The Peptide Ligase Activity of Human Legumain Depends on Fold Stabilization and Balanced Substrate Affinities.

Elfriede Dall,Chiara Cabrele,Fatih Demir,Hans Brandstetter,Sven O Dahms,Pitter F Huesgen,Vesna Stanojlovic,Peter Briza

doi:10.1021/acscatal.1c02057

Elfriede Dall, Chiara Cabrele + Show 6 more

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https://doi.org/10.1021/acscatal.1c02057

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Abstract

Protein modification by enzymatic breaking and forming of peptide bonds significantly expands the repertoire of genetically encoded protein sequences. The dual protease-ligase legumain exerts the two opposing activities within a single protein scaffold. Primarily localized to the endolysosomal system, legumain represents a key enzyme in the generation of antigenic peptides for subsequent presentation on the MHCII complex. Here we show that human legumain catalyzes the ligation and cyclization of linear peptides at near-neutral pH conditions, where legumain is intrinsically unstable. Conformational stabilization significantly enhanced legumain’s ligase activity, which further benefited from engineering the prime substrate recognition sites for improved affinity. Additionally, we provide evidence that specific legumain activation states allow for differential regulation of its activities. Together these results set the basis for engineering legumain proteases and ligases with applications in biotechnology and drug development.

Highlights

Localized to the endolysosomal system, the cysteine protease legumain plays important functions for the processing of antigens for presentation on the MHCII complex.[1−3] Given its specific cleavage after asparagine residues it can develop asparaginyl-endo (AEP) and -carboxypeptidase (ACP) activities in a pH-dependent manner, depending on details of its activation.[4−7] Human legumain is synthesized as an inactive proenzyme consisting of a caspase-like catalytic domain and a C-terminal death domain-like prodomain that are linked by an activation peptide (AP)
To test if mammalian legumain harbors peptide ligase activity, we established a peptide-cyclization assay based on peptides derived from the sunflower trypsin inhibitor (SFTI) precursor sequence and on mass spectrometry detection
We found that human legumain was able to hydrolyze the precursor peptides at pH 4, leading to the formation of linear L-SFTI and L-SFTI(N14) and confirming the suitability of the peptides as legumain substrates (Figure 1B)

Summary

Introduction

Localized to the endolysosomal system, the cysteine protease legumain plays important functions for the processing of antigens for presentation on the MHCII complex.[1−3] Given its specific cleavage after asparagine residues it can develop asparaginyl-endo (AEP) and -carboxypeptidase (ACP) activities in a pH-dependent manner, depending on details of its activation.[4−7] Human legumain is synthesized as an inactive proenzyme consisting of a caspase-like catalytic domain and a C-terminal death domain-like prodomain (legumain stabilization and activity modulation − LSAM − domain) that are linked by an activation peptide (AP). There has been growing evidence that legumain is translocated in the aged brain and thereby facilitates aggregation of proteins critically linked with neurodegenerative diseases, causing neuronal damage.[19−22] The presence of active AEP at cellular compartments outside the acidic endolysosome has been puzzling scientists for many years, as it is inconsistent with legumain’s pH-stability profile. Given the fact that legumain ligase activity is predominantly present at near-neutral pH, the relevance of ligase activity for extralysosomal legumain is obvious, but it is only scarcely studied

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