An Angiogenic Protein from Bovine Serum and Milk — Purification and Primary Structure of Angiogenin‐2

Abstract

Bovine serum and milk contain a basic angiogenic protein that binds tightly to placental ribonuclease inhibitor. It was purified from both sources by ion-exchange and reversed-phase chromatographies. Its amino acid sequence revealed that it is a member of the ribonuclease superfamily. It contains 123 amino acids in a single polypeptide chain, is cross-linked by three disulfide bonds, is glycosylated at Asn33, and is 57 % identical to bovine angiogenin. The amino-terminal and carboxyl-terminal residues are pyroglutamic acid and proline, respectively. The protein has ribonucleolytic activity that is similar to, but somewhat lower than, that of bovine angiogenin, i.e. very low relative to RNase. It is angiogenically potent on chicken chorioallantoic membrane, but less so than angiogenin. The sequence and activities demonstrate that this protein is a second, distinct, member of the angiogenin sub-family of pancreatic ribonucleases, and is referred to as angiogenin-2.

The angiogenins

“…All of the angiogenins isolated to date are very basic proteins, more basic than any other RNases [56]. This property allows for their easy isolation from serum or milk, despite their relatively low abundance [57][58][59][60].…”

“…These are derived from protein and/or gene sequences where needed. They are: ps1,2, the pseudogenes mouse ps-1 (U22517), mouse ps-2 (U22518) [61], with X indicating positions of frame-shifts; m2, mouse Ang-related protein [61]; m4, mouse Ang-4 (consensus of ESTs AA220397, AA277314, AA277790, AA277758, AA469774, AA473916, AA500132, AA596231, AA611252); m3, mouse Ang-3 (U72672) [63]; m1, mouse Ang-1 [58]; rabbit Ang [59]; human Ang [3]; porcine Ang [59]; bov-1, bovine Ang-1 [60,120]; bov-2, bovine Ang-2 [56]; consensus of the active sequences. Residues conserved between active angiogenins are boxed, Cys are in bold and the main residues thought to be involved with the receptor site of angiogenin are shaded.…”

“…Perusal of the EST databanks (GenBank) reveals another murine angiogenin, which for convenience is named Ang-4, a consensus sequence of a number of clones. A second angiogenin from cattle, Ang-2, was recently described [56]. Only one human, rabbit or porcine angiogenin has been seen.…”

“…The underlying RNase activity is reflected by conservation of the catalytic residues His-13, Lys-40 and His-114, and of active site residues such as the RNase-1 signature region -the latter part of region 2. The two angiogenins of cattle which have only 57% identity in sequence additionally differ markedly by a carbohydrate modification on Ang-2 and not Ang-1 [56]. This modification is on the homologous position of Asn-34 of the RNase-1 enzymes of a variety of species, which is the most frequently observed glycosylated site in this family [68,69].…”

Characterization of Bovine Angiogenin-1 and Lactogenin-Like Protein as Glycyrrhizin-Binding Proteins and Their in Vitro Phosphorylation by C-kinase.

“…The N-terminal 18 amino acids (AQDDYRIH-FLTQHYDAKP) of p 15 were 100% identical with the corresponding sequence of bovine angiogenin-1, which belongs both to a class of direct inducers of angiogenesis and to the ribonuclease superfamily. 16) However, p17…”