Enzymatic Activity Restored in Ammodytin L, an Inactive Phospholipase A2 Homologue, Increases its Toxic and

Membrane Damaging Activities

Toni PetanJozef Stefan Institute

Slovenia

Ammodytin L (AtnL), a Ser-49 group IIA sPLA2 homologue

Ser-49/Lys-49 homologues Inability to bind Ca2+

Myonecrosis in vivo Ca2+-independent membrane

damage in vitro

Protein-membrane interactions on the sarcolemma

Lomonte et al., 2003, Toxicon 42.

S49/K49 sPLA2 (in)activity controversy

“sPLA2s with no or very low catalytic activity” – (still) a

controversial issue?

First recombinant BthTX I proved inactivity of K49 sPLA2s

(Ward et al., (2002) Biochem. J. 362)

K49D mutant of BthTX I remained inactive

=> Lys-49 is not the only residue responsible for inactivity

Our aim

Confirm the inactivity of AtnL Restore a working catalytic

machinery in AtnL Determine its impact on the

toxic and membrane damaging activities

Ammodytin L (AtnL)

Substitutions in the Ca2+-binding loop are common in S49/K49 sPLA2s

His28

Ser49

Asn33

Inactive Ser-49 sPLA2

(AtnL)

Leu31

Tyr28

Val31

Asp49

Gly33

Active Asp-49 sPLA2

(AtxA)

Ca2+

Ca2+

LW mutant

Tyr28

Val31

Asp49

Gly33

LV mutant

Two enzymatically active mutants of AtnL

Tyr28

Trp31

Asp49

Gly33

Ca2+

Trp-31 enhances the activity of LW on PC-rich vesicles

POPC 10% PS/ PC 30% PS/ PC POPS POPG

AtnL 0 0 0 0 0

LV ~0.005 0.5 43 90 225

LW 0.22 8.3 96 90 184

specific enzymatic activity, v0 (mol min-1 mg-1)

0

10

20

30

40

50

0 20 40 60 80 100Anionic Phospholipid (mol %)

LW

/LV

Act

ivit

y R

atio

LW binds very well to PC vesicles

100 nM extruded LUVs deposited on a Pioneer L1 chip Running buffer: HBSS w/o Ca2+, Mg2+

50 nM toxins, POPG vesicles100 nM toxins, POPC vesicles

LW displays high cytotoxicity towards C2C12 myoblasts and myotubes

8

44

7

61

11

64

44

72

88

30

0

10

20

30

40

50

60

70

80

90

100

500 nM PLA2 (myoblasts) 500 nM PLA2 (myotubes)

Cyt

oto

xici

ty (

%)

AtnL AtnI2 LV AtxA LW

i.p. LD50

(g/kg)

AtxA 20

AtnL >10,000

LV >7000

LW 2200

LW has a higher toxicity in mice than AtnL

neurotoxic

myotoxic

(mouse LD50 values of recombinant proteins)

LV and LW cause calcein release in a Ca2+-dependent manner

• Good correlation with enzymatic activity• No release in the absence of calcium for any of the toxins

=> crucial role of enzymatic activity

02

8

28

20

25

37

~1

0

5

10

15

20

25

30

35

40

AtnL LV LW AtxA

Cal

cein

Rel

ease

(%

) POPC 10% POPG/POPC(500 nM sPLA2)(3 μM sPLA2)

LW, no Ca2+

0

5

10

15

20

25

30

0 200 400 600Time (s)

Calc

ein

Rele

ase

(%

)

25 nM50 nM100 nM250 nM500 nM

LW and LV cause calcein release from 50% PG/PC vesicles in the presence or absence of calcium

29

24

19

0

60

35 36

74

0

10

20

30

40

50

60

70

80

AtnL LV LW AtxA

Cal

cein

Rel

ease

(%

)

50% PG/PC, no calcium 50% PG/PC

500 nM toxins, after 5 min

8

0

13

22

-5

0

5

10

15

20

25

30

AtxA LV LW AtnL

% L

ipid

mix

ing

NBD/Rh FRET lipid mixing assay with 500 nM toxin, 50% POPG/POPC vesicles, HBSS

All sPLA2s induced lipid mixing only in the presence of Ca2+!

0

10

20

30

40

0 200 400 600Time (s)

% L

ipid

Mix

ing

250 nM

500 nM

AtxA

-10

0

10

20

30

40

0 200 400 600

Time (s)

Lip

id M

ixin

g (

%)

500 nM3100 nM6200 nM

AtnL

sPLA2-induced lipid mixing is dependent on enzymatic activity

50% PG/PC vesicles

DLS analysis of particle size distribution

15 min

1 min

5 min

250 nM AtnL, no Ca2+,100 nm 50% PG/PC

0 min 100 nM AtxA, no Ca2+

100 nm 50% PG/PC

15 min

0 min

500 nM LW, Ca2+,100 nm 50% PG/PC

15 min

1 min

5 min

0 min

Conclusions

S49 sPLA2 homologues are enzymatically inactive

Besides S49/K49, calcium binding loop residues are

crucial for inactivity

the substrate binding and catalytic networks of

S49/K49 sPLA2 homologues are well conserved

Calcein release, Ca2+-dependent

Vesicle fusion,Ca2+-dependent

Calcein release,Ca2+-independent

AtnL

AtxA

LW, LV

LW and LV are more potent than AtnL in causing membrane damage

Conclusions

LW and LV require a lower threshold of anionic

lipid for membrane damage

higher enzymatic activity of LW on PC-rich

membranes,

=> All these factors might have enabled LW, rather

than LV, to express the highest cytotoxicity for

C2C12 cells as well as much higher toxicity in

vivo in comparison with wild-type AtnL.

Thank you for your attention!

Zala Jenko

Petra Prijatelj

Jernej Šribar

Lidija Kovačič

Uroš Logonder

Jože Pungerčar

Igor Križaj

Department of Molecular and Biomedical Sciences,Jožef Stefan Institute, Ljubljana

Gregor Anderluh

Andrej Bavdek

SPR Center,

Biotechical Faculty, Ljubljana

Michael H. Gelb

Farideh Ghomashchi

Jim Bollinger

University of Washington, Seattle, USA

SLOVENIAN BIOCHEMICAL SOCIETY

Calcein release examples

-505

1015202530354045

0 400 800 1200

Time (s)

Calc

ein

Rele

ase

(%

) AtxA

LV

LW

AtnL 05

1015202530354045

0 200 400 600

Time (s)

Cal

cein

Rel

ease

(%

)

AtnL

LV

LW

AtxA

LW

3000 nM toxins, POPC500 nM toxins, 10% POPG/POPC

Calcein release by LW and LV from 10% PG/PC vesicles detected only in the presence of Ca2+

Calcein leakage could not be detected in the absence of calcium for any of the toxins!

05

1015202530354045

0 200 400 600

Time (s)

Cal

cein

Rel

ease

(%

)

AtnL

LV

LW

AtxA

LW20

25

37

~10

5

10

15

20

25

30

35

40

AtnL LV LW AtxA

Ca

lcei

n R

elea

se (

%)

u

0

10

20

30

40

50

60

En

zymatic A

ctivity

500 nM toxins

Mutagenesis of AtnL

AtnL: 28H..L.N33...S49

AtxA: 28Y..V.G33...D49 (74% identity to AtnL)

Two mutants of AtnL:

AtnL(YVGD): 28Y..V.G33...D49 (in short LV)

AtnL(YWGD): 28Y..W.G33...D49 (in short LW)

0102030405060708090

100110

AtxA AtxA(V31W)

activi

ty (

mol

/(m

in x

mg)

)

POPC

HEK293

Petan et al., (2005) Biochemistry, 44.

calcium binding loop

Precautionary step: Introduction of Trp-31 in the LW mutant to increase interfacial binding affinity