Mechanism by which the lectin actinohivin blocks HIV infection of target cells

Abstract

Various lectins have attracted attention as potential microbicides to prevent HIV transmission. Their capacity to bind glycoproteins has been suggested as a means to block HIV binding and entry into susceptible cells. The previously undescribed lectin actinohivin (AH), isolated by us from an actinomycete, exhibits potent in vitro anti-HIV activity by binding to high-mannose (Man) type glycans (HMTGs) of gp120, an envelope glycoprotein of HIV. AH contains 114 aa and consists of three segments, all of which need to show high affinity to gp120 for the anti-HIV characteristic. To generate the needed mechanistic understanding of AH binding to HIV in anticipation of seeking approval for human testing as a microbicide, we have used multiple molecular tools to characterize it. AH showed a weak affinity to Man␣(1-2)Man, Man␣(1-2)Man␣(1-2)Man, of HMTG (Man8 or Man9) or RNase B (which has a single HMTG), but exhibited a strong and highly specific affinity (K d ‫؍‬ 3.4 ؋ 10 ؊8 M) to gp120 of HIV, which contains multiple Man8 and/or Man9 units. We have compared AH to an alternative lectin, cyanovirin-N, which did not display similar levels of discrimination between high-and low-density HMTGs. X-ray crystal analysis of AH revealed a 3D structure containing three sugar-binding pockets. Thus, the strong specific affinity 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. Thus, AH is a good candidate for investigation as a safe microbicide to help prevent HIV transmission.action mechanism ͉ anti-HIV ͉ gp120 ͉ high-mannose type glycan ͉ cyanovirin-N

Actinohivin: specific amino acid residues essential for anti-HIV activity

“…Our results define the amino acid residues essential for the specific interaction between AH and Man-a(1-2)-Man/HMTG/gp120, consistent with X-ray structures, 12 and should facilitate development of highly active AH derivatives for use in anti-HIV microbicides.…”

“…Each segment forms a structural module composed of four or five b-strands, a p-helix and a long loop, as shown in Figure 4a. 12 A valley (10 Å in length, 15 Å in The three modules of AH assemble in a similar manner to the b-trefoil fold 13 that proteins belonging to CBM13 commonly possess, and this fold is also found in RTB and XBD. Although primary sequence homology between AH, RTB and XBD is low, overall three-dimensional structures are generally similar to each other.…”

“…9,10 This inhibitory effect of AH is brought about by potent binding to HIV gp120 through specific interaction with Man-a(1-2)-Man units of HMTGs on gp120. 11,12 AH is composed of 114 amino acid residues and contains three tandem repeats (amino acids 1-38, 39-76 and 77-114; segments 1, 2 and 3, respectively), with each segment containing well-conserved LD-QXW motifs. These profiles suggest that AH belongs to the carbohydrate-binding module family 13 (CBM13).…”

Solution Structure of the Monovalent Lectin Microvirin in Complex with Manα(1–2)Man Provides a Basis for Anti-HIV Activity with Low Toxicity

“…Together, these data demonstrate that structural changes do not occur for residues outside the binding site. Intermolecular interproton distance restraints between MVN and mannobiose used in the calculations were derived from three-dimensional 12 C-filtered/ 13 C-separated intermolecular NOE experiments (Fig. 4A).…”

“…To exploit this barrier as a therapeutic target, carbohydrate-binding proteins, known as lectins, have emerged as promising anti-HIV agents (3,9,10). A number of lectins and at least one monoclonal antibody, 2G12, potently block HIV infection at the initial stage of membrane fusion by binding carbohydrate structures present on gp120 (11)(12)(13)(14), providing support for this approach.…”