Protein Adducts Formed by Aldehyde Products of Spermine Oxidation

Abstract

Polyamines including spermidine and spermine are positively charged molecules present in virtually all living cells, where they function as key regulators of gene expression, cell growth and proliferation, apoptosis, autophagy, and cellular stress responses [1]. Their metabolism involves tightly controlled synthesis from ornithine (via putrescine) and S‑adenosylmethionine, which donates aminopropyl groups, together with catabolic pathways mediated by amine oxidases. Polyamine degradation yields aminoaldehydes such as spermine dialdehyde and 3‑aminopropanal, along with hydrogen peroxide. These aminoaldehydes can release acrolein, a highly reactive protein‑modifying aldehyde [2]. To our knowledge, no mass‑spectrometry‑based study has directly characterized protein adducts formed by spermine dialdehyde. We therefore attempted to generate this dialdehyde using pea seedling amine oxidase incubated with spermine. MALDI‑MS showed that the expected dialdehyde had decomposed, as 3‑aminopropanal (m/z 74) was detected in the reaction mixture. Incubations of model peptides (e.g., angiotensin II and a tryptic bovine serum albumin digest) and proteins (e.g., β‑galactosidase and alcohol dehydrogenase) with spermine in the presence of amine oxidase, or with the products formed during spermine oxidation, yielded acrolein‑derived Schiff bases on lysine residues, Michael adducts on cysteine residues, and additional amino‑acid modifications. The modification patterns closely matched those generated by acrolein itself and by acrolein released from 3‑aminopropanal. We also observed protein crosslinking, yielding covalent oligomers detectable by SDS‑PAGE and gel‑permeation chromatography. Further characterization of these crosslinks is in progress. REFERENCES 1. Schibalski, R.S. et al. (2024) Am. J. Physiol. Cell Physiol. 327, C341–C356. 2. Šebela, M. and Rašková, M. (2023) Molecules 28, 7429.