Human IFN-α Protein Engineering: The Amino Acid Residues at Positions 86 and 90 Are Important for Antiproliferative Activity

Abstract

Human IFN-α is a family of structurally related proteins that exhibit a wide range of antiproliferative activities. To understand the structural basis for these different antiproliferative activities, eight recombinant human IFN-α hybrids (HY) of α21a/α2c (HY-4, HY-5) and mutants (site-directed mutagenesis (SDM)-1, 2 and cassette mutagenesis (CM)-1, 2, 3, and 4) have been expressed, purified, and characterized. The data showed that the amino acid region 81–95 is important for antiproliferative activity. Site-directed mutagenesis and cassette mutagenesis studies showed that if serine (S) 86 and asparagine (N) 90 were replaced by tyrosine (Y), the antiproliferative activity was increased. We have also observed that if Y86 was replaced by isoleucine (I), the antiproliferative activity was comparable. However, if Y86 was replaced by aspartic acid (D), lysine (K), or alanine (A), the antiproliferative activity was substantially decreased. Our results indicate that Y and/or I at position 86 and Y at position 90 are very important in antiproliferative activity of human IFN-α. Circular dichroism spectra showed that the amino acid replacements at position 86 did not change the secondary structure. Thus the biological activity changes among those mutants do not appear to be due to conformational changes. The results also suggest that hydrophobic residue(s) at position 86 may be important for the interaction of the molecule with its receptor. The competitive binding data correlated with the antiproliferative activity. The N-terminal region of the molecule and the hydrophobic residues (including Y and I) on the C-helix region at positions 86 and/or 90 are important for binding and antiproliferative activities of human IFN-αs.

Two Interferons Alpha Influence Each Other during Their Interaction with the Extracellular Domain of Human Type Interferon Receptor Subunit 2

“…This could explain the tenfold lower antiproliferative activity of CM3 than that of IFN-α2c in 48 hours antiproliferative assay. It did however not explain the 800-fold lower ability of CM3 (with 6-histidine tag) to compete with IFN-α2c (with 6-histidine tag) for receptor binding site (17).…”

“…Our previous results showed that hybrid IFNs with N-terminal portion derived from IFN-α21b competed poorly with IFN-α2b for cellular binding (12,17). CM3 is one of these hybrids, and also has a mutation in helix C (Y86K).…”

“…Recombinant IFN-α2c, IFN-α2c-6-histidine-tag, and hybrids IFN-α21b (1-75) (82-95)/IFNα2c (76-81) (96-166) with Y86K (designated CM3), and CM3-6-histidine-tag were generated as previously described (17,24). Specific activities of IFNs on MDBK were 4.9 ×10 8 (IFNα2c), 3.2×10 8 (IFN-α2c-6-histidine-tag), 2.8×10 8 (CM3), and 3.5×10 8 (CM3-6-histidine-tag) IU/mg protein.…”

“…Specific activities of IFNs on MDBK were 4.9 ×10 8 (IFNα2c), 3.2×10 8 (IFN-α2c-6-histidine-tag), 2.8×10 8 (CM3), and 3.5×10 8 (CM3-6-histidine-tag) IU/mg protein. Preparation and purification of IFNs was described previously (17,24). Ellman assays (25) under native and denaturating conditions were performed to assess free cysteine residues.…”

“…Moreover, differences between IFN hybrid molecules (derived from IFN-α2c and IFN-α21b) in competition for receptor binding site were reported (17).…”