Proteomic analysis of human endosulfatases HSulfs, key enzymes in the modulation of the sulfation pattern of heparan sulfate

Les 6-O-endosulfatases HSulf-1 et HSulf-2 (HSulfs) catalysent l'hydrolyse régio-sélective du groupe 6-O sulfate des résidus glucosamine sulfatés de l'héparane sulfate (HS), assurant ainsi une modification post-synthétique unique de ce glycosaminoglycane constitutif des protéoglycanes de la surface cellulaire. Par cette action, les HSulfs modulent les propriétés d'interaction de HS avec un grand nombre de ligands, faisant de ces enzymes des acteurs cruciaux dans de nombreux processus physiopathologiques. De nombreuses études ont souligné le rôle physiologique de HSulf-2 ainsi que son implication dans des processus pathologiques, en particulier du cancer. HSulf-2 est ainsi proposée comme une cible thérapeutique prometteuse dans la recherche anti-cancéreuse. Néanmoins, à l'heure actuelle, aucune structure cristallographique d'endosulfatase Sulf-2 n'a été décrite. Dans le présent travail de thèse, nous présentons la première caractérisation par spectrométrie de masse (MS) de HSulf-2. Une masse moléculaire moyenne de 133115 g.mol-1 a été déterminée pour l'enzyme entière par MS MALDI-TOF. Cette masse expérimentale plus élevée que la masse théorique (98169,86 g.mol-1) déduite de la séquence d'acides aminés, souligne la contribution significative de modifications post-traductionnelles (MPTs) à la masse moléculaire totale de HSulf-2. La séquence protéique de HSulf-2 a été confirmée par analyse nano-LC-MS/MS, ce qui a permis de localiser quatre sites de N-glycosylation (N108, N147, N174 et N217) sur les sept sites potentiels de la chaîne longue. Outre cette N-glycosylation, nous avons également identifié une nouvelle MPT de type O-glycosylation. En combinant différentes approches d'électrophorèse (SDS-PAGE / C-PAGE) et de MS (MALDI-TOF et HILIC-ESI-MS) nous avons montré que cette PTM est constituée d'une chaîne de glycosaminoglycane (GAG) appartenant à la famille des chondroïtine sulfate/dermatane sulfate. L'analyse par MALDI-TOF de la forme modifiée HSulf-2 dans laquelle les deux seuls sites d'ancrage possibles de ce GAG ont été substitués, a permis de déduire une contribution globale des MPTs de 34964 g.mol-1, dont 24727 g.mol-1 pour la seule chaîne de GAG. Cette dernière est greffée de manière covalente au niveau du domaine HD de la chaîne courte. Elle représente une modification inédite pour une enzyme, premier exemple connu d'un protéoglycane de ce type. Plusieurs N-glycosylations réparties sur les deux chaînes longue et courte comptent pour les 10237 g.mol-1 restant. L'ensemble de ces résultats fournissent les bases bioanalytiques solides pour la poursuite de la caractérisation structurale des enzymes HSulfs, visant à comprendre le rôle des MPTs, à élucider l'organisation des deux chaînes de HSulf-2, et à en déchiffrer leur rôle dans la liaison au substrat et dans la formation de la poche catalytique afin de développer un inhibiteur spécifique des HSulfs.The human 6-O-endosulfatases HSulf-1 and -2 (HSulfs) catalyze the regio-selective hydrolysis of the 6-O-sulfate group of glucosamine residues within sulfated domains of heparan sulfate (HS), thereby ensuring a unique and original post-biosynthetic modification of the cell surface proteoglycans. Through their activity, the HSulfs enzymes modulate the interaction properties of HS and they are thus involved in crucial physiological processes as well as in pathological conditions, particularly in cancer. Therefore, HSulf-2 is considered as a valuable therapeutic target in cancer research. There is no crystallographic structure available for HSulfs so that their structural organization in two chains remains poorly understood. In this study, we report the first characterization by mass spectrometry (MS) of HSulf-2. An average molecular weight of 133115 g.mol-1 was determined for the whole enzyme by MALDI-TOF MS, i.e. higher than the naked amino acid backbone (98170 g.mol-1), highlighting a significant contribution of post-translational modifications (PTMs). The HSulf-2 protein sequence was determined by nano-LC-MS/MS, revealing four glycosylated sites at Asn 108, 147, 174 and 217. Besides, a unique O-glycosylation has been evidenced. By combining electrophoresis methods (SDS-PAGE / C-PAGE) and MS analysis (MALDI-TOF / HILIC-ESI-MS), we have identified this PTM as a glycosaminoglycan (GAG) of chondroitin sulfate/dermatan sulfate type. The MALDI-TOF analysis of the HSulf-2 modified form in which the two GAG binding sites have been impaired indicated a contribution of 34964 g.mol-1 for the whole PTMs, including 24727 g.mol-1 for the GAG chain only. This GAG chain is covalently linked to the HD domain within the C-terminus of HSulf-2. It is an unprecedented post-translational modification of an enzyme, providing the first example of a catalytic proteoglycan. N-glycans dispatched on both HSulf-2 long (N-terminus) and short (C-terminus) chains account for the remaining PTMs mass contribution 10237 g.mol-1. Overall, these results provide a bioanalytical platform for further structural investigations of the HSulf enzymes, aiming at deciphering the role PTMs and of each chain in the substrate binding and specificities and in the catalytic activities, and for the discovery of specific HSulfs inhibitors

Mass spectrometry analysis of the human endosulfatase Hsulf-2

The human 6-O-endosulfatases HSulf-1 and -2 catalyze the region-selective hydrolysis of the 6-O-sulfate group of the glucosamine residues within sulfated domains of heparan sulfate, thereby ensuring a unique and original post-biosynthetic modification of the cell surface proteoglycans. While numerous studies point out the role of HSulf-2 in crucial physiological processes as well as in pathological conditions particularly in cancer, its structural organization in two chains and its functional properties remain poorly understood. In this study, we report the first characterization by mass spectrometry (MS) of HSulf-2. An average molecular weight of 133,115 Da was determined for the whole enzyme by MALDI-TOF MS, i.e. higher than the naked amino acid backbone (98,170 Da), highlighting a significant contribution of post-translational modifications. The HSulf-2 protein sequence was determined by Nano-LC-MS/MS, leading to 63% coverage and indicating at least four N-glycosylation sites at Asn 108, 147, 174 and 217. These results provide a platform for further structural investigations of the HSulf enzymes, aiming at deciphering the role of each chain in the substrate binding and specificities and in the catalytic activities. Keywords: HSulf-2, 6-O-Endosulfatase, Sulfatase, Heparan sulfate, Formylglycine, Mass spectrometr

A Nucleolar Isoform of the Drosophila Ubiquitin Specific Protease dUSP36 Regulates MYC-Dependent Cell Growth

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Discrimination of sulfated isomers of chondroitin sulfate disaccharides by HILIC-MS

International audienceChondroitin sulfate (CS) glycosaminoglycans are biologically active sulfated polysaccharides that pose an analytical challenge for their structural analysis and functional evaluation. In this study, we developed a hydrophilic interaction liquid chromatography separation method and its on-line coupling to mass spectrometry (MS) allowing efficient differentiation and sensitive detection of mono-, di-, and trisulfated CS disaccharides and their positional isomers, without requiring prior derivatization. The composition of the mobile phase in terms of pH and concentration showed great influence on the chromatographic separation and was varied to allow the distinction of each CS without signal overlap for a total analysis time of 25 min. This methodology was applied to determine the disaccharide composition of biological reaction media resulting from various enzymatic transformations of CS, such as enzymatic desulfation of CS disaccharides by a CS 4-O-endosulfatase, and depolymerization of the CS endocan by chondroitinase lyase ABC

Extracellular endosulfatase Sulf-2 harbours a chondroitin/dermatan sulfate chain that modulates its enzyme activity

Sulfs represent a class of unconventional sulfatases, which differ from all other members of the sulfatase family by their structures, catalytic features and biological functions. Through their specific endosulfatase activity in extracellular milieu, Sulfs provide an original post-synthetic regulatory mechanism for heparan sulfate complex polysaccharides and have been involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their in vivo functions. Here we show that human Sulf-2 (HSulf-2) features a unique polysaccharide post-translational modification. We identified a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate binding domain. We found that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity in vitro and in vivo using a mouse model of tumorigenesis and metastasis. In addition, we showed that mammalian hyaluronidase acted as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a unique proteoglycan enzyme and its newly-identified GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute in clarifying the conflicting data on the activities of the Sulfs and introduce a new paradigm into the study of these enzymes

Extracellular endosulfatase Sulf-2 harbors a chondroitin/dermatan sulfate chain that modulates its enzyme activity

International audienceSulfs represent a class of unconventional sulfatases which provide an original post-synthetic regulatory mechanism for heparan sulfate polysaccharides and are involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their in vivo functions. Here we show that human SuIf-2 (HSulf-2) harbors a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate-binding domain. We demonstrate that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity in vitro and in vivo. In addition, we show that mammalian hyaluronidase acts as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a proteoglycan-related enzyme and its GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute to clarifying the conflicting data on the activities of the Sulfs

Structure and functional impact of glycosaminoglycan modification of HSulf-2 endosulfatase revealed by atomic force microscopy and mass spectrometry

International audienceAbstractThe human sulfatase HSulf-2 is one of only two known endosulfatases that play a decisive role in modulating the binding properties of heparan sulfate proteoglycans on the cell surface and in the extracellular matrix. Recently, HSulf-2 was shown to exhibit an unusual post-translational modification consisting of a sulfated glycosaminoglycan chain. This study describes the structural characterization of this glycosaminoglycan (GAG) and provides new data on its impact on the catalytic properties of HSulf-2. The unrevealed nature of this GAG chain is identified as a chondroitin/dermatan sulfate (CS/DS) mixed chain, as shown by mass spectrometry combined with NMR analysis. It consists primarily of 6-O and 4-O monosulfated disaccharide units, with a slight predominance of the 4-O-sulfation. Using atomic force microscopy, we show that this unique post-translational modification dramatically impacts the enzyme hydrodynamic volume. We identified human hyaluronidase-4 as a secreted hydrolase that can digest HSulf-2 GAG chain. We also showed that HSulf-2 is able to efficiently 6-O-desulfate antithrombin III binding pentasaccharide motif, and that this activity was enhanced upon removal of the GAG chain. Finally, we identified five N-glycosylation sites on the protein and showed that, although required, reduced N-glycosylation profiles were sufficient to sustain HSulf-2 integrity.</jats:p

The chondroitin sulfate/dermatan sulfate 4-O-endosulfatase from marine bacterium Vibrio sp FC509 is a dimeric species: Biophysical characterization of an endosulfatase

International audienceSulfatases catalyze hydrolysis of sulfate groups. They have a key role in regulating the sulfation states that determine the function of several scaffold molecules. Currently, there are no studies of the conformational stability of endosulfatases. In this work, we describe the structural features and conformational stability of a 4-O-endosulfatase (EndoV) from a marine bacterium, which removes specifically the 4-O-sulfate from chondroitin sulfate/dermatan sulfate. For that purpose, we have used several biophysical techniques, namely, fluorescence, circular dichroism (CD), FTIR spectroscopy, analytical ultracentrifugation (AUC), differential scanning calorimetry (DSC), mass spectrometry (MS), dynamic light scattering (DLS) and size exclusion chromatography (SEC). The protein was a dimer with an elongated shape. EndoV acquired a native-like structure in a narrow pH range (7.0-9.0); it is within this range where the protein shows the maximum of enzymatic activity. The dimerization did not involve the presence of disulphide-bridges as suggested by AUC, SEC and DLS experiments in the presence of β-mercaptoethanol (β-ME). EndoV secondary structure is formed by a mixture of α and β-sheet topology, as judged by deconvolution of CD and FTIR spectra. Thermal and chemical denaturations showed irreversibility and the former indicates that protein did not unfold completely during heating