A Potent Novel Anti-HIV Protein from the Cultured Cyanobacterium Scytonema varium

Abstract

A new anti-HIV protein, scytovirin, was isolated from aqueous extracts of the cultured cyanobacterium Scytonema varium. The protein displayed potent anticytopathic activity against laboratory strains and primary isolates of HIV-1 with EC50 values ranging from 0.3 to 22 nM. Scytovirin binds to viral coat proteins gp120, gp160, and gp41 but not to cellular receptor CD4 or other tested proteins. This unique protein consists of a single 95-amino acid chain with significant internal sequence duplication and 10 cysteines forming five intrachain disulfide bonds.

Topology of the disulfide bonds in the antiviral lectin scytovirin

“…The amino acid sequence of SVN with three feasible disulfide pairings is shown in Figure 1(A). The topology of disulfides in SVN was originally assigned by matching the masses of its tryptic fragments obtained by LC-MS, 1 as well as of fragments of expressed SD1. 2,5 Starting with assignment of species ionizing as m/z ¼ 1318.6 (residues 30-43; C32-C38; SD1), m/z ¼ 1553.6 (residues 79-95; C80-C86; SD2), and m/z ¼ 3157.5 (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] to [51-59]; C7-55), the remaining two species, termed here ''cystine clusters'' of m/z ¼ 2511.0 and m/z ¼ 2719.1, each containing two disulfides, C20-C26/C32-C38 (SD1) and C68-C74/C80-C86 (SD2), were assigned by process of elimination.…”

“…The larger fragment was identified by N-terminal sequencing and MS to be comprised of residues 1-58, and it contained the first six half-Cystines (C7, C20, C32, C26, C38, and C55). This fragment was isolated in two forms, a single-Chain polypeptide comprised of residues 1-58 (found m/z ¼ 6133.5, Table I), and its hetero-dimeric product, residues [1][2][3][4][5][6][7][8] to , resulting from cleavage of the peptide bond Trp8-Asn9 (Table I). The smaller fragment, identified by N-terminal sequencing and MS to be comprised of residues 59-95, contained the remaining four half-Cystines paired in two disulfides (C68, C80, C74, C86; found m/z ¼ 3592.4).…”

“…As can be seen from fragment assignments [ Fig. 2(A), Table II], a heterodimeric peptide, residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]-[51-60], containing C7-C55, has been identified (fragment 10). Its Edman degradation yielded two N-terminal sequences, including di-PTH-Cystine in Cycle 7, confirming the presence of a disulfide bridge.…”

“…Scytovirin (SVN) is a potent antiviral lectin isolated from the cyanobacterium Scytonema varium, 1 reported to exhibit significant activity against human immunodeficiency virus (HIV). 1,2 Such potency makes SVN a promising candidate for development as a component of a female-Controlled, anti-HIV virucide.…”

“…However, the two structures diverged in their assignments of the other disulfide bonds. The disulfides reported on the basis of mass spectrometry 1,5 and NMR 3 indicated the pairing Cys20-Cys26 and Cys32-Cys38 in SD1, and Cys68-Cys74 and Cys80-Cys86 in SD2 [ Fig. 1(A), Mode A], whereas in the crystal structures they were reported to be Cys20-Cys32 and Cys26-Cys38 in SD1 and Cys68-Cys80 and Cys74-Cys86 in SD2 [ Fig.…”