Actinohivin: specific amino acid residues essential for anti-HIV activity.
Actinohivin (AH) is a microbial lectin containing 114 amino acids, which inhibits human immunodeficiency virus (HIV) infection. This effect is brought about by its specific binding to Man-α(1-2)-Man unit(s) of high-mannose type glycan (HMTG) bound to HIV gp120. The recently determined crystal structure of AH suggests that three repeated segments (the residue numbers 1-38, 39-76 and 77-114 for segments 1, 2 and 3, respectively) form three sugar-binding pockets to accommodate Man-α(1-2)-Man units. The strong specific binding of AH to gp120 is considered to be due to multivalent interaction of the three sugar-binding pockets with three HMTGs of gp120 via the 'cluster effect' of lectin. It remains to be seen which residues of the sugar-binding pockets are essential for acceptance of Man-α(1-2)-Man. To identify the amino acid residues critical for anti-HIV effect, we performed mutational analysis. Mutant AHs were subjected to enzyme-linked immunosorbent assay testing for gp120-binding activity and to syncytium formation assay. As a result, it was revealed that Asp15, Tyr23, Leu25, Asn28 and Tyr32 in segment 1, Tyr61 in segment 2 and Tyr99 in segment 3 are essential for anti-HIV activity. The conserved residues, Asp53, Leu63, Asn66 and Tyr70, in segment 2 and, Asp91, Leu101, Asn104 and Tyr108, in segment 3 are also necessary. Furthermore, aromatic residues at positions 23 and 32 are required for creation of potency. These data will be useful for predicting the detailed mechanism of AH-Man-α(1-2)-Man/HMTG/gp120 interaction by computational analysis and for possible development of more potent microbicides for prevention of HIV transmission.
Takahashi
Atsushi
A
Faculty of Pharmacy and College of Science and Engineering, Iwaki Meisei University, Fukushima, Japan. tanakaha@msn.com
Inokoshi
Junji
J
Tsunoda
Masaru
M
Suzuki
Kaoru
K
Takenaka
Akio
A
Sekiguchi
Takeshi
T
Omura
Satoshi
S
Tanaka
Haruo
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019380:Anti-HIV Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D004797:Enzyme-Linked Immunosorbent Assay; D006678:HIV / Q000187:drug effects*; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D006801:Humans; D008358:Mannose / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding
D019380:Anti-HIV Agents; D001426:Bacterial Proteins; D015699:HIV Envelope Protein gp120; C424136:actinohivin protein, actinomycete; C514793:gp120 protein, Human immunodeficiency virus 1; D008358:Mannose