Expression and role of mannose receptor/terminal high-mannose type oligosaccharide on osteoclast precursors during osteoclast formation S Morishima 1,2 , I Morita 1 , T Tokushima 1 , H Kawashima 3 , M Miyasaka 3 , K Omura 2 and S Murota 1 1 Cellular Physiological Chemistry, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-Ku, Tokyo 113-8549, Japan 2 Oral Restitution, Division of Oral Health Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-Ku, Tokyo, Japan 3 Laboratory of Molecular and Cellular Recognition, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka, Japan (Requests for offprints should be addressed to I Morita; Email: morita.cell@tmd.ac.jp) Abstract Osteoclasts are formed from hematopoietic precursors via cell–cell fusion. We have previously reported that man- nose residues are expressed on the outer membranes of monocytes during osteoclast differentiation. In the present study, we have attempted to demonstrate the pattern of expression levels of terminal high-mannose type oligo- saccharide and to show that the mannose receptor is expressed on osteoclast precursor cells. Osteoclasts were formed using three different systems, namely mouse bone marrow cell culture, co-culture of mouse spleen cells with stromal cells, and RAW264·7 cell cultures. During osteo- clast differentiation, the expression of terminal high- mannose type oligosaccharide gradually increased and then peaked at the stage of fusion in all three systems. Expression of the mannose receptor gradually increased during osteoclast differentiation in bone marrow cells and the co-culture system. In contrast, that in RAW264·7 cells had already been detected in the absence of the soluble receptor activator of NF-B ligand and did not change during osteoclast differentiation. To ascertain whether expression of high-mannose type oligosaccharide is in- volved in tartrate-resistant acid phosphatase (TRAP)- positive multinucleated cell (MNC) formation, glyco- sidase inhibitors were used on RAW264·7 cell culture. Castanospermine, an inhibitor of glucosidase I, inhibited the TRAP-positive MNCs, and deoxymannojirimycin, an inhibitor of -mannosidase I, increased the TRAP- positive MNC formation. These results indicate that the binding of terminal high-mannose and mannose receptor is important for the process of cellular fusion in osteoclast formation. Journal of Endocrinology (2003) 176, 285–292 Introduction Terminal high-mannose type oligosaccharide is very rare on the cell surface of mammalian cells, in spite of its being widely recognized that terminal high mannose in a lysosomal hydrolase is important for the recognition of GlcNAc phosphotransferase. Terminal high-mannose type oligosaccharide on the cell surface mediates cell–cell fusion reactions such as sperm–egg fusion (Primakoff et al. 1987, Blobel et al. 1990, 1992) and myoblast fusion (Kaufman et al. 1985, Rosenberg et al. 1985, Menko & Boettiger 1987, Rosen et al. 1992). The involvement of terminal high-mannose type oligosaccharide has also been reported in the events of viral infections such as the influenza virus (Anders et al. 1990) or human immuno- deficiency virus (HIV) (Lifson et al. 1986, Matthews et al. 1987). For example, the mast cell function-associated antigen was found to bind terminal high-mannose residues in the influenza virus specifically (Binsack & Pecht 1997). Osteoclasts, the bone-resorbing cells, are multinucleated giant cells that develop from hematopoietic cells of the monocyte/macrophage lineage via cell–cell fusion (Udagawa et al. 1990). Using a mouse spleen cell co- cultured system, we have previously demonstrated that terminal high-mannose type oligosaccharide is expressed on the outer membranes of monocytes under pathophysio- logical conditions, and that it is also involved in osteoclast formation via cellular membrane fusion events (Kurachi et al. 1994). The expression of terminal high-mannose type oligo- saccharide proceeds as follows: (Glc) 3 (Man) 9 (GlcNAc) 2 is first transferred from dolichol pyrophosphate to asparagine residues on proteins, and three glucoses are moved by glucosidases I and II to afford a terminal high-mannose type oligosaccharide, (Man) 9 (GlcNAc) 2 . The terminal high-mannose type glycan is transformed into complex type one via processing by mannosidases I and II (Oki et al. 1999). 285 Journal of Endocrinology (2003) 176, 285–292 0022–0795/03/0176–285  2003 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology.org