Native Structural and Functional Proteoform Characterization of the Prolyl-Alanyl-Specific Endoprotease EndoPro from Aspergillus niger

Abstract

The prolyl-alanyl-specific endoprotease (EndoPro) is an industrial enzyme produced in Aspergillus niger . EndoPro is mainly used for food applications but also as a protease in proteomics. In-depth characterization of this enzyme is essential to understand its structural features and functionality. However, there is a lack of analytical methods capable of maintaining both the structural and functional integrity of separated proteoforms. In this study, we developed an anion exchange (AEX) method coupled to native mass spectrometry (MS) for profiling EndoPro proteoforms. Moreover, we investigated purified EndoPro proteoforms with complementary MS-based approaches, including released N-glycan and glycopeptide analysis, to obtain a comprehensive overview of the structural heterogeneity. We showed that EndoPro has at least three sequence variants and seven N-glycosylation sites occupied by high-mannose glycans that can be phosphorylated. Each glycosylation site showed high microheterogeneity with ∼20 glycans per site. The functional characterization of fractionated proteoforms revealed that EndoPro proteoforms remained active after AEX-separation and the specificity of these proteoforms did not depend on N-glycan phosphorylation. Nevertheless, our data confirmed a strong pH dependence of EndoPro cleavage activity. Altogether, our study demonstrates that AEX-MS is an excellent tool to characterize complex industrial enzymes under native conditions.

Structural and time‐resolved mechanistic investigations of protein hydrolysis by the acidic proline‐specific endoprotease from Aspergillus niger

“…For AnPEP, seven N-glycosylation sites were predicted, and at each of these (Asn residues 100,162,226,244,328,355,446) additional electron density was observed, which is consistent with previous biochemical studies showing that all sites are occupied by highmannose glycans (Sebela et al, 2009;van Schaick et al, 2021). A total of 20 sugar residues could be fitted into the electron density (Figure 1a).…”

Studying protein structure and function by native separation–mass spectrometry

“…Finally, structural characterization of other biotechnological proteins, including industrially produced enzymes, may also benefit from the native separation approaches hyphenated to MS. For instance, a prolyl-alanyl-specific protease used for proteomics applications was analysed with AEX, followed by identification with online MS detection, as well as fraction collection for functional assignment. Proteoforms with a different level of phosphorylated glycans remained active after AEX separation, permitting their isolation and further study of the specificity in a proteoform-specific manner 164 .…”

Fungal secondary metabolites in food and pharmaceuticals in the era of multi-omics

“…Genome mining tools have also been used to identify the uncharacterized natural product by estimating the genetic potential of strain through scanning the gene of interest (Ziemert et al 2012 ). In genome sequencing of Aspergillus niger , a total of 200 new proteases have been checked during annotation; among them, two have been commercialized as prevention of haze in beer production and others applied for the production of sports drinks (van Schaick et al 2021 ). The application of whole-genome sequencing technology in the SM secreting fungi or its gene clusters bypasses the need of DNA hybridization, library construction, screening, and chromosome editing techniques (Cacho et al 2015 ).…”