Dual site model for the organogenic activity of angiogenin.

Abstract

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...

The amino acid sequence of human ribonuclease 4, a highly conserved ribonuclease that cleaves specifically on the 3′ side of uridine

“…The extremely tight-binding inhibitor of pancreatic RNase (Blackburn et al, 1977;Lee et al, 1989), binds avidly to RNase 4 (Shapiro et al, 1986a), as it does to angiogenin (Lee et a]., 1989) and RNase 2 (Shapiro and Vallee, 1991). This signals a coinciding need for close control of the activities of these enzymes and implies a specific biological function for the relatively rarc RNase 4, and perhaps even Tor RNase 1, which has yet to be found.…”

“…As in the case of angiogenin (Hallahan et al, 1991), these changes might involve non-active-site regions that are related to other, as yet unknown: aspects of RNase-4 function. The deletion at postitions 77 and 78 is superficially easy to accommodate in the RNase structure, as these residues protrude from the 8-sheet structure and do not participate in the hydrogen bonding characteristic for 8 sheets (Wlodawer et al, 1982).…”

“…This enzyme exists in many sizes and with a range of properties, due in part to the covalent attachmcnt of carbohydrates (Beintema et al, 1988a(Beintema et al, , 1988b. The 'non-secretory' RNase (EDN) which is found in spleen (Delaney, 1963;Yasuda et al, 1990), kidney and urine (Delaney, 1963;lwama et al, 1981;Cranston et al, 1980), eosinophils , liver (Sorrentino et al, 1988) and placenta (Shapiro and Vallee, 1991), has also been sequenced (Beintema ct al., 1988b) and is a very different, though homologous, enzyme. Two additional RNase family members whosc sequences havc been determined are ECP , Rosenberg et al, 1989a, which has been isolated thus far only from granulocytes, and angio- genin (Strydom et al, 1985) which is found in tumor-cellconditioned medium (Fett et al, 1985) as well as in plasma (Shapiro et al, 1987).…”

Antitumor effect of B16 melanoma cells genetically modified with the angiogenesis inhibitor RNasin

“…19 Of special interest are the studies performed by site -specific mutagenesis to identify those residues significant for ribonuclease and angiogenic activity of the molecule. In most instances, the efficacy of angiogenin in performing both activities is correlated, although Hallahan et al 27 have suggested that the RNase active site in angiogenin is essential, but not sufficient, for its angiogenic activity, and that an additional second site on the molecule is required.…”

What maintains the high intra-follicular estradiol concentration in pre-ovulatory follicles?

“…This allows for an optimal recovery of these proteins at the purification stage. The following is a sample of papers that used this protocol to recover biotinylated proteins: [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25]. Following the pre-adsorption phase, the column was then washed with 16 ml of 10 % acetic acid and 0.1 M NaHPO 4 until the pH of the flow-through was >6.8.…”