D Petton

V Jegaden

Appere

David Lucas
email: d.lucas@metrabrest.com

Pascale Capellmann

Bertrand Bouard

Marie-Laure Parod

Dominique Jegaden

Vanessa Appere

Évaluation métrologique et biométrologique de l'exposition aux HAPs dans les centres de contrôle technique automobile

Introduction :

Les hydrocarbures aromatiques polycycliques (HAPs) regroupent plusieurs centaines de molécules retrouvées de manière ubiquitaire dans l'environnement [START_REF] Manoli | Profile analysis of ambient and source emitted particlebound polycyclic aromatic hydrocarbons from three sites in northern Greece[END_REF]. De nombreux HAPs présents dans l'atmosphère existent simultanément sous forme gazeuse et particulaire avec généralement une prépondérance des HAPs gazeux sur les particulaires. Leur impact sanitaire et leur comportement dans l'environnement, diffèrent selon la forme considérée [START_REF] Unvin | An assessment of occupational exposure to polycyclic aromatic hydrocarbons in the UK[END_REF].

L'exposition professionnelle à ces composés est associée à un excès de risque de cancer de la vessie, de la peau et du poumon [START_REF] Rota | Occupational exposures to polycyclic aromatic hydrocarbons and respiratory and urinary tract cancers: an updated systematic review and a meta-analysis to 2014[END_REF]. Outre leurs propriétés cancérigènes, les HAPs présentent un caractère mutagène dépendant de la structure chimique des métabolites formés. Des données expérimentales qu'ils pouvaient aussi entraîner une diminution de la réponse du système immunitaire, augmentant ainsi les risques d'infection.

Cette famille chimique compte plus d'une centaine de composés dont 16 sont considérés comme prioritaires, en raison d'effets préoccupants sur la santé par, l'United States Environmental Protection Agency (US-EPA 1984) [START_REF]Substance Details -Polycyclic organic matter -16-PAH[END_REF].

Selon le règlement CLP de l'Union Européenne (1272/2008 et 790/2009), huit HAP particulaires sont classés cancérogènes de catégorie 1B (substance dont le potentiel cancérogène pour l'être humain est supposé) :

Benzo(a)anthracène (BaA), Benzo(b)fluoranthène (BbF), Benzo(j)fluoranthène (BjF), Benzo(k)fluoranthène (BkF), Benzo(a)pyrène (BaP), Benzo(e)pyrène (BeP), Chrysène (Chr), Dibenzo(a,h)anthracène (DahA) [START_REF]Informations sur les substances[END_REF].

Un seul HAP gazeux est classé cancérogène de catégorie 2 par l'Union Européenne (substance suspectée d'être cancérogène pour l'homme) et en groupe 2B (potentiellement cancérogène chez l'homme) pour le CIRC: Naphtalène (Naph [START_REF]Informations sur les substances[END_REF][START_REF] Naphtalène | Fiche toxicologique[END_REF]. L'exposition professionnelle aux HAP concernerait près de 1,5 millions de salariés en France selon l'étude SUMER 2010, dont plus de 800 000 exposés aux émissions de moteur diesel [START_REF] Nikolova-Pavageau | Cartographie des expositions aux hydrocarbures aromatiques polycycliques (HAP) par secteur d'activité : focus sur la surveillance biologique des expositions professionnelles[END_REF]. Les modalités de recueil des urines (fin du poste le 4ème jour de la semaine pour l'analyse du 1-OHP et des métabolites gazeux et fin de semaine, le lendemain, en début de poste (jour 4 fin de poste + 16heures) pour l'analyse du 3-OHBaP) respectant les récentes recommandations en terme de surveillance biologique des HAPs, l'inclusion de salariés nonfumeur uniquement, nous permettent de limiter les biais d'interprétation de ces valeurs [START_REF] Benzo | Biotox INRS juillet[END_REF][START_REF] Barbeau | Comparison of gaseous polycyclic aromatic hydrocarbon metabolites according to their specificity as biomarkers of occupational exposure: Selection of 2-hydroxyfluorene and 2-hydroxyphenanthrene[END_REF].

Pour le 1-OHP, il existe une valeur américaine de l'ACGIH de 1.8 µg/g de créatinine urinaire ajustée sur le ratio pyrène/,BaP et une recommandation de valeur en milieu professionnel sur la base Biotox de l'INRS et issu d'un article récent (< 1 µmol/mol créatinine) [START_REF] Siwińska | Kapka L Association between urinary 1-hydroxypyrene and genotoxic effects in coke oven workers[END_REF]. Il s'agit de la valeur maximale pour laquelle aucun effet génotoxique n'a pour l'instant été rapporté dans la littérature scientifique. L'ensemble des valeurs de notre étude y sont inférieures et même au tiers de cette recommandation. De même, si nous comparons à la VBI population générale allemande BAR DFG (0.3 µg/g de créatinine urinaire ou 0,15 µmol/mol créatinine) nous y sommes inférieur [START_REF]Hydrocarbures aromatiques polycycliques[END_REF].

Pour le 3-OHBaP, la recommandation sur le site biotox est de 0,4 nmol/mol créatinine urinaire et correspond à la concentration susceptible d'être retrouvée en fin de semaine pour une personne exposée chaque jour dans l'air à une concentration de BaP de 150 ng/m3 (recommandation CNAM). Pour la population générale, Nikolova-Pavageau notait comme valeur de VBI disponibles, la valeur 90 ème percentile de l'étude de Lafontaine, soit 0,05 ng/g de créatinine chez les non-fumeurs et 0,14 pour les fumeurs [START_REF] Nikolova-Pavageau | Cartographie des expositions aux hydrocarbures aromatiques polycycliques (HAP) par secteur d'activité : focus sur la surveillance biologique des expositions professionnelles[END_REF]. Dans toutes nos analyses, le taux urinaire de 3-OHBaP est indétectable et donc largement en dessous de ses valeurs. Ils ont ainsi eu la possibilité de réaliser un tableau par activités [START_REF] Nikolova-Pavageau | Cartographie des expositions aux hydrocarbures aromatiques polycycliques (HAP) par secteur d'activité : focus sur la surveillance biologique des expositions professionnelles[END_REF]. Il nous est difficile d'y comparer les données issues de notre étude car le secteur aux activités les plus proches de celle des salariés de contrôle technique serait la réparation, maintenance d'aéronefs, distribution de carburants. Mais, dans ces postes, le contact cutané aux HAPs est majoré dans la distribution de carburant. De plus, l'exposition professionnelle aux HAPs des activités de maintenance est soit à du kérosène, soit aux huiles et graisses de moteurs d'avions, mais non aux particules diesel et autres produits de combustion de carburants pour véhicules automobiles.

La faible population incluse est une des faiblesses de notre étude. Ceci rend difficile de conclure avec certitude.

En terme de recommandations pour le suivi en service de santé au travail des salariés de contrôle technique automobile, il ressort de notre étude qu'il est souhaitable de :

-Mieux informer et sensibiliser les salariés sur les moyens de protection individuelle et collective.

-Lors des visites en entreprise, l'efficacité des moyens de ventilation générale et les captations à la source des fumées d'échappement est à vérifier.

-Lors des visites médicales, la promotion du sevrage tabagique est à réaliser.

-Des mesures biométrologiques du 1-oh pyrène et 3-OHBaP urinaires en respectant les modalités de recueil des urines (fin du poste le 4ème jour de la semaine pour l'analyse du 1-OHP et des métabolites gazeux et fin de semaine, le lendemain, en début de poste (jour 4 fin de poste + 16heures) pour l'analyse du 3-OHBaP) sont à privilégier.

-Des données biométrologiques seront intéressantes après le renforcement du contrôle des émissions polluantes (test des "5 gaz").

  Dans sa récente publication (15/09/2017) sur la prévention des risques liés aux gaz d'échappement, l'INRS notait les particules fines riches en HAPs particulaires et listait les salariés des contrôles techniques dans les situations professionnelles entrainant une exposition[START_REF] Courtois | Prévention des expositions liées aux émissions des moteurs thermiques[END_REF].Dans l'analyse des données métrologiques professionnelles en HAPs collectées sur 20 ans dans la base exporisq-HAP, l'exposition aux fumées de véhicules étaient notées pour les mécaniciens véhicules poids lourds et légers mais pas pour les techniciens de contrôle technique. Ces salariés mettant en régime élevé les moteurs thermiques dans le cadre des contrôles pollution, une exposition majorée a été suspectée[START_REF] Maitre | Exporisq-HAP database: 20 years of monitoring French occupational exposure to polycyclic aromatic hydrocarbon mixtures and identification of exposure determinants[END_REF].Au vue des incertitudespersistantes sur l'exposition réelle des contrôleurs techniques aux HAPs, et notamment aux agents cancérogènes, nous avons décidé de réaliser une étude spécifique sur ce sujet. Moyens et méthodes : Un groupe de travail interne au service de santé au travail en Iroise a été créé en 2016. Il comprenait des médecins du travail, Ingénieur Hygiène Sécurité et environnement IHSE, Assistantes techniques en Prévention Santé Travail ATPST. Nous avons recensé les entreprises de contrôles techniques adhérentes au service et établi la liste de leurs salariés déclarés. En vue de la rédaction du protocole de l'étude, nous avons sollicité le support du service du Toxicologie Professionnelle et Environnementale de l'Institut de Biologie et Pathologie du CHU de Grenoble. Les critères d'inclusion étaient : -Salarié volontaire d'un centre de contrôle technique ayant accepté son inclusion dans l'étude -Salarié non fumeur La période d'inclusion s'est étendue du 01 mars au 30 avril 2017. Les responsables des entreprises ont été informés de l'étude un mois avant son début. S'ils acceptaient que leur centre soit inclus, nous avons sollicité leurs salariés pour les informer et obtenir leur consentement. lors des interventions étaient équipés d'une pompe de prélèvement atmosphérique équipée de deux supports (cassette et tube) pour analyse en chromatographie liquide haute performance (HPLC) avec une détection par fluorescence. Les prélèvements ont été réalisés sur la totalité ou une durée représentative des postes de travail (coupure pendant la durée de la pause déjeuner). Ont été mesurés sur les prélèvements 17 HAPs (le fluoranthène et le pyrène sont à la fois particulaires et gazeux) dont : 7 HAP présents sous forme gazeuse : Naphtalène (Naph), Acénaphtène (Acen), Fluorène (Fluo), Phénanthrène (Phe), Anthracène (Ant), Fluoranthène (Flua), Pyrène (Pyr). 12 HAP présents sous forme particulaire : Fluoranthène (Flua), Pyrène (Pyr), Benzo(a)anthracène (BaA), Chrysène (Chr), Benzo(j)fluoranthène (BjF), Benzo(e)pyrène (BeP), Benzo(b)fluoranthène (BbF), Benzo(k)fluoranthène (BkF), Benzo(a)pyrène (BaP), Dibenzo(a,h)anthracène (DahA), Benzo(g,h,i)pérylène (BghiP) et Indéno(1,2,3,c,d)pyrène (IP). En parallèle, une observation des activités des contrôleurs techniques lors des visites techniques a été réalisée Une visite du centre avec questionnement sur le système de ventilation et de captage des gaz d'échappement à la source a également été réalisée en systématique. Ces visites étaient menées par un IHSE ou une ATPST. Les analyses de ces prélèvements ont été réalisées dans le laboratoire du CHU de Grenoble. Les résultats sont exprimés en ng/m 3 pour chaque HAP quantifié (CMR ou non CMR), et en benzo(a)pyrène équivalent toxique (somme des HAP cancérogènes de l'Union Européenne pondérés par leur TEF (Toxic Equivalent Factor)). -Biométrologie Seuls les salariés non-fumeurs ont été concernés, afin de pouvoir corréler le plus précisément possible les résultats avec l'exposition professionnelle. Les prélèvements urinaires ont été réalisés de la manière suivante : -à la fin du poste le 4ème jour de la semaine pour l'analyse du 1-OHP et des métabolites gazeux -en fin de semaine, le lendemain, en début de poste (jour 4 fin de poste + 16heures) pour l'analyse du 3-OHBaP Un infirmier ou médecin du service recueillait les urines sur le site de travail en respectant les recommandations du laboratoire d'analyse. Les analyses de ces prélèvements ont également été réalisées dans le laboratoire du CHU de Grenoble. Les résultats métrologiques ont été communiqués de manière anonymisée aux employeurs. Pour les résultats biométrologiques , les salariés étaient convoqués de manière individuelle par le médecin du travail de l'entreprise qui leur remettait et expliquait les données. Résultats : La population incluse comporte 14 salariés de 9 entreprises. Ce sont exclusivement des hommes d'une moyenne d'âge de 41 ans, tous non-fumeurs. Tous les prélèvements atmosphériques et urinaires ont pu être analysés. Les centres employaient de un à quatre salariés. Huit étaient ouvert vers l'extérieur, 4 avaient des extracteurs de gaz d'échappement. 93% des salariés bénéficiaient de gants de protection contre les risques mécaniques (7 nitriles, 3 latex, 2 cuir et un Polyuréthane), mais aucun n'avait à disposition des masques respiratoire. Les activités observées comprenaient des contrôles périodiques (véhicules diesel et essence à peu près répartis de façon égale), des visites complémentaires et des contre-visites. L'ensemble des résultats se trouvent dans les tableaux suivants. Tableau 1 pour les Concentrations atmosphériques des HAPS sous forme particulaire en ng/ m3 , tableau 2 pour les Concentrations atmosphériques des HAPS sous forme gazeuse en ng/m 3 , tableau 3 pour les concentrations urinaires en 3-hydroxybenzo(a)pyrène et tableau 4 pour les autres résultats de biométrologie. Le schéma 1 montre la proportion des principaux HAPS au sein des analyses atmosphériques. Les moyennes des résultats sont sur la dernière ligne de chaque tableau. Discussion : Les analyses atmosphériques réalisées au sein des 9 centres de contrôle technique ont montrées que les concentrations atmosphériques individuelles d'HAP présents sous forme particulaire sont très faibles et très nettement inférieures à celles des HAPS présents sous forme gazeuse (facteur 100). Le mélange est donc représenté par 99 % d'HAPS sous forme gazeuse et 1% d'HAPS sous forme particulaire. En ce qui concerne les HAPS Gazeux, les concentrations sont assez homogènes (facteur 1 à 4) comprises entre 173 et 759 ng/m 3 . Nous n'avons pas noté de différence significative en fonction du nombre de véhicules entretenus (nombre de contrôles anti-pollution ni de la présence/absence d'un système d'extracteur). Le naphtalène est la plupart du temps majoritaire au sein du mélange. Ces concentrations (150 à 695 ng/m 3 ) sont très largement inférieures à la VLEP 8h du naphtalène (50 mg/m 3 ou 50 000 000 ng/m 3 ). Ceci est cohérent avec les analyses dans d'autres secteurs d'activité avec 50 à 90% de naphtalène dans les analyses atmosphériques [7]. Quant aux HAPs cancérogènes, ils sont en concentrations souvent indétectables et au maximum de 2,5 ng/m 3 . Le benzo(a)pyrène est retrouvé en concentrations extrêmement faibles et au maximum de 0.38 ng/m 3 , niveau très inférieur à la valeur maximale recommandée par la CNAM de 150 ng/m 3 sur 8h [10]. Il ne nous a pas paru pertinent de calculer les concentrations de BaPeqT dans la mesure où les niveaux d'HAP cancérogènes pris en compte dans le calcul étaient souvent indétectables, et que les niveaux de BaP chez les contrôleurs techniques étaient compris entre < 0,09 ng/m 3 et 0,38 ng/m 3 (soit 0,25% de la valeur CNAM). Dans ces secteurs d'activité, les niveaux de BaPeqT sont généralement augmentés de 30% par rapport au seul BaP [9]. Nous pourrions ainsi considérer que globalement les concentrations de BaPeqT représentent 1,3 fois celles du BaP seul soit 0.49 au maximum. Pour l'évaluation de l'imprégnation des salariés, nous avons chois d'utiliser les deux composés pour lesquels il existe à ce jour une valeur biologique d'interprétation en milieu professionnel : le 1-hydroxypyrène (1-OHP), métabolite du pyrène (gazeux et particulaire) et le 3-hydroxybenzo(a)pyrène (3-OHBaP), métabolite du Benzo(a)pyrène.

  En ce qui concerne les métabolites des HAP présents sous forme gazeuse dans les fumées et émissions, naphtalène(1-et 2-naphtols), fluorène (1-2-3-9-fluorénols) et phénanthrène (1-2-3-4-9-phénanthrols), nous ne disposons que de valeurs en population générale et non en milieu professionnel. Toujours dans la revue de Nikolova-Pavageau, les auteurs notaient que l'utilisation du 95 ème percentile des valeurs des études NHANES et ECMS pouvaient être utiles comme valeur de VBI [7]. Les concentrations urinaires en métabolites gazeux des salariés inclus sont toutes également similaires à celles observables en population générale non fumeuse non exposée professionnellement, hormis pour 3 salariés dont les valeurs de 1-OH phénanthrène sont légèrement plus élevées indiquant une faible exposition professionnelle au phénanthrène pour l'un et sans corrélation avec l'activité pour les autres. Ces analyses biométrologiques sont considérées comme utiles et intéressantes pour confirmer l'exposition de salariés, justifier et vérifier l'efficacité des mesures de prévention [7]. Nikolova-Pavageau a également réalisée une revue bibliographique des données de surveillance biologique d'exposition professionnelle aux HAPs publiées entre 2008 et 2017.

  faible exposition professionnelle aux HAPS présents dans les gaz d'échappement chez les contrôleurs techniques. C'est une exposition aux HAP présents sous forme gazeuse plus qu'aux HAP présents sous forme particulaire et les résultats biologiques sont cohérents avec les résultats atmosphériques. Les valeurs relevées tant en métrologie qu'en biométrologie sont comparables avec celles de la population générale. Le risque sanitaire, notamment cancérigène, lié à l'exposition aux HAPs dans cette catégorie professionnelle ne semble donc pas supérieur à celui de la population générale. Du fait de la cancérogénicité de certains HAP, il est recommandé de maintenir les mesures de prévention collective (système d'aspiration à la source des gaz d'échappement pendant les Tableau1-Concentrations atmosphériques des HAPS particulaires en ng/m

  Concentrations urinaires du 1-hydroxypyrène et des métabolites gazeux en µmol/ mol de créatinine 1OHP : 1-hydroxypyrene, < inférieur au seuil de détection du laboratoire Tableau 4. Concentrations urinaires de 3 hydroxy-benzo-a-pyrène en nmol/ mol de créatinine

	Durée en min Créatinine g/l Créatinine g/l Naph ng/m 3 1-OH P	Acen ng/m 3 1-OH	Fluo ng/m 3 Phe ng/m 3 Ant ng/m 3 Flua ng/m 3 Pyr ng/m 3 Somme HAP gazeux 1-OH naphtol 1-OH fluorène 2-OH fluorène 3-OH fluorène 1-oh 9-OH phénantrè fluorène ne 3 Oh BaP	2-OH phénantrè ne	3-OH phénantrè ne	4-OH phénantrè ne	9-OH phénantrè ne
	1.3	2.26		0,02	0,24		3,52	< Sd	<		<	0,11		0,09	quantifiabl 0,15		0,04	Ip ng/m 3 Somme 0,03 <	Sommes <
	1.88 2.15		0,04	0,91		4,36	0,1 < Sd	0,15	0,19		<	es ng/m 3 0,1		0,03	<	HAP <	HAP <
	1.33	2	477	367,15 0,04	7,94 0,18		16,72 5,5	<v	7,6 0,05	0,82 0,08	0,13	0	0,39 0,15	400,6 0,61		<	<	particulai <	Cancérog <
	1.4 0.89		0,03	0,25		4,78	< Sd	<		<	0,04		0,15	0,49		<	<	res <	ènes <
	0.9 1.78		0,09	0,24		7,38	< Sd	<		<	0,08		<	0,1		<	<	quantifia <	quantifia <
	2.39 2.36		0,01	<			1,01	0,03 < Sd	0,11	0,07		<	<		<	<	bles <	bles <
	477 1.02 1.6 1.89 0.7	255	0,26 337,86 0,13 0,05	0,43 0,38 7,04 0,14	<sd	0,28 22,9 1,82 < Sd 4,9 < Sd	<sd 12,5 < <	0,53 0,86 < <	<sd 0,57 0,07 0,15	<sd 0,41 < <	0,2 0,51 382,2 <	<sd	0,07 <	0,29	< <	0,25	ng/m 3 2,24 < <	ng/m 3 1,02 < <
	255 0.96 2.43	423	<sd 593,86 0,05	0,31 0,13 92,9	<sd	<sd 61,77 1,14 < Sd	<	<sd 8,7	<sd 0,99 <	<sd 0 0,07	<sd 0,18 0,17	<sd 758,5 0,16	<sd	<	<sd	<	<sd	<	0,31	<sd <
	423 1.99 2.53 528 465 444 1.5 3 1.3 2.01 1.66 2.39 1.65 1.66	528 465 444 492	<sd 0,04 420 <sd <sd <sd 180,4 0,03 412 0,06 0,03 695,4 0,02	0,24 0,09 9,2 0,15 0,15 <sd 88,7 0,1 14,7 0,33 61,57 15,4 0,08	<sd <sd <sd <sd	<sd 21,35 0,9 < Sd <sd <sd <sd 84,8 4,19 < Sd 22,2 1,38 < Sd 5,53 0,04 13,7 <sd < <sd <sd <sd 12,1 < 11,7 < < Sd 18,52 18,5 8,57 < < Sd	0,28 0,89 0,08 <sd <sd <sd 1,06 < 0,74 < 0,1 0,94 <	<sd 0,24 0,13 <sd <sd <sd 0 0,07 0,23 0,06 0,06 0,59 0,05	<sd 0,5 0,09 <sd <sd <sd 0,32 < 0,34 < < 0,62 <	0,18 465,9 0,09 <sd <sd <sd < 367,4 0,23 461,9 0,24 750 0,07	<sd <sd <sd <sd	0,03 < < < <	0,23 <sd <sd <sd	0,04 < 0,04 < <	<sd <sd <sd <sd	< < < < <	0,93 0,15 0,15 <,d	< 0,46 <sd <sd < < < <sd <
	492 473 459 Moyenne 473 459 2 Moyenne Tableau 3. < Sd 0,82 <sd <sd 219,6 150,1 375,1 0,03 0,05 : inférieur au seuil de détection 1,5 0,48 0,17 0,08 12,1 4,56 28,6 5,4	<sd <sd 0,2	0.45 <sd 0,2 29,3 14,26 32,4 4,49 4 ,6	0,32 <sd <sd 6,2 3,1 10,45 0,015 0,02	0,84 <sd 0,38 0,97 0,56 0,87 0,037 0,044	0,34 <sd 0,23 1,27 0,2 0,34 0,1	0,13 <sd 0,14 1,52 0,31 0,51 0,08 0,05	0,38 0,11 0,27 271 173,1 447,8 0,2 0,24	<sd <sd <sd 0,014 0,01	0,79 <sd 0,58	0,007 0,01	0,4 <sd 0,44	< <	5,95 0,6 2,61	2,45 0,11 1,43 < <
	Moyenne Médiane	0,26 367,1	0,37	0,022 12,1	0,1 22,2			0,03 11,7	0,22 0,89	0,06 0,23	0,03 0,39	0,13 400,6	< sd	0,21	0,12	1,44	0,6
	Médiane	0,54	0,275		0,2	0,24			0,32	0,455	0,285	0,135	0,2	< sd	0,435	0,4	0,765	1,02
	Naphtalène (Naph), Acénaphtène (Acen), Fluoranthène (Fluo), Phénanthrène (Phe), Anthracène
	(Ant), Fluoranthène (Flua), Pyrène (Pyr)
	Fluantrène (Flua), pyrène (Pyr) Benzo(a)anthracène (BaA), Chrysène (Chr), Benzo(j)fluoranthène (BjF), Benzo(e)pyrène (BeP), Benzo(b)fluoranthène (BbF),
	Benzo(k)fluoranthène (BkF), Benzo(a)pyrène (BaP), Dibenzo(a,h)anthracène (DahA), Benzo(g,h,i)pérylène (BghiP) et Indéno(1,2,3,c,d)pyrène (IP). Tableau 2 Concentrations atmosphériques des HAPS gazeux en ng/m 3


Pierre Lafite
email: pierre.lafite@univ-orleans.fr

Sami Marroun

Gaël Coadou

S Montaut

S Marques

M Schuler

P Rollin

A Tatibouët

R Daniellou

Hassan Oulyadi

S Marquès

S-glycosyltransferase UGT74B1 can glycosylate both Sand O-acceptors: mechanistic insights through substrate specificity

Keywords: Glucosyltransferase, glycosylation, enzyme mechanism, sulfur, Arabidopsis thaliana GT, Glycosyltransferase, PATH: Phenylacetothiohydroximate, PPTH: Phenylpropanothiohydroximate, PAH: Phenylacetohydroximate, PPH: Phenylpropanohydroximate, CTP: Chlorothiophenol

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Glycosyltransferases (GT) (EC: 2.4.x.x) catalyze the transfer of a sugar from an activated donor to an acceptor [START_REF] Lairson | Glycosyltransferases: Structures, Functions, and Mechanisms[END_REF]. Nucleotide-sugar utilizing GTs, known as Leloir enzymes, are the most abundant group, and the molecular mechanisms that lead to the formation of the glycosidic [START_REF] Xie | Exploring the Catalytic Promiscuity of a New Glycosyltransferase from Carthamus tinctorius[END_REF] bond have been widely studied, for retaining or inverting GTs, considering the stereochemistry of the anomeric carbon of the glycoside [START_REF] Ardèvol | Reaction Mechanisms in Carbohydrate-Active Enzymes: Glycoside Hydrolases and Glycosyltransferases. Insights from ab Initio Quantum Mechanics/Molecular Mechanics Dynamic Simulations[END_REF].

In the case of inverting GTs, a SN2 reaction takes place, involving a single displacement step with the formation of an oxocarbenium ion-like transition state. Moreover, a catalytic base residue located in the active site can generally increase the nucleophilicity of the acceptor-attacking atom to generate the glycosidic linkage.

Whereas the canonical O-and N-glycoconjugates are involved in a wide range of biological processes, [START_REF] Varki | Biological roles of oligosaccharides: all of the theories are correct[END_REF][START_REF] Spiro | Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds[END_REF] the more unusual S-or C-glycosides also exhibit many biological roles, for instance in protein glycosylation [START_REF] Lafite | Rare and unusual glycosylation of peptides and proteins[END_REF]. Moreover, the nature of the atom in the glycosidic linkage can influence the chemical and physical properties of the glycoconjugates. Especially, S-glycosides are of particular interest because they are structural analogs of their O-counterparts, while being much more resistant to chemical and/or enzymatic hydrolysis [START_REF] Dere | Synthesis of Glycosylthiols and Reactivity Studies[END_REF][START_REF] Codee | Thioglycosides in sequential glycosylation strategies[END_REF][START_REF] Driguez | Thiooligosaccharides as Tools for Structural Biology[END_REF][START_REF] Brito-Arias | Synth. Charact. Glycosides[END_REF]. However, though GT engineering has been extensively investigated to increase substrate promiscuity and specificity towards sulfur containing acceptors [START_REF] Chang | Glycosyltransferase structural biology and its role in the design of catalysts for glycosylation[END_REF][START_REF] Palcic | Glycosyltransferases as biocatalysts[END_REF][START_REF] Hancock | Engineering of glycosidases and glycosyltransferases[END_REF][START_REF] Ati | Enzymatic synthesis of glycosides: from natural O-and Nglycosides to rare C-and S-glycosides[END_REF], little is known still about the influence of the nucleophilic atom on substrate specificity. Even though several examples of GTs exhibiting dual or triple activity with either O-, S-, N-, or C-containing acceptors have been reported [START_REF] Wen | UGT74AN1, a Permissive Glycosyltransferase from Asclepias curassavica for the Regiospecific Steroid 3-O -Glycosylation[END_REF][START_REF] Brazier-Hicks | Characterization and engineering of the bifunctional N-and O-glucosyltransferase involved in xenobiotic metabolism in plants[END_REF][START_REF] Gantt | Probing the Aglycon Promiscuity of an Engineered Glycosyltransferase[END_REF][START_REF] Falcone Ferreyra | Identification of a bifunctional maize C-and O-glucosyltransferase[END_REF][START_REF] Wang | Cytotoxic quassinoids from Ailanthus altissima[END_REF][START_REF] Wang | The glycosyltransferase involved in thurandacin biosynthesis catalyzes both O-and S-glycosylation[END_REF][START_REF] Chen | Probing the Catalytic Promiscuity of a Regio-and Stereospecific C-Glycosyltransferase from Mangifera indica[END_REF][START_REF] Gandia-Herrero | Detoxification of the explosive 2,4,6-trinitrotoluene in Arabidopsis: discovery of bifunctional Oand C-glucosyltransferases[END_REF], the molecular mechanism underlying this promiscuity was never discussed. However, Gutmann et al. have managed to identify active site motifs that could turn an O-GT into a C-GT [START_REF] Gutmann | Enzymatic C-glycosylation: Insights from the study of a complementary pair of plant O-and C-glucosyltransferases[END_REF][START_REF] Gutmann | Switching between O-and C-Glycosyltransferase through Exchange of Active-Site Motifs[END_REF].

In this context, we have recently reported the UDP-sugar donor specificity of UGT74B1, making this versatile GT a strong candidate for biosynthesis of S-glycoconjugates [START_REF] Marroun | UGT74B1 from Arabidopsis thaliana as a versatile biocatalyst for the synthesis of desulfoglycosinolates[END_REF]. Along with ThuS and SunS, involved in S-glycosylation of bacteriocin peptides [START_REF] Stepper | Cysteine S-glycosylation, a new post-translational modification found in glycopeptide bacteriocins[END_REF][START_REF] Oman | Sublancin is not a lantibiotic but an S-linked glycopeptide[END_REF][START_REF] Wang | Substrate selectivity of the sublancin S-glycosyltransferase[END_REF][START_REF] Hsieh | Synthesis of the Bacteriocin Glycopeptide Sublancin 168 and S-Glycosylated Variants[END_REF], UGT74B1 from Arabidopsis thaliana is one of the only S-GTs involved in the natural biosynthesis of the most historically known S-glycosides, namely glucosinolates [START_REF] Grubb | Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis[END_REF][START_REF] Kopycki | Kinetic analysis of Arabidopsis glucosyltransferase UGT74B1 illustrates a general mechanism by which enzymes can escape product inhibition[END_REF]. Herein, we have studied UGT74B1 and identified the mechanism that leads to its specificity for S-glycosylation versus O-glycosylation, using a diverse panel of acceptor substrates.

Materials and methods

Chemicals

All used chemicals and buffers were of the highest purity available. HPLC solvents, phenols, thiophenols, pyruvate kinase, lactate dehydrogenase, phosphoenolpyruvate, NADH were purchased from Sigma-Aldrich (Merck). Molecular biology and microbiology chemicals were purchased from ThermoFisher. UDP-α-D-glucose was obtained from Carbosynth Ltd (UK). 1 H NMR and 13 C NMR spectra of synthesized compounds were recorded on Bruker Avance II 400 or Bruker DPX 250 spectrometers. Assignments are based on DEPT 135 sequence and on homo-and heteronuclear correlations. Chemical shifts are reported in parts per million (ppm) from tetramethylsilane as the internal standard. Coupling constants (J) are reported and expressed in Hertz (Hz); splitting patterns are designed as br (broad), s (singlet), d (doublet), dt (doublet of triplets), t (triplet) and m (multiplet). High-resolution mass spectra (HRMS) were obtained with a Maxis Bruker 4G instrument from the "Fédération de Recherche" ICOA/CBM (FR2708) platform in the electrospray ionization (ESI) mode. Infrared spectra of compounds were recorded with a Thermo Scientific Nicolet iS10 spectroscope.

Preparation of PAH and PPH was adapted from published literature [START_REF] Yoganathan | N-Methylimidazole-catalyzed Synthesis of Carbamates from Hydroxamic Acids via the Lossen Rearrangement[END_REF]. 1,1'-Carbonyldiimidazole (1.2 equiv.) was added to phenylpropanoic acid or phenylacetic acid (6 mmol, 1 equiv.) dissolved in CH3CN (20 mL) under argon atmosphere and the mixture was stirred at 22 °C for 1h. A solution of NH2OH (50 wt. % in H2O, 2 ml, 5 equiv.) was then added and the reaction mixture was stirred at 22 °C for 20h. After concentration under reduced pressure, H2O (15 mL) was added to the residue and the aqueous solution was extracted with EtOAc (3 x 15 mL). The combined organic phase was washed with brine (1 x 25 mL), dried over Na2SO4, filtered and evaporated under reduced pressure. The residue was purified by Reveleris ® column chromatography on C18 silica gel (H2O 100% to CH3CN 100%) to afford the desired products as a slightly greenish powders. PAH (Figure S1): 682 mg, 75% yield. 1 H NMR (250 MHz, DMSO-d6): δ 10.59 (s, 1H, OH), 8.77 (s, 1H, NH), 7.28-7.14 (m, 5H, H-Ar), 3.26 (s, 2H, CH2). [START_REF] Ohtsuka | A base-mediated self-propagative Lossen rearrangement of hydroxamic acids for the efficient and facile synthesis of aromatic and aliphatic primary amines[END_REF][START_REF] Jia | Consecutive Lossen rearrangement/transamidation reaction of hydroxamic acids under catalyst-and additive-free conditions[END_REF] PPH (Figure S2): 761 mg, 65% yield). 1 H NMR (400 MHz, DMSO-d6): δ 10.36 (s, 1H, OH), 8.70 (s, 1H, NH), 7.27-7.17 (m, 5H, H-Ar), 2.80 (t, 2H, J = 7.7 Hz, H-3), 2.25 (t, 2H, J = 7.7 Hz, H-2). [START_REF] Ohtsuka | A base-mediated self-propagative Lossen rearrangement of hydroxamic acids for the efficient and facile synthesis of aromatic and aliphatic primary amines[END_REF][START_REF] Jia | Consecutive Lossen rearrangement/transamidation reaction of hydroxamic acids under catalyst-and additive-free conditions[END_REF].

1.2 Cloning and expression of UGT74B1.

UGT74B1 was cloned as a histidine-tagged protein in E. coli and purified as previously reported [START_REF] Marroun | UGT74B1 from Arabidopsis thaliana as a versatile biocatalyst for the synthesis of desulfoglycosinolates[END_REF].

Site-directed mutagenesis were carried out using QuikChange II XL Site-Directed Mutagenesis kit (Agilent) using WT-UGT74B1 plasmid as DNA template. Primers used for site-directed mutagenesis are presented in Table S1 in Supplementary data. Mutagenesis products were directly transformed into XL10-Gold ultracompetent cells (Agilent) by heat shock method. Each mutant sequence DNA was sequenced (Eurofins genomics) and confirmed to be identical to the known wild-type enzyme sequence DNA except the targeted codon (either His22 and Asp113) replaced by alanine.

Enzymatic assay (Enzyme-coupled)

Determination of enzymatic glycosylation for thiohydroximates PATH, PPTH and hydroximates PAH, PPH were done using a tri-enzymatic assay that couples UDP formation with NADH consumption, using pyruvate kinase and lactate dehydrogenase as enzymatic mixture. Reactions conditions were identical to those reported previously [START_REF] Marroun | UGT74B1 from Arabidopsis thaliana as a versatile biocatalyst for the synthesis of desulfoglycosinolates[END_REF]. Kinetics data were analyzed and fitted using Prism 4 (GraphPad).

Enzymatic assay (HPLC separation)

For thiophenols and phenols, an HPLC separation methodology was used to quantify enzymatic activity.

A reaction (200 µL total volume) containing 1mM UDP-glucose, acceptor (20 µL in 100% MeOH -final concentrations ranging from 50 to 5000 µM) in 20 mM Tris buffer pH 8.0 was started by addition of 1 µM UGT74B1. The reaction was left for 15 min at 37°C, then 200 µL of quenching reagent (CH3CN:HCOOH, 10:1) was added to the mixture. Proteins were precipitated by centrifugation for 10 min at 10,000 rpm, and the supernatant was analyzed by reverse-phase HPLC on a Zorbax Eclipse XDB-C18 column 4.6 × 150 mm (Agilent) on an Agilent 1220 Infinity II LC System. The mobile phase was delivered at a rate of 1 mL/min with a gradient from A (0.1% HCOOH in H2O) to B (0.1% HCOOH in CH3CN) (10% B for 4 min, 10% to 60% B in 10 min, 60% to 100% B in 2 min.). The column effluent was monitored at 250 nm.

NMR structural study of desulfo-glucotropaelin

NMR spectra of glucosylated PATH product were recorded in DMSO-d6 solution at 313K on a Bruker AVIII 500 spectrometer operating at 500.13 MHz for 1 H and 125.13 MHz for 13 C. 1D and 2D experiments 13 C HMQC and HMBC) were run under TopSpin (version 3.2, Bruker Biospin, Karlsruhe) with a BBFO { 1 H, X} probe and a z gradient coil giving a maximum gradient of 50 G cm -1 . 1 H and 13 C chemical shifts were referenced to the solvent residual signals of DMSO-d6 (δ 2.49 for 1 H and 39.70 ppm for 13 C).

(1D 1 H, 2D 1 H-1 H COSY, 1 H-1 H NOESY, 1 H-

Saturation transfer difference

STD spectra of the mixture of UGT74B1 (100µM) with UDPG (1 mM) in phosphate buffer pH=7.3 were undertaken on a Bruker Avance III 600 equipped with CPTXI-cryoprobe. The experiments were acquired with the standard Bruker stddiffesgp.3 sequence by using trains of E-Burp-1 90° selective pulses. Selective pulses were applied at 0 ppm for the on resonance STD excitation, and -17.00 ppm for the difference spectrum, with respect to H2O at 4.70 ppm. The spectra were measured with 8080 scans after eight dummy scans and lasted approximately 39 hours.

pKa determination by NMR

Thiohydroxamic and hydroxamic acids, and CTP were first characterized by NMR on a Bruker Avance III 600 equipped with a CPTXI-cryoprobe. Then, 1 H chemical shifts of these compounds were monitored for pH in order to determine their pKa in a 90:10 mixture of phosphate buffer pH 7.3 and methanol (CD3OH).

The method consists of making 1D 1 H acquisitions following successive additions of acid or base. Labile protons are not detectable under these experimental conditions (phosphate buffer) because they undergo rapid exchange. The chemical shift of the Hα protons closest to the deprotonation site is the one which is followed by this method. A titration curve was established by following the chemical shift of the chosen proton as a function of the pH. The obtained data were fitted with an asymmetrical sigmoidal function, which gave the value of the pKa.

pKa calculations

Phenols and thiophenols pKa were calculated using ACD/Labs software.

Results

UGT74B1 is able to glycosylate O-acceptors

Phenylacetothiohydroximate (PATH) and phenylpropanothiohydroximate (PPTH) (Fig. 1.A) were prevsiously reported to undergo efficient S-glycosylation, with second order catalytic rate kcat/KM above 10 5 min -1 .mM -1 (Table 1) [START_REF] Marroun | UGT74B1 from Arabidopsis thaliana as a versatile biocatalyst for the synthesis of desulfoglycosinolates[END_REF]. The O-containing analogues of PATH and PPTH, respectively phenyacetohydroximate (PAH) and phenylpropanohydroximate (PPH) were synthesized using slightly modified reported methods [START_REF] Yoganathan | N-Methylimidazole-catalyzed Synthesis of Carbamates from Hydroxamic Acids via the Lossen Rearrangement[END_REF]. Both hydroximates were used in UGT74B1 glycosylation assays following the same procedure, and their catalytic constants were determined (Table 1). Both O-acceptors have µM range KM similar to those calculated for thiohydroximates PATH and PPTH. However, the catalytic turnover is dramatically decreased when swapping the sulfur atom for the oxygen atom, as kcat of a hydroximate is 100-fold lower than the one for the corresponding thiohydroximate. UGT74B1 can thus bind hydroximates as efficiently as thiohydroximates, as KM values lie in the same order of magnitude. However, the reactivity of the oxygen atom in hydroximates is much lower than that of sulfur atom in PATH and PPTH, yielding lower catalytic rate kcat and catalytic efficiency kcat/KM. Figure 1: (A

UGT74B1 variant

Acceptor KM (µM) kcat (min -1 ) kcat/KM (min -1 .mM -1 )

Wild-type

PATH a 3

UGT74B1 requires an acidic acceptor

UGT74B1 belongs to the CAZy GT1 family [START_REF] Lombard | The carbohydrateactive enzymes database (CAZy) in 2013[END_REF], and we have previously built an homology model using several GT1 templates, and identified residues surrounding the active site [START_REF] Marroun | UGT74B1 from Arabidopsis thaliana as a versatile biocatalyst for the synthesis of desulfoglycosinolates[END_REF]. A NMR structural Like most GT1 reported structures, UGT74B1 exhibits a catalytic dyad formed of a carboxylic acid amino acid (Asp113) in close contact with an histidine (His22), which is located in the suitable distance to deprotonate the acceptor (Fig. 3). It has to be noted that hydroximates and thiohydroximates can exist under two tautomeric forms (Fig 1 .B). Yet, deprotonation of both tautomers will eventually lead to the nucleophilic attack of either oxygen or sulfur, depending on the substrate considered. This mechanism is similar to the triad observed in a serine protease mechanism. Both residues were also identified as the potential catalytic dyad in the homology model built by Kopycki et al. [START_REF] Kopycki | Kinetic analysis of Arabidopsis glucosyltransferase UGT74B1 illustrates a general mechanism by which enzymes can escape product inhibition[END_REF]. However the role of these residues is still unclear as some GT1 do not possess these two amino acids [START_REF] Dong | Understanding substrate selectivity of human UDPglucuronosyltransferases through QSAR modeling and analysis of homologous enzymes[END_REF] and several studies have demonstrated that unlike O-glycosylation, the histidine residue was not involved catalytically in N-glycosylation reactions [START_REF] Brazier-Hicks | Characterization and engineering of the bifunctional N-and O-glucosyltransferase involved in xenobiotic metabolism in plants[END_REF][START_REF] Patana | Mutation analysis in UGT1A9 suggests a relationship between substrate and catalytic residues in UDP-glucuronosyltransferases[END_REF].

In contrast to hydroxyl that needs to be deprotonated to avoid the formation of a highly unstable positively charged oxonium ion (R1-OH + -R2), amines can readily form a positively charged intermediate without preliminary deprotonation [START_REF] Brazier-Hicks | Characterization and engineering of the bifunctional N-and O-glucosyltransferase involved in xenobiotic metabolism in plants[END_REF]. In order to elucidate the role of these two residues in the catalytic mechanism of UGT74B1, we have generated two mutants in which either His22 or Asp113 were replaced with the non-reactive alanine. Both mutants were expressed and purified. However Asp113Ala could not be purified to homogeneity on Immobilized Metal-Affinity Chromatography column, unlike His22Ala that could be purified in quantity as high as WT UGT74B1 (typically 10 mg. l -1 culture) (Fig. S8-A). His22Ala mutation abolished enzymatic activity, as no activity could be detected in our assay (Fig. S8-B). This indicates that this residue is critical to deprotonate thiol acceptors before the nucleophilic attack (like in other GT1 O-glycosylation mechanism).

Unlike His22, the contribution of Asp113 in promoting this deprotonation is unclear as Asp113Ala mutant still exhibited enzymatic activity. The Michaelis Menten fit of PATH or PPTH concentration dependence (Fig. S8-B) gave respective Michaelis constants KM of 27 µM and 59 µM, higher than the value calculated for WT UGT74B1 (Table 1). However, the catalytic turnover kcat could not be determined, as Asp113Ala mutant could not be obtained as a pure enzyme. Maximum velocity Vmax was determined to be 1.2 and 2.4 µM.min -1 for each substrate, indicating that Asp113Ala residual activity is not insignificant, considering the low amount of impure enzyme introduced in the reaction mixture.

These mutation studies indicated that the acceptor acidity is crucial in UGT74B1 activity, and to assess this hypothesis we determined the pKa of sulfur-(PATH, PPTH) and oxygen-(PAH, PPH) containing acceptors. Concerning pKa values, no recent data were available for thiohydroximates in the literature [START_REF] Nagata | Studies on Thiohydroxamic Acids and Their Metal Chelates. II. Structures of Thiohydroxamic Acids[END_REF].

Acidity constants of all four substrates were thus determined by NMR.

Protons located on the alpha-carbon (Hα) of the 4 substrates were used as probes for pKa determination (Fig. 4-A). Chemical shifts of both protons were determined in a range of buffered solutions, and plotting of δ=f(pH) gave a sigmoidal curve fitting, which inflexion point corresponding to experimental pKa (Fig. 4-B). This methodology was successfully used for other acidity constant determinations [START_REF] Bezençon | pKa determination by 1H NMR spectroscopy -An old methodology revisited[END_REF][START_REF] Popov | Guidelines for NMR measurements for determination of high and low pKa values (IUPAC Technical Report)[END_REF] The calculated values for all 4 substrates were PATH:

GT activity is correlated to the pKa of the nucleophile acceptor

pKa comparison of thiohydroximates and hydroximates indicates that a low pKa (PATH and PPTH) gives a higher enzymatic activity than a high pKa (PAH and PPH). This can be understood as thiohydroximates can easily form the corresponding thiolates after His22 deprotonation, and thus are more reactive than their hydroximate counterparts. However, because the pKa values do not cover a wide range of values (pKa values gap between 5 and 9), the correlation between pKa and enzymatic activity is not obvious.

To evaluate this potential correlation, we have used simple acceptors, that cover a more extended pKa range (Fig. 1 and Table 2). The selected compounds were O-containing chloro-substituted phenols (4chlorophenol CP, 2,3-2,4-and 2,5 dichlorophenols DCP, 2,3,4-2,4,5-2,4,6-trichlorophenols TCP) and S- containing thiophenols (4-chlorothiophenol CTP and thiophenol TP), presenting a calculated pKa range between 5.5 and 9.0. To validate the in silico calculation of pKa, pKa determination by NMR was applied to CTP (Fig. S12), giving a value similar to the calculated value (respectively 5.8 and 5.5). Chlorophenol.

Acceptor

Because of their lower solubility, phenols can interfere with the tri-enzymatic assay used that couples UDP formation by UGT74B1 with NADH consumption. The latter is determined spectrophotometrically at 340 nm. We have set up an activity assay based on HPLC separation of phenols and the corresponding glycosylated products. When validated by using PATH and PPTH as substrates, this assay gave similar catalytic constants as those presented in Table 1 (data not shown).

All eight phenols and thiophenols where used as acceptors in UGT74B1 reaction, and their catalytic constants where determined (Table 2). All compounds are glycosylated by UGT74B1 when using UDP-α-glucose as sugar donor. They all exhibit similar KM values (100-200 µM), higher than those determined for hydroximates or thiohydroximates, probably because phenols might establish less stabilizing interactions in UGT74B1 active site. The observed rates fit those reported by Braziers-Hicks et al., as 2,4,5-TCP is one order of magnitude less active than 4-CTP [START_REF] Brazier-Hicks | Characterization and engineering of the bifunctional N-and O-glucosyltransferase involved in xenobiotic metabolism in plants[END_REF].

The resulting Brønsted analysis of glycosylation provides evidence of a correlation between enzymatic efficiency kcat/KM and pKa of the acceptor (Fig. 5). Unlike other studies on nucleophile pKa dependence of GT activity [START_REF] Lee | Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase[END_REF], the corresponding slope calculated βnu=-0.64 (R 2 =0.91) for UGT74B1 is negative. As βnu value can give an estimate of the change in effective charge on the nucleophile towards the transition state A similar enzymatic mechanism can be proposed for thiohydroximates and hydroximates, although no data is available between pKa values of 5 and 9. Brønsted analysis gives also a negative slope βnu=-0.31, although shallower than for phenols. During catalysis, thiohydroximates and hydroximates accumulate less negative charge on nucleophilic atoms, and are more sensitive to general acid/base mechanism than phenols.

In both cases, the Brønsted analyses do not demonstrate that the nature of the nucleophilic atom is critical for catalysis, as there is a continuum in linear correlation -especially for phenolic derivatives.

Discussion

In Brassicaceae plants, UGT74B1 was originally identified as a key enzyme in the biosynthesis of glucosinolates, catalyzing the S-glucosylation of thiohydroximates [START_REF] Grubb | Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis[END_REF][START_REF] Halkier | Biology and biochemistry of glucosinolates[END_REF]. The enzyme was annotated as a S-glycosyltransferase in genomic data, like other GTs where the specificity of atom that form the glycosidic bond is highlighted [START_REF] Lombard | The carbohydrateactive enzymes database (CAZy) in 2013[END_REF]. However many GTs have been reported to be able to use acceptors containing diverse nucleophilic atoms, which calls into question the canonic specificity of GTs towards one particular atom [START_REF] Wen | UGT74AN1, a Permissive Glycosyltransferase from Asclepias curassavica for the Regiospecific Steroid 3-O -Glycosylation[END_REF][START_REF] Brazier-Hicks | Characterization and engineering of the bifunctional N-and O-glucosyltransferase involved in xenobiotic metabolism in plants[END_REF][START_REF] Gantt | Probing the Aglycon Promiscuity of an Engineered Glycosyltransferase[END_REF][START_REF] Falcone Ferreyra | Identification of a bifunctional maize C-and O-glucosyltransferase[END_REF][START_REF] Wang | Cytotoxic quassinoids from Ailanthus altissima[END_REF][START_REF] Wang | The glycosyltransferase involved in thurandacin biosynthesis catalyzes both O-and S-glycosylation[END_REF][START_REF] Chen | Probing the Catalytic Promiscuity of a Regio-and Stereospecific C-Glycosyltransferase from Mangifera indica[END_REF][START_REF] Gandia-Herrero | Detoxification of the explosive 2,4,6-trinitrotoluene in Arabidopsis: discovery of bifunctional Oand C-glucosyltransferases[END_REF][START_REF] Gutmann | Switching between O-and C-Glycosyltransferase through Exchange of Active-Site Motifs[END_REF].

Nevertheless, the corresponding studies did not dissected in detail the molecular mechanism underlying this broader specificity. More precisely, the influence of nucleophile's nature on enzyme reactivity was not examined into detail.

UGT74B1 was shown to glycosylate S-and O-containing acceptors, such as hydroximates, the structure of which is closely related to the natural acceptor thiohydroximates. The ratio of catalytic efficiency between both families of analogues is of 2 orders of magnitude (10 5 and 10 3 min -1 .mM -1 range respectively for thiohydroximates and hydroximates). This catalytic efficiency towards O-acceptors is not negligible, as other GT1 enzymes exhibited similar kinetic constants towards their "natural" substrates [START_REF] Brazier-Hicks | Characterization and engineering of the bifunctional N-and O-glucosyltransferase involved in xenobiotic metabolism in plants[END_REF][START_REF] Wang | Structure, mechanism and engineering of plant natural product glycosyltransferases[END_REF]. Thus, UGT74B1 can efficiently catalyze O-and S-glycosylation in vitro when using appropriate acceptors, but is solely catalyzing the latter in vivo because only endogenous thiohydroximates are efficiently binding to the enzyme and exhibit low pKa.

To assess this versatility of UGT74B1 towards O-and S-acceptors, a range of chloro-substituted thiophenols and phenols were also used as substrates by UGT74B1. More interestingly, when plotting the logarithm of the second order rate kcat/KM vs. pKa values of (thio)phenols, a linear correlation could be fitted, and Brønsted-type relationship was demonstrated, with a negative βnu value (Fig. 5). The acceptor nucleophilic atom shows a decrease in effective charge during the catalysis towards the transition state. A transition state where the proton abstraction by His22 is significantly advanced, whereas the glycosidic bond formation is not, can explain this negative βnu value. As a lower absolute value for βnu value can be estimated in the case of hydroximates and thiohydroximates, these substrates are less prone to general base catalysis by His22 [START_REF] Ye | The Mechanism of RNA Strand Scission: An Experimental Measure of the Brønsted Coefficient,βnuc[END_REF][START_REF] Macauley | O-GlcNAcase catalyzes cleavage of thioglycosides without general acid catalysis[END_REF].

UGT74B1 is not the first example of a GT able to generate several glycosidic linkages, when considering the nature of the atom involved in the bond. However, the Brønsted-type relationship for (thio)phenol glycosylation does not exhibit a break between O-and S-acceptors. The influence of pKa on the reactivity overtakes the nature of the nucleophilic atom. UGT74B1 reactivity is therefore mostly driven by the predisposition of the nucleophile chemical property to be energetically close to the transition state, and thus decreasing the activation energy. The enzyme can accommodate and use several nucleophiles as acceptors, as long as they satisfy the chemistry underlying the catalysis.

The dissected mechanism of UGT74B1 can be used as a paradigm for other GTs able to use several acceptors of similar shape, size, and properties, bearing different nucleophilic atoms, and redefine the conventional nomenclature that classifies a GT according to the nature of the nucleophilic atom.

  Figure 1: (A) UGT74B1 substrates used in this study. (B) Tautomeric equilibrium and deprotonation of thiohydroximates and hydroximates.

  study of glucosylated PATH (namely desulfo-glucotropaelin[START_REF] Marroun | UGT74B1 from Arabidopsis thaliana as a versatile biocatalyst for the synthesis of desulfoglycosinolates[END_REF][START_REF] Ibrahim | Advanced NMR-Based Structural Investigation of Glucosinolates and Desulfoglucosinolates[END_REF]) product solution was undertaken in DMSO-d6 (Fig S3-S7). This allowed for a complete assignment of protons signals for desulfoglucotropaelin, showing reverse anomeric carbon stereochemistry between UDP-α-D-glucose donor and product confirming the mechanism predicted by the homology model. Moreover, a Saturation Transfer Difference (STD) experiment was used to probe UDP-α-D-glucose binding to UGT74B1. In this experiment, the signals of the UDP-α-D-glucose atoms are modulated in intensity depending on how closely they bind to UGT74B1 (Fig.2). The relative ranking of this modulation shows that UDP-α-D-glucose is strongly bound to UGT74B1 by its aromatic base and less by its glucosyl moiety. This result is in full agreement with binding modes predicted through molecular docking where the aromatic ring is stabilized by a strong and close range π-stacking interaction[START_REF] Marroun | UGT74B1 from Arabidopsis thaliana as a versatile biocatalyst for the synthesis of desulfoglycosinolates[END_REF].

Figure 2 :

 2 Figure 2: (A) 1D 1 H NMR spectrum of the mixture of UGT74B1 and UDP-α-D-glucose and (B) the corresponding STD-NMR (600MHz, T=298K, phosphate buffer pH 7.3). (C) Epitope mapping of UDP-α-D-glucose.

Figure 3 :

 3 Figure 3: Proposed SN2 mechanism of glycosylation reaction catalyzed by UGT74B1 (X=O or S). Asp113 and His22 are depicted as putative components of the catalytic dyad, as identified with the homology model of UGT74B1.

4 . 4 ;

 44 PPTH: 4.6; PAH: 9.2; PPH: 9.4. (See Fig. S9 to S11) The values for thiohydroximates, are close to those (between 4.0 and 5.6) reported by Nagata et al. [39].

Figure 4 :

 4 Figure 4: PATH pKa determination by NMR. (A) 1 H NMR spectrum of PATH. Arrow indicates protons and corresponding signals used for pKa determination. (B) Evolution of chemical shift with pH and pKa determination.

[ 43 ]Fig. 3 ) 5 :

 4335 Figure 5: Brønsted plot of kcat/KM for all tested UGT74B1 substrates. Values for thiohydroxamic and hydroxamic acids are depicted as black squares. Data corresponding to thiophenol and phenol derivatives are marked as white circles.

Table 1 :

 1 Catalytic constants of UGT74B1 (wild-type and mutated) towards thiohydroxamic and hydroxamic acids. Values were obtained at 37°C, pH 8.0.

			.1 ± 0.6	280 ± 58	90 x 10 3
		PPTH a	2.2 ± 0.5	326 ± 95	148 x 10 3
		PAH	1.1 ± 0.2	3.9 ± 1.3	3.5 x 10 3
		PPH	0.9 ± 0.2	4.3 ± 1.2	4.8 x 10 3
	His22Ala	PATH PPTH	n.d. b n.d. b	n.d. n.d.	n.d. n.d.
	Asp113Ala	PATH PPTH	27.2 ± 4.5 58.9 ± 11.4	1.2 ± 0. 1 c 2.4 ± 0.1 c

PATH : Phenylacetothiohydroximate ; PPTH : Phenylpropanothiohydroximate ; PAH : Phenylacetohydroximate ; PPH : Phenylpropanohydroximate. a From reference 31. b n.d. non detectable. c : Vmax in µM.min -1

Table 2 :

 2 Catalytic constants and pKa of thiohydroxamic acids, hydroxamic acids, phenol and thiophenol derivatives. Values were obtained at 37°C, pH 8.0. a measured by NMR; b calculated using ACD/Labs.

		pKa a	KM (µM)	kcat (min -1 )	kcat/KM (min -1 .mM -1 )
	PATH	4.4 a	3.1 ± 0.6	280 ± 58	90 x 10 3
	PPTH	4.6 a	2.2 ± 0.5	326 ± 95	148 x 10 3
	PAH	9.2 a	1.1 ± 0.2	3.9 ± 1.3	3.5 x 10 3
	PPH	9.4 a	0.9 ± 0.2	4.3 ± 1.2	4.8 x 10 3
	4-CTP	5.8 a /5.5 b	215 ± 13	139.0 ± 5.6	647
	TP	6.6 b	105 ± 24	10.9 ± 1.5	104
	2,4,6-TCP	6.0 b	132 ± 24	14.5 ± 1.3	110
	2,4,5-TCP	6.8 b	130 ± 45	6.1 ± 1.1	47
	2,3,4-TCP	7.0 b	147 ± 6	6.6 ± 0.9	45
	2,5-DCP	7.2 b	132 ± 23	4.2 ± 0.2	32
	2,3-DCP	7.4 b	195 ± 26	1.9 ± 0.3	10
	2,4-DCP	7.4 b	148 ± 5	2.2 ± 0.3	15
	4-CP	9.0 b	149 ± 43	0.6 ± 0.2	3

CTP: Chlorothiophenol; TP: Thiophenol; TCP: Trichlorophenol; DCP: Dichlorophenol; CP:

Acknowledgments & funding sources

The authors thank G. Jousset and A. Duvergé for technical help in producing and characterizing UGT74B1 wild-type and mutants' activities. PL, RD, MS, AT, SMar, GC and HO thank the Labex SynOrg (ANR-11-LABX-0029) for financial support. SMar, GC and HO thank the Région Normandie, CNRS, the University and INSA of Rouen for financial support. SMarq and SMarr were funded by French Ministry of Research.

Declaration of interest

The authors declares that there is no conflict of interest.


Emilie Lesvignes
email: cedarc.emilie.lesvignes@hotmail.fr

Eric Robert
email: eric.robert@mnhn.fr

Boris Valentin

Michèle Ballinger

Alain Bénard

Fantine Bellanger

Béatrice Bouet

François Bougnères

Alexandre Cantin

Laurent Costa

Cécile Dardignac

Sophie David

Colas GUÉRET Patrick Dubreucq

Emmanuel Guy

Isabelle De Miranda

Vanesse Romero-Godofredo

Thomas Sagory

Médard Thiry

Benoit Touchard

Régis Touquet

Laurent Valois

Lydia Zotkina

Using digital techniques to document prehistoric rock art: first approaches on the engraved panels of the Paris Basin shelters

Keywords: preservation, prehistoric rock art, photographic techniques, 3D, relevés, Paris Basin

In the sandstone boulder fields of the southern Paris Basin, over two thousand small engraved shelters have been identified. A new collective research programme aims to investigate on this broad subject and focus above all on Mesolithic contexts, which constitute a large proportion of the engravings in the shelters, among other earlier or later periods represented.

Since 2017, several field campaigns led to the documenting of rock shelters at various scales, their environment and a selection of their engraved panels, focusing on issues of relative chronology. Photographic techniques, digital technologies and recording processes are used to meet three objectives: preserving the sites by means of digital archiving, raising awareness of this heritage, and carrying out new studies.

The documenting methods, digitisation rationale, and use of the different media, along with their contributions and limits, is discussed below.

Introduction

The group research programme, Art rupestre préhistorique dans les chaos gréseux du Bassin parisien. Etude, archivage et valorisation1 (PCR ARBap) is funded by the French Ministry of Culture (DRAC Île-de-France). Launched in 2017 and directed by B. Valentin, it aims to investigate the decorated shelters in the sandstone boulder fields of the southern Paris Basin (Fig. 1) [START_REF] Valentin | PCR ARBap (Art rupestre préhistorique dans les chaos gréseux du Bassin parisien : étude, archivage et valorisation) : tout premiers résultats[END_REF]. Over two thousand engraved shelters have been identified to date, notably thanks to the relevés and inventories carried out by an Association called GERSAR (Groupe d'Etudes, de Recherches et de Sauvegarde de l'Art Rupestre2 ), founded in 1975. The shelters are distributed over a vast geographical area, visible in Fig. 1, between Rambouillet and Fontainebleau, in which the density of engraved shelters varies greatly according to their location, from 1 shelter to more than 50 shelters for 10 km square area. These shelters may contain only few engraved features, but for the most part, dozens or even hundreds of engravings, which are generally quite deep and are often found over almost the entire inner surfaces of the small caves [START_REF] Bénard | Symbole et mystères. L'art rupestre du sud de l'Île-de-France[END_REF]. In general, geometric themes predominate, notably grid patterns (Fig. 2 a.), with a few more or less schematic figurative depictions (Fig. 2 b.). Current research tends to attribute most of these engraved shelters to Mesolithic contexts [START_REF] Guéret | Fontainebleau rock art (Ile-de-France, France), an exceptional rock art group dated to the Mesolithic? Critical return on the lithic material discovered in three decorated rock shelters[END_REF]. However some rare but important engraved panels have been attributed to earlier periods such as the Upper Palaeolithic [START_REF] Bénard | L'abri orné de la Ségognole, Noisy-sur-École, Seine-et-Marne. Description des gravures et proposition d'attribution chronoculturelle[END_REF], and to later periods such as Neolithic, Metal Ages, Middle-Age and Modern period, whose figurative motifs are easily recognizable.

The programme on prehistoric engravings has three objectives: preserving, studying, and raising awareness of this heritage (Robert et al., submitted). These objectives are all closely linked, as study requires the preservation of the sites, and promoting and raising awareness of the heritage necessarily requires study. This outdoor heritage is extremely fragile. A simple entry into the shelters is not without risk for the conservation of the engravings, as sandstone is a very fragile rock, which disintegrates little by little into sand. The environment in which it is situated is very popular for tourism, rock climbing, hiking, etc. The open-air decorated shelters (Fig. 3), which are hidden and little known of, are often vandalized and degraded both by people and the passing of time (cf. Fig. 5). Conservation is therefore a major aspect of our programme.

Figure. 3

Open-air rock art shelter (La Touche aux Mulets 1). Photo E. Lesvignes/PCR ARBap.

Documenting methods and techniques

These three objectives require the particularly abundant and diverse documentation of the engravings and their contexts, whether in the form of graphic, photographic or three-dimensional media. Moreover, the whole decorated shelters are extremely numerous. Different documenting techniques and methods have been used to compile data acquired at several different scales, through a series of multiple approaches: photography and macro-photography, photogrammetry and 3D imaging, video, topography, aerial photography using drones, LiDAR (Light Detection and Ranging). In parallel, a GIS (Geographic Information Systems) programme is developed to combine the results of all or part of these media.

Photography

Photography has been applied to archaeology ever since they both emerged in the 19th century, the former as a technique and the latter as a science [START_REF] Lesvignes | Usages et apports de la photographie en archéologie. Archéo Situla[END_REF]. It is a means of documenting and defining the different stages of archaeological operations and is of particular use in establishing inventories and representing the sites and collections, while contributing a unique perspective. Applied to rock art, photography is a key means of contextualizing and representing graphic elements. Given the considerable number of often similar shelters and engravings within this programme, it is essential that they are clearly represented, located and contextualized (Fig. 4). Photography is also used to produce precise and accurate analytical material with which to carry out rock art relevés and image analysis (in particular colour evaluations) [START_REF] Fritz | The hidden meaning of forms: methods of recording Palaeolithic parietal art[END_REF][START_REF] Robert | Applications of digital photography in the study of Palaeolithic cave art[END_REF]. However, the creation of this material is subject to constraints of framing and distance, orthogonality with respect to the average plane of the subject, resolution, and lighting. Respecting these principles is a matter of technical choice and equipment (type of camera and lens, settings, type of lighting, etc.), which varies according to the subject photographed. Photography also allows the evolution of the state of conservation of the photographed subjects to be assessed and is particularly useful in documenting the progression over time of such or such a phenomenon, alteration, destruction, etc. As such, reviewing old photographic archives is an important opportunity to ensure the continued conservation of the sites (Fig. 5). In these sandstone contexts, photography is not the easiest to implement for several reasons, which we shall return to below.

Photogrammetry

Photogrammetry, based on photographic techniques, allows three-dimensional images to be produced by triangulating points (in this case, pixels) and correlation algorithms between the photographic views in order to reconstruct the volume of a subject [START_REF] Egels | La photogrammétrie, principes généraux et terminologie[END_REF][START_REF] Pierrot-Deseilligny | Évolutions récentes en photogrammétrie et modélisation 3D par photo des milieux naturels[END_REF].

Nowadays it is very widely used in the context of rock art, offering significant advantages in terms of cost, usability, texturing, and making it the subject of numerous applications (e.g. [START_REF] Chandler | The development and application of a simple methodology for recording rock art using consumer-grade digital cameras[END_REF][START_REF] Domingo | Latest developments in rock art recording: towards an integral documentation of Levantine rock art sites combining 2D and 3D recording techniques[END_REF][START_REF] Berquist | A new aerial photogrammetric survey method for recording inaccessible rock art[END_REF]Carero-Pazos et al., 2018;[START_REF] Strasser | Palaeolithic cave art from Crete, Greece[END_REF].

This technique allows us to create reliable digital archives, offering virtual copies of the shelters and their engraved panels (Fig. 6). Until the recent popularization of 3D tools, moulding techniques were used to create these copies and unfortunately may have been abused. While very precise, moulding techniques pose many problems when used in the context of rock art. In the specific context of our studies, several engraved panels currently present significant deterioration marks made by moulding (traces and prints, residues, surface films, tearing of the substrate, pollution of the rock, etc.) (some white residues are visible on Fig. 4 e.). The serious consequences visible today have incited us to focus on non-invasive techniques. Beyond the conservation aspect, 3D modelling provides us with useful data on the relief and contexts of the engravings and helps us to analyse certain figures which are difficult to study because of their location and access. In addition, it allows us to place the engraved figures in a volumetric context, and not only in a two-dimensional plane. This 3D modelling is used to complement a photographic approach when carrying out relevés of the engraved figures. We also took into account the microscopic scale in the degree of resolution of the three-dimensional acquisitions. At present, macrophotography remains the best way to obtain accurate information and a good texture. New investigations into the 3D documenting of microscopic and use-wear information are also underway within the programme [START_REF] Plisson | From 2D to 3D at macro-and microscopic scale in rock art studies[END_REF][START_REF] Zotkina | Le bison de La Grèze (Marquay, Dordogne, France) sous le microscope[END_REF].

Regardless of their scale, from the macro to the shelter in its context, the three-dimensional models created have been georeferenced with topographic laser tacheometer readings (X, Y, and Z coordinates) (Fig. 7).

Figure. 7

Topography recording of the calibration points for 3D reconstruction (La Ségognole 3). Photo E. Lesvignes/PCR ARBap.

Topography

Documenting the topography of the shelters and the sectors they are located in allows us to georeference the three-dimensional models to give them an orientation and a position in space. This technique also makes it possible to create links between a delineated 3D model (a shelter for example) and the wider sector in which it is located, in which there may be other documented models (other neighbouring shelters). This information allows precise locations to be established within a very large area, makes it possible to document the geometry of the shelters and their location within the environmental landscapes, and enables the scaling of the documented subjects to provide realistic metric references. Finally, this topographic information represents the basic data on which we can rely to situate the other documentary (photography and photogrammetry) and scientific (relevés and analyses) approaches. This combination forms a precious documentary substrate upon which to contextualize the decorated sites [START_REF] Boche | De l'art pariétal à ses contextes : bilan des applications pour l'enregistrement, l'intégration et l'analyse des informations spatiales[END_REF].

For the external surfaces, in addition to the topography, we also carried out photography using drones, and LiDAR (Fig. 8) to document spatial and physical data on a medium to large scale. Drones are used for photography and photogrammetry, while LiDAR is a remote laser sensing technique allowing for the acquisition of high-resolution, three-dimensional data for mapping purposes. Post-acquisition data processing allowed us to distinguish the points on the ground and those representing the surrounding vegetation. Once isolated, the points identified as part of the ground are used to produce a very detailed model of the topography through the creation of a Digital Elevation Model (DEM). These techniques allow us to produce precise material with which to carry out spatial analyses and geographical and topographical observations over extensive areas [START_REF] Georges-Leroy | Apport du LiDAR à la connaissance de l'histoire de l'occupation du sol en forêt de Haye[END_REF][START_REF] Trouvé | Montmorency (Val-d'Oise, 95) : une forêt passée au laser[END_REF]. In the context of the shelters of the Paris Basin, it will be used for a variety of purposes: establishing the distribution of the shelters and sandstone boulders, large-scale geological observation, locating historic quarries which have destroyed engraved shelters, and creating maps (exposure, slope, remains, etc.). Thanks to this multi-scale body of data, the shelters and their engravings can be apprehended within their contexts and environments, from the level of the territory itself down to the engraved panel, like an optical zoom.

Compiled in a Geographic Information System, this data represents our raw material with which to answer several scientific questions. Offering a shared mapping interface, this GIS format favours a dynamic and collaborative approach by making a wide body of data and documentation available to the whole team as a single living document. This allows for the storage, processing, analysis, management and presentation of spatial, geographic, archaeological and contextual data. This type of unifying document makes it possible to compare and contrast the data resulting from the different approaches and scales of documentation, to foster new perspectives, and to gain a better overview of the different, but closely related, data [START_REF] Boche | Le SIG comme outil fédérateur de la recherche interdisciplinaire : application à la grotte Chauvet-Pont d'Arc. Actes du colloque des Journées Informatique et Archéologie de Paris (JIAP 2010)[END_REF].

Technical constraints: photography and photogrammetry

The shelters formed in the sandstone boulder fields are usually small in size; both in height and in width. Their depth is variable. The interior space of the shelters is often small and very constraining. The relief is varied and sometimes very uneven. Significant difficulties were encountered in framing, positioning the photographer, lighting and, to the least extent possible, installing a tripod, given the available space and the fragility of the sandstone. In addition, photo coverage for 3D modelling the interior of a shelter requires access, within reasonable focal distance, to all the areas and reliefs involved, which is not always easy (cf. Fig. 7).

The small size of the shelters also lead to strong contrasts between the interior darkness and the intensity of the outside light (cf. Fig. 9 b.). This created difficulties in two areas. Firstly, problems were encountered in measuring the colour temperatures and associated colour values. At the same time, obstacles were encountered in lighting up the engravings when they were exposed to daylight.

While framing is less essential in photogrammetry than in photography, the physical installation, the topography of the shelters, and the strong light contrasts presented the same difficulties. Furthermore, the powerful frontal lighting required for photogrammetry is little compatible with the constraints of these small and fragile shelters.

It is necessary to constantly adapt to these particular constraints, which differed in each case, by making technical compromises and, at best, having recourse to a wide range of photographic equipment and portable lighting, which is not always possible.

An adapted methodology of relevé

In the context of the documentation and analysis of the engravings, and to minimize our presence in the shelters, we are conducting methodological tests to develop a strategy adapted to each shelter, panel and engraving. This involved the complementary use of both photography and photogrammetry.

The "classic" method is derived from proven procedures established over the course of numerous studies in Palaeolithic art, and is based on the analytical logic of never breaking the link with the direct observation of the decorated panel [START_REF] Fritz | The hidden meaning of forms: methods of recording Palaeolithic parietal art[END_REF]. However, a significant amount of time is required for the observation and production of photographic relevé material, in particular to take into account the reliefs and the morphology of the elements to be represented in 2D. For this reason, 3D tools offer a significant advantage to allow a better speed of execution, and also for working off site and limiting our own presence in the shelters. Furthermore, digital tools and technologies are increasingly used to meet the needs of rock art research (e.g. [START_REF] Aujoulat | Contribution de la saisie tridimensionnelle à l'étude de l'art pariétal et de son contexte physique[END_REF][START_REF] Bourdier | Technologies 3D appliquées à la sculpture pariétale magdalénienne[END_REF][START_REF] Feruglio | Un outil de relevés 3D partagé en ligne : premières applications pour l'art et la taphonomie des parois ornées de la grotte de Cussac (ArTaPOC/programme LaScArBx)[END_REF][START_REF] Fritz | Restauration virtuelle de l'art pariétal paléolithique: le cas de la grotte de Marsoulas[END_REF][START_REF] Robert | Applications 3D pour la contextualisation et le relevé d'art en grotte ornée[END_REF][START_REF] Robert | Applications of digital photography in the study of Palaeolithic cave art[END_REF]Jalandoni et al. 2018).

The choice of documenting method for creating the analytical material results from its feasibility, since the small size and the morphology of the shelters did not always favour the use of photography alone. It was the photographic conditions that determined the use of one or other method: when there was not sufficient space to allow for the orthographic photography of the subject (Fig. 9), we favoured photogrammetry. Photogrammetry offers a methodology which allows the subject to be approached by overlapping oblique views. This makes it possible to overcome the framing constraints related to photography and to produce an orthoimage from the 3D model suitable for an analytical reading and the production of a relevé. It is also these highly practical aspects that has formed the basis of our methodology. The methodological tests that are currently being developed make use of both of these techniques and involve carrying out observations and relevés on both types of media. With photographic media, the relevé is carried out on site, facing the wall (Fig. 10 a.). With the 3D models, the readings are carried out with and without prior observation of the subject in order to compare the results and assess the limits of this work at a later date off site (Fig. 10 b.). We also tested a blind reading of a 3D model by a person who had never seen the engravings before. The next step is to carry out an onsite control of the observations and readings made in the laboratory. Finally, a comparison was drawn between the readings made on photographic media and those taken from the three-dimensional models, which has thus far revealed some relatively significant differences. Continuing these tests in order to achieve a reliable methodological strategy necessarily requires carrying out new, more localized modellings with a very high degree of resolution, for purely analytical purposes.

It should be noted that at present no software allows for a relevé to be carried out directly on a 3D model, even if innovative methodological developments have been proposed, notably by the research team at Grotte de Cussac [START_REF] Feruglio | Un outil de relevés 3D partagé en ligne : premières applications pour l'art et la taphonomie des parois ornées de la grotte de Cussac (ArTaPOC/programme LaScArBx)[END_REF] by volume selection, by recent innovations with Blender software (Pinçon et al., submitted) or on the Gavrinis cairn [START_REF] Cassen | Complementarity of acquisition techniques for the documentation of Neolithic engravings: lasergrammetric and photographic recording in Gavrinis passage tomb (Brittany, France)[END_REF]. It is therefore currently necessary to carry out a manual relevé from readings on a computer screen and from an orthoimage taken from the 3D model before reintroducing it as a superimposed texture (Fig. 11). However, the tests made on the orthoimages drawn from the 3D models have revealed several limits, in terms of the technical constraints, resolution and readability of the engraved lines. Regarding the technical aspects, aside the choice of photographic equipment, the main difficulty lies in the type of lighting and its orientation, which is particularly difficult to accomplish in these cramped spaces. In terms of resolution, it is the quantity of photos and the object focal distance that determines the accuracy and zoom capacity of a model.

A three-dimensional model created to meet the objectives of preserving and raising awareness of the sites does not necessarily come close to meeting the requirements of an analytical reading. Thus, the off-site interpretation of the results, based exclusively on a 3D model, sometimes leads to information gaps and does not always allow for the analysis of the fine details of the superimpositions or the overlapping of the engraved lines, whose interpretation constitutes one of our main scientific objectives. Other limits have also been encountered in terms of the wall texture, notably in the poor rendering of alteration phenomena and geomorphological deposits.

Despite developing this methodological strategy, the diversity of the subjects inevitably lead to technical compromises that had to be adapted to each site, shelter and engraved panel, according to their location and context, their state of conservation and fragility, the observation conditions, etc.

Usage

Techniques of photography, macro-photography and 3D modelling are combined to produce several types of document with which to answer the scientific issues involved in the study of rock art. These issues concern both the chronology of the figurative and abstract depictions, and the internal structuring of the grid patterns.

Given the large number of decorated sites and the considerable documentation involved, a classification and archiving strategy was also developed to safeguard the data. The elements of nomenclature, classification and archiving are part of a long-term perspective and are necessary regarding the large number of sites, shelters and engravings. Also designed as part of a transdisciplinary approach, the classification of the documentation functions as an indexing by site rather than by discipline or recording technique. The tree structure is divided first by site, then by shelter and finally by technique, thematic or discipline. The nomenclature of the files is systematic, and their precise metadata are entered, on the one hand to guarantee a follow-up of the recorded information, and on the other hand to make the link with the database of the Regional Archaeology Service of Ile de France (French Ministry of Culture and Communication). It is a question of guaranteeing the durability of data, beyond the preservation of the heritage itself.

All documentation is stored on two servers. Huma-Num server, which is supported by the CNRS (the French National Center for Scientific Research), Aix-Marseille University and the Campus Condorcet. Huma-Num is a French Very Large Research Infrastructure ("Très Grande Infrastructure de Recherche", TGIR) with international reach devoted to Social Sciences and Humanities. This service provides a shared, secure and professional storage space with high access availability. The second server is located at the National Centre for Prehistory (CNP), which is a research department of the French Ministry of Culture and Communication specialising in the multidisciplinary study, conservation and archiving of decorated caves and rock art sites.

The objectives of preserving and promoting these decorated shelters imply the multiple use of this documentation, in an attempt to respond to the issues of this very dense and fragile open-air rock art. The difficulty lies in finding a balance between preservation and promotion, so that knowledge of this heritage does not drive new potentially destructive visits.

The three-dimensional models were first archived in their digital form, allowing for the subsequent production of replicas, in particular through computer numerically controlled milling. These digital documents can also be used to create virtual reality and augmented reality interactive programmes or scripted videos. In terms of virtual and augmented reality, the documentation (photos, relevés, pan shots, videos, 3D models, etc.) forms a body of resources that can contribute to the creation of virtual discovery trails. This type of interactive and educational resource requires a specific kind of text and script. The fragility of the sites, their difficult access, their very wide distribution over a large geographical area and the sometimes difficult reading of the engravings can all be overcome through the use of this type of virtual discovery trail, provided that it is designed in close connection with the research objectives.

Photography also contributes to preserving the sites in their current state of conservation by means of regular and targeted recordings and through the classification and archiving strategy. In addition, reviewing old photographic archives enables conservation monitoring to be carried out over several decades. The photographic documentation also allows publics to discover and see interior shelters and engraved panels without any contact, near by the sites or even at great distances, as well as raising awareness of the sites through original perspectives and points of view, writing with light and presenting narrative subjects. A photographic approach in natural light reveals the shelters and their engravings as they can be observed today and taking into account their current environment (Fig. 12), which is also a way to approach perceptions and representations during times. Virtual tours based on panoramic photos (360°) goes beyond the photographic frame and allows to explore the inside of these decorated shelters and their surroundings.

Thanks to these productions, several projects are underway to promote the sites among the general public, in particular in schools, through mediation, workshops, events, exhibitions, conferences, and publications. Beyond providing an archaeological knowledge of this heritage, these educational projects raise awareness of these prehistoric sites and engravings, which is essential to their preservation.

Figure.

12 Photographic approach of the shelters and their engravings in natural light, long exposure (La Ségognole 4). Photo E. Lesvignes/PCR ARBap.

Conclusion

The different digital methods and techniques employed in the context of this programme are a real means of meeting the objectives of preserving, studying and raising awareness of the sites.

The production of multi-scale data provides the necessary documentation to support numerous approaches and address the scientific issues involved in this programme, while ensuring the archiving and preservation of the sites.

However, the implementation of these techniques and the methodological experiments involved have demonstrated the limitations of some of these tools, in particular 3D modelling for research objectives, for which the level of resolution and texture rendering have proven insufficient or even ineffective. As such, the consistent use of conventional photographic and macrophotographic documenting processes allows these scientific objectives to be achieved and shows that it is essential that these different methods are continuously applied in parallel. This documentation, which can also be used for educational purposes, comprises images that reveal an extremely fragile and often hidden heritage. The density and richness of this open-air rock art, which is comparable to that of the most internationally renowned rock art sites, incites us to protect it by educating the public and raising awareness of the sites.

Figure. 1

 1 Figure. 1 Location and repartition of the decorated shelters in the sandstone boulder fields of the southern Paris Basin. Doc. E. Lesvignes/PCR ARBap from A. Bénard/GERSAR.

Figure. 2

 2 Figure. 2 a. Engraved inner surfaces of a shelter, geometric themes (Abri Leuillet). b. Schematic figurative depictions, cervids (Mont-Rouget). Photos E. Lesvignes/PCR ARBap.

Figure. 4

 4 Figure. 4 From the shelters context to the engravings (La Ségognole). a. Panoramic photomontage of the site. b. Ségognole 4 rock shelter. c. Small entrance of the Ségognole 4 rock shelter. d. Interior of the Ségognole 4 rock shelter and localisation of the engraved panel. e. One of the engraved panels of the Ségognole 4 rock shelter, located on the floor. Photos and doc. E. Lesvignes/PCR ARBap.

Figure. 5

 5 Figure. 5 Schematic cervid depiction (Sables du Cul du Chien). a. 1985. b. Today. Photos a. Archives GERSAR; b. E. Lesvignes/PCR ARBap.

Figure. 6

 6 Figure. 6 3D model of a decorated shelter, views at different scales and points of view (La Butte du Puits). Model F. Bougnières and B. Touchard/Summum 3D/PCR ARBap.

Figure. 8

 8 Figure. 8 LiDAR view of a sector in the south of the Paris basin (Gorges Houx). LiDAR S. David/ONF.

Figure 9 .

 9 Figure 9. a. Oblique view of an engraved panel on the floor. b. Situation of the panel: the lack of distance above the panel does not allow orthographic photography. Photos and doc. E. Lesvignes/PCR ARBap.

Figure 10 .

 10 Figure 10. a. Classic method of relevé, on a layer placed on an ortho-photographic document, on site and face to the wall. b. Example of desk situation for the relevé with 3D tools, facing the model and taking the reading on an orthoimage from the 3D model. Photos a. E. Lesvignes/PCR ARBap; b. E. Robert/PCR ARBap.

Figure 11 .

 11 Figure 11. Engraved panel of Segognole rockshelter. a. Relevé from 3D model readings on a computer screen and made on an orthoimage from the model. b. Relevé superimposed to the orthoimage. Model A. Lureau/PCR ARBap; relevé and doc. E. Robert/PCR ARBap.

Prehistoric rock art in the sandstone boulder fields of the Paris Basin. Study, archiving and promotion.

Group for the Study, Research and Preservation of Rock Art

Acknowledgements

We would like to thank, for their participation and support: DRAC Île-de-France (ministère de la Culture), Centre national de Préhistoire (ministère de la Culture), Office National des Forêts, Parc naturel régional du Gâtinais français, Centre archéologique de Pincevent, Musée départemental de Préhistoire d'Île-de-France, UMR 7041 ArScAn -Ethnologie préhistorique, Université Paris 1, Museum national d'Histoire naturelle, UMR 7194 HnHp -NOMADE. We would also like to thank Sarita Mardon for the translation of this article.