubated with various concentrations
of dimethylsuberimidate (0.05, 0.1, 0.35, or 5 mglml). The reaction was
carried out in the dark for 1.5 h and stopped by the addition of SDS-PAGE
sample treatment buffer and boiling for 1min. The samples were subjected
to 3-8% polyacrylamide gradient SDS-PAGE.
Amino Acid Analysis
Amino acid analysis was performed as described by Spackman et al. [lv.
Carbohydrate Analysis
The purified chromoprotein was subjected to SDS-PAGE and electrophoretically transferred to nitrocellulose paper. The nitrocellulose paper was
washed for 30 min with 10 mM sodium phosphate buffer containing 100 mM
sodium chloride and 0.05% Tween 20, pH 7.5 to block nonspecific binding
sites. It was then transferred to buffer (10m M Tris pH 7.4, 500 mM NaC1, 1
mM MgCI2, 1mh4 CaC12, 0.02% NaN3). After staining with FITC-conjugated
concanavalin A (Miles, Elkart, IN) and washing, glycoproteins were visualized under ultraviolet (W)light [B]. The total percent carbohydrate was
measured by the method of Dubois et al. [19] using mannose as the reference
standard.
Immunoanalysis
The proteins, in 3 p1 of hernolymph, from different insects species (see
below) were subjected to SDS-PAGE electrophoresis and electrophoretically
4
Joneset al.
transferred to nitrocellulose paper. The nitrocellulose paper was then incubated with primary antibody directed against H. zea blue chromoprotein [6].
After washing, the blot was incubated with 1z51-proteinA (Amersham, Arlington Heights, IL), and crossreactivity was detected by autoradiography.
UVlVisible Spectra
The Wlvisible absorbance spectrum of the T. ni blue chromoprotein was
obtained on a Perkin-Elmer Lambda 3 Wlvisible spectrophotometer. The
native protein (2 mglml) containing the chromophore was examined for its
absorption spectrum from 300 to 800 nm. The chromophore was extracted
from the protein by n-butanol, and the absorption spectrum of the isolated
chromophore was obtained. For reference, the spectrum of commercial biliverdin IX (Sigma, St. Louis) was also recorded.
Lipid Analysis
Lipid from 2 mg of purified chromoprotein was extracted with chloroform/
methanol according to Bligh and Dyer [20]. The extracted material was dried
under a stream of nitrogen, dissolved in hexane, and subjected to thin-layer
chromotography [hexane:diethyl ether:acetic acid (80:20:1)]. Ten micrograms
each of 1,2-dipentadecanoin and 1,2-diheptadecanoylphosphatidylcholine,
pentadecanoic acid, and tripentadecanoin were added as internal standards.
After completion of thin-layer chromatography, the phospholipid, diacylglycerol, triacylglycerol, and free fatty acid fractions were scraped off the plate,
eluted with diethylether and transesterified in 14%boron trifluoride in methanol. The resulting fatty acid methyl esters were analyzed by gas-liquid
chromotography as described by Haunerland and Bowers [6].
RESULTS
Purification and Molecular Size
The hemolymph of T. ni fifth instar larvae has a characteristic blue-green
color. Preliminary experiments suggested the presence of a blue and a yellow
component, which are separable by density gradient ultracentrifugation (the
yellow component floated higher than the blue component). The yellow
component possessed properties indicating that it is the major hemolymph
lipoprotein, lipophorin [21]. In an attempt to learn more about the blue
component a purification scheme based on its floatation behavior was
devised.
The protein obtained was analyzed by SDS-PAGE and was found to be
homogenous (Fig. 1)with an apoprotein M, of 150,000. Gel filtration chromatography was used to estimate the native molecular weight. As shown in
Figure 2, the protein eluted as a single peak at the position of an M, 320,000
protein (compared to the elution pattern of known standards). The apoprotein stoichiometry of T. ni chromoprotein was confirmed as a dimer by
chemical crosslinking with dimethylsuberimidate followed by SDS-PAGE
(Fig. 3). At increasing cross1inker:protein ratios, no more than a single band
with a molecular size significantly larger than the monomer was observed.
Chromolipoprotein From Hernolyrnph of Trichoplusia ni (Hubner)
5
kDa
x
10-3
200
116
94
67
54
MW
Fig. 1. SDS-PACE of purified biliverdin-binding protein from Trichoplusia ni (left) and molecular weight markers (right). The protein has a monomer molecular weight of 150,000.
A
1.0
,
280
0
20
40
60
80
100
120
fraction
Fig. 2. Elution profile of chromoprotein from an AcA 22 (LKB) gel filtration column. The
protein was eluted with PBS at a flow rate of 10 ml/h. AZa0 i s the absorbance at 280 nm
wavelength; Vo = void volume; V, = inclusion volume.
6
Joneset al.
KDa
~10-3
200
116
94
64
M
W
1
2
3
4
’
Fig. 3. Chemical crosslinking of subunits. The purified protein was incubated with the
following final concentrations of dimethylsuberirnidate: 0.0, 0.05, 0.1, 0.35, and 0.5 mg/ml,
lanes 1-5, respectively. The left side contains molecular weight markers. Arrow indicates
position of dimers.
The band of slightly higher molecular weight than uncrosslinked apoprotein
is probably due to intrachain crosslinks.
Carbohydrate, Lipid, and Amino Analysis
Following separation of T. ni blue VHDL by SDS-PAGE, transfer to nitrocellulose paper and probing with FITC-concanavalin A revealed that the
apolipoprotein possesses mannose-containing oligosaccharide moieties. The
total carbohydrate content was estimated to be 1.5%.An equilibrium density
of 1.26 g l m l was determined by measuring the refractive index of chromoprotein-containing fractions obtained following KBr density gradient ultracentrifugation. Lipid analysis indicated that the native protein contains 10.6% lipid.
These facts are consistent with classification of T. ni chromoprotein as a
VHDL. The relative distribution of lipid classes was 4.7% triacylglycerol,
22.6% diacylglycerol, 14.6% free fatty acid, and 58% phospholipid. Amino
acid analysis revealed that the protein is rich in AsplAsn and GlulGln (Table
1). This is consistent with the acidic nature of this protein (unpublished
Chromolipoprotein From Hernolymph of Trichoplusia ni (Hubner)
7
TABLE 1. Amino Acid Composition of
Chromoprotein From T . ni*
Amino acid
Asp/Asn
Thr
Ser
GluiGln
Pro
GlY
Ala
Val
Met
Ile
Leu
TYr
Phe
LYS
His
Are
mol Yoa
11.5
6.2
8.4
12.3
4.0
6.1
7.7
7.3
3.4
4.9
9.6
5.5
6.2
8.6
1.4
3.5
*Data derived from analyses of samples
hydrolyzed for 24 h in 6 N HCI, in vacuo, at 110°C.
aCys and Trp were not determined.
data). Also, the content of tyrosine, phenylalanine, and methionine suggests
that this chromoprotein is distinct from the other major larval hemolymph
storage proteins [22].
Immunological Study
Immunoblotting experiments revealed that antiserum directed against the
biliverdin-containing chromoprotein from H. zeu crossreacted with purified
T. ni chromoprotein. This antibody recognized only the blue VHDL from T.
ni hemolymph. The presence of a second lower molecular weight band in
lane 4 of the immunoblot may correspond to a breakdown product of the T.
ni chromoprotein preparation since it was not observed when freshly collected hemolymph was run. In contrast, the antibody failed to crossreact
with purified M. sextu insecticyanin- or chromoprotein-containing fractions
from P. polyxenes of Eurnorphu sp. hemolymph (Fig. 4).
A UVlvisible absorbance scan of purified T. ni blue VHDL revealed the
presence of two major absorbance peaks in the visible range (380 nm and 675
nm) (Fig. 5). However, when the chromophore was extracted with n-butanol
and scanned, a much lower absorbance around 675 nm (relative to that at
380 nm) was observed. This phenomenon is similar to that reported by
Haunerland and Bowers [6] for H. zeu blue chromoprotein. The spectrum
obtained for the isolated chromophore was very similar to that obtained
when a biliverdin standard was scanned. As suggested by Haunerland and
Bowers [6], the hyperchromic shift around 675 nm observed upon complexation with the protein may be due to interaction with the lipid moiety of the
particle.
8
Jonesetal.
KDa
x 10-3
200
116
94
67
54
M
W
1
2
3
4
5
COOMASSI E
6
7
1 2 3 4 5 6 7
I MMUNOBLOT
Fig. 4. lrnrnunoblot of the larval hernolyrnph from different insect species. Antiserum directed against H. zea chrornoprotein was used as the primary antibody. lodinated protein A
was used to locate the position of primary antibody-antigen complexes. From left to right the
lanes were loaded as follows: 1) M. sexta (10 pg purified insecticyanin), 2) P. polyxenes
(partially purified blue chromoprotein), 3) Eomorpha (3 PIhernolyrnph), 4) T: ni (10 pg purified
blue chrornoprotein), 5) 7: ni (3 pl whole hernolyrnph), 6) H. zea (3 pI whole hernolyrnph), 7)
M.sexfa (3 pi whole hernolyrnph).
DISCUSSION
The coloration provided by chromoproteins often serves an important
camouflage function. In M . sextu the biliverdin-binding protein, insecticyanin, is synthesized in epidermal tissue [1,23] and is found in hemolymph
during all life stages [24]. The green coloration of M . sextu larvae is a result of
chromophores present in epidermal tissue. Insecticyanin-bound biliverdin
and yellow carotenes, which are transported to epidermal cells by the hemolymph lipoprotein, lipophorin, combine to yield a green color. When
carotenes are not available in the diet, the animal appears blue rather than
green.
Binding chromophore may not be the sole function of certain biliverdincontaining chromoproteins. Recent work of Haunerland and Bowers [6]
suggest that the H. zeu chromoprotein may function in lipid transport or as a
storage protein. Furthermore, these authors suggested that H. zeu chromoprotein is the first member of a new class of hemolymph protein.The results
of the present study are in agreement with this hypothesis. We have isolated
a hemolymph chromoprotein from T. ni larval hemolymph with properties
similar to those described for the H. zed chromoprotein. Trichoplusiu ni chromoprotein was isol-atedby a rapid differential density gradient ultracentrifugation method in < 12 h. The chromoprotein possesses 10.6% noncovalent
Chromolipoprotein From Hemolymph of Trichoplusia ni (Hubner)
9
1.0
0.75
w
0
5m
0.5
a
%
a
0.2 5
J
425
5 50
675
800
WAVELENGTH Cnm)
Fig. 5. UV lisible absorption spectra of the purified T: ni chrornoprotein (-),
n-butanol
extracted chrornophore (- - - - -), and biliverdin standard in butanol (--). The concentration
of chrornoprotein was 2 rng/rnl.
lipid and therefore falls into the very high density class of lipoproteins. A
native M, of 320,000 was estimated by gel permeation chromatography, and
an apoprotein M, of 150,000 was determined by SDS-PAGE. The apoproteins
possess covalently bound oligosaccharide moieties, as judged by lectin-binding studies. The amino acid composition of T. ni chromoprotein is distinct
from that of the other insect blue chromoproteins, M . sextu insecticyanin [2]
and L. rnigrutovia cyanoprotein [7l.
The exact molecular weight and subunit composition of the protein has
not been determined. The H. zeu protein behaves as a trimer or tetramer on
native PAGE [6]. The T. ni protein behaves similarly on native PAGE (data
not shown), but is eluted from a gel permeation column in the region
expected for a dimer of two M, 150,000 apoproteins plus lipid. Furthermore,
only crosslinked dimers were observed after chemical crosslinking procedures. However, the presence of lipid might isolate apoproteins and prevent
crosslinking between all apoproteins. For the present, we prefer to leave
open the question of the structural organization of the intact protein. Physical
studies on the H. zeu protein, currently underway, should provide a more
exact solution to this problem.
Immunological studies revealed that the T. ni chromoprotein is related to
H. zeu chromoprotein. In contrast, no crossreaction with Eumorpha sp. hemolymph of P. pdyxenes fractions enriched in blue chromoprotein or isolated
M. sextu insecticyanin was observed. Investigation of the properties of the
10
Joneset al.
blue proteins from these species is currently in progress and should allow
for determination of their potential relationship with other known biliverdinbinding proteins.
It is interesting that biliverdin-binding proteins from insects representing
the same order (M. sextu, P. brussicue, and T. ni, or H. zeu) are so different in
physicochemical and immunological properties. We have extended the work
of Haunerland and Bowers [6] and have found support for their suggestion
of a new class of insect hemolymph protein. However, the results of the
present study and unpublished observations on Hylophoru cecropia (R.O.
Ryan and J.H. Law) suggest this class of chromoprotein may be restricted to
the Noctuidae. Indeed, the report [25]of a blue chromoprotein in Spodopteru
littoralis with characteristics similar to the H. zeu and T ni proteins supports
this notion. Trichoplusiu ni chromoprotein has properties very similar to the
H. zeu protein. The exact function of these hemolymph and fat body chromoproteins remains to be determined. A larval honeybee hemolymph protein sharing many of the properties of the H. zeu and T. ni chromoproteins,
but lacking a chromophore, has been reported [26]. Thus, it is important to
explore the potential of such proteins as lipid transport vehicles, storage
proteins, or carriers of special ligands, rather than focusing upon their color.
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2. Riley CT, Barbeau BK, Keim PS, Kezdy FJ, Heinrikson RL, Law JH: The covalent protein
structure of insecticyanin, a blue biliprotein from the hemolymph of the tobacco hornworm, Munducu sextu (L.). J Biol Chem 259, 3680 (1984).
3. Goodman WG, Adams B, Trost JT: Purification and characterization of a biliverdinassociated protein from the hemolymph of Munducu sextu. Biochemistry 24, 1168 (1985).
4. Holden HM, Law JH, Rayment I: Crystallization of insecticyanin from the hemolymph of
the tobacco hornworm, Munducu sextu L. in a form suitable for a high resolution structure
determination. J Biol Chem 261, 4217 (1986).
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(1986).
7. Chino H, Abe Y, Takahashi K: Purification and characterization of a biliverdin-binding
cyanoprotein from the locust hemolymph. Biochim Biophys Acta 748, 109 (1983).
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(1987).
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and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon Press, Oxford, Vol10, pp 307346 (1985).
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green? In: Bioregulators for Pest Control. Hedin PA, ed. ACS Symposium Series No. 276,
pp 511-521 (1985).
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hemolymph proteins of HeIiotkis zeu. Arch Insect Biochem Physiol3, 87 (1985).
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13. Ryan RO, Wang XY, Willott E, Law JH: Major hemolymph proteins from larvae of the
black swallowtail butterfly, Pupilio polyxenes. Arch Insect Biochem Physiol 3, 539 (1986).
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16. Davies GE, Stark GR: Use of dimethylsuberimidate, a crosslinking reagent, in studying
the subunit structure of oligometric proteins. Proc Natl Acad Sci USA 66, 651 (1970).
17. Spackman DH, Stein WH, Moore S: Automatic recording apparatus for use in the chromatography of amino acids. Anal Chem 30, 1190 (1958).
18. Ryan RO, Anderson DR, Grimes WJ, Law JH: Arylphorin from Munducu sexfa: Carbohydrate structure and immunological studies. Arch Biochem Biophys 243, 115 (1985).
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20. Bligh EA, Dyer WJ: A rapid method of total lipid extraction and purification. Can J
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21. Chino H: Lipid transport: Biochemistry of hemolymph lipophorin. In: Comprehensive
Insect Physiology, Biochemistry and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon
Press, Oxford, Vol10, pp 115-136 (1985).
22. Riddiford LM, Law JH: Larval serum proteins of Lepidoptera. In: The Larval Serum
Proteins of Insects. Scheller K, ed. Georg-Thieme Verlag, New York, pp 75-85 (1983).
23. Riddiford LM: Changes in translatable mRNAs during the larval-pupal transformation of
the epidermis of the tobacco hornworm. Dev Biol92, 330 (1982).
24. Trost JT, Goodman WG: Hernolymph titers of the biliprotein, insecticyanin, during development of Munducu sexfa. Insect Biochem 26, 353 (1986).
25. Rahbe Y: Stockage et mobilisation de la tyrosine au cours d u developpement larvaire de
Spodopfera liftoralis [LPpidoptere Noctuidae] . Doctoral thesis. Institut National Agronomique, Paris-Grignon (1984).
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