Plasma membrane association and preliminary characterization of Drosophilasperm surface glycosidases

Previous studies have identified beta-N-acetylglucosaminidase (GlcNAc'ase) and alpha-mannosidase activities on the Drosophila melanogaster sperm surfa ce which may have a role in fertilization. The aim of this study was to inv estigate their linkage to the sperm plasma membrane. We verified that glyco sidases are not peripherally adsorbed to the cell surface by evaluating the ir resistance to release by KI, by buffered salt solutions of high ionic st rength or alkaline buffers. Glycosidases were released from the sperm surfa ce by detergents and, only to a minor extent, by mild proteolysis. Differen tial detergent solubilization pointed out that Triton X-114 was the most ef fective releasing agent for GIcNAc'ase and CHAPS for mannosidase. Na activi ty was released from the membrane by a phosphatidylinositol-specific phosph olipase C (PI-PLC). The released forms were quite hydrophilic in phase sepa ration experiments with Triton X-114. This finding indicates the presence o f a hydrophobic domain limited to a single transmembrane helix or/and the p resence of an extensive glycosilation. The use of a Con-A binding assay dem onstrated that both the enzymes are glycosilated. The molecular weight of t he released glycosidases estimated by gel filtration was 158 kDa for GlcNAc 'ase and 317 kDa far mannosidase. These results suggest that Drosophila mel anogaster GlcNAc'ase and mannosidase are mannosylated integral membrane pro teins that would function as exoenzymes with their active sites accessible in the extracellular space. Mol. Reprod. Dev. 52:166-173, 1999. (C) 1999 Wi ley-Liss, Inc.