Cholesterol Binding in <i>Schistocerca gregaria </i>and Insect Cell Lines

Cholesterol Binding in Schistocerca gregaria and Insect Cell Lines

Ravi Kant Upadhyay*, Ravi Dhart and H C Agarwal Department of Zoology, University of Delhi, Delhi 110 007, India

Revised received: 06 December, 1999; accepted: 27 April, 2000

Cholesterol binding affinities in dillerent tissue homogenates of Schistocerca gregaria (Forskal) and insect cell line are reported. In the first set of experiment, binding could be observed in haemolymph, ovary, testis, fat body, midgut, mucin and serum protiens of both sexes of Schistocerca gregoria. The binding assays were conducted in the presence of labelled and unlabelled cholesterol. The total number of binding sites (Rt) are found to be 4.77 x I

o ·

⁸ mole/L while extracted proteins from 1st and lind peaks show 7.10 x 10·⁹ and 12.8 x I

o ·

⁹ mole/L. The binding sites in delipidated mucin have shown some higher binding (2.97 x I

o ·

⁸ mole/L) than the non-/ipidated mucin ( 1.38 x I

o ·

⁸ mole/L). Insect cell lines Sf-9 and BmN cells have kd value of 1.494 X I

o ·

^{8 and 4.08 X I}

o ·

^{8 mole/}Lin crude homogenate.

Introduction

After ingestion it is essential that sterols are absorbed by the gut wall to fulfill the sterol requirement of an insect. In isolated as well as in intact tissues, these sterols are certainly absorbed. In sterol absorption, it is essential that these sterols have a specific binding affinity with the tissue proteins.

However , relatively little information is available on cholesterol binding affinity in insects.

The cholesterol binding specificity has been studied by many researchers in mammalian proteins'·³ Nemeez and Schroeder studied the affinity of the recombinant fatty acid binding proteins from the rat liver. A fatty acid binding protein was isolated by Veerkamp et af.5. However, binding specificity of JHBP Manduca sexta by monoclonal antibodies has been studied by Goodman et aF Until now, four intracellular proteins which bind with lipophilic agents have been found in insects. Recently, from the midgut cytosol of Manduca sexta revealed the presence of two fatty acid binding proteins⁷; MFB I and MFB2. MFB I has a single affinity binding site,

*

Author for correspondence

Address: Department of Zoology, Institute of Basic Sciences, Khandari Campus, Dr 8 R Ambedkar University, Agra 282004, India

t

National Institute of Immunology, Aruna Asaf Ali Road, ew Delhi I 10067, India

with a dissociation constant< I 00 mM. Recheri et al.s studied, equilibrium constants for the binding of fatty acids with fatty acid binding proteins from adipocyte.

intestine, heart and liver using a flourescent probe. In the present investigation cholesterol binding affinity was calculated in the crude midgut homogenates as well as in the purified proteins. Affinity was also calculated for the ovary, testis, haemolymph, serum, fat body and insect cell line.

Materials and Methods

Maintenan ce of lnsect Cultures

Insects used for this study were the locust, Locus/a migratoria Linn and Schistocerca gregoria.

Insects were reared under crowded conditions in aluminium cages (approximately 37 x 37 x 37cm\

The cages were provided with a 25 watt bulb and a photoperiod regime of 12L: 12D was maintained. The temperature in the cages varied from 32 to 36 °C and the relative humidity was between 60-80 per cent. The insects were fed on a diet of green grass, leaves of maize (Zea mays), jai (Avena sativa) or jowar (Sorghum vulgare) in the laboratory. Adult locusts, of 8-15 days after the last moult were used for experiments.

Ch emicals

Cholesterol ³H ( Sp. Act. 7.45 Ci/mmol) was obtained from the Radiochemical Centre Amersham,

846 ^J SCI IND RES VOL 59 OCTOBER 2000

England. N' -2-hydroxymethylpiparizine-N' -2 hy- droxyethylpiperazine N' -2 ethane sulhponic acid (Hepes); tris (hydroxymethyl aminomethane (Tris)- HCI, N,N,N,N,-dimethylarninetetraacetic acid (ED- T A), 2,5-diphenyloxazole (PPO), I ,-4-bis(5-phenylo- xazole-2YL) benzene (POPOP), N,N-methylene-bis- acrylamide, N,N-tetramethylenediamine (TEMED) and Sepharose CL 6B-200 were from Sigma Chemical Co, USA.

Culture Media and Cell Lines

Sterile TNMFH (Grace's medium), RL 86 medium, cell lines Sf-9 (Spodoptera frugiperda ovarian follicle cells), and BmN (Bombyx mori cells), cell culture flasks T-25, petridish 60mm and 90mm diameter (Corning, USA) were used for the cultures.

Seeding the Cells

Suspended cells were cultured in Grace's medium supplemented with 5% of fetal calf serum (Sigma Co. USA) and a physiological dose of antibiotics from a gentamycin stock solution of I 0

!lg/mL and I 0 !lL of streptomycin 20 units/!lL were made and added in culture. Cells were supplemented regularly with medium after every third day and half of the medium was changed for every subculture.

Assay for Radioactivity

The radioactivity was estimated by a Packard Tricarb Liquid Scintillation Spectrophotometer. The samples in scintillation vials were taken in triplicate in aliquots of fixed volume and added 5 mL toluene based scintillation fluid (5 g of 2,5-diphenyloxazole (PPO) and 50 mg of I ,4-bis[2-(5-phenyloxazolyl) benzene (POPOP) and counted for 5 minutes each.

Appropriate background corrections were made and the quenching correction was also done in the individual samples by channel ratio method.

Protein Estimation

Samples were prepared for protein estimation using ultracentrifugation and TCA precipitation method. For protein estimation, Lowry's method⁹ was used.

Cholsterol Binding Assay

Cholesterol binding affinity was calculated using Scatchard Analysis¹⁰"·b. The affinity binding para-

meters included (i)

Rt

= Total receptor concentration, (ii) Kd =Dissociation constant, (iii) Ka =Association constant, (iv) N = Non-specific binding and (v) SB = Specific binding.

(I) Cholesterol binding assay with Spodoptera frugiperda ovarian follicle cells (Sf-9) and Bombyx mori ovarian nuclear cells (BmN)

In vitro experiments were conducted with Sf-9 cells and BmN cell homogenates. Cells were cultured in Grace's medium supplemented with 5% FCS and physiological dose of antibiotics, 20 !lL gentamycin (10 !lgl!lL) and amphotericin-B 10 ~LL (20 !lgl!lL) in T-25 flasks. Cell proliferation and confluency were observed routinely in every sub-culture and cells were harvested at the time of S-phase. Freshly harvested cells were centrifuged at 4°C at 1000 x g (Hermie BHG Z 360K) for 15 minutes and pellet was sonicated at 45 Hz for 3 min in ice chamber (Ultrasonic Instrument Co.). The freshly prepared crude cell homogenate was used for cholesterol binding assay.

Ligand Preparation for Cholesterol Bindin g Assay

(a) ³H-cholesterol (Sp. activity 7.45 Ci/mmol) was diluted by propylene glycol to prepare a solution of 36.2 xI

o -

¹⁰ mole/L concentration.

(b) The stock solution of unlabelled ligand was prepared by dissolving crystalline cholesterol in absolute alcohol to give concentrations of 1.81 x I

o - s

mole/L, I .81 x I 0-⁶ mole/L and I .81 x I Q-⁷ mole/L.

The working unlabelled ligand was prepared by further dilution of the stock solution to give 1.81 X I Q·

8 mole/L and I .81 x I

o -

^{9 mole/L solutions. The}

following experiments were conducted:

(a) In first experiment, binding sites were saturated by incubating 100 !lL of cell homogenate with increasing concentrations (36.2 x I 0·¹⁰ to 36.2

X I

o -

⁹ mole/L) of labelled ligand in 1.5 mL Eppendorf tubes (Tarson) at 30°C for 2h. Total assay volume was maintained at 200 !lL with PBS (pH 7 .4) in each tube.

After incubation tubes were centrifuged at 4°C for 30 minutes (Piasto Crafts Superspin R). Supernatant was removed and pellet was washed and rewashed and centrifuged. Scintillation counting was done on a Beckman Model for I minute for each vial.

(b) For competitive binding unlabelled ligand was used in excess for saturating the 50% of the total

binding sites and tubes were preincubated for 30 minutes.

In other experiments, unlabelled and labelled

ligands were used in increasing and decreasing concentrations of ligand to determine the competitive binding.

Gel Filtration Chromatography

Elution of the Gel Filtration Column and Estimation of Radioactivity

Sepharose CL-6B-200 Column was used for the purification of cholesterol binding proteins. PBS buffer (pH 7 .6, 50rnM) was used for all the subsequent elutions. The column was held errect with a stand tightly holding with the clips. Flow rate of 22- 24 mL/h

, was maintained by using

a peristaltic pump for a continuous buffer supply

.

Accessories were connected with a fraction collector (Pharmacia FRAC-1 00) and fractions were collected at fixed interval of time at a constant flow rate of 20-24 mL/h

.

Single aliquot was taken for radioactive determination from each fraction and samples were mixed with 5 mL of scintillation cocktail. Samples were counted in a liquid scintillation counter (Aioka Model in USIC) for one mjnute each.

(II) In vivo and in vitro Binding of

³

M-

Cholesterol with Midgut Cholesterol Binding Proteins

Preparation of Midgut Tissue Homogenate

Adult male and female

Schistocerca were

maintained on cotton soaked with water for 24 h the locusts were dissected under cold PBS

(pH 7

.4, 50

rnM)

within

30

minutes after 24 h for collecting the tissue. Midgut ti

ssue was

washed thoroughly with cold buffer. The tissue was meshed and vortexed. The tissue was homogenized in Kontes hand homogenizer.

The homogenate was soni

cated

in

an ice

chamber 50 Hz for

I

0 minutes (Ultrasonic Co.). Sonicated tissue was centrifuged at

I ,20,000 x

g for 45 minutes in a Sorvall A 1256 Rotor at 4°C. Supern

atant

was removed very carefully without disturbing the pellet

and

the volume was mea

sured.

A known quantity of radiolabelled cholesterol was

added to

the supernatant and mixed

. Supernatant

was incubated for

I

h at 30°C with constant shaking.

Protein

s

were estimated according to Lowry

's

method. From supernatant three aliqouts of lO ll

each

were taken for estimation of radioactivity.

Study of

³

M-Cholesterol Binding Affinity in Tissue Proteins in vitro

Cytosolic preparations from midgut, ovary, testis and haemolymph were examjned for characterization of binding sites using

³

H-cholesterol. The bindin

g

sites were saturated in presence of increasing concentration of labelled ligand (36.2 x

I

0-

¹⁰

moi/L - 3.62 x J0·

⁸

mole/L.

³

H cholesterol was incubated with 50 IlL of cytosolic homogenate in a total volume of 200 IlL An incubation time of 60 min was used in all subsequent determinations of binding. The bound cholesterol was separated from the free cholesterol by using cold centrifuge at 25

,000 g for 45 min.

The saturability of binding

³H

cholesterol was explored by adding increasing concentrations of

³

H- cholesterol to cytosolic preparations with an excess of unlabelled cholesterol added to the incubation tube.

The specific binding was calculated by substracting non-specific binding from total binding.

Transformation of these data to a Scatchard plot yielded a straight line (Figure 1), demonstrating a single type of high affinity binding site. Dissociation

2~01

~

""' 1.'01

['"'

j

^{o.•o• .}

0.001

Figure I - Elution pattern of PBS extractable proteins of S.

gregaria midgut chromatographed on a Sepharose CL-68 column. Seven mL of midgut supernatant was incubated with 5 J..l Ci of ³H - cholesterol for 60 min at 30°C with constant shaking. The column was eluted with PBS buffer. Fractions of 5.5 mL size were collected (a) Absorbance at 280 nm, (b) Absorbance at 640 nm, (c) J..lg/protein/200 mL fraction, and (d) Specific activity dpm/J..lg protein

848 J SCI IND RES VOL 59 OCTOBER 2000

0.190

{a) fo ^{d)

0.5 •

0.14 0

. .

0.090

-

0.3 ~ •

..

0.040

70 90 5 15 25

o.1,1"';; o- ' ---,3;';;o----'---;':5 o----'----=': - - - ' - U

35 0.300

(b) {<>)

0.6,---~

0.200

. _.

0.100

7 21 35 49 63

{c) ^0.070 {f)

0.050

0.020

o.2,~-;';-15--'-+.2 5,.--L-,}3!;---1-5--:4':-5 J._c:':55_j o.o o !-1 -:!--!---!--!--~:::I Bound l.Ox 10-10

mol<>/L

Figure 2 -Saturation curve of S. gregaria homogenatcs with varying quantities of ³H-cholestrol (3.62 x 10'¹¹¹- 36.02 x 10'¹¹¹ mole/L). B=Bound; F=Free cholesterol

(a) midgut homogenate, (b) Pool+ (c) Pool++ (d) mucinx (c) mucinxx (f) mucinxxx

constant Kd value

is determin ed

from the slope of

lin e. The line

was

draw n by meth od of least squares.

The x-axis

intercept value on Scatchard plot

(Figures

2, 3 and

4) was

meas ured to ca lculate the

co

nce ntration

of

total no. of binding sites in the

reaction

mixture.

The

di ssoc iation

constant

was estimated us ing

midgut

cytoso l preparati ons

from adult Schistocerca gregaria

(Tabl e

I). An

es timate

from

the slop e of

curve obt

ained

from

th e binding

data

yielded

an dissociation

constant of about 1.64 x 10·

⁷

(F igure

2a),

thereby sugges ting th at prote in present in the

cytosol

functi ons as a high affinity ca rri er in

in vivo. Purifi

ed

choleste

rol bindin g prote ins revealed two types of binding sites (a) high affinity bindin g site and (b) one low affinity bindin g site (Figures

2b and 2c).

The

receptor

concentrati on

was

meas ured

for

both of the

affinities

by usin g th e

x-axis

intercept.

All affinity

binding parameters ca

lculated from th

e purified proteins a re dipi cted in Tabl e

I.

In o rder to stud y th e saturati on the mucin preincubted

in

excess

of

choles tero l

and

bile salt s.

There

was

hi gher saturation

of the

binding s ites

was obse

rved in presence

of cholesterol

( 1 .38 x

I

o ·x

mo le/L) than in presence the bile sa lts ( 1.24 x I0-

⁹

mole/L). Mucin showed

a

lesser affinity than the

0.700 (o) O.llor - -- - - - -(-e)

0.500 . .

0.300

0.100,4'--'-~44,..-t--,8t-4-'-"7l2~4::!1._.,.!-"~4 ...J...J o.soo, - - - - (b) 0.400 •

0.)00

0.200

..

~

~ 0.100,~<---;;-'-;';--'--};_...:::::t,L_._

- 4 24 1.4 64 84

u 0.920 c g

CD 0.720 •

·.

0.100

0.600 •

0.400

(d) 0.100

0.250 0.)80

(h)

0.260

o.2 oat - - - -

.

••~•

--.--._,._ __ _

0.140 0.150

Bound 1.0 x 1.0-¹⁰ mote /L

Figure 3 - Saturation curve of S. gregaria tissue homogenates with varying quantities of ³H-cholesterol. 50 J..lL of tissue homogenate was incubated with increasing quantities of ³H- cholesterol (3.62 x 10'¹¹¹- 36.2 x 10·¹¹¹ mole/L), (a) male serum proteins, (b) fat body female (c) fat body female, (d) fat body male, (c) haemolymph male, (f) haemolymph female, (g) ovary, and (h) testis

0.1 ,";;----'---::-1;:---L---:f-:-_..l--.J..-_L-.L__j

10 30 50 70 90

Bound 1.0x 10-10mote/ L

Figure 4 - Saturation curve of cell homogenates of Sf-9 and BmN cells with varying quantities of ³H-cholesterol, 50 J..lL of cell homogenate were incubated with im;reasin<> quantities of

3H-cholesterol "'

Table 1- Cholesterol binding affinity constants determined from the Scatchard plot analysis in tissues of Schistocerca gregoria, Sf-9 and BmN cells

Tissues Total binding Dissociation Association Non-specific Specific

sites (Rt) X I oR constant (K d) x I 0⁷ constant (Ka) x I 0-⁷ binding (NB) x I 0 Binding (SB) x 10-~

mole/ L mole/ L mole I L mole/ L Mole/ L

Midgut 4.77 1.64 0.60 203 .2.52

Pool+ .710 15.6 6.4 .166 0.47

Pool++ 1.28 44.8 2.23 .0925 .854

Mucin* 2.97 32.8 0.30 .1213 1.71

Mucin** 1.38 62 1.61 1.38 2.79

Mucin*** .124 44.0 2.27 .106 1.86

Serum (M) 1.45 31.3 3.19 5.25 5.122

Serum (F) 0.83 33.0 3.02 3.493 3.540

Fat body(M) 1.99 31.2 3.20 3.28 4.791

Fat body (F) 10.6 2.0 0.50 .1124 8.437

Haem(M) 1.23 8.3 12.1 0.67 1.89

Haem( F) 0.71 40.0 2.5 4.750 3.09

Testis 1.84 17.3 .058 .123 1.82

Ovary 3.00 62.8 1.58 .189 .03

Sf-9 cells 1.27 14.94 6.69 3.641 5.860

BmN cells 1.22 40.8 2.45 .281 1.40

* Mucin was extracted in PBS, lyophilized, delipidated with acetone and dissolved in 5% aCI.

** Mucin was extracted in presence of unlabelled cholesterol,lyophilized and dissolved in I% Triton X-1 00.

*** Mucin was extracted in presence of bile salts 50j..iL sodium taurocholate ( 18.5 j..lmole), SOj..iL deoxycholate (24.0 j..lmolc) and SO j..iL glycholatc (2 j..lmolc).

M denotes Male, and F Female

midgut homogenate. Affinity difference against the association of cholesterol in midgut and midgut mucin was calculated from the assay.

Binding affinity and receptor concentration was also calculated for testis, ovary and haemolymph. The dissociation constant for all these tissues was estimated using cytosol preparations from the binding data . The concentration of cholesterol association with binding proteins obtained from the intercept Bmax (Rt) was estimated to be 0.60 x I 0⁷ Limo I e.

Discussion

The dose dependent experiments (Table I) showed that dissociation constant from the midgut supernatant and from the purified fractions give

similar incorporation in the presence of labelled ligand. This indirectly suggest that binding proteins are similar in both the assays. For the affinity binding assay, tissue proteins were saturated with increasing concentration of ³H cholesterol. Competitive binding of ³H cholesterol was studied in the presence of I 00- fold excess of unlabelled ligand. Non specific binding amounted to about 20-35% of the total binding which also consists of a low affinity specific binding. When isolated midgut portions were incubated in the presence or absence of bile salts a thick mucous material was found secreted from the midgut wall.

This insoluble material contained protein which probably function for the entrance of cholesterol in the epithelial cells¹¹• This was confirmed as a cholesterol carrier mucoprotein complex by Mayer ^cl

850 ¹ SCI IND RES VOL 59 OCTOBER 2000

at. ¹²• The isolated midgut mucous protein when added to the cholesterol containing medium, a low affinity was obtained. This has been discussed here after. The delipidated mucin exihibited a greater binding of ³H- cholesterol than the mucin isolated in the presence of cholesterol as well as in the presence of bile salts. The Kd value was calculated for the delipidated mucin (3.28 x I 0-⁸ mole/L) showed a higher saturation of the binding sites in the presence of bile salts ( 1.24 X I

o -

⁹

mole/L) than the cholesterol ( 1.38 X I

o - s

^mole/L).

The dose dependent experiments showed that dissociation constant form supernatant and from purified fractions have similar values in the presence of radiolabel ligand. This indirectly suggests that the binding protein is same in both the assays.

Competitive binding of ³H-cholesterol was studied in presence of I 0-fold excess of unlabelled ligand. Non- specific binding was higher than the specific one, which is amounted to about 40% of the total binding, consisting of both true and non-specific binding and a low affinity binding.

Recheri et al. ¹³ studied binding affinity in mammalian system. equlibirium constants were measured at 37

o c

^for palmitate, stearate, oleate, linoleate, linolenate and arachidonic acid binding to six FABPs from intestine, heart and adipose and liver from different species. Equilibrium contants for each fatty acid were found to be extremely sensitive to the tissue of origin of the FABP but largely independent of species differences. The measured values of dissociation constants Kd ranged from about 2 to I 000 nm, depending upon the tissue origin of the FABP and FA. L-FABP gave Kd of 0.4

+

0.2) mm and 1.4

+

0.5 sites/monomer.

When purified unlabelled proteins (Figures I a, I b and I c) of S. gregaria midgut were analyzed for affinity binding, the binding affinity from· both of the regions gave dissociation constant (Kd) of I .56 x I

o -s_

4.48 X I

o - s

^mole/L (Figures 2b and 2c). The presence of high molecular weight protein ("" 32-50 kd) from the first region of protein elution gave higher binding than the second region. The association constant (Ka) from the first peak was higher 1.66 X I

o -

⁹ than the second peak 0.925 x I 0-⁹ mole/L.

The affinity binding experiments may help in understanding the distinctive mechanism of lumenal absorption of cholesterol and its loading at the cell surface for further transfer to other tissues as a bound

ligand or a cell synthesized ligand protein. Present study provide evidence for a cytoplasmic binding protein in all the analysed tissues. In BmN cells the initial binding of cholesterol to membrane receptors has been ruled out. None the less, the BmN cell binding proteins can be ()resumed to be receptor because of its specific binding, and saturability. There are indications that cholesterol binding proteins serves as a specific carrier molecule in cells.

Cholesterol binding proteins in BmN cells have dissociation constant of 1.5 x I

o - s

^mole/L, association constant of 6.66 x I 0⁷ Llmole while the total receptor concentration is calculated I .22 x I

o -s

^mole/L

Similarly cytosol of Drosophila Kc cells contains a protein that binds JH with specificity, and high affinity¹⁴ with a Kd of 1.56 x I0-⁸ mole/U^4.

Affinity experiments showed that cholesterol bound in vitro to a low molecular weight protein.

Other prelimjnary results have also demonstrated that in cholesterol fed insects in vivo it also bind to a low molecular weight protein. It has already been shown that lipoprteins bind with lipids¹⁵ released by the fat bodyl⁶·17 and these lipoproteins are same in different insects which has been addressed here and is the first report of its kind.

An analysis of Sctchard plot obtained for the binding of cholesterol to haemolymph proteins in the present study, showed that cholesterol bind specifically with haemolymph proteins. The affinity of haemolymph proteins was quite high. An apparent dissociation constant (Kd) of 1.428 x I

o -s

^{mole/L was}

calculated for cholesterol binding. In plant feeding insects, conversion of phytosterol to cholesterol in the haemolymph cells before the release of sterol into the circulation medium¹⁸-20 may be necessary step in the transport of sterols as haemolymph proteins²¹·23

showed low affinity for cholesterol in the present study.

From the above facts it is clear that low affinity binding obtained in all the tissues in vitro which associated cholesterol is indeed due to the presence of cholesterol binding proteins. Essentially these proteins cause intracellular binding and subsequently deliver the cholesterol from the midgut lumen. In the midgut it is possible that the cholesterol is transferred spontaneously from the mjdgut to haemolymph as protein bound non-specifically. From cholesterol binding with the purified proteins of the midgut, and

insect cell lines receptors are present on the cell surface. Once cholesterol binds to the receptor with a weak affinity and the complex remain intact on solubilization and get its way for transportation from one organ to another. This study needs to be carried further in order to shows some other important facts.

Acknowledgements

The authors express their sincere gratitude to the Head, Department of Zoology, University of Delhi, and Director N.J.I., New Delhi, for providing the necessary facilities for carryingout this work. The financial support from the Council of Scientific and Industrial Research, New Delhi, ts gratefully acknowldged.

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