Insect Cells Contain an Unusual, Membrane-bound β-N-Acetylglucosaminidase Probably Involved in the Processing of Protein N-Glycans

• Published in: The Journal of biological chemistry 1995-07, Vol.270 (29), p.17344-17349
• Main Authors: Altmann, Friedrich, Schwihla, Herwig, Staudacher, Erika, Glössl, Josef, März, Leopold
• Format: Article
• Language: English
• Subjects: Acetylglucosaminidase - physiology; Animals; Carbohydrate Sequence; Cell Line; Cell Membrane - enzymology; CULTIVO DE CELULAS; CULTURE DE CELLULE; GLICOPROTEINAS; GLYCOPROTEINE; LEPIDOPTERA; Molecular Sequence Data; Polysaccharides - metabolism; Proteins - metabolism; Spodoptera - enzymology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.29.17344

The β-N-acetylglucosaminidase activity in the lepidopteran insect cell line Sf21 has been studied using pyridylaminated oligosaccharides and chromogenic synthetic glycosides as substrates. Ultracentrifugation experiments indicated that the insect cell β-N-acetylglucosaminidase exists in a soluble and a membrane-bound form. This latter form accounted for two-thirds of the total activity and was associated with vesicles of the same density as those containing GlcNAc-transferase I. Partial membrane association of the enzyme was observed with all substrates tested, i.e. 4-nitrophenyl β-N-acetylglucosaminide, tri-N-acetylchitotriose, and an N-linked biantennary agalactooligosaccharide. Inhibition studies indicated a single enzyme to be responsible for the hydrolysis of all these substrates. With the biantennary substrate, the β-N-acetylglucosaminidase exclusively removed β-N-acetylglucosamine from the α1,3-antenna. GlcNAcMan5GlcNAc2, the primary product of GlcNAc-transferase I, was not perceptibly hydrolyzed. β-N-Acetylglucosaminidases with the same branch specificity were also found in the lepidopteran cell lines Bm-N and Mb-0503. In contrast, β-N-acetylglucosaminidase activities from rat or frog (Xenopus laevis) liver and from mung bean seedlings were not membrane-bound, and they did not exhibit a strict branch specificity. An involvement of this unusual β-N-acetylglucosaminidase in the processing of asparagine-linked oligosaccharides in insects is suggested.