Résumé
The extracellular matrix (ECM) is dominated in-vivo by macromolecular crowding and resultantexcluded volume effects [1]. It is composed of a large quantity of various macromolecules which fillthe interstitial space within cells forming a hydrated gel [2]. ECM is a highly dynamic structure. It isconstantly regenerated, remodeled and degraded to maintain tissue homeostasis, through the actionof metalloenzymes such as collagenase, hyaluronidase (Hyal) and elastase [3]. The presentinvestigation is part of a large multidisciplinary project, X-Crowd, aiming to scrutinize the kinetics ofthese enzymes in a realistic picture. For this purpose, crowded environments mimicking the ECM invitroare used. The crowding environment was simulated using dextran at two different molecularweights (40 and 476 kDa), respecting so the ratio between enzyme and crowder size.We have first studied the activity of Hyal, a glycosidase responsible for the degradation of hyaluronicacid (HA), a large polysaccharide responsible for skin hydration and cartilage lubrication. Capillaryelectrophoresis (CE), thanks to its miniaturized dimensions, was advantageously used to monitor theenzymatic reaction, after optimizing the injection step, taking into account the media viscosity andcomplexity. To better understand the effect of dextran on the catalytic activity of Hyal, a smallsubstrate, decasaccharide (10-mers), was firstly used. Results were compared to those obtained withthe high molecular weight natural substrate, HA. Moreover, the interaction between Hyal and theDextran was characterized using microscale thermophoresis (MST), a biophysical miniaturizedtechnique based on fluorescence detection. Hyal was thus labeled with ATTO-647 and studied in thepresence of dextran with different buffer compositions and pH conditions. The inhibition study of thisenzyme by a referenced inhibitor firstly, and a home-made inhibitor secondly, was also carried out indilute and crowded media. Finally, ionic mobility spectrometry (IMS) was used to evaluate the effectof crowding on the folding state and conformational dynamics of Hyal. CE combined to IMS and MSTallowed to disentangle the impact of crowding on Hyal kinetics, folding state and interactions.Therefore, this reaffirms the significance of conducting biological assays under conditions that closelymimic in vivo environments. The approach we propose ensures the development of efficient and morereliable bioactive compounds during the development process.AcknowledgementThe authors acknowledge the Agence Nationale de la Recherche (ANR) for financial support of the XCrowdproject (ANR-21-CE44-0020) and QUALICHIM (APR-IA-PF 2021-00149467) for financial supportof ICOA.References[1] A.S. Zeiger, F.C. Loe, R. Li, M. Raghunath, K.J. Van Vliet, PLoS ONE. 2012, 7 e37904.[2] A.D. Theocharis, S.S. Skandalis, C. Gialeli, N.K. Karamanos, Adv. Drug Deliv. Rev. 2016, 97 4–27.[3] C. Chantrain, Y.A. DeClerck, Med. Sci. 2002, 18, 565–575.