Abstract
Human pancreatic ribonuclease (HPR), a member of RNase A superfamily, has a high activity on double stranded (ds) RNA. By virtue of this activity HPR appears to be involved in the host-defense against pathogenic viruses. To delineate the mechanism of dsRNA cleavage by HPR, we have investigated the role of glutamine 28 and arginine 39 of HPR in its activity on dsRNA. A non-basic residue glycine 38, earlier shown to be important for dsRNA cleavage by HPR was also included in the study in the context of glutamine 28 and arginine 39. Nine variants of HPR respectively containing Q28A, Q28L, R39A, G38D, Q28A/R39A, Q28L/R39A, Q28A/G38D, R39A/G38D and Q28A/G38D/R39A mutations were generated and functionally characterized. The far-UV CD-spectral analysis revealed all variants, except R39A, to have structures similar to that of HPR. The catalytic activity of all HPR variants on single stranded RNA substrate was similar to that of HPR, whereas on dsRNA, the catalytic efficiency of all single residue variants, except for the Q28L, was significantly reduced. The dsRNA cleavage activity of R39A/G38D and Q28A/G38D/R39A variants was most drastically reduced to 4% of that of HPR. The variants having reduced dsRNA cleavage activity also had reduction in their dsDNA melting activity and thermal stability. Our results indicate that in HPR both glutamine 28 and arginine 39 are important for the cleavage of dsRNA. Although these residues are not directly involved in catalysis, both arginine 39 and glutamine 28 appear to be facilitating a productive substrate-enzyme interaction during the dsRNA cleavage by HPR.
Highlights
Human pancreatic ribonuclease (HPR) is a member of an ancient superfamily of proteins, called RNase A superfamily [1]
HPR exhibits a remarkably high activity against double stranded RNA. This high dsRNA cleavage activity of HPR suggests the enzyme to be playing a role in host defense
In this study we have investigated the role of glutamine 28, glycine and arginine in the dsRNA cleavage activity of HPR
Summary
Human pancreatic ribonuclease (HPR) is a member of an ancient superfamily of proteins, called RNase A superfamily [1]. Unlike the other members of the family, HPR displays substantial activity on double stranded (ds) RNA even under conditions in which dsRNA maintains stable secondary structure [2]. This activity is unrelated to digestion and is thought to be involved in the host-defense against pathogenic viruses [2,3]. The members of RNase A superfamily cleave single stranded (ss) RNA by a transesterification reaction, which requires linear arrangement of the 29oxygen atom, 59oxygen atom and the intermediate phosphorus atom [8] This ‘inline’ orientation of phosphodiester bond is not possible in dsRNA, which adopts helical secondary structure [9]. We have reported that lysine 6, arginine 32, lysine 62 and lysine 74 do not play a direct role in the dsRNA cleavage activity of HPR, lysine 6, lysine 74 and lysine 62 appear to be involved in general catalysis, structural integrity and stability and DNA helix unwinding activity of HPR [12]
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