# N‐Truncated Superoxide Dismutase‐1 in Cerebrospinal Fluid Is Folded and Active

## TL;DR

This study shows that a common N-terminal truncation in SOD1 in cerebrospinal fluid does not cause misfolding and remains active, unlike harmful C-terminal truncations linked to ALS.

## Contribution

The study reveals that N-truncated SOD1 in CSF is folded, active, and not associated with ALS pathology.

## Key findings

- N-truncated SOD1 in CSF retains full enzymatic activity and remains folded in a β-barrel structure.
- Truncated SOD1 is mainly found in heterodimers with native SOD1 and is absent in human plasma.
- N-terminal truncation does not induce misfolding or contribute to ALS, unlike C-terminal truncations.

## Abstract

Mutations in the antioxidant enzyme superoxide dismutase‐1 (SOD1) are a well‐established cause of amyotrophic lateral sclerosis (ALS). The mutations promote SOD1 misfolding, resulting in protein aggregation and motor neuron degeneration. SOD1 is normally a structurally stable enzyme, and the mechanisms underlying SOD1 misfolding remain poorly understood. Approximately one third of SOD1 in cerebrospinal fluid (CSF) exhibits an N‐terminal truncation, the biological significance of which remains unclear. This is remarkable given the dramatic effects ALS‐linked C‐terminal truncations have on the enzyme. In this study, we identified the truncation site and investigated its impact on SOD1 stability and enzymatic activity. Edman degradation revealed the cleavage site between Asn‐26 and Gly‐27, generating a 26‐residue peptide that was confirmed by mass spectrometry. We analyzed postmortem tissues from different parts of the central nervous system (CNS), including the choroid plexus, and found only trace amounts of N‐terminally truncated SOD1. Biochemical characterization of the SOD1 in CSF was done by size exclusion chromatography, ion exchange chromatography, and mass spectrometry. Our findings demonstrate that SOD1 in CSF retains full enzymatic activity, that the N‐terminally truncated variant is mainly present in heterodimers with native SOD1 subunits, and that the dimer remains folded and active, with both fragments of the truncated SOD1 fixed after proteolysis. Truncated SOD1 was absent in human plasma. In mice, only transgenically expressed human SOD1 underwent truncation in CSF, whereas endogenous murine SOD1 remained intact. Lastly, the N‐terminal truncation does not induce misfolding, unlike the destabilizing effects observed with C‐terminal truncations. The location where the truncation takes place and the underlying mechanism could not be identified. Whether the N‐truncated SOD1 variant contributes to ALS pathogenesis remains to be determined.

Superoxide dismutase 1 (SOD1) mutations cause amyotrophic lateral sclerosis (ALS) through mechanisms involving protein misfolding and aggregation. While C‐terminal truncations of SOD1 promote neurotoxicity, our study reveals that approximately one‐third of SOD1 in cerebrospinal fluid (CSF) is N‐terminally truncated—a striking observation given the structural importance of this region. The cleavage site was between Asn‐26 and Gly‐27. Both fragments stay fixed within the folded β‐barrel architecture and retain full enzymatic activity. Importantly, truncated SOD1 was abundant in CSF but nearly absent in CNS tissue, and its presence was independent of ALS status. We found no correlation between N‐terminal truncation and misfolded SOD1 levels, suggesting no contribution to ALS.