Interaction of human pancreatic ribonuclease with human ribonuclease inhibitor. Generation of inhibitor-resistant cytotoxic variants.
Mammalian ribonucleases interact very strongly with the intracellular ribonuclease inhibitor (RI). Eukaryotic cells exposed to mammalian ribonucleases are protected from their cytotoxic action by the intracellular inhibition of ribonucleases by RI. Human pancreatic ribonuclease (HPR) is structurally and functionally very similar to bovine RNase A and interacts with human RI with a high affinity. In the current study, we have investigated the involvement of Lys-7, Gln-11, Asn-71, Asn-88, Gly-89, Ser-90, and Glu-111 in HPR in its interaction with human ribonuclease inhibitor. These contact residues were mutated either individually or in combination to generate mutants K7A, Q11A, N71A, E111A, N88R, G89R, S90R, K7A/E111A, Q11A/E111A, N71A/E111A, K7A/N71A/E111A, Q11A/N71A/E111A, and K7A/Q11A/N71A/E111A. Out of these, eight mutants, K7A, Q11A, N71A, S90R, E111A, Q11A/E111A, N71A/E111A, and K7A/N71A/E111A, showed an ability to evade RI more than the wild type HPR, with the triple mutant K7A/N71A/E111A having the maximum RI resistance. As a result, these variants exhibited higher cytotoxic activity than wild type HPR. The mutation of Gly-89 in HPR produced no change in the sensitivity of HPR for RI, whereas it has been reported that mutating the equivalent residue Gly-88 in RNase A yielded a variant with increased RI resistance and cytotoxicity. Hence, despite its considerable homology with RNase A, HPR shows differences in its interaction with RI. We demonstrate that interaction between human pancreatic ribonuclease and RI can be disrupted by mutating residues that are involved in HPR-RI binding. The inhibitor-resistant cytotoxic HPR mutants should be useful in developing therapeutic molecules.
Gaur
D
D
Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110067, India.
Swaminathan
S
S
Batra
J K
JK
D019943:Amino Acid Substitution; D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D002417:Cattle; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010926:Placental Hormones / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D010926:Placental Hormones; C022052:placental ribonuclease inhibitor; D012259:Ribonuclease, Pancreatic