Oxidative Folding and N-Terminal Cyclization of Onconase

Abstract

Cyclization of the N-terminal glutamine residue to pyroglutamic acid in onconase, an anti-cancer chemotherapeutic agent, increases the activity and stability of the protein. Here, we examine the correlated effects of the folding/unfolding process and the formation of this N-terminal pyroglutamic acid. The results in this study indicate that cyclization of the N-terminal glutamine has no significant effect on the rate of either reductive unfolding or oxidative folding of the protein. Both the cyclized and uncyclized proteins seem to follow the same oxidative folding pathways; however, cyclization altered the relative flux of the protein in these two pathways by increasing the rate of formation of a kinetically trapped intermediate. Glutaminyl cyclase (QC) catalyzed the cyclization of the unfolded, reduced protein, but had no effect on the disulfide-intact, uncyclized, folded protein. The structured intermediates of uncyclized onconase were also resistant to QC-catalysis, consistent with their having a native-like fold. These observations suggest that, in vivo, cyclization takes place during the initial stages of oxidative folding, specifically, before the formation of structured intermediates. The competition between oxidative folding and QC-mediated cyclization suggests that QC-catalyzed cyclization of the N-terminal glutamine in onconase occurs in the endoplasmic reticulum, probably co-translationally.The N-terminal glutamine residue of peptides and proteins undergoes non-enzymatic spontaneous cyclization resulting in the formation of pyroglutamic acid (1). This is a slow process requiring day(s) for completion, depending on the conditions (1). Examples of peptides and proteins with an N-terminal pyroglutamic acid include gonadotropin-releasing hormone, thyrotropin-releasing hormone, neurotensin, etc, for which biological activity depends on the presence of pyroglutamic acid at their N-termini (2,3). Loss or modification of the N-terminal pyroglutamic acid residue leads to a decrease in biological activity (4,5). This implies the existence of an enzyme that accelerates this cyclization in vivo (6)(7)(8). Indeed, enzymes with glutaminyl cyclase (QC) activity have been isolated from many sources (9-13), and the cDNA of QC was identified in many organisms (14)(15)(16) The anti-cancer activity of ONC (due to the absence of a specific intracellular inhibitor such as one that inhibits the activity of RNase A) is related to its ribonuclease activity (19,20,(26)(27)(28)(29).In this study, we examine the influence of the cyclization of N-terminal glutamine on the reductive unfolding and oxidative folding profiles (rates and pathways) in onconase. We also investigate the effect of formation of the native structure (which is coupled to disulfide-bond formation) on the QC-catalyzed cyclization of the protein.Experimental reductive unfolding (30-39) and oxidative folding studies (40-53) of proteins, in vitro and in vivo, have contributed to a better understanding of the relationship between protein structure and fol...

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“…This may be spontaneous, like during the recombinant production (Boix et al, 1996; Leland et al, 1998; Natomista et al, 1999) or catalyzed by glutaminyl cyclase. Most probably the cyclization takes place in the endoplasmic reticulum during the initial stages of oxidative folding and may be co-translational mechanism (Welker et al, 2007). …”

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