Monika Haugg

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.

7 – Evolutionary Reconstructions in the Ribonuclease Family

Publisher Summary This chapter provides an overview of the ribonuclease (RNases) family. RNases of the bovine pancreatic superfamily play a central role in the development of ideas and technologies to study protein structure and catalysis. This is because RNase is small, present in large amounts in the pancreas of ruminants, stable under a wide range of conditions, and therefore amenable to full chemical analysis. It was among the first proteins to be sequenced, the first protein to be examined by NMR spectroscopy, and the first to be unfolded and refolded in the laboratory. This chapter also discusses the use of protein engineering for understanding the evolution. It explains concepts related to experimental paleomolecular geobiology. It also describes collection of additional seminal RNase sequences from recently diverging artiodactyls. An overview of reconstructing evolution of biomolecular behavior in the RNase superfamily is also presented in this chapter. The chapter elaborates in detail about the repair of damaged pseudogenes by gene conversion. The chapter concludes with a discussion on physiological functions of seminal RNase.