Enzymatic access to the rare ΔUA (1→4) Glc 3, 6, N-sulfated heparin disaccharide, implications for heparin quality control

Background

Heparin is a naturally occurring glycosaminoglycan found in animal tissues, primarily sourced from porcine intestinal mucosa, though it may occasionally be derived from bovine lungs [1]. The extraction of heparin, however, is challenging, complicated further by the potential contamination of pharmaceutical heparin. Along with supply chain risks and growing demand, this has spurred an interest in alternative sources of heparin, including bioengineered heparin.

The distinct chemical profile of heparin, determined by factors like molecular weight ranges and sulfation structures, can lead to variations in its biological effects and safety in clinical use. This underscores the importance of reliable methods for detecting and analyzing heparin [2,3]. Approaches that utilize controlled depolymerization of heparin with heparinases facilitate both the structural and sequence analysis of the resulting disaccharides and oligosaccharides.

Key findings

Heparin digestion profile by Bacteroides eggerthii (BeHepI) and Pedobacter heparinus (PhHepI)

The preprint authors digested unfractionated heparin by using Heparin lyase I from Bacteroides eggerthii (BeHepI). They noted an increase in absorbance at 232 nm, corresponding to the formation of unsaturated uronic acid (see Figure 2A). Size exclusion chromatography (SEC) profiles revealed distinct digestion patterns (see Figure 2B): BeHepI generated lower molecular weight products (<3.6 kDa), while heparinase I from Pedobacter heparinus (PhHepI) produced less digested, higher molecular weight products (4.2-6.0 kDa), suggesting a higher cleavage efficiency of BeHepI.

Heparin chromatography (BPC) profiles and mass spectra following fondaparinux digestion by BeHepI and PhHepI

The authors then compared the digestion products of fondaparinux, a heparin mimetic, by BeHepI and PhHepI enzymes using liquid chromatography coupled with mass spectrometry (LC-MS). BeHepI produced a wider variety of fragments (see Figure 3), including specific disaccharides, demonstrating its ability to cleave key regions of fondaparinux, particularly those containing modifications like 3-O-sulfation. In contrast, PhHepI showed a more limited profile, with lower efficiency in processing these critical regions.

Structural analysis of fondaparinux digestion products by BeHepI using nuclear magnetic resonance (NMR)

By obtaining a 1H-13C HSQC nuclear magnetic resonance spectrum for fondaparinux digested with BeHepI, the authors could observe the presence of unsaturated products resulting from cleavage (see Figure 4). Moreover, they noted differences in the digestion efficiency of iduronic acid (IdoA) and glucuronic acid (GlcA), confirmed by the progressive loss of the corresponding peaks.

Crystallographic structure of BeHepI’s active site in complex with GA

The authors were able to obtain the crystal structure of BeHepI in complex with the disaccharide GA* at the active site (see Figure 5). Figure 5A displays the overall structure of the enzyme, while figure 5B and figure 5C provide detailed views of the active site, highlighting the interactions between the bound disaccharide and enzyme residues. Figure 5D emphasizes the presence of a C4-C5 unsaturated double bond in the uronic acid, resulting from β-eliminative cleavage, corroborating LC-MS and NMR data on cleavage specificity. In this preprint, the authors provide a detailed structural analysis of BeHepI, showing the enzyme binding to the disaccharide GA*. Unlike the heparinase from Pedobacter heparinus (PhHepI), the structural model of BeHepI includes two additional loops (Loop 1 and Loop 2) around the binding site, located between residues Tyr93-Lys99 (Loop 1) and Leu213-Gly232 (Loop 2). Loop 1 is particularly important because it facilitates interactions with the 3-O-sulfate group of the disaccharide GA*, enhancing BeHepI’s affinity and specificity for this structural modification. The presence of these loops expands the positive electrostatic interaction surface of BeHepI, making it more effective at cleaving highly sulfated oligosaccharides, a characteristic that PhHepI lacks.

Why I think this preprint is important

This preprint is relevant as it presents BeHepI as a valuable tool for heparin quality control and structural analysis. BeHepI uniquely targets 3-O-sulfated glucosamine regions essential for heparin’s anticoagulant activity where traditional heparinases are less effective. This specificity enables precise disaccharide production and accurate characterization of heparin’s active sequences, enhancing standardization and pharmaceutical reliability, and addressing global quality demands for heparin.

Suggestions and questions for the authors:

Q1) Perhaps you could add a ‘Highlights’ section to your article summarizing the main findings and emphasize the study’s relevance. Highlights enable readers and reviewers to quickly grasp the study’s significance and innovation.

Q2) Could you include a comparison of the fragments generated by BeHepI with the expected fragments from different sulfation patterns to confirm the enzyme’s specificity.

Q3) BeHepI’s efficiency was tested under specific conditions and with synthetic substrates like fondaparinux, a heparin mimic. However, the enzyme’s observed efficiency and specificity may vary depending on different heparin samples and sources. If possible, I would really like to see a comparison with heparins from bovine and porcine sources.

Q4) Considering that loops 1 and 2 are unique to BeHepI, it could be inferred that they play a crucial role in increasing the enzyme’s specificity for certain heparin regions. In this sense, would you consider – in a follow-up study – to genetically engineering PhHepI to incorporate the exact amino acid sequences of loops 1 and 2, in order to assess whether this modification enhances its specificity for particular heparinase activity?

Q5) For industrial applications, large-scale production and stability of BeHepI are critical factors. While this study demonstrated BeHepI’s efficacy in a laboratory setting, aspects such as cost, storage stability, and reproducibility could be discussed in the manuscript as this is important to appreciate its potential use of BeHepI in industrial heparin quality control processes.

References

[1] Beurskens, D. M. H., Huckriede, J. P., Schrijver, R., Hemker, H. C., Reutelingsperger, C. P., & Nicolaes, G. A. F. (2020). The Anticoagulant and Nonanticoagulant Properties of Heparin. Thrombosis and Haemostasis. doi:10.1055/s-0040-1715460.

[2] Baytas SN, Linhardt RJ. Advances in the preparation and synthesis of heparin and related products. Drug Discov Today. 2020 Dec;25(12):2095-2109. doi: 10.101.

[3] Li B, Zhao H, Yu M. Techniques for Detection of Clinical Used Heparins. Int J Anal Chem. 2021 May 6;2021:5543460. doi: 10.1155/2021/5543460. PMID: 34040644; PMCID: PMC8121598.

Tags: heparin, heparin cleavage, heparinase, quality control, structural analysis

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