Actinohivin

1. Discovery, producing organism and structure1-4,6)
Actinohivin (AH was isolated from a cultured broth of an actinomycete strain using a syncytium
formation assay system established by our group and identified to be an anti-HIV protein.3) The
producing organism was recognized as a new genus and named Longispora albida K97-0003T.6)
AH consists of a 114-amino-acid chain that exhibits internal sequence triplication.2) (Segments
1-3,; 1-38, 39-76 and 77-114, respectively).2) The gene encoding AH has been cloned and a production system of recombinant AH in Escherichia coli has been established.1)
1–38 ASVTIRNAQTGRLLDSNYNGNVYTLPANGGNYQRWTGP
39–76 GDGTVRNAQTGRCLDSNYDGAVYTLPCNGGSYQKWLFY
77–114 SNGYIQNVETGRVLDSNYNGNVYTLPANGGNYQKWYTG

Longispora albida K97-0003T

Proposed 3D modeling of actinohivin
The modeling protocol was executed by the automatedcomputer program FAMS (K. Ogata and H. Umeyama, J.
Mol. Graph. Model. 18, 258–272, 305–306 (2000)).

2. Physical data2)
White powder. 114 amino acids; mol wt 12,524. Isoelectric point 8.3.
3. Biological activity2, 5-9)
AH potently inhibits the T-cell line (T) and macrophage (M)-tropic syncytium formation inco-incubation of HeLa/T-env/Tat and HeLa/CD4/Lac-Z cells and of HeLa/M-env/Tat and HOS/
CD4/CCR5/Lac-Z cells. AH is a potent inhibitor against the infection of T- and M-tropic HIV-1
strains, including strains resistant to drugs inhibiting reverse transcriptase and protease, and HIV2 strains.

4. Mechanism of action7-15)
AH binds to gp120 from both the T- and M tropic HIV-1 in a concentration dependent manner.
AH does not bind to non-glycosylated gp120, gp120 treated with α(1-2) mannosidase and cellswith CD4 and co-receptors. Therefore, sugar chains of gp120, especially Manα(1-2)Man terminal units of high-mannose type sugar chains, play a crucial role in AH binding to gp120. It wasalso found that AH has low affinity to a Manα(1-2)Man, a high-mannose type sugar chain or
glycoproteins with low density of high-mannose type sugar chains. Conversely, it binds strongly
only to glycoproteins such as gp120 having many high-mannose type sugar chains. Cyanovirin-N, a 101 amino acid anti-HIV lectin from a cyanobacterium, has high affinity not only to aglycoprotein with single high-mannose type sugar chain but also to a high-mannose type sugar
chain.
Deletion of one or two segment (s) among three segments of AH caused severe decrease of antisyncytium formation activity. AH requires cooperative binding of three segments to high-mannose type sugar chains of gp120 for exhibiting high activity. Consequently, AH exhibits a highaffinity by the so-called ‘cluster effect’ of lectin only when three high-mannose type sugar chainsbind to three segments of AH. Thus, AH binds only to glycoproteins having many high-mannose
type sugar chains such as gp120 to show much higher specificity than cyanovirin-N.
Comparison between AH and cyanovirin-N in affinity tosugar chains and glycoproteins7)

5. Mutants with improved activities8)
A mutant of AH, His-Seg 1 trimer, of which segments 2 and 3 are replaced with segment 1, exhibited 2-fold high syncytium formation inhibitory activity. His-TEV-AH dimer/RTB-L containing two AH molecules shows about 20-fold high anti-syncytium formation activity comparedwith that of His-TEV-AH. Furthermore, His-TEV-AH dimer/RTB-L had 2-20-fold anti-HIV
activity against various primary isolates, including strains resistant to inhibitors of reverse transcriptase and protease.

Binding affinities of actinohivin, cyanovirin-N and concanavalin Ato known high-mannose type glycoproteins5)
0–4

0–4
0–4?

0–4
0–4?

0–4?
?

0.6
0.6
0.50.80.8

0.8
0.8
0.8
0.8
0.8
0.8
0.6
0.6
0.6
0.6
0.40.5
0.6
0.6
0.6
0.6
0.6
0.6
0.30.4
0.4
0.4
0.4
0.4
0.3
0.4
0.4
0.20.4
0.3
0.4
0.4
0.4
0.3
0.4
0.2
0.2
0.2
0.2
0.2
0.1
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0
0
0
0
00.1
0.1
0.1
0 2
4 6 8 10 12
0 0.5
1.5 2.0 2.5 3.0
0 0.2 0.4 0.6 0
0 2
4 6 8 10 12
2.5 3.0
0 0.2 0.4 0.6
0.8 1.0 1.2
000 0Actinohivin
000 0 2000 A ( g/ml)(g/ml)
ConcanavalinA(g/ml)Cyanovirin-N
468
10
120.5
1.0
1.5
2.0
2.5
3.0
0 0.2 0.4 0.6g/ml)
Concanavalin
Cyanovirin-N(g/ml)
2 1.0
4 1.5
6 2.0
8 2.5
10
1210 1.2
125 1.0 1.5 2.0 2.5 3.0
0.5
1.0
1.5
2.0
2.5 3.0
3.0
0.2 0.4
0.4 0.6
0.6 0.8
0.8 1.0
1.0 1.2
1.2
00 0.2
22 0.444 0.666 0.888 1.01012000 0.5
00 0.20.8

0.8

0.60.60.8
0.5
0.5
0.6
0.4
0.4

ctinohivin ( g/ml)

ActinohivinAg/ml)
Concanavalin
ActinohivinAg/ml)
g/ml)
Concanavalin
Cyanovirin-NA((µ((g/ml)
g/ml)
Actinohivin((µ(g/ml)
Concanavaling/ml)
mannose
rgp120µg/ml)
Concanavalin
Cyanovirin-N Ag/ml)
Cyanovirin-N(( (g/ml)

Ribonuclease B (Man 7 or 8)

rgp120mannose BB (Manrgp120
rgp120
Ribonuclease B (Man 7galactoseor 8)
Ribonuclease(Man 7 or 8)
Ribonuclease
Thyroglobulin(Man 7–9)7 or 8)
Man 7–9)
Ovalubmin (Manrgp120
5 or 6)N-acetylglucosamine
galactose
Thyroglobulin(ManThyroglobulin (Man 7–9)
Thyroglobulin
Ovalubmin (Man(Man5 or7–9)
6)
Ovalubmin (Man(Manor7–9)
6)
Thyroglobulin(Man7–9)
Ovalubmin
55 or6)N-acetylglucosamine

Ribonuclease B (Man 7 or 8)
Ribonuclease
B (Man 7 or 8)
mannosemannoseOvalubmin (Man 5 or 6)
galactosegalactose
Ovalubmin (Man 5 or 6)
N-acetylglucosamine
N-acetylglucosamine

Syncytium formation inhibiting activities of AH and AH dimers8)

Anti-retroviral activities of actinohivin by MAGI assay5)
Virus
T-tropic HIV-1
IIIB
NL4-3
O18A
(primary isolate)M-tropic HIV-1
JR-CFS
HIV-2
ROD
EHO

IC50 (µM)
0.002
0.016
0.11
0.038
0.014
0.004

Cyanovirin-N

mannosemannosegalactosegalactos
N-acetylglu
N-acetyl

Anti-HIV activities of AH and His-TEV-AH dimer/RTB-L against clinical isolates8)
IC50 (nM)
HIV strain
307
36
Bal
214
251
NL4-3
182
242
158

resistancesensitive
sensitive
sensitive
sensitive
RTI/PI
sensitive
NNRTI/PI
RTI/NNRTI/PI
RTI/NNRTI/PI

AH
620>10,000344423343014010His-TEV-AH
dimer/RTB-L
35446729762

RTI: reverse transcriptase inhibitor NNRTI: non-nucleotide RTI PI: protease inhibitor

6. References
1. [774] J. Inokoshi et al., Biochem. Biophys. Res. Commun. 281, 1261-1265 (2001)
2. [775] H. Chiba et al., Biochem. Biophys. Res. Commun. 282, 595-601 (2001)
3. [794] H. Chiba et al., J. Antibiot. 54, 818-826 (2001)
4. [844] A. Matsumoto et al., Int. J. Syst. Evol. Bacteriol. 53, 1553-1559 (2003)
5. [857] H. Chiba et al., Biochem. Biophys. Res. Commun. 316, 203-210 (2004)
6. [883] A. Takahashi et al., Arch. Biochem. Biophys. 437, 233-240 (2005)
7. [1056] H. Tanaka et al., Proc. Natl. Acad. Sci., USA 106, 15633-15638 (2009)
8. [1074] A. Takahashi et al., J. Antibiot. 63, 661-665 (2010)
9.			
F. Zhang et al., ChemBioChem. 15, 2766-2773 (2014)10.			
K. Suzuki et al., Acta Crystallogr. D. Biol. Crystallogr. 69, 1818-1825 (2013)
11. [1133] M. M. Hoque et al., Acta Crystallogr. D. Biol. Crystallogr. 68, 1671-1679 (2012)12.			
K. Suzuki et al., Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 68, 10601063 (2012)
13. [1095] A. Takahashi et al., J. Antibiot. 64, 551-557 (2011)
14. [1081] N. Matoba et al., PLoSOne 5, e11143 (2010)15.			
B. Hoorelbeke et al., Antimicrob. Agents Chemother. 54, 3287-3301 (2010)