1

Subject: Molecular Biodiversity and DNA

Analysis

Topic: Superoxide

dismutase:

types and importance in

plants and human and

fungi. Submitted to: Dr. M. Ishfaq

Submitted by: Jannat Iftikhar

MS16-01

Department of botany

University of the Punjab

Lahore.

Contents 1. Reactive oxygen species (ROS)

2. Role of ROS

3. Superoxide dismutase (SOD)

4. Discovery and nomenclature

5. Types of SOD

6. SOD in human

7. SOD in plants

8. SOD in fungi

9. Evolution of SOD

10. Extraction of SOD from Arabidopsis (Plant)

11. Extraction of SOD from Cheatomium

thermohilum (Fungi)

12. Conclusion2

Reactive oxygen species

(ROS) ROS are chemically reactive chemical

species containing oxygen.

ROS are generated as by-productsduring mitochondrial electrontransport.

ROS are formed as necessaryintermediates of metal catalyzedoxidation reactions.

Types of ROS includes: superoxide;hydrogen peroxide; hydroxyl radical;hydroxyl ion; and nitric oxide.

3

Role of ROS

ROS play important

role in

Apoptosis

Gene expression

Activation of cell

signaling.

Serve as both

intra- and

intercellular

messengers.

ROS also

responsible for

Aging

Carcinogenic

Cell death

4

Superoxide dismutase

Superoxide dismutase are

metalloproteins found ubiquitously in

all aerobic organisms. (Fridovich & McCord,

1969)

Superoxide dismutase (SOD)

catalyzes the conversion of two

superoxide anions into a molecule of

hydrogen peroxide (H2O2) and oxygen

(O2).

5

Discovery

Irwin Fridovich and Joe M. McCord,

discovered the enzymatic activity of

copper, zinc superoxide

dismutase(SOD).

Subsequently, Fridovich's research

group also discovered the

manganese-containing and the iron-

containing SODs from E.coli and the

mitochondrial MnSOD (SOD2), now

known to be an essential mammalian

protein. (Fridovich,1975) 6

7

SOD Nomenclature (Culotta et. al.,2006)

Types of SODs

There are three major families of

superoxide dismutase, depending on

the metal cofactor:

Cu/Zn (which binds both copper and

zinc), (Richardson et. al., 1975)

Fe and Mn types (which bind either

iron or manganese), (Borgstahl et. al., 1992)

Ni type, which binds nickel. (Barondeau

et. al., 2004)

8

SOD in Human

Three forms of superoxide dismutase

are present in humans.

SOD1, located in cytoplasm

SOD2, located in mitochondria

SOD3 is extracellular

The genes are located on

chromosomes 21 (Levnon et. al.,1985) ,

chromosome 6 (Creagan et. al., 1973) , and

chromosome 4 (Hendrickson et. al., 1990) ,

respectively.9

10

Genomic organization of the three known members of the human SOD

enzyme family. SOD3 was placed in the middle in order to demonstrate

areas of amino acid sequence homology between SOD1 and SOD3.

SOD2 has no significant amino acid sequence homology with either

SOD1 or SOD3. The size of each exon and intron, in base pairs, is

shown in association with that fragment.

SOD1

SOD1, contains copper (Cu) in its

reactive center.

SOD1 has molecular mass of about

32,000 Da. (Chang et. al. 1988)

It is found in the cytoplasm, nuclear

compartments, and lysosomes of

mammalian cells. (Crapo et. al.,1992)

11

Crystal structure of the human SOD1 enzyme

(rainbow-color N-terminus = blue, C-terminus =

red) complexed with copper (orange sphere) and

zinc (grey sphere).

SOD1, contains copper (Cu) in its reactive

center.

12

SOD2

This isoform of SODs has manganese

(Mn) as a cofactor and has been

localized to mitochondria of aerobic

cells (Mn-SOD or SOD2).

It exists as a homotetramer with an

individual subunit molecular weight of

about 23,000 Da.

13

SOD2, contains manganese (Mn) in its

reactive center.

Active site of human mitochondrial Mn superoxide

dismutase (SOD2)

14

SOD3

SOD3 is the most recently discoveredand least characterized member of theSOD family.

The enzyme exists as a homotetramer ofmolecular weight 135,000 Da with highaffinity for heparin.

SOD3 was first detected in humanplasma, lymph, ascites, andcerebrospinal fluids. (Markland, 1982)

The expression pattern of SOD3 is highlyrestricted to the specific cell type andtissues.

15

SOD3, contains zinc (Zn) in its reactive

center.

Crystallographic structure of the tetrameric human

SOD3 enzyme (cartoon diagram) complexed with

copper and zinc cations (orange and grey spheres

respectively).16

Role of SOD in Human

SOD1 enzyme is an important

constituent in apoptotic signaling

and oxidative stress.

SOD2 confer protection against cell

death.

This protein plays an anti-apoptotic role

against oxidative stress,

ionizing radiation,

and inflammatory cytokines.

SOD3 is thought to protect

the brain, lungs, and other tissues

from oxidative stress.17

Clinical Significance of SOD in

HumanSOD is involved in a number of

diseases and pathologies:

ALS, Down’s syndrome, and

premature aging are some of the

pathological conditions that develop

due to altered SOD activity and ROS

concentration.

SOD plays in cardiovascular and

pulmonary diseases.

18

SOD in Plants

There are three well-known and well-

studied classes of SOD metallic

coenzymes that exist in plants.

Fe SOD

Mn SOD

Cu-Zn SOD

19

Fe SOD

They are thought to be the most

ancient SOD metalloenzymes.

They are found within both

prokaryotes and eukaryotes.

Fe SODs are most abundantly

localized inside plant chloroplasts,

where they are indigenous.

Fe SOD is inactivated by H2O2 and is

resistant to KCN inhibition.

20

Fe SOD

There are two Fe SOD groups.

The first group is a homodimer formedfrom two identical 20 kDa subunitsproteins, with 1-2 gram atom of iron inthe active centers.

The second Fe SOD group found inmost higher plants, is a tetramer offour equal subunits with a molecularweight of 80-90 kDa. . This groupcontains 2-4 grams of iron atom in theactive center.

21

Out of 43 families investigated, the Fe

containing superoxide dismutase was

found in three families: Ginkgoaceae,

Nymphaeaceae and Cruciferae

(Brassicaceae) (Salin and Bridges, 1981).

22

Mn SOD

Second, Mn SODs consist of a

homodimer and homotetramer species

each containing a single Mn(III) atom

per subunit.

They are found predominantly in

mitochondrion and peroxisomes.

23

Mn SOD

The enzyme is not inhibited by KCN or

inactivated by H2O2.

Plant Mn SODs have approximately

65% sequence similarity to one

another and these enzymes has also

high similarity to bacterial Mn SODs (Bowler, 1994).

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Cu-Zn SOD

Third, Cu-Zn SODs have electrical

properties very different from those of

the other two classes.

These are concentrated in

the chloroplast, cytosol, and in some

cases the extracellular space.

25

Cu-Zn SOD

There are two different groups of this enzyme. The first group consists of cytoplasmic and periplasmic forms which are homodimeric.

Homodimer has molecular weight of 32,500.

The second group is chloroplastic and extracellular and are homotetrameric. (Bordo et al., 1994)

Cu-Zn enzyme is sensitive to cyanide.

26

27

(Alscher et al., 2002.)

Importance of SODs for

Plants FeSOD is essential for chloroplast

development in Arabidopsis. (Husodo et. al., 2008)

affect the efficiency of microspore embryogenesis in Triticosecale. (Dubaset. al., 2014)

Cu-ZnSOD improves tolerance against cold and drought stresses.

MnSOD involved in heat-stress tolerance during grain filling of rice. (Takeshi et. al., 2015)

28

Importance of SODs for

Plants Cu-Zn superoxide dismutase enhance

in-vitro shoot multiplication intransgenic plum. (Faize et. al., 2013)

It improves the recovery ofphotosynthesis in sugarcane plantssubjected to water deficit and lowsubstrate temperature. (Chistina et. al.,2013)

It is a protective enzyme againstozone injury in snap beans(Phaseolus vulgaris L.) (Bennet et. al.,1982)

29

SOD in Fungi

C. neoformans, only two SODs were

identified,

one cytosolic Cu/ZnSOD (SOD1) (Hwang et al., 2003)

one mitochondrial MnSOD (SOD2). (Martchenko et al., 2004)

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SOD in Fungi

Four genes encoding putative Sods

have been identified in the A.

fumigatus genome. (Lambou et. al., 2010)

a cytoplasmic Cu/ZnSOD (AfSod1p)

a mitochondrial MnSOD (AfSod2p),

a cytoplasmic MnSOD (AfSod3p)

a AfSod4 displaying a MnSOD C-

terminal domain.

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During growth, AfSOD1 and AfSOD2

were highly expressed in conidia

AfSOD3 was only strongly expressed

in mycelium.

AfSOD4 was weakly expressed

compared with other SODs.

32

Role of SOD in Fungi

Superoxide dismutases (SODs), whichprovide protection against oxidativestress, exhibit an essential role for fungalcell survival, especially during hostinvasion.

The CuZn superoxide dismutase fromSclerotinia sclerotiorum is involved withoxidative stress tolerance, virulence, andoxalate production. (Selvakumar et. al., 2012)

Function of SODs has been investigatedyeast pathogens Candida albicans.(Lamarre et al., 2001)

33

Cu-Zn SOD involvement in virulence

is shown in Candida albicans. (Hwang et

al., 2002)

Mn-containing SODs were shown to

be involved in protection against

various stresses in C. albicans. (Hwang

et al., 2003).

We can also make phylogenetic tree

based on Manganese superoxide

dismutase of pathogenic fungi.

34

Evolution of SODs

The appearance of SOD enzymes

was triggered by the proliferation of

photosynthetic organisms that began

to produce oxygen about 2 billion

years ago.

Two major kinds of superoxide

dismutase appeared in prokaryotes at

that time, copper/zinc-containing

SODs and iron/manganese-containing

SODs.35

Three hypothesis explains the

presence of Cu-Zn SOD in

prokaryotes Evolves independently in prokaryotes

and eukaryotes.

Originated in eukaryotes and then

gene is transferred to prokaryotes.

Originated in prokaryotes and then

transferred to eukaryotes.

36

Cellular Extract Preparation for

SOD (Kuo et. al., 2013)

This protocol is to demonstrate how to

prepare the cellular extract for the

identification and characterization of

SODs in plants.

37

Materials and Reagents

1. Nitroblue tetrazolium (NBT) solution

2. N,N,N’,N’-Tetramethylethylenediamine(TEMED) (Sigma-Aldrich, catalog number: T9281)

3. Grinding buffer

4. Riboflavin solution

5. KCN (Sigma-Aldrich, catalog number: 60178)

6. H2O2(Sigma-Aldrich, catalog number: 349887)

38

Equipment

1. A light box (white light)

2. Centrifuge

3. Protein gel cassette

39

Procedure

A. Arabidopsis cellular extract

preparation

B. SOD activity staining

C. Identification of different SOD

species

40

Arabidopsis Cellular Extract

Preparation1. Arabidopsis seedlings were grown at 23°C

with 16 h of light at 60–100 μmol/m2/s. Nine-day-old seedlings were collected and weighted.

2. Seedlings were homogenized with ice-cold Grinding buffer (tissue weight/buffer volume = 1 mg/3 μl). Note that the tissue and extract should be kept at 4°C during all extraction processes.

3. Centrifuge at 16,000 x g at 4°C for 10 min.

4. The supernatant is the resulting cellular extract, and the amount of protein was quantified by Bradford method (1976).

41

SOD Activity Staining

Proteins or cellular extract (15 to 25 μg) was subjected to 10% native-PAGE at 4°C.

Wash the gel with distilled water for 3 times.

Incubate with NBT solution in dark with shaking for 15 min at room temperature (RT).

Pour off the NBT solution, wash the gel with distilled water for 3 times.

Incubate with Riboflavin solution in dark with shaking for 15 min at RT. 42

SOD activity staining

Pour off the Riboflavin solution, wash

the gel with distilled water for 3 times.

Gel was illuminated with a white-light

box for 10-15 min at RT. During

illumination, immerse gel in a thin

layer of distilled water to avoid drying

the gel.

White SOD activity bands appear in

the blue background.

43

Identification of different SOD

Species

44

SOD activity verification in Arabidopsis thaliana. KCN is an

inhibitor of CuZnSOD activity, whereas H2O2 inhibits both

CuZnSOD and FeSOD activities. MnSOD activity is not inhibited

by either treatment.

Extraction of SOD from Fungi (C.

thermophilum) (Guo et. al., 28)

A thermostable superoxide dismutase

(SOD) from the culture supernatant of

a thermophilic fungus Chaetomium

thermophilum strain CT2 was purified

to homogeneity by fractional

ammonium sulfate precipitation, ion-

exchange chromatography on DEAE-

sepharose, phenyl-sepharose

hydrophobic interaction

chromatography.45

Reagents

Yeast extract

Casein

Coomassie brilliant blue

materials for gel electrophoresis

DEAE-Sepharose fast flow, phenyl-

sepharose,

sephacryl S-100-sepharose

Standard protein makers (14.4–97.4

kDa)46

Organism and growth

conditions Chaetomium thermophilum CT2 was

isolated and preserved on potato

dextrose agar (PDA) medium.

C. thermophilum CT2 was grown in

shake cultures at 50 C in 20 flasks

each with 50 mL liquid medium

containing (g/L): casein, 40.0; glucose,

10.0; yeast extract, 4.0;

K2HPO4.3H2O, 1.0; MgSO4.7H2O,

0.5; dissolved in distilled and tap water

(3:1). 47

Preparation of the crude

enzyme After incubation for 8 d in liquid

medium the culture fluid was filtered

and centrifuged at 8000 xg for 15 min,

4 C, and the supernatant was used for

the purification of SOD.

48

All procedures of the SOD purification

were carried out at 4 C. These buffers

were used:

(A) 50 mM Tris-HCl (pH 7.5)

(B) containing 50% saturation

ammonium sulfate.

49

Fractional ammonium sulfate

precipitation Solid ammonium sulfate was added to

the supernatant to 90% saturation.

After 12 h, the precipitate was collected by centrifugation (10 000 g, 15 min), dissolved in buffer A and dialyzed overnight against three changes of the same buffer.

Insoluble material was removed by centrifugation (10 000 g, 15 min) and the supernatant was put on a DEAE-Sepharose column.

50

Ion exchange chromatography

on DEAE-Sepharose column Ion exchange chromatography on

DEAE-Sepharose column (1X 20 cm)

equilibrated with buffer A.

After the column was washed with five

column volumes of buffer A, a 200 mL

linear gradient of NaCl (0–0.3 M in

buffer A) was applied at a flow rate of

45 mL/h.

51

Phenylsepharose hydrophobic

interaction chromatography The sample from the DEAE-Sepharose

column with 50% saturation ammonium sulfate added was applied to a phenylsepharose column (13-20 cm) previously equilibrated with buffer B.

After the column was washed with five column volumes of buffer B, SOD was eluted with a 160 mL linear gradient of ammonium sulfate from 50–0% saturation at a flow rate of 45 mL/h.

Fractions with SOD activity were pooled and concentrated for determination of purity and properties.

52

To identify the type of SOD, duplicate

gels were incubated with

10 mM KCN,

10 mM H2O2 and

10 mM NaN3 during activity staining to

inactivate Cu, ZnSOD, MnSOD or

FeSOD, respectively (Asada et al 1975,

Britton et al 1978).

53

Conclusion

They are very important against ROS.

They act as first line defense against

ROS.

There expression and regulation of

both SOD and ROS should be

controlled.

54

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