1 3 Identification and first insights into the structure and biosynthesis 4 of chitin from the freshwater sponge Spongilla lacustris (Linnaeus, 1759) 5 6 7 Hermann Ehrlich a,c,⇑ Q1 , Oksana V. Kaluzhnaya b,c , Eike Brunner c , Mikhail V. Tsurkan d , 8 Alexander Ereskovsky e , Micha Ilan f , Konstantin R. Tabachnick g , Vasilii V. Bazhenov a , Silvia Paasch c , 9 Martin Kammer c , René Born h , Allison Stelling i , Roberta Galli i , Sergei Belikov b , Olga V. Petrova j , 10 Victor V. Sivkov j , Denis Vyalikh k , Sebastian Hunoldt c , Gert Wörheide l,m,n,⇑ 11 a Institute of Experimental Physics, TU Bergakademie Freiberg, 09599 Freiberg, Germany 12 b Limnological Institute SB RAS, 664033 Irkutsk, Russia 13 c Institute of Bioanalytical Chemistry, TU Dresden, 01069 Dresden, Germany 14 d Leibniz Institute of Polymer Research & Max Bergmann Centre for Biomaterials, 01062 Dresden, Germany 15 e Mediterranean Institute of Biodiversity and Ecology, CNRS, IRD, Aix-Marseille University, 13007 Marseille, France 16 f Department of Zoology, Tel Aviv University, Tel Aviv 69978, Israel 17 g P.P. Shirshov Institute of Oceanology, Russian Academy of Sciences, Moscow, Russia 18 h Institute of Materials Science, TU Dresden, 01062 Dresden, Germany 19 i Carl-Gustav-Carus Klinikum, TU Dresden, 01069 Dresden, Germany 20 j Department of Mathematics Komi SC UrD RAS, Syktyvkar, Russia 21 k Institute of Solid State Physics, TU Dresden, 01062 Dresden, Germany 22 l Department of Earth and Environmental Sciences, Palaeontology & Geobiology, Ludwig-Maximilians-Universität München, Richard-Wagner-Str. 10, 80333 München, Germany 23 m GeoBio-Center, Ludwig-Maximilians-Universität München, Richard-Wagner-Str. 10, 80333 München, Germany 24 n Bayerische Staatssammlungen für Paläontologie und Geologie, Richard-Wagner-Str. 10, 80333 München, Germany 25 27 article info 28 Article history: 29 Received 29 January 2013 30 Received in revised form 24 June 2013 31 Accepted 27 June 2013 32 Available online xxxx 33 Keywords: 34 Sponges 35 Skeleton 36 Chitin 37 Chitin synthase 38 Bioanalytics 39 40 abstract 41 This work demonstrates that chitin is an important structural component within the skeletal fibers of the 42 freshwater sponge Spongilla lacustris (Linnaeus, 1759). Using a variety of analytical techniques ( 13 C solid 43 state NMR, FT-IR, Raman, NEXAFS, ESI-MS, Morgan-Elson assay and Calcofluor White Staining); we show 44 that this sponge chitin is much closer to a-chitin, known to be present in other animals, than to b-chitin. 45 Genetic analysis confirmed the presence of chitin synthases, which are described for the first time in a 46 sponge. The presence of chitin in both marine (demosponges and hexactinellids) and freshwater sponges 47 indicates that this important structural biopolymer was already present in their common ancestor. 48 Ó 2013 Published by Elsevier Inc. 49 50 51 1. Introduction 52 Chitin is the second most abundant polysaccharide found in 53 nature. It usually occurs in complexes with other polysaccharides, 54 pigments, and/or proteins. Interactions between chitin and other 55 molecules and elements are often the key to tuning the rigidifica- 56 tion of biological tissues. From a structural point of view, chitin 57 performs the same function among invertebrates as cellulose does 58 in the plant kingdom. Furthermore, chitin is an important player in 59 biomineralization (Ehrlich, 2010) and possesses templating activ- 60 ity in both biosilica and calcium-based biominerals (Ehrlich et al., 61 2010c). Originally, this structural biopolymer was known to be a 62 part of the skeletal structures of various invertebrates. It was first 63 described by Odier (1823) as the main element of insect exoskel- 64 etons (Phylum Arthropoda). Depending on its source, chitin occurs 65 in a two main allomorphs, namely the a and b forms. These forms 66 can be differentiated from each other by infrared and solid-state 67 NMR spectroscopy in conjunction with X-ray diffraction. A third 68 allomorph, c-chitin, has also been described. However a detailed 69 analysis shows that it seems to be just a variant of the family 70 (see for review Ehrlich, 2010).There are no reports of chitin occur- 71 ring in bacteria; however, chitin is an important structural compo- 72 nent of the cell walls of yeast and fungi (Nguyen et al., 1998; Peter, 73 2002) and diatoms (Brunner et al., 2009b). Today, chitin is predom- 74 inantly associated with the skeletal structures of arthropods. 75 Meanwhile, its presence in other invertebrates – including sponges 1047-8477/$ - see front matter Ó 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.jsb.2013.06.015 ⇑ Corresponding author at: Institute of Experimental Physics, TU Bergakademie Freiberg, 09599 Freiberg, Germany. Q2 E-mail address: hermann.ehrlich@physik.tu-freiberg.de (H. Ehrlich). Journal of Structural Biology xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi YJSBI 6420 No. of Pages 10, Model 5G 11 July 2013 Please cite this article in press as: Ehrlich, H., et al. Identification and first insights into the structure and biosynthesis of chitin from the freshwater sponge Spongilla lacustris (Linnaeus, 1759). J. Struct. Biol. (2013), http://dx.doi.org/10.1016/j.jsb.2013.06.015