Biochem. J. (2013) 455, 307–318 (Printed in Great Britain) doi:10.1042/BJ20130354 307 A novel fluorescence assay and catalytic properties of Crh1 and Crh2 yeast cell wall transglycosylases Marian MAZ ´ A ˇ N* 1 , Noelia BLANCO† 1 , Krist´ ına KOV ´ A ˇ COV ´ A*, Zuzana FIR ´ AKOV ´ A*, Pavel ˇ REHULKA‡, Vladim´ ır FARKA ˇ S* 2 and Javier ARROYO† 2 *Institute of Chemistry, Center for Glycomics, Department of Glycobiology, Slovak Academy of Sciences, 84538 Bratislava, Slovakia, †Departamento de Microbiolog´ ıa II, Facultad de Farmacia, Universidad Complutense de Madrid, IRYCIS, 28040 Madrid, Spain, and ‡Institute of Molecular Pathology, Faculty of Military Health Sciences, University of Defence, Tˇ rebeˇ ssk´ a 1575, CZ-500 01 Hradec Kr´ alov´ e, Czech Republic The mechanical properties of fungal cell walls are largely determined by composition and mutual cross-linking of their macromolecular components. Previous work showed that the Crh proteins are required for the formation of cross-links between chitin and glucan at the Saccharomyces cerevisiae cell wall. In the present study, the proteins encoded by CRH1 and CRH2 were heterologously expressed in Pichia pastoris and a sensitive fluorescence in vitro soluble assay was devised for determination of their transglycosylating activities. Both proteins act as chitin transglycosylases; they use soluble chitin derivatives, such as carboxymethyl chitin, glycol-chitin and/or N-acetyl chito- oligosaccharides of DP (degree of polymerization)  5 as the oligoglycosyl donors, and oligosaccharides derived from chitin, β -(1,3)-glucan (laminarin) and β -(1,6)-glucan (pustulan), fluorescently labelled with sulforhodamine or FITC as acceptors. The minimal number of intact hexopyranose units required by Crh1 and/or Crh2 in the molecule of the acceptor oligosaccharide was two and the effectivity of the acceptor increased with the increasing length of its oligosaccharide chain. Products of the transglycosylation reactions were hybrid molecules composed of the acceptor and portions of carboxymethyl chitin attached to its non-reducing end. Both proteins exhibited a weak chitinolytic activity in different assays whereby the ratio of endo- compared with exo-chitinase activity was approximately 4-fold higher in Crh1 than in Crh2. The pH optimum of both enzymes was 3.5 and the optimum temperature was 37 ◦ C. The results obtained in vitro with different fluorescently labelled oligosaccharides as artificial chitin acceptors corroborated well with those observed in vivo. Key words: cell wall cross-link, chitin, Crh protein, family 16 of glycoside hydrolases (GH16), glucan, transglycosylation. INTRODUCTION Fungal cells are surrounded by the cell wall, an essential structure that determines the cell shape, mediates cellular interactions, protects the cells from adverse effects of the environment and provides protection against internal turgor pressure that would cause bursting of the cell [1]. The yeast cell wall consists of three principal polysaccharides: β -(1,3)-glucan, the major structural component, β -(1,6)-glucan and chitin. Although chitin is a minor component representing 1–2 % of the cell wall dry weight, this polysaccharide is essential for cell survival. Additionally, mannoproteins (approximately 25–50 %) are present as an ex- ternal layer of the cell wall [2,3]. Individual polymer components of the cell wall are mutually linked by covalent bonds, thus creat- ing large macromolecular complexes. The backbone of these com- plexes is formed by β -(1,3)-glucan chains, branched at random by short chains of β -(1,6)-glucan extended by chitin linked by β -(1,4) glycosidic bonds at their non-reducing ends. In addition, GPI (glycosylphosphatidylinositol)-anchored mannoproteins are linked to the complex via β -(1,6)-glucan side chains [3–5]. Despite its apparent rigidity, the cell wall structure needs to accommodate continuously to morphological changes during cell growth. Therefore a balance between the biosynthesis of the wall constituents and their breakdown and reorganization is essential for both cell integrity and proper morphogenesis [6,7]. The biosynthesis of the respective cell wall components takes place at different locations in the cell. The mannoproteins are synthesized in the endoplasmic reticulum, further glycosylated in the Golgi apparatus and transported by exocytosis to the cell wall, whereas the insoluble cell wall constituents chitin [8], β -(1,3)-glucan [9] and probably β -(1,6)-glucan [10,11] are formed at the plasma membrane and extruded into the cell wall. Moreover, the synthesis of some of these polymers, such as β -(1,3)-glucan and β -(1,6)-glucan, seems to be co-ordinated [12]. The final stage of cell wall construction, the creation of a supramolecular structure through covalent cross-links between the individual components exported by the cells into the ex- tracytoplasmic space, must therefore take place outside the plasma membrane, catalysed by enzymes located in the cell wall. In analogy to the plant system [13], the most probable candidates for this function are the polysaccharide hydrolases/transglycosylases capable of creating covalent bonds between individual polysac- charide molecules by transglycosylation. The transglycosylation Abbreviations used: CH3, N-triacetyl chitotriose; CH4, N-tetra-acetyl chitotetraose; ChitOS, chito-oligosaccharide; CM, sodium-carboxymethyl; DP, degree of polymerization; Gly-chitin, [O-(2-hydroxyethyl)] chitin, glycol-chitin; GPI, glycosylphosphatidylinositol; HEC, hydroxyethyl cellulose; L4, laminaritetraose; L5, laminaripentaose; LamOS, laminarioligosaccharide; MU, methylumbelliferyl; MU-Ch2, 4-MU-N-acetyl-β-D-N,N ′ -diacetylchitobioside; MU-Ch3, 4-MU-N-acetyl-β-D-N,N ′ ,N ′′ -triacetylchitotriose; P4, pustulotetraose; PustOS, pustulo-oligosaccharides; SR, sulforhodamine; RBV, Remazol Brilliant Violet; TXG, tamarind seed xyloglucan; XET, xyloglucan endotransglycosylase; XTH, xyloglucan hydrolase; WT, wild-type. 1 These authors contributed equally to this work. 2 Correspondence may be addressed to either of these authors (email chemvfar@savba.sk or jarroyo@farm.ucm.es). c  The Authors Journal compilation c  2013 Biochemical Society