Structural and Morphological Diversity of (1f3)--D-Glucans Synthesized in Vitro by Enzymes from Saprolegnia monoı ¨ca. Comparison with a Corresponding in Vitro Product from Blackberry (Rubus fruticosus) † Ludovic Pelosi, ‡ Tomoya Imai, ‡ Henri Chanzy, Laurent Heux, Eric Buhler, and Vincent Bulone* Centre de Recherches sur les Macromole ´ cules Ve ´ ge ´ tales (CERMAV-UPR CNRS 5301), Joseph Fourier UniVersity of Grenoble, B.P. 53, 38041 Grenoble cedex 9, France ReceiVed January 13, 2003; ReVised Manuscript ReceiVed March 25, 2003 ABSTRACT: Detergent extracts of microsomal fractions from Saprolegnia monoı ¨ca and blackberry (Rubus fruticosus) cells were incubated with UDP-glucose to yield in Vitro (1f3)--D-glucans. The insoluble products were analyzed by conventional and cryo transmission electron microscopy, X-ray diffraction, and 13 C CP/MAS NMR, and their molecular weights were determined by light scattering experiments. All the products were microfibrillar, but for the detergent extracts from S. monoı ¨ca, important morphological differences were observed when the pH of the synthesizing medium was modified. At pH 6, the product had a weight average degree of polymerization ( DP w ) exceeding 20 000 and consisted of endless ribbon- like microfibrils. The microfibrils obtained at pH 9 had a length of only 200-300 nm, and their DP w was ∼5000. Of all the in Vitro (1f3)--D-glucans, the one from R. fruticosus had the shortest length and the smallest DP w . Crystallographic and spectroscopic data showed that the three in Vitro samples consisted of triple helices of (1f3)--D-glucans and contained substantial amounts of water molecules in their structure, the shortest microfibrils being more hydrated. In addition, the long microfibrils from S. monoı ¨ca synthesized at pH 6 were more resistant toward the action of an endo-(1f3)--D-glucanase than the shorter ones obtained at pH 9. These results are discussed in terms of molecular biosynthetic mechanisms of fungal and plant (1f3)--D-glucans, and in relation with the possible existence of several (1f3)--D-glucan synthases in a given organism. The interpretation and discussion of these observations integrate the current knowledge of the structure and function of (1f3)--D-glucans. (1f3)--D-Glucans are abundant polysaccharides synthe- sized by plants, fungi, yeasts, some algae, and bacteria, mainly as structural or storage material, but also for specialized functions such as, for instance, plant cell division, defense responses of the plant cell to microbial attack, etc. (1). The name (1f3)--D-glucan is in fact a generic name that encompasses a number of glucans containing either exclusively -(1f3) linkages or -(1f3) linkages together with a variable proportion of -(1f4) or -(1f6) glycosidic bonds. Depending on their origin, these polysaccharides are known under a number of common appellations, namely, callose, Curdlan, scleroglucan, pachyman, lentinan, laricinan, laminaran, paramylon, etc. (1). Several of these glucans are attracting strong interest as they not only lead to the formation of potentially useful hydrogels (2) but also present specific immunomodulating, antitumor, antiviral, anticoagu- latory, and wound healing activities (3, 4). The biological activities of (1f3)--D-glucans appear to be related to several factors: a high molecular weight, a highly ordered structure, and the presence of glucosyl side units connected to the main chain by -(1f6) linkages (3, 5). The ordered structure of (1f3)--D-glucans is in itself quite remarkable and has been investigated in great detail for Curdlan, a linear (1f3)--D-glucan produced by a mutant of Alcaligenes faecalis (2). Depending on the preparation conditions, three allomorphs of Curdlan have been identified by X-ray fiber diffraction methods (6-10) and 13 C cross- polarization/magic-angle-spinning nuclear magnetic reso- nance (CP/MAS NMR) 1 spectroscopy (11-13). The anhy- drous allomorph is obtained after the samples had been subjected to vacuum heat annealing. It is characterized by the intertwining of three glucan chains having a right-handed six-fold conformation, a fiber repeat of 0.58 nm, and a P6 3 space group (8-10). If the annealing is performed under hydrothermal conditions, a crystalline hydrate containing two molecules of water per glucosyl residue is obtained (14). † This work was supported by a Grant-in-Aid to V.B. from Hercules Inc. (Wilmington, DE). T.I. was a recipient of fellowships from the French Ministry of Research and from CNRS. * To whom correspondence should be addressed: Laboratoire de Physico-Chimie Biologique, UMR CNRS 5013, Ba ˆtiment Chevreul 4 e `me e ´tage, Universite ´ Claude Bernard Lyon I, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne cedex, France. Phone: (+33) 4 72 43 15 42. Fax: (+33) 4 72 43 15 43. E-mail: Vincent.Bulone@univ-lyon1.fr. ‡ These authors contributed equally to this work. 1 Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]- 1-propanesulfonate; cmc, critical micelle concentration; CP/MAS NMR, cross-polarization/magic-angle-spinning nuclear magnetic resonance; DMSO, dimethyl sulfoxide; DP n , number average degree of polymer- ization; DP w , weight average degree of polymerization; TEM, trans- mission electron microscopy. 6264 Biochemistry 2003, 42, 6264-6274 10.1021/bi0340550 CCC: $25.00 © 2003 American Chemical Society Published on Web 04/29/2003