Enzymically active angiogenin/ribonuclease A hybrids formed by peptide interchange

Abstract

The primary structures of the blood vessel inducing protein human angiogenin and human pancreatic ribonuclease (RNase) are 35% identical. Angiogenin catalyzes the limited cleavage of ribosomal RNA (18 and 28 S), yielding a characteristic pattern of polynucleotide products, but shows no significant activity toward conventional pancreatic RNase substrates [Shapiro, R., Riordan, J. F., & Vallee, B. L. (1986) Biochemistry 25, 3527-3532]. Angiogenin/RNase hybrid enzymes--wherein particular regions of primary structure in RNase are replaced by the corresponding segments of angiogenin--serve to explore the structural features underlying angiogenin's characteristic activities. Herein we show that synthetic angiogenin peptides, Ang(1-21) and Ang(108-123), form noncovalent complexes with inactive fragments of bovine RNase A--RNase(21-124) (i.e., S-protein) and RNase(1-118), respectively--with regeneration of activity toward conventional RNase substrates. Maximal activities for the Ang(1-21)/S-protein complex (Kd = 1.0 microM) are 52%, 45%, and 15% toward cytidine cyclic 2',3'-phosphate, cytidylyl(3'----5')adenosine, and yeast RNA, respectively. In contrast, activities of the RNase(1-118)/Ang(108-123) hybrid (Kd = 25 microM) are 1-2 orders of magnitude lower toward cyclic nucleotides and dinucleoside phosphates. However, substitution of phenylalanine for Leu-115 in Ang(108-123) increases activity up to 100-fold. Both His-13 and His-114 in the angiogenin peptides are required for activity since their substitution by alanine yields inactive complexes. Importantly, the pattern of polynucleotide products formed during cleavage of ribosomal RNA by the Ang(1-21)/S-protein hybrid shows a striking resemblance to that formed by angiogenin, demonstrating that the hybrid retains features of both angiogenin and RNase A.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystal structure of bovine angiogenin at 1.5-A resolution.

“…The replacement of Phe-120 in RNase A by Leu-115 in hAng has been thought to be another factor contributing to the lower enzymatic activity of Ang (28). bAng, like RNase A, contains a Phe at this position in the sequence that is located similarly in the three-dimensional structure.…”

Ribonuclease activity and substrate preference of human eosinophil cationic protein (ECP)

“…None of the sixteen dinucleoside phosphates (NpN ') and neither uridine and cytidine 2' ,3'-cyclic phosphates was degraded at an appreciable rate by ECP under the conditions used; in each assay bovine RNase A was included as a positive control on the method used. Again this is characteristic of the non secretory class of RNases which have been found to degrade dinucleoside phosphates very slowly and to be virtually inactive in the hydrolysis of nucleoside 2' ,3 ' -cyclic phosphates [ 15,161. This inactivity with small substrates may be due in part to the absence of an aromatic residue (Phe or Tyr) at the site equivalent to position 120 of the bovine RNase A sequence; in human angiogenin the replacement of leucine by phenylalanine at this position increases the activity of this protein against small substrates up to lOO-fold [31].…”

Folding and activity of hybrid sequence, disulfide-stabilized peptides.

“…5). Hybrid sequence peptides of RNase with angiogenin, which confer modified activity with RNAs, have also been made (27). In this case, the intent was to transfer substrate specificity rather than to stabilize a particular secondary structure, as in our case.…”