A HPLC-based glycoanalytical protocol allows the use of natural O-glycans derived from glycoproteins as substrates for glycosidase discovery from microbial culture Li Liu & Tharmala Tharmalingam & Eva Maischberger & Simone Albrecht & Mary E. Gallagher & Raúl Miranda-CasoLuengo & Wim G. Meijer & Pauline M. Rudd & Jane A. Irwin Received: 5 February 2013 / Revised: 31 May 2013 / Accepted: 4 June 2013 # Springer Science+Business Media New York 2013 Abstract Many disorders are characterised by changes in O- glycosylation, but analysis of O-glycosylation has been limited by the availability of specific endo- and exo-glycosidases. As a result chemical methods are employed. However, these may give rise to glycan degradation, so therefore novel O-glycosidases are needed. Artificial substrates do not always identify every glyco- sidase activity present in an extract. To overcome this, an HPLC-based protocol for glycosidase identification from mi- crobial culture was developed using natural O-glycans and O- glycosylated glycoproteins (porcine stomach mucin and fetuin) as substrates. O-glycans were released by ammonia- based β-elimination for use as substrates, and the bacterial culture supernatants were subjected to ultrafiltration to separate the proteins from glycans and low molecular size molecules. Two bacterial cultures, the psychrotroph Arthrobacter C1-1 and a Corynebacterium isolate, were examined as potential sources of novel glycosidases. Arthrobacter C1-1 culture contained a β-galactosidase and N-acetyl-β-glucosaminidase when assayed using 4-methylumbelliferyl substrates, but when defucosylated O-glycans from porcine stomach mucin were used as substrate, the extract did not cleave β-linked galactose or N-acetylglucosamine. Sialidase activity was identified in Corynebacterium culture supernatant, which hydrolysed sialic acid from fetuin glycans. When both culture supernatants were assayed using the glycoproteins as substrate, neither contained endoglycosidase activity. This method may be ap- plied to investigate a microbial or other extract for glycosidase activity, and has potential for scale-up on high-throughput platforms. Keywords Arthrobacter . Corynebacterium . Glycan analysis . Exoglycosidase . Endoglycosidase . O-glycan Abbreviations 4-MU 4-methylumbelliferyl PSM Porcine stomach mucin GU Glucose unit NP-HPLC Normal phase HPLC X-Neu 5-bromo-4-chloro-3-indolyl-α-D-N- acetylneuraminic acid sodium salt Introduction O-glycosylation, one of the most important post-translational modifications made to many proteins, is initiated by enzymatic addition of a single monosaccharide to serine/threonine via a hydroxyl group. Glycosylation initiated with the addition of α- N-acetylgalactosamine (GalNAc) is the most abundant form in nature, and is referred to as mucin-like O-glycosylation as this is found in mucin glycoproteins. Aberrant O-glycosylation L. Liu : T. Tharmalingam : S. Albrecht : P. M. Rudd Dublin-Oxford Glycobiology Group, National Institute for Bioprocessing Research & Training, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland T. Tharmalingam : E. Maischberger : M. E. Gallagher : J. A. Irwin (*) School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland e-mail: jane.irwin@ucd.ie R. Miranda-CasoLuengo : W. G. Meijer School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland Present Address: L. Liu Glycomics and Glycan Bioengineering Research Center, Department of Food Science & Technology, Nanjing Agricultural University, Nanjing, China Glycoconj J DOI 10.1007/s10719-013-9483-9