USOO9215869B1

(12) United States Patent Adang et al.

(54) NON-CADHERIN POLYPEPTIDE POTENTITATORS OF CRY PROTEINS

(75) Inventors: Michael J. Adang, Athens, GA (US); Rui Zhang, Gainsville, FL (US); Gang Hua, Athens, GA (US) (73) Assignee: UNIVERSITY OF GEORGIA RESEARCH FOUNDATION, INC.,

Athens, GA (US) (*) Notice:

Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 186 days.

(21) Appl. No.: 13/090,830 (22) Filed:

Apr. 20, 2011

Related U.S. Application Data (60) Provisional application No. 61/325,944, filed on Apr. 20, 2010.

(51) Int. Cl. (2006.01)

AOIN37/18

(52) U.S. Cl. CPC ...................................... A0IN37/18 (2013.01)

(58) Field of Classification Search None See application file for complete search history. (56)

References Cited U.S. PATENT DOCUMENTS

7,011,975 B1 8, 101,568 B2

3/2006 Adang et al. 1/2012 Adang et al.

(10) Patent No.: (45) Date of Patent:

US 9,215,869 B1 Dec. 22, 2015

Fillinger etal (“Efficacy and efficiency of new Bacillus thuringiensis var. israielensis and Bacillus sphaericus formulations against Afrotropical anophelines in Western Kenya Tropical Medicine and International Health v8(1) Jan. 2003 pp. 37-47).* Vectobac product sheet (retrieved from http://publichealth. valentbiosciences.com/docs/resources, vectobac-Wolg-specimen-la bel.pdf on Jan. 8, 2015, 2 pages).* Chen, J. et al. “Synergism of Bacillus thuringiensis toxins by a fragment of a toxin-binding cadherin.” Proc. Natl. Acad. Sci., Aug. 28, 2007, pp. 13901-13906, vol. 104, Issue 35, USA. Nakanishi, K. et al., “Aminopeptidase Nisoforms from the midgut of Bumbyx mori and Plutella xylostella—their classification and the factors that determine their binding specificity to Bacillus thuringiensis Cry1A toxin.” FEBS Letters 519, Apr. 23, 2002, pp. 215-220, FEBS 26077.

Yaoi, K. et al., “Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N.” FEBS Letters 463, Nov. 16. 1999, pp. 221-224, FEBS 23056. Park, Y. et al., “Enhancement of Bacillus thuringiensis Cry3Aa and Cry3Bb Toxicities to Coleopteran Larvae by a Toxin-Binding Frag ment of an insect Cadherin.” Applied and Environmental Microbiol ogy, Mar. 27, 2009, pp. 3085-3092, vol. 75, Issue 10. Park, Y. et al., “Cadherin Fragments from Anopheles gambiae Synergize Bacillus thuringiensis Cry4Ba's Toxicity against Aedes aegypti Larvae. Applied and Environmental Microbiology, Oct. 2, 2009, pp. 7279-7282, vol. 75, Issue 22. Masson, L. et al., “The Cry IA(c) Receptor Purified from Manduca sexta Displays Multiple Specificities,” J. Biol. Chem. Sep. 1, 1995, pp. 20309-20315, vol. 270, Issue 35, USA. Pigott, C.R., et al., “Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity.” Microbiology and Molecular Biology Review, 2007, pp. 254-281, vol. 71, Issue 2. Zhang, R. et al., “A 106-kDa aminopeptidase is a putative receptor for Bacillus thuringiensis Cry11 Ba toxin in the mosquito Anopheles gambiae.” Biochemistry, Oct. 28, 2008, pp. 11263-11272, vol. 47. Issue 43.

* cited by examiner Primary Examiner — Karlheinz, R Skowronek Assistant Examiner — Ronald Niebauer

FOREIGN PATENT DOCUMENTS WO

WO 2009/124258

* 8/2009

OTHER PUBLICATIONS

Hua et al (Anopheles gambiae cadherin AgCadl binds the cry4ba toxin of bacillus thuringiensis israelensis and a fragment of agcad1 synergizes toxicity’ Biochemistry 2008 v47 pp. 5101-5110).* Zhang et al (Synergistic and inhibitory effects of aminopeptidase peptides on bacillus thuringiensis cry1 lba toxicity in the mosquito anopheles gambiae' Biochemistry 2010 v49 pp. 85 12-8519).* Insecticide definition retrieved from http://organic.about.com/od/ organicdefinitionsij/g/Insecticide.htm on Nov. 14, 2013, 1 page.

(74) Attorney, Agent, or Firm — Faegre Baker Daniels LLP (57)

ABSTRACT

The Subject invention relates in part to fragments of non cadherin Cry toxin binding proteins, wherein the fragments potentiate, or act as Synergists with, the insecticidal activity of Cry proteins. In some preferred embodiments, the binding protein (a Cry protein receptor on insect midgut cells) is an aminopeptidase. In preferred embodiments, the fragment comprises a Cry protein binding region. 1 Claim, 7 Drawing Sheets

U.S. Patent

Dec. 22, 2015

US 9,215,869 B1

Sheet 1 of 7

ka 50 -

843, 935

40 30 25 a.

20 r

Helix: 34%

3-stand: 20% Turns and UnOrdered: 46%

“is

8xieg:

is

.

is

Hex. 53%

3-stand: 20%

sode -tooge -isogo

Turns and Unordered 27%

is

U.S. Patent

Dec. 22, 2015

Sheet 2 of 7

US 9,215,869 B1

Fig. 2 A

OO

6O

4. O

O

Cry11Ba Cry11Ba with (4-g/ml) APN2t ta th ta4th

100 -

80 -

60

4.O

-:

20 -

|Cry11Ba (0.5 kg/ml)ap

Cry11Ba with

Backs.

tb only

U.S. Patent

Dec. 22, 2015

Sheet 3 of 7

US 9,215,869 B1

Fig. 3

4

a total binding to Nor-specific birding

awa E

K. : 16.7 + 4.8 nM Rs. 0.95

v Specific binding

5

arra

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Kic 26.4 it 3.6 nM Rs. 0.94

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83

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Biotin-APN2ta (nM)

Biotin-APN2tb (nivl)

2 20

100 -

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--

80

60

80 t

W \:

y N\

Y---

S Biotin-AM2t e - ApN2ia APN2.

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10

Competitor (M)

60 20 -

t

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Biot. APN2te 8 + APN2ta c + A2th

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Competitor (M)

O

CO

U.S. Patent

Fig. 4

Dec. 22, 2015

Sheet 4 of 7

US 9,215,869 B1

A 336

421

506

590

APN2ta

tafe taf)e.2 ta/e3

B

5 ka

N

2

&S.

S.

5

40 or 30 25 m 20 x 15 M.

C

100 80 so s

60

4.O

r

2O -

i k

s

s wa

s

ca

a

O

U.S. Patent

Dec. 22, 2015

Sheet 5 Of 7

US 9,215,869 B1

Biotin-APN2a

s

20

--a

O v

-tatDe tatDe2

A

talDe3

O.OO

-ve

y:

O.

O.

1

as y

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100

Competitor (uw) 0.8

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Total Binding

O w

-- ta -- tale

A.

-- tale2

- to -- T.

-- tale3

u-3-- --

-.

O2

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20

40

60

8O

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Biotin-APN2ta (nM)

to '-Cry1 Ba (2.5 nM) 30 -

e

25 2) -

15 -

b

O -

O

*-Cry1 Ba

'-Cry11 Ba with APN2t

a

tale1 ta|De2 af)e3

20

U.S. Patent

Dec. 22, 2015

Sheet 6 of 7

US 9,215,869 B1

Fig. 6 A

591

662 676

761

843 APN2to

thf)e. tbfe2 tbfe3

B

Y-

CY

co

5

g

g

g

N

ka

C

Se

C

d

D

OO 80 >

sO

cN

60

s

C s

SS 40

e

S

S

U.S. Patent

Dec. 22, 2015

Sheet 7 Of 7

US 9,215,869 B1

Fig. 7 A

2O

Biotin-AN2th

OO

89 2 60 SS 40

A

20 -

e O v

+ to -- De -- bide2

A

-- fe3

\

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t

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r

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w

+ to -- De

:

52 title3

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-

-

15

2

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2O

40

60

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s

'-Cry11 Ba (2.5 nM)

50

C

s

C

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C

30

a

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v-

b

as

- .

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10

b

O

8

v

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'I-Cry Ba

*-Cry! Ba with APN2t

th

thfelt to/De2 to/Del3

US 9,215,869 B1 1.

2

NON-CADHERN POLYPEPTIDE POTENTITATORS OF CRY PROTEINS

region between 'Arg and 77Leu. (Chen et al., 2009).

CROSS-REFERENCE TO RELATED APPLICATION

The subject application claims priority to U.S. provisional application Ser. No. 61/325,944, filed Apr. 20, 2010. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

10

This invention was made with government Support under National Institutes of Health Grant RO1 AI 29092. The U.S.

Government has certain rights in this invention.

15

BACKGROUND

The bacterium Bacillus thuringiensis israelensis (Bti) has been used worldwide as an important mosquito control agent for decades (Lacey, 2007). The active ingredient of Bti is a parasporal crystal complex composed of four Cry proteins (Cry4Aa, Cry4Ba, Cry 10Aa and Cry 11 Aa) and two cytolytic proteins (Cyt1 Aa and Cyt2Ba) (Porter et al., 1993). Concerns about potential mosquito resistance development to Bti have led to discoveries of other mosquitocidal toxins with high potency. Cry 11Ba produced by B.t.jegathesan (Bteg) is the single most effective toxin against mosquitoes to date. Cry 11 Ba shares 58% similarity to Cry 11 Aa and is 7- to 34-fold more toxic to mosquito larvae than the related Cry11 Aa (Delécluse et al., 1995). The resolved structures of Cry proteins show a conserva tive 3D-topology, suggesting a common mode-of-action. (Boonserm et al., 2005; Boonserm et al., 2006; Galitsky et al., 2001: Grochulski et al., 1995; Li et al., 1991; Morse et al.,

2001). Two models regarding the intoxication process of tox ins are proposed reviewed in (Pigott and Ellar, 2007). The colloid-osmotic lysis model Suggests that proteolytically activated toxins bind cadherin, oligomerize and then bind glycosylphosphatidylinositol (GPI)-anchored aminopepti dase (APN) and GPI-anchored alkaline phosphatase (ALP) to induce toxicity (Bravo et al., 2004). An alternative model proposes the activation of intracellular signaling pathways by toxin monomer binding to cadherin without the need of the toxin oligomerization step to cause cell death (Zhang et al., 2006). Whether toxicity is independent of toxin oligomeriza tion remains arguable, the toxin-receptor interaction has been elucidated in both models as the major determinant of toxin specificity. APN has long been implicated as a Cry 1 toxin binding protein in a number of lepidopteran species reviewed in (Pigott and Ellar, 2007). As a glycoprotein, APN interacts with Cry toxins through either glycan moieties or amino acid residues. For example, Cry1Ac has been shown to bind an N-acetylgalactosamine (GalNAc) moiety on APNs from Manduca sexta (Burton et al., 1999), Heliothis virescens (Luo et al., 1997) and Lymantria dispar (Valaitis et al., 1997). In contrast, Cry1Aa and Cry1Abare believed to bind APN only in a carbohydrate-independent manner (Masson et al., 1995; Nakanishi et al., 2002). Yaoi et al. (1999) localized a Cry1Aa binding site on Bombyx mori APN to the region between

region (S-P') on AgAPN2 that is essential for toxin observed an enhancing effect of another fragment ('GV") on Cry11 Ba toxicity. This is the first report that a

binding and blocking toxicity. Unexpectedly, we also

25

non-cadherinfragment of a Cry toxin-binding protein can act as a synergist of Cry toxicity to pest insects (Chen et al., 2007; Parket al., 2009a; Parket al., 2009b). BRIEF SUMMARY

30

The Subject invention relates in part to fragments of non cadherin Cry toxin binding proteins, wherein the fragments potentiate, or act as Synergists with, the insecticidal activity of Cry proteins. In some preferred embodiments, the binding protein (a Cry protein receptor on insect midgut cells) is an aminopeptidase. In preferred embodiments, the fragment comprises a Cry protein binding region.

35

BRIEF DESCRIPTION OF THE FIGURES

40

45

FIG. 1: Purified AgAPN2ta and -2tb fragments demon strate partially folded structure. FIG. 1(A) Schematic repre sentation of the truncations of AgAPN2. FIG. 1 (B). SDS PAGE of purified AgAPN2ta and -2tb. FIG. 1(C) Far-UVCD spectrum (190-240 nm) of AgAPN2ta peptide. FIG. 1(D) Far-UV CD spectrum (190-240 nm) of AgAPN2tb peptide. FIG. 2: AgAPN2ta inhibits and AgAPN2tb enhances Cry 11 Ba toxicity to An... gambiae larvae. Soluble Cry 11 Ba alone or with APN inclusions at a toxin?peptide molar ratio of 1:100 were diluted in plastic plates containing 2 ml of deion

ized water and tested against ten early 4" instar larvae of An.

50

55

60

'Ile and 'Pro. This region contains amino acid residues

RXXFPXXDEP conserved among APNs from different spe cies, and thus has been suggested as a common Cry1Aa binding region (Nakanishi et al., 2002; Nakanishi et al., 1999). Recently, a 1 12-kDa APN (AaeAPN1) in Aedes aegypti has been identified to bind Cry 11 Aa through the

Unlike the Cry1Aa binding site near the N-terminus, The Cry 11 Aabinding region was located to the C-terminal region of AaeAPN1. In our previous study, we identified a 106-kDa APN (AgAPN2) as a Cry11 Babinding protein and putative receptor in An... gambiae (Zhang et al., 2008). The 70-kDa partial AgAPN2 expressed in E. coli binds Cry 11 Ba with high affinity and blocks Cry 11 Ba toxicity towards mosquito lar vae. This APN fragment shows no similarity to the Cry1Aa binding site. Collectively, the data provide evidence that a few primary amino acid sequences on APNs are probably key factor in determining toxin specificities. To further characterize interactions between Cry 11 Ba and 70-kDa Ag APN2t we divided the peptide into two fragments of similar size. We showed that one fragment inherited the inhibitory effect of the 70-kDa peptide. By using a combina tion of in-frame deletions and binding assays, we located a

65

gambiae. Each treatment was in triplicate and the bioassays were conducted three times. Larval mortality was recorded after 24h. FIG. 2(A) Mean percent mortality (ESE) of larval mosquitoes treated with 0.5ug/ml Cry 11 Ba when APN inclu sion bodies were absent or present. FIG. 2(B) Mean percent mortality (SE) of larval mosquitoes treated with 4 ug/ml Cry 11 Ba when APN inclusion bodies were absent or present. An asterisk indicates a significant difference between larval mortality with Cry 11 Ba treatment alone and that with Cry 11Ba plus peptide treatment at the same toxin dose (one way ANOVA, P