Mutagenesis of aspartic acid-116 enhances the ribonucleolytic activity and angiogenic potency of angiogenin.

Harper, Jay; Vallée, Bert L.

doi:10.1073/pnas.85.19.7139

Cited by 40 publications

(38 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, abolition of the enzymatic activity of angiogenin by chemical modification (7), mutagenesis (8,9), or binding of placental RNase inhibitor (10) concomitantly diminishes angiogenic activity. Further, mutations that enhance the catalytic activity of angiogenin simultaneously increase angiogenicity (11). These results clearly show that the ribonucleolytic activity of angiogenin is necessary for biological activity.…”

mentioning

confidence: 52%

Dual site model for the organogenic activity of angiogenin.

Hallahan

Shapiro

Vallée

1991

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

108

105

View full text Add to dashboard Cite

The residues that are indispensable for the ribonucleolytic activity of angiogenin are also known to be essential for its angiogenic activity. We now demonstrate that residues in another region of the protein, devoid of catalytic residues, are additionally required for angiogenesis. Endoproteinase Lys-C or a baby hamster kidney cell protease cleaves angiogenin at the peptide bond either between Lys-60 and Asn-61 or between Glu-67 and Asn-68, respectively. The two polypeptide fragments resulting from either cleavage remain linked by disulfide bonds. These two derivatives and des-(Asn"'-Glu 7)-angiogenin-n which both bonds are cleavedretain their ribonucleolytic activities toward tRNA, 18S and 28S rRNA, and dinucleoside phosphates but are no longer angiogenic on the chicken embryo chorioallantoic membrane. C and A2 (12-14), (ii) the highaffinity, specific binding of angiogenin to endothelial cells (15), (iii) the isolation of an endothelial cell surface protein that binds angiogenin (16), and (iv) the capacity of certain enzymatically inactive angiogenin mutants to inhibit angiogenesis (9). The last item suggests in addition that the receptor-binding region of angiogenin is, at least in part, distinct from the catalytic site.Ifa region of the angiogenin molecule functions exclusively in receptor binding, it might be possible to alter that region selectively, thereby affecting angiogenesis but without altering enzymatic activity. We have been able to achieve precisely this dissociation of activities by specific proteolysis of the peptide bond either between Lys-60 and Asn-61 or between Glu-67 and Asn-68 in angiogenin to generate angiogenin K and angiogenin E, respectively. Angiogenin E and K, as well as des-(Asn61Glu67)-angiogenin in which both bonds are cleaved, retain enzymatic activity yet are neither angiogenic nor capable of activating second messenger-regulated pathways in endothelial cells. Moreover, all three derivatives fail to compete with angiogenin in the angiogenesis assay. This indicates that the region from Lys-60 to Asn-68 encompassing the two cleavage sites may form part of a cell-surface receptor binding site separate and distinct from the enzymatic active site of angiogenin. MATERIALS AND METHODSAngiogenin was obtained from recombinant expression systems in either Escherichia coli (17) or baby hamster kidney (BHK) cells (18). In the latter case, purification to homogeneity was achieved by CM-52 cation-exchange chromatography followed by Mono S (Pharmacia) HPLC. In some instances, the final preparation obtained had been partially cleaved by a BHK cell protease (see Results). The cleaved derivative, denoted angiogenin E, was purified by C18 HPLC using a Synchropak RP-P column (250 x 4.6 mm; SynChrome, Lafayette, IN). Elution was achieved with a 54-min linear gradient from 30o to 42% solvent B at 0.8 ml/min at ambient temperature, where solvent A was 0.1% CF3COOH in water and solvent B was 2-propanol/acetonitrile/water, 3:2:2 (vol/vol) containing 0.08% CF3COOH. Angiogenin K (19) was prepare...

show abstract

mentioning

confidence: 52%

Dual site model for the organogenic activity of angiogenin.

Hallahan

Shapiro

Vallée

1991

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

108

105

View full text Add to dashboard Cite

show abstract

“…Mutational studies on hAng have quantified the contributions of various C-terminal residues to the low ribonucleolytic activity of the protein and to the stability of the B 1 subsite blockage (76)(77)(78)(79)(80). Analysis of the Q117G-and Q117A-hAng mutants has proved to be straightforward: tRNA cleavage activity is increased 30-and 14-fold, respectively, dinucleotide specificity is unchanged, and structural perturbations are localized to the side chain in question (31,78,80).…”

Section: Resultsmentioning

confidence: 99%

Biological and Structural Features of Murine Angiogenin-4, an Angiogenic Protein^,

et al. 2007

View full text Add to dashboard Cite

Murine angiogenin-4 (mAng-4) is a member of the pancreatic ribonuclease superfamily that is expressed in some endodermally derived organs. We now show that mAng-4 is angiogenic using a thoracic aorta assay never before applied to the angiogenins. mAng-4, human angiogenin (hAng), and murine angiogenin-1 (mAng-1) stimulate the proliferation of IGR1 melanoma cells but do not stimulate the proliferation or migration of bovine corneal endothelial cells or primary mouse embryonic fibroblasts. In addition, we report the 3-D structure of mAng-4 at 2.02-Å resolution. The structure shows that the residues forming the putative B 1 , P 1 , and B 2 RNA-binding subsites occupy positions similar to their hAng counterparts. The B 1 subsite is obstructed by Glu115 and Ile118. The obstruction is stabilized by a novel salt bridge between the C-terminal carboxyl group and the side chain of Arg99. Through mutational studies, we identify residues critical to the angiogenic function of mAng-4. The effect of H12A and H112A mutations in the catalytic site indicates that ribonucleolytic activity is essential to angiogenesis. The consequences of a nearby E115A mutation are consistent with a significant role for Glu115 in the attenuation of enzymatic activity but also suggest that sufficient suppression of catalysis is necessary for angiogenesis. The effect of an R32A mutation in the putative nuclear localization sequence indicates that this residue is crucial for angiogenesis. In the putative cell-binding segment, the replacement of Lys59 with Asn (its counterpart at position 61 of hAng) does not abrogate enzymatic activity but abolishes angiogenic activity, the reason for which is unclear.

show abstract

“…However, ANG has no known effect by itself on cell proliferation, migration, or other physiological events associated with angiogenesis. Its unusual ribonucle- olytic specificity toward ribosomal RNA (9,10,12,35) and its angiogenic capacity appear to be interrelated since chemical modification or site-directed mutagenesis of amino acid residues involved directly (9,36) or indirectly (37) in RNase catalysis have resulted in the abolition or enhancement of both the enzymic and the angiogenic activities. This apparent relationship suggests that RNA in vivo might be a target for ANG and points to a potential intracellular function of ANG.…”

Section: Resultsmentioning

confidence: 99%

Specific binding of angiogenin to calf pulmonary artery endothelial cells.

Badet

Soncin

Guitton

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

121

106

View full text Add to dashboard Cite

Specific binding of angiogenin (ANG) to calf pulmonary artery endothelial cells was demonstrated. Cellular binding at 4TC of 125-Ilabeled human recombinant ANG was time and concentration dependent, reversible, and saturable in the presence of increasing amounts of the unlabeled molecules. The interaction was shown to be specific since a large excess of unlabeled ANG reduced labeled ANG binding by >80%, whereas similar doses of RNase A, a structurally related protein, had no effect. Scatchard analyses of binding data revealed two apparent components. High-affinity sites with an apparent dissociation constant of 5 x 10-9 M were shown to represent cell-specific interactions. The second component, comprising low-affinity/high-capacity sites with an apparent dissociation constant of 0.2 x 10-6 M, was essentially associated with pericellular components. High-affinity ANG binding sites varied with cell density and were found on other endothelial cells from bovine aorta, cornea, and adrenal cortex capillary but not on Chinese hamster lung fibroblasts. Divalent copper, a modulator of angiogenesis, was found to induce a severalfold increase in specific cell-bound radioactivity. Placental ribonuclease inhibitor, a tight-binding inhibitor of both ribonucleolytic and angiogenic activities of ANG, abolished 1251-labeled human recombinant ANG binding only in the absence of copper.

show abstract

Mutagenesis of aspartic acid-116 enhances the ribonucleolytic activity and angiogenic potency of angiogenin.

Cited by 40 publications

References 20 publications

Dual site model for the organogenic activity of angiogenin.

Dual site model for the organogenic activity of angiogenin.

Biological and Structural Features of Murine Angiogenin-4, an Angiogenic Protein^,

Specific binding of angiogenin to calf pulmonary artery endothelial cells.

Contact Info

Product

Resources

About

Mutagenesis of aspartic acid-116 enhances the ribonucleolytic activity and angiogenic potency of angiogenin.

Cited by 40 publications

References 20 publications

Dual site model for the organogenic activity of angiogenin.

Dual site model for the organogenic activity of angiogenin.

Biological and Structural Features of Murine Angiogenin-4, an Angiogenic Protein,

Specific binding of angiogenin to calf pulmonary artery endothelial cells.

Contact Info

Product

Resources

About

Biological and Structural Features of Murine Angiogenin-4, an Angiogenic Protein^,