MOLECULAR-CLONING AND CHARACTERIZATION OF A NOVEL HUMAN RIBONUCLEASE (RNASE K6) - INCREASING DIVERSITY IN THE ENLARGING RIBONUCLEASE GENE FAMILY

The discovery of Ribonuclease k6 (RNase k6) was an unexpected result o f our ongoing efforts to trace the evolutionary history of the ribonuc lease gene family. The open reading frame of RNase k6, amplified from human genomic DNA, encodes a 150 amino acid polypeptide with eight cys teines and histidine and lysine residues corresponding to those found in the active site of the prototype, ribonuclease A. The single-copy g ene encoding RNase k6 maps to human chromosome 14 and orthologous sequ ences were detected in both primate and non-primate mammalian species. A single mRNA transcript (1.5 kb) was detected in all human tissues t ested, with lung representing the most abundant source. At the cellula r level, transcripts encoding RNase k6 were detected in normal human m onocytes and neutrophils (but not in eosinophils) suggesting a role fo r this ribonuclease in host defense. Of the five previously identified human ribonucleases of this group, RNase k6 is most closely related t o eosinophil-derived neurotoxin (EDN), with 47% amino acid sequence id entity; slight crossreactivity between RNase k6 and EDN was observed o n Western blots probed with polyclonal anti-EDN antiserum. The catalyt ic constants determined, K-m = 5.0 mu M and k(cat) = 0.13 s(-1), indic ate that recombinant RNase k6 has similar to 40-fold less ribonuclease activity than recombinant EDN. The identification and characterizatio n of RNase k6 has extended the ribonuclease gene family and suggests t he possibility that there are others awaiting discovery.