HEG-5, a novel glycoprotein with hemagglutinating activity, was firstly isolated and purified from the cultured mycelia of Hericium erinaceus CZ-2. SDS-PAGE, Native-PAGE and MALDI-TOF-MS proved that HEG-5 was a single band with the molecular weight of approximately 14.4 kDa. HEG-5 had the protein: polysaccharide ratio of approximately 10:1 (%/%) and contained ID-glucose, L-rhamnose, D-galactose and D-mannose with a molar ratio of 1.00:1.09:2.45:7.14 in polysaccharide fraction. HEG-5 was an acidic glycoprotein with a PI value of 6.3 and the higher content of acidic amino acids (Asp, 12.42 +/- 0.25% and Glu, 12.24 +/- 0.26%) in protein fraction. FT-IR and NMR spectra revealed that HEG-5 contained the protein and carbohydrate portions with (1 -> 4)-linked beta-galactose residues and beta-linked glucose residues. Circular dichroism (CD) demonstrated that HEG-5 was a beta-sheet predominant glycoprotein. Hemagglutination assay proved it was a thermo-unstable glycoprotein. The HEG-5 structural novelty was finally presented by protein sequencing and modeling by using MALDI-TOF-MS, NCBI blast search and online SWISS-MODEL Workspace service. (C) 2014 Elsevier Ltd. All rights reserved.