# Insights into extracellular matrix enzymes: assessing purity, oligomerization, and enzymatic activity using capillary electrophoresis and mass spectrometry

Andelini Magdalena, Beaufour Martine, Talhi Anissa, Nasreddine Rouba, Launay Alexandra, Piazza Francesco, M. Cadene, Nehmé Reine

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## To cite this version:

Andelini Magdalena, Beaufour Martine, Talhi Anissa, Nasreddine Rouba, Launay Alexandra, et al. "Insights into extracellular matrix enzymes: assessing purity, oligomerization, and enzymatic activity using capillary electrophoresis and mass spectrometry." SEP2025, ASFEP, Mar 2025, Paris, France. hal-05377038

HAL Id: hal-05377038

https://hal.science/hal-05377038v1

Submitted on 21 Nov 2025

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# Insights into extracellular matrix enzymes: assessing purity, oligomerization, and enzymatic activity using capillary electrophoresis and mass spectrometry

**Magdalena Andelini**¹, Martine Beaufour², Anissa Talhi¹, Rouba Nasreddine¹, Alexandra Launay¹, Francesco Piazza², Martine Cadène², Reine Nehmé*¹ (reine.nehme@univ-orleans.fr)

¹Olnstitut de Chimie rganique et Analytique, Université d'Orléans, CNRS FRA2097, UMR7311, Orléans, France.
2 Centre for Molecular Biophysics, CNRS Orléans, Université d'Orléans, CNRS FRA2097, UPR4301, Orléans, France.

The extracellular matrix (ECM) is a complex network of macromolecules that play essential structural and functional roles in tissue organization and cellular processes¹,². It undergoes continuous remodelling, mediated by **matrix metalloproteinases** (MMPs), **serine proteases** e.g. human neutrophil elastase (HNE), and **hyaluronidases** (Hyal)³,⁴. These enzymes also contribute to pathological tissue remodelling, leading to inflammation, tumor progression, and metastasis³,⁵. Understanding their molecular characteristics, mechanisms, and the development of potential inhibitors remains a significant challenge⁶. Despite growing interest, there is limited literature detailing their structures, spatial conformations, oligomerization and physicochemical properties. Additionally, existing literature lacks information regarding the quality and purity of samples used in catalytic and affinity assays.

The aim of this work is to clarify the effect of macromolecular crowding on hyaluronidase inhibitors, **assess the purity** of enzyme preparations used, and gain insight into the degree of oligomerization of ECM enzymes.

## Extracellular matrix (ECM)

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<figure><img src="image_4.png" /></figure>

Collagen

<figure><img src="image_5.png" /></figure>

Elastin

<figure><img src="image_6.png" /></figure>

Fibronectin

<figure><img src="image_7.png" /></figure>

Proteoglycans

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Hyaluronic acid

### Enzymes of the ECM:

* Serine proteases

* Matrix metalloproteinases

* **Hyaluronidases (Hyal)**

<figure><img src="image_9.png" /></figure>

### Mechanism of action

Hyaluronidase (Hyal)

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Hyaluronic acid (HA)

Anti-inflammatory

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

Low molecular weight products

Inflammatory

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

Tetrasaccharide (Tet) (final product)

## Influence of crowding on hyaluronidase inhibition

Hyaluronidase inhibitors play an integral role in maintaining hyaluronic acid homeostasis. Thus, developing new inhibitors offers a powerful tool to prevent or treat pathologies.

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

### Conditions for capillary electrophoresis-based enzymatic assay:

Incubation buffer (for enzymatic reaction): ammonium acetate 2 mM pH 4
All reactions done in triplicates.

**BGE:** ammonium acetate 50 mM pH 9; anodic injection (13 nL); separation +15 kV; detection λ=200 nm; Capillary: 60cm x 50cm x 50µm

<figure><img src="image_18.png" /></figure>

Biotinylated 4-sulphated chondroitin tetrasaccharides (CS-A)

Crowding caused a 22% increase in inhibition

## What are the isoforms of Hyal present in BTH samples?

## Hyaluronidase kinetics as a function of pH

Proteomics analyses by QTOF MS/MS
identified no Hyals in BTH samples

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

Complementary approach for Hyal
identification: kinetic studies

Enzymatic reactions run at various pH values and various incubation
times and controlled by CE-UV

<figure><img src="image_21.png" /></figure>

Electropherograms of BTH enzymatic reaction
degradation products

<figure><img src="image_22.png" /></figure>

CPA of tetrasaccharide as a function of the
pH of the incubation buffer

✓ Presence of Hyal1 and Hyal2 indirectly confirmed in BTH samples

<figure><img src="image_23.png" /></figure>

Hyal2 was characterized by
MS to obtain structural
information

UHPLC-ESI-QTOF MS
(quality control)

Hyal2 is pure

QTOF MS/MS
(proteomics analysis)

<figure><img src="image_24.png" /></figure>

Native MS analysis
feasible

<figure><img src="image_25.png" /></figure>

MO²VING

## Liquid Native MALDI (NALIM) Mass Spectrometry

In NALIM-TOF MS, **liquid deposits** are used, avoiding the transition of the sample through the solid state, which can lead to a loss of native conformation. NALIM allows to acquire structural information, such as **oligomers distribution**, while conserving the noncovalent interactions⁸.

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Liquid deposits

Human recombinant Hyaluronidase-2 (Hyal2)

<figure><img src="image_27.png" /></figure>

Buffer exchange

<figure><img src="image_28.png" /></figure>

Improved S/N and
signal intensity

<figure><img src="image_29.png" /></figure>

+ imidazole (to dissociate
non-specific interactions
due to His-tag)

<figure><img src="image_30.png" /></figure>

Oligomers not
dissociated

<figure><img src="image_31.png" /></figure>

Hyal2 oligomers
are specific

<figure><img src="image_32.png" /></figure>

NALIM also preformed on HNE (no oligomers detected)
and MMP-1 (dimer detected)

## Conclusions & perspectives

* Significant influence of a crowding agent (dextran 476 kDa) observed on the inhibition of BTH

* Hyal1 and Hyal2 indirectly identified in BTH samples, but other proteins observed via proteomics analysis

* Quality control confirmed good quality of Hyal2 (and HNE and MMP-1)

* Oligomerization of Hyal2 and MMP-1 observed for the first time

* Continue developing Hyal inhibitors, in crowded media, for potential in vivo use

* Next: characterization of Hyal1 isoform

## References

[1] Jabłońska-Trypuć et al., *J. Enzyme Inhib. Med. Chem.* 2016, 31, 177–183.

[2] Karamanos et al., *FEBS J.* 2021, 288, 6850–6912.

[3] Bosman F. T. and Stamenkovic I., *J. Pathol.* 2003, 200, 423–428.

[4] Cox T. R. and Erler J. T., *Dis. Model. Mech.*, 2011, 4, 165–178.

[5] Pardo A. and Selman M., *Int. J. Biochem. Cell Biol.*, 2005, 37, 283–288.

[6] Girish K. S., *Curr. Med. Chem.*, 2009, 16, 2261–2288.

[7] Wolf et al., *ACS Biomater Sci Eng.* 2019;5(8):3753-3765.

[8] Beaufour et al., *J Am Soc Mass Spectrom.* 2018, 29(10):1981-1994.

## Acknowledgements

Authors gratefully acknowledge the
financial support from ANR-21-
CE44-0020 (X-Crowd) and Le
Réseau Thématique “Physique et
Chimie du Vivant” (RT2708).
Authors also thank Guillaume
Gabant (MO2VING plateform) for
performing proteomic experiments.