~view of Literatureshodhganga.inflibnet.ac.in/bitstream/10603/18653/7/07_chapter 2.pdf · such as the Bible, Vedas, Iliad and Odyssey. The Chinese, Romans, Greeks, Egyptians and Sumerians

~view of Literature

Review of Literature

Since ancient times, plants have been an exemplary source of medicine. The use .of

plants for the treatment of various human ailments is mentioned in ancient manuscripts

such as the Bible, Vedas, Iliad and Odyssey. The Chinese, Romans, Greeks, Egyptians

and Sumerians developed their own characteristic coHection of Materia Medica. Indian

literature like Ayurveda, Charak Samhita, Susruta Samhita, Siddha and Unani have

mentioned the use of plants for therapeutic purposes. India has about 4500 plant species

and among them, several thousands have been claimed to possess medicinal properties.

Recent research has validated the medicinal properties of plants mentioned in ancient

literature or used traditionally for different diseases including diabetes. Indian plants, which

are the most effective and most commonly studied in relation to diabetes are: Allium.cepa,

Allium sativum, Aloe vera, Cajanus cajan, Coccinia indica, Ficus, Ocimum, Pterocarpus,

Gymnema, Momordica charantia, Tinospora, Trigone/fa foenum and Brassica juncea. A

large number of these plarits exhibiting antidiabetic activity in experimental animals or in

clinical studies have been thoroughly investigated and exhibit varying degree of hypoglycemic

and anti-hyperglycemic activities (Grover eta/., 2002). As present study is conducted on

Momordica charantia, its medicinal potentials and active principles are reviewed henceforth.

1. 1 Momordica charantia and its therapeutic potentials

Momordica charantia (family Cucurbitaceae; bitter gourd, English; Karela, Hindi) that

grows in tropical regions of Asia, East Africa, Amazon and South America is used as a

vegetable and medicine. The plant is a slender, climbing annual vine with long-stalked

leaves and yellow, solitary male and female flowers borne in the leaf axils and bears warty

gourd shape fruits. The Latin name Momordica means "to bite" (referring to the jagged

edge of the leaf, which appears as if they have been bitten).

Momordica fruits, leaves and roots are used in "Ayurvedic Medicine" for curing various

diseases (Chopra eta/., 1956) and reports about its medicinal use in the western world has

been put forth by Ainsile (1826). Concentrated fruit or seed extracts as well as whole herb

powders in capsules and tinctures are becoming more widely available in the US and are

employed by natural health practitioners for diabetes, viral infections, colds and flu, and

psoriasis etc. The fruit of bitter gourd is routinely used as a folklore medicine for the treatment

of rheumatism, gout, dysmenorrhoea, leprosy, piles, jaundice, liver and spleen dismders

(Nadakarni, 1954). In vitro clinical studies have demonstrated the relatively low toxicity of

all parts of the bitter melon plant when ingested orally (Piatel, 1993). However, toxicity and

even death in laboratory animals has been reported when extracts are injected intravenously

or intraperitoneally (Foley, 1976).

Whole plant of Momordica, its fruits and seeds are documented to possess antibacterial,

anthelmintic, antibiotic, antidiabetic, anti-inflammatory, antileukemic, antimicrobial,

4


antimutagenic, antioxidant, antitumor, antiviral, antiulcer, aphrodisiac, astringent, carminative,

cytostatic, cytotoxic, hypocholesterolemic, hypotensive, hypotriglyceridemic, hypoglycemic,

immunostimulant, insecticidal, laxative, purgative, refrigerant, stomachic, styptic, toxic,

vermifuge activities etc. (Duke, 1986).

The seeds have demonstrated the ability to induce abortions in rats and mice. Two

abortifacient proteins, a- and f3-momorcharins have been isolated from seeds of Momordica

charantia (Ng eta/., 1988). The roots have been documented to possess uterine stimulant

. effect in animals and momorchochin, an abortifacient protein of 32 kDa glycoprotein have

been isolated from tubers of Momordica cochinchinensis (Yeung eta!., 1987). The fruit and

leaf of bitter melon have been reported to exert an in vivo antifertility effect in female animals

(Saksena, 1971; Stepka eta/., 1974); in male animals, these were reported to affect the

-testicular function (Dixit eta/., 1978; Naseem eta/., 1998).

A novel phytochemical isolated from Momordica charantia has been shown to possess

ability to inhibit an enzyme named guanylate cyclase. This enzyme is linked to the

pathogenesis and replication of psoriasis and also with leukaemia and cancer (Vesely et

a/., 1977; Claflin et a/., 1978; Takemota et a/., 1980, 1982). Another group of proteins

having cytotoxic activity are ribosome inactivating proteins (RIPs) isolated from fruits and

seeds of Momordica. These include a- and f3-momorcharin {Fong et a/., 1996).

y-momorcharin (Pu eta/., 1996), momordin (Terenzi eta/., 1996) and charantin {Prakash et

a/., 2002). Ribosome inactivating proteins (RIPs)-arrest protein synthesis by virtue of their

N-glycosidase activity, which cleaves the adenine at position 4324 of ribosomal RNA. RIPs

are endowed with a host of other activities including antiproliferative, antitumor,

immunomodulatory and antiviral activities (Ng et a/., 1992). Momordin has clinically

demonstrated cytotoxic activity against Hodgkin's lymphoma in vivo (Bolognesi eta/., 1996).

Other in vivo studies have shown cytostatic and antitumor activity of the entire plant of

bitter melon (West eta/., 1971; Jilka eta/., 1983). Hot water extract of the entire plant have

also been reported to inhibit the development of mammary tumors in mice (Nagasawa,

. 2002). Investigations using in vitro approach have also demonstrated the anti-cancerous

and anti-leukemic activity of Momordica against numerous cell lines including liver cancer,

human leukemia, melanoma and solid sarcomas (Takemoto eta/., 1982; Singh et at., 1998).

Antimutagenic activity of M. charantia green fruits is attributed to acylglucosylsterols

and is shown to reduce the number of micronucleated poly-chromatic erythrocytes induced

by the well know'"\ mutagen mjtomycin C by about 80% (Guevara eta/., 1990). Analgesic

effect of M. charantia· seed extract has also been reported in mice and rats (Biswas eta/.,

1991 ). However, no evidence of the presence of antimalarial activity has been detected

(Amorim eta/., 1991; Ueno eta/., 1996).

5


In vitro antiviral activity of bitter melon against numerous viruses including Epstein Barr,

herpes and HIV virus has also been documented. Leaf extracts of the plant are shown to

increase resistance to viral infections as well as enhanced immunostimulant effect in humans

and animals by increasing interferon production and natural killer cell activity (Cunnick et

a/., 1990). Two proteins, namely a- and ·J3-momorcharin, have been reported to inhibit the

HIV virus in vitro (Ng eta/., 1992). An inhibitor of HIV virus called MAP30 (Momordica anti

HIV protein of 30 kDa) has been isolated and purified to homogeneity from the seeds and

fruits of M. charantia (Lee-Huang eta/., 1990). MAP30 has been shown to inhibit HIV in T

lympocytes and monocytes and does not enter healthy cells. It inhibits HIV integrase (Lee

Huang eta/., 1995). In treating HIV infections, the protein is administrated alone or in

conjunction with conventional AIDS therapies (Bourinbaiar and Lee-Huang, 1995). Antiviral

activity of MAP-30 has also been demonstrated in infections of the Herpes simplex virus

(Bourinbaiar and Lee-Huang, 1996).

Various protease inhibitors viz., trypsin and elastase inhibitors (Hameto eta/., 1995;

Miura and Funatsu, 1995), serine proteinase inhibitors (Hara eta/., 1989; Hayashi et a/.,

1994) have been isolated and characterized from the seeds of M. charantia. A number of

galactose binding lectins, isolated and characterized from M. charantia, (Li, 1980; Komath

eta/., 1996) exhibited different biological activities like inhibition of protein synthesis (Barbieri

eta/., 1979) and lipogenic activity (Ng et at., 1986b). A 7419 Da protein which inhibits the

activity ofan acidic amino acid-specific endopeptidase of Streptomyces griseus, has been

isolated from M. charantia seeds (Ogata eta/., 1991 ).

In addition to these properties, leaf extracts of bitter melon have clinically demonstrated

broad-spectrum antimicrobial activity (George and Pandalai, 1949; Khan and Omoloso,

1998). Various water, ethanol and methanol extracts of the leaves have shown in vitro

antibacterial activities against E. coli, Salmonella, Shigella, Pseudomonas, Streptobacillus

and Streptococcus (Hussain and Deeni., 1991; Omoregba et at., 1996). Momordica was

among the 6 out of 50 Puerto Rican plants, which were active against Mycobacterium

tuberculosis in vitro (Frame et at., 1998). The growth of the Helicobacter pylori {stomach

ulcer-causing bacteria) have shown to be inhibited by fruit extract of M.charantia (Yesilada

et a/., 1999). Lalt>oratory investigation on the repellency of some plant oils to the red flour

beetle, Tribolium castaneum Herbst revealed that M.charantia has got repellent activity of

th,e class IV (Mohiuddin eta!., 1987).

1.1.1 Hypoglycemic Potential of M. charantia

M. charantia is a common folklore remedy for diabetes. Fruit extract of M. charantia

was first documented to have hypoglycemic activity by Rivera (1941, 1942). Later, extracts

of fruit pulp, seed, leaves and whole plant of Momordica have been shown to possess

6


hypoglycemic activity in animal models of diabetes (Sharma eta/., 1960, Gupta and Seth,

1962; Jose eta/., 1976; _Kedar and Chakrabarti, 1982; Ali eta/., 1993).

Leatherdale eta/. (1981) have demonstrated hypoglycemic activity of Momor:dica fruit

in both normal and diabetic humans, suggesting that the Karela fruit mimics insulin action

in humans. The dried fruit powder, when administrated orally in mild and moderate chronic

diabetics, reduced blood and urine glucose 4ev~ls. It has also been shown that oral

administration of the fruit juice improves glucose tolerance in both non-insulin dependent

diabetics in human (leatherdale eta/., 1981; Akhtar, 1982; Welihinda eta/., 1986) and

diabetic rats (Ali eta/., 1993; Sarkar eta/., 1996). M. charantia showed potent hypoglycemic

effect when administrated subcutaneously to gerbils, langurs and humans (Khanna et a/.,

1981 ). Aqueous extract of M. charantia reduced hyperglycemia by 50% in streptozotocin

(STZ) diabetic mice (Bailey et al., 1985; Day et a/., 1990). Ethanol extract of M. charantia

showed an anti-hyperglycemic as well as hypoglycemic effect in normal and STZ diabetic

rats (Chandrasekar eta/., 1989; Shibib eta/., 1993). Singh and coworkers (1989) have

shown that oral administration of acetone extract of fruit powder of M. charantia for 15-30

days to alloxan-diabetic rats lowered the blood sugar and serum cholesterol levels to normal

range and the blood sugar was found normal even after 15 days of discontinuation of the

treatment. Oral administration of aqueous extract of M. charantia, but not of ethanolic

extract, exhibited anti-hyperglycemic and hypoglycemic effect in cyproheptadine-induced

hyperglycemic and normoglycemic mice, respectively (Cakici eta/., 1994).

Wong and co-workers (1985) have isolated a lectin and saponin enriched fraction from

saline extract of seeds by differential acetone precipitation and demonstrated an in vitro

antilipolytic activity in isolated rat adipocytes. A saponin (steryl glycosides) isolated from

M. charantia seeds exhibited antilipolytic activity. The saponins acted as a non-competitive

inhibitor of corticotrophin, glucagon and epinephrine in lipolysis and antagonized cAMP

induced lipolysis in rat's adipocytes culture (Ng eta/., 1986a). An acid-ethanol extractable

fraction of M. charantia seed and fruit contains saponins that can be precipitated with

acetone. A saponin containing fraction inhibited both lipolysis and 3H-glucose incorporation

into lipids (Ng et a/., 1987). lnsulinomimetic activities {anti-lipolytic and lipogenic) of a

Momordica lectin belonging to galactose-binding lectins in isolated rat and hamster

adipocytes have beeri demonstrated (Ng eta/., 1986b, 1989). The antilipolytic potency of

these lectins are :third highest, the first two being man nose binding and N-acetyl-glucosamine

binding lectins. Ng eta/. (1986c) have a{so reported the presence of insulin-like molecules

in M. charantia seeds. It resembles insulin in extractability by acid-ethanol, in their acid

stability and ability to stimulate lipogenesis and inhibit lipolysis. The chromatographic

behavior of these molecules suggested that they are small molecular weight peptides,

7


approximately 5 kDa, which are responsible for hypoglycemic effect. However, no

(corresponding) band of this size was visible on SDS-PAG. Thus, the hypoglycemic activity

of M. charantia fruit and seed extracts ,can be attributed to a mixture of steroidal saponins,

insulin-like peptides and lectins.

The pulp juice of M. charantia lowered fasting blood glucose levels in normal rats;

however, the effect was more pronounced with the saponin-free methanol extract of the

pulp juice (Ali efa/., 1 993). Charantin, a peptide isolated from M. charantia lowered fasting

blood sugars in rabbits gradually beginning from first and lasting till the fourth hour and

slowly recovering to the initial level (Lolitkar and Rajarama-Rao, 1 966). Aqueous juice of

M. charantia fruit exerted anti-hyperglycemic and anti-oxidant effect in pancreas of STZ

diabetic mice (Sitasawad eta/., 2000). Oral supplementation with freeze-dried powder of

M. charantia for 14 days, with and without 0.5% cholesterol in the diet, resulted in a consistent

decrease in serum glucose levels in normal rats only in the former group, thus exhibiting

anti-atherogenic effect (Jayasooriya eta/., 2000).

Diamed, a herbal formulation composed of aqueous extracts of three medicinal plants

-Azardirachta indica, Casia auriculata and Momordica charantia, caused antihyperglycemic

action in alloxan-induced diabetic rats. It also prevented a decrease in body weight (Pari et

a/., 2001).

1.1.1.1 Possible mechanisms of hypoglycemic action of M. charantia

Kedar and Chakrabarti (1 982) reported activation of some of P-cells with M. charantia

seed treatment and returning of granulation to normal, thus resulting in insulinomimetic

activity. Aqueous extract of unripe fruits of M. charantia has also been shown to partially

stimulate insulin release from isolated beta-cells of obese-hyperglycemic mice, which differed

from D-glucose and other insulin secretagogues agent in the manner by not being

suppressed by L-epinephrine and in even being potentiated by the removal of Ca2+. This

suggested that the insulin-related action is the result of perturbations of membrane functions

(Welihinda eta/., 1982). The fruit juice significantly increased the number of beta cells in

diabetic rats (Ahmed eta/., 1 998). STZ diabetic rats fed with a diet containing M. .charantia

for 6 weeks did not show beneficial hypoglycemic effect and neither prevented diabetes

related abnormalities in the levels of protein, urea and creatinine {Plate! and Srinivasan,

1 995). In another study, a diet containing M. charantia for 8 weeks did not affect blood

sugar, food intake, growth, organ weight and hematological parameters of normal adult

rats, while it caused a significant hypo-cholesterolemic effect (Plate! eta!., 1 993). In addition,

glycosylated hemoglobin concentration remained unchanged in treated and untreated

diabetic rats. Results of these investigations suggest that viable beta cells are required to

manifest the hypoglycemic activity of M. charantia (Karunanayake eta!., 1 990).

8


Contrary to the above reports are the studies conducted by Sarkar et at. (1996) and

Akhtar eta/. (1981), who reported hypoglycemic action in normal and STZ and alloxan

diabetic animals, without significant change in insulin levels. Thus, this could involve extra

pancreatic mechanisms of bringing down glucose levels. Indeed, experiments in rats have

demonstrated that two important constituents of M. charantia i.e. oleanolic acid 3-D

glucuronide and momordin lc exert anti-hyperglycemic effect by inhibiting glucose transport

at the brush border of the small intestine (Matsuda et at., 1998).

Oral administration of different M. charantia extracts showed a varying pattern of anti- ·

hyperglycemic effect without altering the insulin response suggesting a mechanism of action,

which is independent of intestinal glucose absorption and probably involves an

extrapancreatic effect (Day eta/., 1984). Oral feeding of M. charantia juice to normal rats

prior to glucose loading increased hepatic and muscle glycogen content without altering

the triglyceride content. Fruit juice increased glucose uptake by diaphragm tissue in vitro

without concomitant increase in tissue respiration, suggesting the ability of the M. charantia

extract to directly stimulate glucose uptake (Welihinda and Karunanayake, 1986),

Collectively these investigations suggest that hypoglycemic effect of M. charantia is

not, solely, due to increased secretion of insulin, but also due to increased carbohydrate

utilization. This is supported by studies conducted by Shibib et at., (1993), who attributed

blood glucose lowering potency of ethanolic extract of fruit to depression of glyconeogenic

enzymes glucose-6-phosphatase and fructose1,6-bisphosphatase in the liver and stimulation

of red blood celf and hepatic glucose-6-phosphate dehydrogenase activity. Presence of at

least two inhibitory compounds in M. charantia fruit extract, one inhibiting hexokinase and

other inhibiting the uptake of glucose by intestinal fragments in rats in vitro has been

elucidated (Meir and Yaniv, 1985).

Diabetes is usually accompanied by various complications like retinopathy, neuropathy,

ketoacidosis, retinitis etc. Jones and Cerami (1985) have shown that exposure to higher

concentrations of glucose leads to a non-enzymatic glycosylation of lens proteins resulting

into opacity and cataract. Daily administration of extract ofM. charantia fruit for 2 months to

alloxanized diabetic rats delayed the development of cataract (Srivastava eta/., 1988)

Anti-oxidant activity has also been described in Karela extracts (Dhar eta/., 1999). Shi

eta!. (1996) have shown that fruit extract of Momordica scavenged superoxide and hydroxyl

radicals and inhibited lipid peroxidation.

In clinical trials, fruit juice of Momordica charantia significantly improved glucose . .tolerance

of 73% of patients with maturity-onset diabetes (Welihinda eta/., 1986). Fried Karela fruits

consumed as a daily supplement to the diet produced a small but significant improvement

in glucose tolerance in diabetic subjects without any increase in serum insulin levels

9


(Leatherdale eta/., 1981 ). Other clinical trials of Karela with human patients did not show

any visible sign of toxicity or any other side effect (Akhtar, 1982; Khanna .et a/., 1981 ). To

ascertain whether M. charantia extract had any possible hepatotoxicity, Tennekoon eta/.,

(1994) performed experiments to study the effects on key hepatic enzymes: Serum

y-glutamyl transferase and alkaline phosphatase concentration was found to be significantly

elevated following oral administration of both th~ fruit and seed extract. However, consistent

significant histopathological changes in the liver were not observed thus ruling out the

possibilities of having hepatotoxins capable of causing cellular changes at the molecular

levels. All these investigations strongly suggest the presence of the compound(s) with

insulinomimetic activities in M. charantia fruits and seeds.

Presence of antimicrobial activities in various parts of M. charantia has been

demonstrated. However, till date no reports demonstrating antifungal activity has been put

forth. Seeds are generally exposed to all kinds of microorganisms during germination, so

presence of protein having antibacterial and antifungal activities have been detected in

seeds. Though, one of such proteins napin-like proteins have been isolated from seeds of

bitter gourd (Neumann et a/., 1996c), no biological activity has been demonstrated for

these proteins either in vivo or in vitro.

Thus, virtually whole plant of M. charantia possesses one or the other therapeutic

activities and some of the active principles involved in these activities have been identified.

Our laboratory has been interested in identifying active principles with hypoglycemic activities

from M. charantia seeds. These investigations have led to the purification, identification

and partial characterization of at least two active components that elicit hypoglycemic effect

in M. charantia seeds (Choudhary, S. K., 1998).

10


1.2 SEED STORAGE PROTEINS

Plant seeds play a vital role in propagation. They serve as a major source of ·dietary

protein for mankind. The amount of prote_ins in seeds varies from -10% (in cereals) to

-40% (in legumes and oilseeds) ofthe,dry weight. Majority of these proteins haye metabolic

or structural roles. There are also one or two groups of proteins in high amounts, which

serve as stores of amino acids for use during germination and seedling growth. These

seed storage proteins have few common properties. First, they are synthesized at high

levels at certain stages of development in specific tissues and act as long-term amino acid

stores as their time of formation is well separated from moments of their major breakdown.

Second, these proteins in mature seeds are present in discrete deposits called protein

bodies. Third, all seed storage proteins exhibit polymorphism, which arise from the presence

of multigene families and in some cases, proteolytic processing and glycosylation.

In general, all seed storage proteins are secretory proteins synthesized with a signal

peptide, which is cleaved as proteins are translocated into the lumen of the endoplasmic

reticulum (ER). Seed storage proteins have been classified by Osborne (1924) on the

basis of their extraction and solubility in water (albumins), dilute saline (globulins),-alcohol/

water mixtures (prolamins) and dilute acid or alkali (glutelins). The major seed storage

proteins include prolamins, globulins and albumins. Since the present research work focuses

on a protein belonging to 2S albumins, therefore this will be reviewed in greater detail as

compared to the other classes of seed storage protein.

1.2.1 PROLAMINS

Prolamins' presence is restricted to one family, the grasses, which include major cereals.

Prolamins usually account for -50% of total grain nitrogen except in oats and rice where

11 S globulin-like proteins are major storage protein and prolamins are present only at low

concentrations (-5 to 10% ).

Prolamins of triticeae (barley, wheat and rye) have been best studied. Prolamins of

these species are highly polymorphic mixtures of components whose molecular weight

(M,) ranges from -30,000 to 90,000 (Shewry eta/., 1995). These prolamins are classified

into three groups (Miflin eta/., 1983) based on their amino acid composition. These are the

S-rich, S-poor and high molecular weight (HMW) prolamins.

The S-rich prolamins are major prolamins among all three species and consist of at

least two families in each species: the J3- and y-hordeins of barley; two types of y-secalin of

rye; and the a-g·liadins, y-gliadins and low molecular weight glutenin subunits of wheats

(Shewry eta/., 1995). The S-poor prolamins include c-hordein of barley, the w-secalins of

rye and the cu-gliadins of wheat. Both S-rich and S-poor prolamins have repeat sequences

11


at N-terminus while the S-poor prolamins have repeated sequence at their C-terminus. As

the name indicates S-rich prolamins have cysteine residues while S-poor lack these. The

HMW prolamins have extensive repeated sequences flanked by non-repeated N- and

C-terminal domains (Shewry eta/., 1993). Prolamins related to triticeae are also present in

other cereals. These include oats, where avenins have repeats rich in proline and glutamine

(Chestnut eta/., 1989). The prolamins of maize are known as the zeins and grouped as a

, 13-. y- and 8- zeins. The 13-. y- and 8-zeins belong to prolamin superfamily oftriticeae while

a-zeins which account for -75% to 80% of total prolamins in maize are classified into two

groups with slightly different Mr -19,000 and 22,000 (Marks eta/., 1985).

Secretory proteins assume their folded conformations within the .lumen of the ER, which

is also the site of disulfide bond formation. Studies on other systems demonstrate that ER

luminal proteins assist in these processes. Roden et a/. (1982) have shown that protein

disulfide isomerase (POl) associated with the ERin developing wheat endosperms, facilitates

disulfide bond formation of prolamins. Molecular chaperones of the HSP70/BiP family are

present in developing endosperms of rice (Li eta/., 1993), wheat (Giorini and Galini, 1991)

and maize (Boston eta/., 1991 ). In developing cereal endosperms, two routes of protein

body formation appear, one in which protein body forms from the vacuole and the other

from the ER. Prolamins of rice (Krishnan eta/., 1986) and maize (Larkins and Hurkman,

1978) are retained within lumen of ER and distended to form protein bodies while rice

glutenins are of vacuolar origin.

1.2.2 GLOBULINS

The globulins are the most widely distributed group of storage proteins; they are present

in dicots, monocots (including cereals and palms) and also in fern spores (Templeman et

a/., 1987). Globulins are divided into two groups based on their sedimentation coefficients:

the 7S vicilin-type globulins and the 11 S legumin-type globulins. The 11 S legum ins are

major storage proteins in most legumes and in other dicots like members of brassicaceae,

compositeae and cucurbitaceae and some cereals like oats and rice. The mature protein

consists of six subunit pairs that interact non-covarantly. Each of these subunit pairs consists

of an acidic subunit of Mr -40,000 and a basic subunit of Mr -20,000, linked by a single

disulfide bond. Each subunit is synthesized as a precursor protein that is proteolytically

cleaved after disulfide bond formation {Boulter, 1981 ). Legum ins are not glycosylated, an

exception being the 12S globulin ~f lupin (Duranti eta/., 1988). The study of edestin, an

11 S globulin from hempseed showed that the subunits were arranged in an open ring

structure, oriented alternately up and down, in the disk with a diameter of 145A and thickness

of -90A (Patel eta/., 1994).

12


7S globulins are trimeric proteins ofMr -1"50,000 to 190,000 that lack cysteine residues

and hence -cannot form disulfide bond. They vary in subunit compositions because of

differences in the extent of post-translational processing (proteolysis and glycosylation).

The vicilin subunits of pea are initially synthesiz.ed as groups of polypeptides of Mr -47,000

and -50,000. However, post-translational proteolysis and glycosylation result in the .formation

of subunits with M values between 1.2,500 and 33,000 (Gatehouse eta!., 1984; Casey et r '

a!., 1993). On the other hand, 7S phaseolin of P. vulgaris consists of highly glycosylated

subunits with Mr values between -43,000 and 53,000 (Hall eta/., 1977; Bellini and Chrispeels,

1978) and does not undergo proteolysis. The three dimensional structures of 7S globulins

determined using X-ray crystallography (Lawrence eta/., 1990; Ko eta/., 1993) showed

that these proteins are disk shaped, with diameters of -90A and thicknesses of 30- 40A.

N-linked glycosylation of the 7S phaseolin subunits occurs in the ER lumen as a

cotranslational event (Bellini eta/., 1983; Vitale eta/., 1993).

The assembly of 11 S globulins is a highly regulated event where monomeric proteins

are initially assembled in the lumen of the ER into trimers. The trimer is then transported to

the storage vacuole where they assemble into hexameric form. The assembly requires

specific proteolytic cleavage of the subunits present in the trimers (Dickinson eta/., 1989).

A vacuolar protease responsible for processing of 11 S globulins has been identified from

several species like soya bean (Scott eta/., 1992) and castor bean (Hara-Nishimura et a/.,

1993). This protease specifically recognizes asparagines processing site.

In leguminous plants, the 7S and 11 S globulins appear to be in the same protein bodies

with no spatial separation (Harris eta!., 1993).

1.2.3 2S ALBUMIN SEED STORAGE PROTEINS

A group of seed storage proteins having sedimentation coefficients of -2 are defined

as 2S albumins (Youle and Huang, 1981). They are widely distributed in dicotyledons and

occur in diverse plant species. The amount of 2S protein in the total seed storage proteins

ranges from 20% in peanut to 62% in sunflower and mustard, accounting for 20-25% of

total seed dry weight. The 2S albumins possess characteristics distinct from other classes

of seed storage proteins, which include low molecular weight, high solubility in water, high

cysteine content and extremely high nitrogen content (Youle and Huang, 1981 ).

The 2S albumins have been characterized in Ricinus communis {Castor bean; Youle

and Huang, 1978; Sharief and Li, 1982), G;ssypium hirsutum (Cottonseed; Youle and

Huang, 1979; Galau eta/., 1992), Brassica spp. (Mustard; Lonnerdal and Jansen, 1972;

Ericson eta!., 1986), Lupin us spp. (Lupin, Gerritsen, 1956; Lilley and Inglis, 1986), Raphanus

sativus (Radish; Laroche-Raynal et at., 1984; Monsalve eta/., 1994) Bertholletia excelsa

.(Brazil Nut; Ampe eta/., 1986) Hellianthus annus (Sunflower; Kortt and Caldwell, 1990),

13


Arabidopsis thaliana (Krebbers eta/. , 1988), Cucurbita pepo (Pumpkin; Hara-Nishimura et

a/., 1993) and Momordica (Karela; Chang eta/., 1995).

A significant part of the research about 2S albumins has been performed on Brassica

napus and therefore, these are generally referred to as napins.

1.2.3.1 STRUCTURE

Lonnerdal and Janson ( 1972) were the first to isolate and report low molecular weight

proteins from seeds of Brassica napus (napins) having molecular weight of about 12,000-

14,000 Da, rich in glutamines, highly basic (pl=11) and composed of two subunits (9,000 and

4,000 Da) bound together with two disulfide bridges. Crouch eta/. (1983) have characterized

napin eDNA clones of Brassica napus and predicted a precursor polypeptide of 178 amino

acids, consistent with the 21 ,000 Da in vitro translation product. Comparison of the deduced

amino acid sequence of this precursor (schematically depicted in Fig . 1; Crouch eta/., 1983)

with published amino acid compositions (Lonnerdal and Janson, 1972) of mature nap in subunits

suggest that both the large and the small subunits are present in one precursor polypeptide

and that other regions of the precursor are removed during processing.

Ericson et a/.,(1986) have shown that the 178 amino acid long precursor of napin, is

subsequently processed through proteolytic events and generate two mature napin chains

of 86 and 29 residues. Similarly, the eDNA of Ricinus communis 2S albumin encodes a

precursor of 258 amino acids residues (Mr 34,000 Da; Irwin and Lord, 1990), while the

Region

Net Charge Residue

1 2 3 4 Signal Negative Small Subunit Negative

5 Large Subunit

Fig.1 Regions of the putative napin precursor polypeptide based on am ino acid composition differences. Shaded areas are the putative subunits of the mature protein.

mature protein (Mr 10,900 Da) consists of two subunits of Mr 7,000 and 4,000 Da linked by

disulfide bonds (Sharief and Li, 1982).

Six different napins have been reported in B.napus on the basis of size, by column

chromatography (Lonnerdal and Janson; 1972). Crouch eta/. , (1983) were able to obtain

two clones of napin by differential colony hybridization whose nucleotide sequences

demonstrated 95% homology. The southern blots and reconstruction experiments suggested

that napins are multigene products encoded by a gene family containing over 16 members

(Scofield and Crouch , 1987). The genomic (napA, napB, BngNAP1 and gNa) and eDNA

(pN1 , pN2 and pNAP1) clones for napin variants have been identified and characterized

(Jossefson eta/., 1987; Scofield and Crouch, 1987; Baszczynski and Fallis, 1990; Byczynska

14


and Barciszewski, 1999). This -is further confirmed by the presence of multiple forms of

napins in Brassica napus and Sinapis alba (yellow mustard) (Neumann .eta/., 1996a,d).

Napin variants show high degree of homology, which makes their separation and purification

very difficult. Analysis of napin variants by MALO! mass spectrometry rev.ealed that napin

protein BngNAP1 and napA as well as gNa correspond well with reported gene sequences

(Gehrig eta/., 1996). Three other short chains, termed BngNap1 ', BngNAP1A and gNa'

and long chains BngNAP1 B, BngNAP1C and gNaA differ by 1-4 r:esidues from sequences

of BngNAP1 and gNa, respectively. The mass spectrometric analysis also indicated that

most of the napin chains were present as partially truncated forms at either or both termini

(Gehrig et a/., 1996).

Monsalve eta/. (1991) have isolated low molecular weight (LMW) nap ins (12.5 kDa)

nla and nib from the B. napus seeds, which possess common structural featur:es of the 2S

albumins and showed only 54% similarity to major napins (14.5 kDa). Thus, these

investigators established the existence of a new distinct group of napins LMW-napins.

Different isoforms of 2S albumin proteins are also reported in other plant species. In

case of radish, two-dimensional electrophoresis of in vitro translated products of napin

mRNA showed presence of eight distinct precursors (Laroche-Raynal and Delsey, 1986).

Napins of radish are classified into two subfamilies based on existence of two different

sized precursors. Sequences of the two subfamilies are very well conserved and display an

organization similar to homologous genes from rapeseed and Arabidopsis. Based on

Southern blot analysis, it is established that 2S albumins of radish are also a multigene

family represented by eight to twelve members, of which three eDNA clones have been

characterized (Raynal eta/., 1991 ). Nine 2S albumin proteins from garden and sea radish

seeds (Raphanus sativus and Raphanus raphanistrum, respectively) have been purified

and their amino acid compositions are typical of napin-like proteins and differ only slightly

in basic residues content (Monsalve eta/., 1994).

2S storage proteins of Brazil nut, Bertholletia exce/sa (Bn-2S) exhibit abnormally high

amount of methionine in addition to high le~els of cysteine (Hirs, 1956). Ampe et at., (1986)

have shown that Bn-2S consists of two subunits of 28 residues and 73 residues interlinked

-by two sulfur bridges. They also observed sequence heterogeneity, with multiple large

subunits and one major small subunit, demonstrating that these storage proteins belong to

multigene family with at least six functional structural genes. The small subunit of Brazil nut

napin showed remarkable feature of three repeated tetrapeptide sequences of the type

·Arg-Gix-Gin-Met, which is not found in the corresponding regions of other 2S storage

proteins. The cDNAs coding for two isoforms of 2S albumins of Bertholletia excetsa have

been isolated and characterized (Aitenbach et at., 1987). Two other genes, BE2S1 and

15


BE2S2, have been isolated and characterized by Gander eta/. (1991). They have shown

that 2S genes of B. excelsa have introns located in the coding region of small subunit, in

contrast to 2S genes of B. napus, which contain no introns.

2S albumins in Arabidopsis are encoded by a small gene family, whose four members

(AT2S1, AT2S2, AT2S3, AT2S4) have been isolated and fully characterized. The four genes

are arranged in a tandem array where the intergenic distances are fairly small (1.4, 1. 7 and

1.3 kb). Unlike B. exce/sa, none of the genes of arabidopsis contain an intron in the protein

encoding region (Krebbers eta/., 1988).

Three napin-like complexes have been resolved from Momordica charantia seeds. Each

of three small chains complexes with one large chain and disulfide bonds are involved in

complex formation (Chang eta/., 1995). Neumann eta/., (1996c) have shown presence of

yet another small chain. The small chain as well as large chain shows homology to 2S

albumin proteins of Cucurbita maxima, Ricinus communis and Brassica napus.

Other variant of 2S albumins also occur eg. 2S albumin of pea lack interchain disulfide

bonds (Higgins eta/., 1986), whereas the 2S albumins of sunflowers remain uncleaved

(Kortt and Caldwell, 1990). In addition, in sunflowers and castor bean, some mRNAs encode

two mature albumin proteins, each consisting of one or two subunits (Allen et a/., 1987;

Irwin eta/., 1990).

All the 2S albumins are compact globular proteins with conserved cysteine residues

showing a pattern of eight cysteine residues, --C-C--1-CC-CXC-C-C-. The

structural stability of Brassica napus napin in solution has been studied by microcalorimetry.

The thermodynamic data reveals that the napin is very stable since its transition temperatures

at pH 6 and 3 are 1 00.3°C and 80°C, respectively (Krzyzaniak et a/., 1998). The highly

stable structure of napin is presumably due largely to four disulfide bonds in napins. The

importance of disulfide bonds in stabilizing the foldings of 2S albumins is also reflected by

the significant change in ellipticity of napin measured by CD under reducing conditions

(Schwenke eta/., 1988).

Secondary structure predictions based on the primary structure suggested high content

of a-helix in napin. These predictions are further supported by conclusions drawn from CD

spectroscopy data of napin (Schwenke et a/., 1988). Analysis of CD spectra of pronapin

and napin of Brassica napus have shown presence of 34% and 50% a-helix, respectively,

confirming conformational differences between the two forms {Muren et at., 1996). High

content of a-helix (-50%) have also been reported in 2S protein$ from Sinapis alba (yellow

mustard; Menendez-Arias et at., 1987) as well as from Raphanus raphanistrum and R.

sativus (Monsalve et at., 1994).

16


The proteolysis experiment suggested that most of the cleavage sites were located in

the propeptides, indicating that the portions of mature napins are tightly folded (Muren et

a/., 1996). Menendez-Arias eta/. (1988) have also shown that mature 2S proteins are

resistant towards proteolysis. Three cleavage sites for endopeptidases have been identified

within the small subunit but only one in large subunit, indicating that small subunit is less

tightly folded than the large subunit (Muren eta/. , 1996).

To have a better insight about the structure of napin, one would like to know the exact

three-dimensional (30) structure, which is lacking except the NMR studies conducted on

Bn 1 b nap in-like protein belonging to LMW-napins (Rico et a/., 1996). Bn 1 b consists of

two short polypeptide chains of 3.8 and 8.4 kDa. 1H spectrum of napin Bnlb showed that

light chain is split into two a-helices (I and !1) connected by a two to four residue extended

strand . The C-terminus of the small subunit and theN-terminus of large subunit is situated

close in space suggesting that the two are linked at some stage in the protein expression.

The first a-helix (a helix II) of large subunit runs antiparallel to helices I and 11 followed by

a short loop and helix Ill, which serves as the protein backbone as it anchors the N-

Fig. 2 Structure of Bn 1 b (LMW-napin) of Brassica napus (Rico et a/., 1996)

17


terminus of the light chain and the C-terminus of the heavy chain through the disulfide

bridges {Cys25-Cys5 and Cys 27 and Cys70, respectively). A short loop of eight residues

rich in glutamines is present between helix Ill and helix IV. Helix IV is the shortest helix and

after this helix, there is a long loop without any secondary structure which folds over itself.

The additional disulfide bridges are between residue Cys18 of helix 11 of small subunit and

Cys14 of helix II of large subunit and the residues Cys15 of helix II andCys62 inC-terminal

large loop of large subunit: The arrangement of different consecutive helices and loops

corresponds to a right-handed helical disposition (Rico eta/., 1996).

The disulfide bridge organization of napin Bnlb seems to be same as that for other 2S

albumins, for which an experimental determination of the .location of the disulfide bridges

has been carried out (Nirasawa eta/., 1993; Gehrig and Biemann, 1996). Analysis of the

modeled 3D napin structure indicates that the three a-helices of the napin large subunit

form a cleft in which two a-helices of the small subunit fit well (Rico eta/., 1996).

All napins show high sequence homology and their overlapping CD spectra demonstrate

high similarity in their secondary structure. Therefore, it is suggested that all napins and

napin-like proteins belong to same superfamily with a very similar 3D structure {Rico et al.,

1996).

1.2.3.2 PROCESSING OF 2S ALBUMINS

Storage protein precursors have N-terminal signal peptides, which are responsible for

their cotranslational transport from the cytoplasmic side of the rough endoplasmic reticulum

into the endomembrane system as shown by in vitro translation of globulin mRNA from

Vicia faba (Puchel et at., 1979) and hordein mRNA from Barley (Cameron-Mills et a/.,

1978). Secretary and vacuolar proteins are then transported from ER to Golgi apparatus

where sorting into different vesicles occurs (Chrispeels, 1991 ). As the secretary proteins

do not appear to have any additional targeting information, they appear to be secreted by

default (Bar-Peled eta/., 1996). The sorting information in case of soluble vacuolar protein

appears to reside in primary structure of polypeptides. Three different types of vacuolar

targeting have been directed so far: short targeting peptides at the N-terminus such as in

sporamin (storage protein of sweet potato; Nakamura et al., 1993); short targeting peptides

at the C-terminus as in Brazil nut 2S albumin (Saalbach et at., 1996); and the long internal

sequence stretches, present in mature polypeptides as in legumin and vicilin (von Schaewen

et at., 1993; Saalbach eta! .. 1991 ). · .·.·

Pulse-chase experiments and subcellular fractionation studies, combined with data

obtained through monensin treatment (which blocks traffic at the medial Golgi and affects

the vacuolar pH, Tartakoff. 1983), suggest that processing occurs in the protein storage

vacuole (Chrispeels eta/., 1982; Higgins eta/., 1983; Stinissen eta/., 1985; Holwerda eta/.,

18

•

I .


1990; Matsuoka eta/., 1990) The pro 2S albumin from pumpkin was shown to be in dense

vesicles and processed to mature 2S albumin by vacuolar processing enzymes in the vacuole

(Hara-Nishimura eta/., 1993).

Pulse chase experiments also suggest that 2S albumin processing in Bertholletia .is a

multi-step process (Sun eta/., 1987). Comparison of napin of B.rassica napuswith the 2S

protein arabidin in Arabidopsis thaliana showed that proproteins are more conserved ~han

the mature chains (Krebbers et al., 1988). This suggested that propeptides play important

role in processing of these proteins. However, deletion of major portions of the pr,opeptides,

or even entire propeptides, did not inhibit the transport or processing of napin of Brassica

napus (Muren et a/., 1996) or arabidin of Arabidopsis thaliana (D'Hondt et a/., 1993b) in

transgenic tobacco seeds.

The processing of the 2S albumins has not been investigated in detail but appears to

involve several processing enzymes (D'Hondt eta/., 1993b; Muren eta/., 1996). Different

classes of proteinases that have been isolated from plant seeds include aspartic proteinases

(Sarkkinen eta/., 1992; D'Hondt eta/., 1993a), cysteine proteinases (Hara-Nishimura et

a/., 1991; Scott eta/., 1992; Abe eta/., 1993) and a serine proteinase (Morita eta/., 1994).

Since the internal processed fragment and N-terminal processed fragment end in ari

asparagine in most of the 2S albumins except in few pro 2S albumins like pN1 and napA,

it is possible that vacuolar enzyme recognizes this as a cleavage site. Hara-Nishimura et

a/. ( 1991) have isolated a vacuolar -processing enzyme (VPE) from R. communis that cleaves

on the C-terminal side of an asparagine residue. They also demonstrated in vitro cleavage

of a pumpkin 2S albumin precursor by this thiol protease. Similar cysteinyl protease from

soya bean involved in processing of 11 S globulins has also been isolated (Scott et a/.,

1992). Muren eta/., (1996) have shown that thiol protease is not the only endopeptidase

involved in processing because the mutant protein having mutation in the recognition site

of these peptidases were also processed in a similar manner as wild-type protein. D'Hondt

et a/. (1993a) described a B. napus seed aspartic acid endoprotease activity, which was

able to cleave the -internal pro peptide of arabidin at two sites within its central portion.

Muren and Rask{1996) have also shown that additional endopeptidase, other than aspartic

proteinase, are involved in pronapin processing as processing of pronapin occurs in vitro

by embryo extract even after aspartic proteinase is inhibited. Later, Hiraiwa eta!., (1997)

showed that aspartic proteinases are involved in the degradation of detached propeptides.

It has been proposed that the two propeptides and the C-terminal part of the pmproteins

are exposed at the surface of the protein. The internal propeptide is probably attacked by a

mixture of endoproteases, then carboxy peptidases and amino peptidases remove exposed

portions of the propeptides to the extent that the three-dimensional structure of the protein

19

No. Processing scheme

1 N c

: ·~;----'------'1-------, N

~ ~ NC c . N c !

s Ns C

2 N C

N C

Subunit name (processing

enzyme)

Subunits of legumin like 12S globulins

Severallections

(signal peptidase, VPE) Napin, 2S albumins

(signal peptidase, VPE, unknown enzymes?)


Plant species

Pisum sativum, Viciafaba, Glycine max, Lupinus spec., Avena sativa, Oryza sativa

Brassica napus Berthol/etia exceisa Cucurbita pepo

Fig.3 Diagrammatic representation of seed storage protein processing. N, C, N-and C- terminus of the polypeptide; hatched fragments. signal peptides; PP, propeptides; SS, disulfide bridge(s); small black bars, cleavable oligopeptides; VPE, vacuolar processing enzyme (MOntz, K , 1998).

is maintained (Murem eta/., 1996). Therefore, the presence of propeptide might maintain

pronapin in a transfer-capable state. Propeptide cleavage in the acidic protein storage

vacuole and detachment of small linker and C-terminal peptides might transform pronapin

into the deposition-capable mature 2S albumin (MOntz, 1998).

Little is known about how storage proteins are organised within protein bodies. However,

organization is important for the efficient use of storage space and in facilitating mobilization

during germination. In many dicots, such as pumpkin, sunflower, mustard and castor bean,

2S albtimins are stored together with 11 S globulins (Shewry et al., 1995).

1.2.3.3 REGULATION OF EXPRESSION OF 2S ALBUMINS

One of the common features of all seed storage proteins is that their synthesis is restricted

to the developing seed. Monocotyledons plants express their storage proteins in the endosperm

20


whereas dicotyledons plants preferentially express their storage proteins in the embryo

(Gatehouse and Shirsat, 1993). Their abundance and tight r:egulated synthesis makes storage

protein genes ideal for understanding the mechanisms of tissue-specific and developmental

gene regulation (Goldber-g eta/., 1989). Studies onvarious storage protein ·promoters revealed

complex regulatory network behind the quantitative and tissue-specific expression of the

individual storage protein gene (Schmidt, 1993; Morton eta/., 1995). Various-cis elements have

bee'n identified which are involved in the control of seed-specific ,expression but to assign

particular role to these cis elements are difficult (Thomas, 1993).

Napin gene promoters from B. napus and A. thaliana show high sequence similarity

(Scofield and Crouch, 1987; Conceicao and Krebbers, 1994). Using electrophoretic mobility

shift assay, several elements, which are able to bind nuclear proteins from B. napus have

been identified (Ericson eta/., 1991; Gustavsson eta/., 1991). Deletion studies performed

on napA promoter showed that the promoter region spanning -309 to +44 is sufficient to

direct high levels of correct tissue-specific expression. However, transgenic tobacco plants

harbouring this construct exhibited earlier expression in endosperm when compared to

B.napus (Stalberg eta/., 1993). It has also been shown that a 98 bp deletion from position

-309 to position -211 relative to transcriptional start site resulted in drastic decrease of

activity. This region harbours several elements strongly inducing embryo-specific transcription

and have sequences showing similarities to cis elements known to be important in other

promoters like binding sequences for the SEF-4, GT-1, AG-1 factors and the RY repeat

(EIIerstrom et a/., 1996). A construct with deletion in its 5· end spanning -152 to -126

region completely abolished this activity (Stalberg eta/., 1993). Further, a deletion of 8 bp

from -152 to -144 caused promoter silencing (EIIerstrom eta/., 1996). The sequences in

this region show high similarity to the abscisic-acid-responsive element (ABRE)

(GCCACGTGTCC) involved in ABA responsiveness of the Em gene from Triticum aestirum

(Guiltinan et at., 1990). The sequence between -148 to -139 is crucial for activity of the

napA promoter and harbour a G-box motif with ACGT core which has been suggested to

be important for high-affinity binding of bZIP proteins (Foster et at., 1994). This region also

shows homology to E-box (CANNTG), which is involved in both tissue-specific and

developmental control of the 13-phaseolin promoter (Burow et at., 1992; Kawagoe eta/.,

1994). Ellerstrom et at. (1996) have also shown that deletion of -133 to -121 regions

altered the tissue-specific regulation within the seed and also resulted in an increased ' ' .. / .

activity in non-seed tissue. This region shows homology to the (CA)n elemenL(Morton et<Jt.,

1995). The (CA)n element of the region -133 to -120 is highly conserved in napin and

arabidin promoters and is involved in embryo-specific expression of j3-phaseolin (Burow et

at., 1992) and glutelin promoters (Vellanoweth and Okita, 1993 ; Zheng eta!., 1993). Later,

~ l!'"r;\)~Ji ,.,,_, '\. ~ '""I \.~·!:.~ ' \\~.,$; / J '.\\? ~~ /'

.... , .. ,_~p;'"\ ~.--:fl

r--··· --

21 583.63

V442 Cl

: .· lllll/llllllllllllllllll/111 Ill · TH10188 \.._~-~.~::~-:,_· _· . --. ---- ---:~--- -~--- ·-- .. :--- --

TH


Ezcurra et at. (1999) denominated the two conserved regions as B-box, the ABRE-Iike

element distB (distal B) and the CA-rich -element proxB (proximal B).

Another motif characterized for seed--specific expression is RY-repeat motifs bordering a

G-box at -78 to -50 (RY/G). The RY-repeat of sph motif is highly-conserved in seed promoters

(Baumlein et at., 1992) and is shown to be crucial for seed~specific expr-ession directed by

leguminin and glycinin promoters (Baumlein et at., 1992; lelievre-eta/., 1992). Based on mutation

analysis and gain-of-function analysis of cauliflower mosaic virus 358 minimal promoter, it has

been proposed that the seed-specific activjty of the napA promoter relies on the combinatorial

interaction between the RY/G complex and the B-box ABA-responsive complex during the

ABA response in seed development (Ezcurra eta/., 1999). Interaction of r-egulators FUS3 and

AB13 with RY-promoter element regulates the gene expression during late embryogenesis

and seed development in Arabidopsis (Reidt eta/., 2000).

1.2.3.4 EFFECT OF ABA ON GENE EXPRESSION DURING SEED MATURATION

low levels of storage proteins are found in embryos removed from seed, but addition of

hormone abscisic acid (ABA) to culture restores the high in vivo accumulation of storage

proteins (Crouch and Sunex, 1981 ). The regulation of napin synthesis by ABA is at the

level of mRNA accumulation as depicted by northern blot analysis (Crouch eta/., 1983) .. In

response to ABA treatment, napin transcripts accumulation (Delisle and Crouch, 1989)

and enhanced reporter gene activity driven by napin promoter have been reported (Jiang

et at., 1996). Finkelstein eta/., (1985) have reported that during the phase of rapid embryo

growth preceding desiccation, ABA plays role in maintaining embryogeny and suppressing

germination. However, during desiccation low water content rather than ABA prevents

germination. It is well established that ABA is a key regulator of gene expression during

seed maturation (Marion-Poll, 1997; Phillips eta/., 1997). Transient analysis experiments

revealed promoter elements mediating ABA-responsive gene expression in seed specific

genes (Hattori eta/., 1995; Shen and Ho, 1995; Vasil et at., 1995; Ono eta/., 1996). Further

analysis showed the composite nature of ABA-responsive complexes (ABRCs), consisting

of an ABRE and a coupling element (Roger and Roger, 1992). Various mutants have been

isolated and characterized in Arabidopsis thaliana like Fus3 and abi3, which shows pleiotropic

effects during embryogenesis (Koornneef eta/., 1984; West et a!., 1994, Baumlein et al.,

1994). It has also been established that the ABI3 is a transcriptional activator important for

ABA-dependent dormancy as well as maturation related gene expression in.Arabidopsis

thaliana embryo (Parcy eta/., 1994).

Jasmonic acid (JA) and its methyl ester, methyljasmonate, are commonly referred to as

jasmonates. JA influences several aspects of plant growth and development, including

inhibition of germination (Wilen et at., 1991, 1994). JA also induces accumulation of seed

22


storage and oil-body protein mRNA in B. napus (Wilen eta/., 1991). Experimental evidence

suggests additive interaction of ABA and jasmonates on inhibiting seed germination in

Arabidopsis (Staswick eta/., 1992). The ability of JAto elicit the expression of the gene

coding for napin appears to be dependent on the endogenous concentration of ABA. During

the late stages of seed development, ABA levels were shown to decline and JA may play a

role in the reduction of ABA concentration in the seed thereby allowing the seed to move

expeditiously from ABA-mediated processes into late seed development {Hays eta/., 1999).

1.2.3.5 DIFFERENTIAL EXPRESSION OF 2S ALBUMIN GENES

Gene families ranging in size from a few genes to more than 100 encode seed storage

proteins. Significance of multiple genes encoding same proteins has been explained using

the rbcS genes, which encode the small subunit of ribulose bisphosphate carboxylase.

Dean eta/., (1985) have shown that individual members of rbcS gene families respond

differently to light of different qualities or other developmental stimuli. At present, little is

known about differential expression and its significance in case of storage proteins. In case

of Arabidopsis thaliana, the expression of all four genes encoding 2S albumin seed storage

proteins (at2S1 to at2S4) follows similar temporal profiles throughout development, but

at2S2 and at2S3 are expressed at significantly higher levels than at2S 1 or at2S4. In situ

hybridization analysis demonstrated that tissue specificity differed among the four genes.

The three genes namely at2S2, at2S3 and at2S4 were expressed throughout the embryo

while at2S1 was expressed in the embryo axis but at only-insignificant levels in the~otyledons

(Guerche eta/., 1990). It has been proposed that the different members of the gene family

are likely to be regulated by different combinations of cis-acting elements, but role of

posttranscriptional factors cannot be ruled out (Guerche eta/., 1990). In Brassica napus, a

gene family of at least sixteen members of which six members are sequenced and

characterized encodes napin. S1 nuclease analysis and sub-family specific oligonucleotide

hybridization demonstrated that the expression of one sub-class represented by the nap in

gene gNa peaks and declines earlier than the other members of the family and also highly

expressed representing 20% of nap in mRNA at 26 days after anthesis (Biundy eta!., 1991 ).

1.2.3.6 FUNCTIONS OF 2S ALBUMINS

Seed storage proteins have evolved for long-term amino acid storage in seeds, where

periods of predominant protein formation are temporally separated from times of their major

breakdown. The high N/C ratio resulting from high percentages of amides and argin~nes in

their amino acid composition is consistent with a nitrogen storage function (Muntz, 1998;

Vitale and Raikhel, 1999). 2S albumins have higher content of nitrogen and sulfur as

compared to 7S and HS globulin proteins in most species (Youle and Huang, 1981)_ 2S

albumins have many cysteine and methionine, which serve as sulfur-storage form in seed.

23


During germination, the sulfur is mobilized and utilized not only for amino acid and protein

structure, but also for synthesis of cofactors, coenzymes and membrane sulfolipids (Youle

and Huang, 1981 ). No other biological activity had been attributed to these storage proteins

until recently, when these were shown to possess many other biological activities. Few of

the major activities are discussed below.

1.2.3.6.1 ANTIFUNGAL ACTIVITY

·. Plant seeds are normally sown on substrates that are extremely rich in microorganisms,

but infection remains a rare event. A wide array of antimicrobial peptides or proteins, either

produced in a constitutive or in an inducible manner, is believed to be involved in defense

mechanisms. Many different proteins with antifungal and/or antibacterial activity have been

detected in seeds. These are: chitinases (Roberts and Selitrennikoff, 1986), thionins (Bohlmann

et at., 1988; Fernandez et at., 1972), permatins (Roberts and Selitrennikoff, 1990; Vigers eta/.,

1992), ribosome-inactivating proteins (Roberts and Selitrennikoff, 1986; Leah et at., 1991 ),

chitin-binding lectin (Raikhel eta/., 1993) and other antimicrobial proteins (Hejgaard et a/.,

1991; Broekaert et at., 1992; Cammue et at., 1992; Terras eta/., 1992a, 1992b, 1993a). Most

of these antimicrobial proteins are small, highly basic and cysteine-rich. They are highly divergent

at the level of primary structure and exhibit different antimicrobial activities. Terras et at., ( 1992b,

1993a) were the first to demonstrate that 2S albumins from seeds of a number of Brassicaceae

species inhibit the growth of several plant pathogenic fungi. They also demonstrated that 2S

fractions retain the antifungal activity even after boiling at 1 00°C for 10 min. However, treatment

of 2S fraction with various proteases leads to loss of activity. Antifungal activity of the 2S

albumins are affected by presence of cations and is completely abolished in the presence of 50

mM KCI and 1 mM CaCI2 (Terras eta/., 1992b). Microscopic analysis of fungal growth inhibition

on treatment with 2S albumins revealed a growth inhibition, featuring characteristic claws of

branched swollen hyphae which causes delayed growth of hyphae. Therefore, the antifungal

effect of the 2S albumins might be fungistatic rather than fungicidal (Terras eta/., 1992b). All

2S albumin-like proteins do not possess antifungal activity eg. CMe, a trypsin-inhibitor from

barley, possessing amino acid sequence homology to napin (Garcia-Oimedo eta/., 1987), is

devoid of substantial antifungal activity (Terras eta/., 1993a). The 2S albumins and a- and 13-thionins act synergistically with respect to their antifungal activities by enhancing ability to

permeabilize fungal membranes (Terras eta/., 1993b). This synergism between 2S .albumins

andJhionins is also observed in high ionic strength media (Terras et al., 1993b). Based on 3D

structure of 2S albumin, it has been proposed that the antifungal activity of the 2S albumins

could be due to permeabilization of the fungal plasmalemma close to hyphal-tip. Since strong·

interactions with phospholipid bilayers of mustard napins were observed {Ofiaderra eta/., 1994),

it is likely that fungal membrane permeabilization is induced by electrostatic-contacts between

24


basic protein residues of napin and acidic phospholipids (Rico eta/., 1996). A peptide melittin,

is known to form a pore structure in membrane (Vogel and Jahnig, 1986) has shown presence

of amphipathic a-helix. Presence of such amphipathic a-helices has been predicted in napin,

which could be involved in membrane or membrane protein interactions contributing to the

antifungal action (Neumann eta/., t996b).

1.2.3.6.2 TRYPSIN-INHIBITOR ACTIVITY

Napins purified from seeds of Brassica napus and Sinapis arvensis act as trypsin

inhibitors with K; values 50 JlM and 7 JlM, respectively (Svendsen eta/., 1989, 1994).

Rapeseed napin-rich fraction inhibit trypsin with an IC50 value of 3.0±1.5 Jlm. However,

treatment of these fractions with 1 OmM dithiothreitol largely abolished trypsin inhibitory

activity, suggesting that the separated small and large chains are poor trypsin inhibitors

(Neumann eta/., 1996d).

Usually the inhibitory site of a protease inhibitor is located in a loop of the polypeptide

chain (McPhalen eta/., 1985). Comparing the sequences of these napins with other reported

napins suggested presence of a loop between positions 40 and 60 amino acids of heavy

chains, which might contain the inhibitory site of trypsin inhibitors.

1.2.3.6.3 CALMODULIN-ANTAGONIST ACTIVITY

Seed germination is associated with an increase in calmodulin and a decrease in

calmodulin inhibitory activity (Cocucci and Negrini, 1988). Napins as well as both their

separated subunits have been shown to be calmodulin-antagonist (Newmann eta/., 1996a).

Calmodulin antagonist activity of napin small chains from Raphanus sativus (Hara-Nishimura

et a/., 1993), napin small and large chains from Sinapis alba (Neumann eta/., 1996d) and

Brassica napus (Neumann eta/., 1996a, 1996b), napin small chain from Ricinus communis

(Neumann et al., 1996c) has been established. Calmodulin as well as napin contain similar

a-helix-hinge-ahelix motif in their structure (Neumann eta/., 1996a,c). Therefore, .competitive

inhibition due to the binding of napin to kinases could be responsible for the phenomenon

(Neumann et at., 1996b,c). Brassica napus napin abolishes Ca2•-dependent fluorescence

enhancement of dansylated calmodulin and also inhibits calmodulin-dependent myosin

light-chain kinase (Neumann eta/., 1996b). Similar activity has been reported with Raphanus

sativus napin small chain (Polya et a/., 1993) and Ricinus communis nap in small .chain

(Neumann eta/., 1996c). Napin retains calmodulin antagonist activity even after heat

treatment and chromatography in CH3CN involved in isolati~~. suggesting nonspecific

interaction of denatured basic polypeptides with acidic calmodulin (Polya et at., 1993;

Neumann et a/., 1996a-d). Napins are phosphorylated by Ca2• -dependent plant kinase

(CDPK) in vitro at serine residues in the nsmhelical C-terminus of small subunit (Neumann

et at., 1996b,c) and at serine residue of the C-terminus of amphipathic a-helix on the large

25


subunit {Neumann eta/., 1996c). The functional consequences of the phosphorylation of

napins are unknown. However, phosphorylation of S. alba large subunit greatly decreases

trypsin-catalyzed hydrolysis in this region of large subunit in vitro (Neumann eta/., 1996d).

Therefore, it has been suggested that phosphorylation of napins affect precursor processing,

polypeptide chain folding, interaction with other proteins such as calmodulin and proteolysis

in early germination (Polya eta/., 1993; Neumann eta/., 1996a).

1.2.3.6.4 ALLERGEN

Allergy is a common disease among workers in seed-processing industry. The allergenic

components of the seeds are mainly soluble, low molecular weight proteins, as has been

found in case of wheat (Gomez et a/., 1990), cottonseed (Youle and Huang, 1979),

castorbean (Youle and Huang, 1978), soyabean (Moroz eta/., 1980), mustard (Menendez

Arias eta/., 1988) and brazil nut (Nordlee eta/., 1996). Rapeseed flour could be associated

with several allergenic responses such as the induction of celiac disease, baker's asthma

and many others. Allergy to inhaled rape seed flour is caused by the 2S storage napins ..

These polypeptide components of the seed have been fractionated and the presence of

napin-binding lgE antibodies in the serum of a patient allergic to rapeseed has been

demonstrated (Monsalve eta/., 1997).

Two allergens, Sina I and Braj IE, have been cloned and sequenced from yellow and

oriental mustard seeds respectively (Menendez-Arias et a/., 1987, 1988; Monsalve et a/.,

1993). An epitope mapping of these allergens revealed that hypervariable region in napin

like proteins is involved in recognition by monoclonal antibodies (Menendez-Arias et a/.,

1990; Monsalve eta/., 1993). Recombinant 2S albumin from English walnut (Juglans regia)

is reported to bind lgE, suggesting inherently allergenic nature of this class of proteins

(Teuber eta/., 1998). The 2S proteins have nutritional value due to presence of high content

of essential amino acids, cysteine and methionine, but the allergenic properties of the 2S

proteins deserve consideration in nutritional evaluation of this class of seed proteins as

cautioned by Youle and Huang (1981 ).

Further studies are needed to explore the functional potential hidden in the molecular

structure of napins.

1.2.3. 7 RECOMBINANT 25 ALBUMINS

Recombinant production of proteins is a useful strategy to obtain well-defined and

homogeneous materials for research or industrial purposes. Few attempts have been made

for the recombinant expression of heterodimeric 2S albumins or their precursors. Sina I,

the major allergen from yellow mustard, was produced in E. coli as fusion protein (Gonzalez

de Ia Peria eta/., 1993, 1996). A tow amount of purified and soluble protein was obtained

26


because of the tendency of the molecules to aggregate as inclusion bodies. The expression

of eukaryotic proteins in E. coli leads to accumulation of these proteins as inclusion bodies

{Wilkinson and Harrison, 1991 ). Inclusion bodies formation is favoured on proteins rich in

disulfide bonds, such as Sina I allergen. The recombinant Sina I is recognized by Sina I

specific antibodies. Another allergen Jugr1 (2S albumin protein of English walnut) was

produced in E. coli as fusion protein with the ability. to react with lgE from 16 patients with

walnut food allergy (Teuber eta/., 1998). Another 2S napin gene (napA) has been expressed

in transgenic tobacco (Muren and Rask, 1995) and Baculovirus (Muren and Rask, 1996)

systems. Structural and immunological characterization of this pronapin and the mature

form suggested that there is conformational difference of internal processed fragment in

pronapin (Muren eta/., 1996). The expression of the Arabidopsis tha/iana 2S albumin gene

3 in Saccharomyces cerevisiae showed accumulation of the product in vacuolar bodies

and that 13 kDa protein was processed to 9 and 4 kDa proteins, as obtained in transgenic

tobacco plants (Pal and Biswas, 1995). Recently, the recombinant expression of the eDNA

encoding proBnlb (LMW-napin of B. napus) has been carried out in Pichia pastoris and

shown to be a successful method for high yield production of homogeneous and properly

folded proteins. The recombinant pronapin displayed structural and immunological properties

equivalent to its mature product isolated from rapeseed (Palomares eta/., 2002).

Much of the recent interest in 2S albumins has focussed on their exploitation in genetic

engineering. Altenbach et a/. (1987, 1992) have used the 2S albumin of Brazil nut, to

increase the methionine content of tobacco seeds by upto 30%. The methionine-rich

sunflowers 2S albt.Jmin has been used to increase the methionine content of forage grasses

(Tabe eta/., 1993). Also, the expression of a~tisense RNA in a seed specific manner has

been used to manipulate the quantity and quality of storage components in Brassica napus

seeds (Kohno-Murase eta/., 1994). In addition, the 2S albumins of Arabidopsis have been

used as "hosts" for the synthesis of biologically active peptides, including the pentapeptide

Leu-en kephalin (Vandekerckhove eta/., 1989) and a 28-residue antibacterial peptide from

Xenopus (DeClercq eta/., 1990; Krebbers et a/., 1993).

A detailed understanding of storage protein structure and diversity is an important

prerequisite for attempts to manipulate quality because it indicates the extent to which .the

structure of the napins can be manipulated without affecting the biological properties.

27


1.3 Protein Refolding: a major challenge

The advent of recombinant DNA technology has opened a new era for protein production

both for research and industrial applications. It is now possible to use prokaryotic organisms

to overexpress rare and high value proteins. However, expression of genetically engineered

proteins in bacteria often results in the accumulation of the protein product in inactive

insoluble deposits inside the cell, called inclusion bodies. Several attempts have been made '

to improve the solubility of these proteins by a variety-of means, such as growing the cells

at lower temperatures, co-expressing the protein of interest with chaperones and foldases

and using solubilizing fusion partners, among others. However, expressing a protein as

inclusion body can be advantageous. Large amounts of highly enriched proteins can be

expressed as inclusion bodies. Trapped in insoluble aggregates, these proteins are protected

from proteolytic degradation. If the protein of interest is toxic or lethal to the host cell, then

inclusion body expression may be the best available production method. The challenge is

to take advantage of the high expression level of inclusion body proteins by being able to

convert inactive and misfolded inclusion body proteins into soluble bioactive products

(Rudolph and Ulie 1996; Clark, 1998; Lilie et at., 1998). The general strategy used to

recover active protein from inclusion bodies involves three steps: inclusion body isolation

and washing; solubilization of the aggregated protein and refolding of the solubilized protein.

1.3.1 Inclusion bodies isolation and purification

Inclusion bodies are dense, amorphous protein deposits that are normally found in

cytoplasmic and periplasmic space of bacteria. Cells containing inclusion bodies are usually

disrupted by high-pressure homogenization as a combination of mechanical, chemical and

enzymatic methods (Michaelis et at., 1994; Rudolph, 1995). The resulting suspension is

treated by either low-speed centrifugation or filtration from the particulate containing the

inclusion bodies. Membrane-associate proteins that are released upon cell breakage and

are present as contaminants of inclusion body protein preparations are most difficuit to

remove. Washing steps are performed to remove membrane proteins and other

contaminants. The most common washing steps utilize EDTA and low concentrations of

denaturants and/or weak detergents such as Triton X-1 00, deoxycholate and octylglucoside

(Collins eta!., 1999; Fahey et a/., 2000; Lee et at., 2000).

1.3.2 Inclusion bodies solubilization

A variety of methods are used to solubilize inclusion bodies. The most commonly used

solubilizing agents are denaturants. Solubilization may be accomplished by the complete

disruption of the protein structure {unfolding) or by the disruption of intermolecular

(noncovalent, hydrophobic or ionic) interactions with partial unfolding of the protein. The

existence of native-like structural features of proteins in inclusion bodies ofiL-1~ (Oberg et

28


a/., 1994) and f3-lactamase {Przybycien eta/., 1994) indicates that inclusion bodies form

from a folding intermediate having a considerable native-like secondary structure. Therefore,

proteins can be solubilized in lower denaturant concentrations (1-2 M) with retention of the

existing structure of the protein molecule. This strategy has been used to solubilize cytokines

from E. coli inclusion bodies (Li eta/., 1999). Extremes of pH have also been used to

solublize inclusion bodies. Gavit and Better {2000) used a combination of Jaw pH (= 2.6)

and high temperature (85°C} to solubilize antifungal recombinant peptides from E. coli.

High pH(~ 12) has been used to solubilize growth hormones (Khan eta/., 1998; Patra et

a/., 2000) and proinsulin (Hartman et a/., 1999). Solubilization of inclusion bodies with

retention of secondary structure has been reported by use of surfactant like n-cetyl

trimethylammonium bromide (Kim and Lee, 2000). Proteins having multiple disulfide bonds

are usually solubilized by addition of a reducing agent to maintain cysteine residues in the

reduced state and thus prevent non-native intra- and inter-disulfide bond formation.

Commonly used reducing agents include dithiothreitol (OTT), dithioerythritol (DTE) and (3-

merceptoethanol. Solubilization may also be accomplished by applying high hydrostatic

pressures (1-2 kbar) in the presence of reducing agents and low concentrations of solubilizing

agents (St. John eta/., 1999). However, the majority of the publisheg work on inclusion

body protein refolding has used high denaturant (6-8 M) and protein (1-1 0 mg/ml)

concentration~ in the solubilization step (Huxtable eta/., 1998; Trans-Moseman eta/., 1999;

Fahey eta/., 2000).

1.3.3 Renaturation of the solubilized proteins

When inclusion bodies have been solubilized using lower denaturants concentration in

combination with extremes of pH or using reducing agent, renaturation is accomplished by

removal of excess of reducing agent by dialysis or by changing the pH followed by purification

of protein of interest. However, when high concentrations of denaturants along with reducing

agents are used to solubilize inclusion bodies, renaturation is accomplished by removal of

excess denaturants by either dilution (Katoh and Katoh, 2000) or a buffer-exchange step such

as dialysis (West eta/., 1998}, diafiltration (Varnerin eta/., 1998), gel filtration chromatography

(Fahey eta/., 2000) or immobilization onto a solid support (Rogl eta/., 1998).

In case of disulfide-bonded proteins, renaturation buffers must promote disulfide-bond

formation (oxidation). In vivo folding of proteins is assisted by folding catalysts, such as

disulfide isomerase, and molecular chaperones, which inhibit misfolding and aggregation

(Woycechowsky and Raines, 2000). In vitro refolding of proteins that contain disulfide bonds

is challenging and low refolding yields are often seen due to improper disulfide bond

formation, aggregation and instability of folding intermediates and products (Goldberg et

a/., 1991; Raman eta/., 1996). Various in vitro methods have been developed to induce the

29


formation of disulfide bridges. Out of these, air oxidation is the most economical but is

suitable only when the reduced protein can be brought to a near-native conformation before

oxidation. The rate of disulfide-bond formation through air oxidation may be limited by the

slow mass transfer rate of oxygen in aqueous solutions. Oxidation rates can be accelerated

using an oxide-shuffling system, which consists of mixtures of reduced and oxidized low

molecular weight thiol reagents. The most commonly used oxide-shuffling reagents are

reduced and oxidized glutathione (GSH/GSSG) (Saxena and Wetlaufer, 1970). However,

the pairs cysteine/cystine, DTT/GSSG, Cysteamine/Cystamine and DTE/GSSG have also

been utilized. A third oxidation method uses a two-step approach: the formation .of mixed

disulfides between glutathione and the denatured protein before renaturation, followed by

refolding in the presence of catalytic amounts of a reducing agent to promote disulfide

bond formation and reshuffling (Ejima eta/., 2000).

Refolding yields are enhanced by use of additives like L-Arginine {0.4-1 M) {Fischer et

a/., 1986); low concentrations of denaturants like Urea (1-2M) and Guanidine-HCI {0.5-1.5

M) (Builder and Ogez, 1986); polyethylene glycols (Cleland eta/., 1992); CHAPS and

cosolvents as sugars, alcohols and detergents (Valax and Georgiou, 1991) in the refolding

buffer.

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