Structural changes to ribonuclease A and their effects on biological activity.
Bovine seminal ribonuclease (BS RNase) displays immunosuppressive and antitumor activities on mammalian cells, whereas bovine pancreatic ribonuclease (RNase A) is not cytotoxic. To learn more about the mechanism of BS RNase cytotoxicity, various mutants and hybrid proteins were prepared. A series of RNase A variants substituted with amino acid residues from BS RNase were prepared. Concerning quaternary structure, a significant impact was achieved in the variant TM (Q28L K31C S32C), which forms a dimer joined covalently by two intersubunit disulfide bonds. This variant is more efficient than RNase A but less active than BS RNase. Introduction of cationic residues at positions 55, 62, and 64 or substitution at positions 111 and 113 enhanced the immunosuppressive activity of RNase A but did not confer its antitumor activity. The substitution at positions 28, 31, 32, 55, 62, 64, 111, and 113 in variant T13 exerted the best immunosuppressive and antitumor effect observed among the round of the RNase A variants. Replacement of the active-site histidine residues H12 and H119 with asparagine led to the loss of both catalytic and biological activities. Five previously prepared hybrid enzymes (SRA 1-5), synthesized by introducing 16 amino acid residues from RNase A into BS RNase, exerted the same immunosuppressive activities as did the wild-type BS RNase. However, the substitution at positions 111, 113, and 115 in variant SRA 5 caused a marked decrease in its antitumor effect, indicating that these residues play an important role in antitumor efficiency. A different mechanism of action of RNases on tumor cells and/or on blastogenic transformed lymphocytes has been assumed.
Soucek
J
J
Department of Cell Physiology, Institute of Hematology and Blood Transfusion, Prague, Czech Republic. souc@uhkt.cz
Raines
R T
RT
Haugg
M
M
Raillard-Yoon
S A
SA
Benner
S A
SA
D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D006801:Humans; D007166:Immunosuppressive Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D020014:K562 Cells; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects; D009154:Mutation; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
D000970:Antineoplastic Agents; D007166:Immunosuppressive Agents; D012259:Ribonuclease, Pancreatic