Copper binding to naturally occurring, lactam form of angiogenin differs from that to recombinant protein, affecting their activity

Abstract

Angiogenin is a member of the ribonuclease family and a normal constituent of human plasma. It is one of the most potent angiogenic factors known and is overexpressed in different types of cancers. Copper is also an essential cofactor in angiogenesis and, during this process, it is mobilized from inside to outside of the cell. To date, contrasting results have been reported about copper(ii) influencing angiogenin activity. However, in these studies, the recombinant form of the protein was used. Unlike recombinant angiogenin, that contains an extra methionine with a free terminal amino group, the naturally occurring protein present in human plasma starts with a glutamine residue that spontaneously cyclizes to pyroglutamate, a lactam derivative. Herein, we report spectroscopic evidence indicating that copper(ii) experiences different coordination environments in the two protein isoforms, and affects their RNase and angiogenic activity differently. These results show how relatively small differences between recombinant and wild type proteins can result in markedly different behaviours.

Coordination Environment of Cu(II) Ions Bound to N-Terminal Peptide Fragments of Angiogenin Protein

“…All signals have been assigned (Tables S1–S8 in Supplementary Materials); the range of ppm chemical shift related to peptide backbone N–H protons (7.79 < HN < 8.42) is narrower for both Ang(1–17) and AcAng(1–17) in comparison to that reported for the whole protein (7.74 < HN < 8.62) [42]. However, such a shift is larger than that assigned to completely unfolded protein (8.12 < HN < 8.42) [45], suggesting a partial folding of both peptides.…”

“…Noteworthy, the two proteins, r-Ang and wt-Ang, bind copper ions differently: in the recombinant form, the anchoring site of metal ion is the terminal amino group, whereas the native wild-type protein binds Cu 2+ through His-114 and His-13 [42]. Such amino acids constitute two out of the three catalytic residues of the protein, and the addition of copper ions at physiological pH influences much more the RNase activity and the capillary-like tubes formation in wt-Ang than in r-Ang [42].…”

“…Such amino acids constitute two out of the three catalytic residues of the protein, and the addition of copper ions at physiological pH influences much more the RNase activity and the capillary-like tubes formation in wt-Ang than in r-Ang [42]. A comprehensive characterization of the Ang–copper(II) complex species is therefore a valuable support in the improved understanding of potential mutual biological influences.…”

A Naturally Encoded Dipeptide Handle for Bioorthogonal Chan–Lam Coupling

“…[28] It has recently been suggested that N-terminal cyclization to form pyroglutamate creates anew,additional copper-binding site in the protein angiogenin, despite the fact that this protein does not have ah istidine in the second position of its amino acid sequence. [29] It is,t hus,t empting to speculate for ab iological metal-binding role for pyroglutamate.H owever, the affinity of pyroglutamate-histidine N-terminal sequences is quite modest (K d % 1 mm), far below typical biological copper availability. [28] This does not, of course,p reclude biological roles that involve binding to ametalloprotein.…”

A Naturally Encoded Dipeptide Handle for Bioorthogonal Chan–Lam Coupling

“…Curiously, several of these have pyroglutamate‐histidine N‐terminal sequences, and their Cu‐binding properties have been characterized . It has recently been suggested that N‐terminal cyclization to form pyroglutamate creates a new, additional copper‐binding site in the protein angiogenin, despite the fact that this protein does not have a histidine in the second position of its amino acid sequence . It is, thus, tempting to speculate for a biological metal‐binding role for pyroglutamate.…”