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

Abstract

The beta-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 beta-N-acetylglucosminidase 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 beta-N-acetylglucosaminide, tri-N-acetylchitotriose, and an N-linked biantennary agalactooligosaccharide. Inhibition studies indicted a single enzyme to be responsible for the hydrolysis of all these substrates. With the biantennary substrate, the beta-N-acetylglucosaminidase exclusively removed beta-N-acetylglucosamine from the alpha 1,3-antenna. GlcNAcMan5GlcNAc2, the primary product of GlcNAc-transferase I, was not perceptibly hydrolyzed. beta-N-Acetylglucosaminidases with the same branch specificity were also found in the lepidopteran cell lines Bm-N and Mb-0503. In contrast, beta-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 beta-N-acetylglucosaminidase in the processing of asparagine-linked oligosaccharides in insects is suggested.

The Drosophila fused lobes Gene Encodes an N-Acetylglucosaminidase Involved in N-Glycan Processing

“…It is noteworthy that this enzyme could not hydrolyze Man5Gn, 4 but acted only further "downstream" on MGn and GnGn where it specifically removed GlcNAc-1 (20). Subsequent cell culture experiments in the presence of hex-osaminidase inhibitors (7,11,21) or of recombinant ␤1,4-galactosyltransferase (22,23) corroborated the putative existence of a processing hexosaminidase.…”

“…Incubation was terminated by the addition of 0.18 ml of 20 mM ice-cold sodium borate. Aliquots of 0.05 ml were analyzed by reverse-phase HPLC as described previously (15,20).…”

“…In fact, in 1995, Sf-9 cells (as well as Bm-N and MB0503 cells) were reported to contain a membrane-bound N-acetylglucosaminidase possibly residing in the Golgi apparatus (20). It is noteworthy that this enzyme could not hydrolyze Man5Gn, 4 but acted only further "downstream" on MGn and GnGn where it specifically removed GlcNAc-1 (20).…”

Human Neutrophils Secrete Bioactive Paucimannosidic Proteins from Azurophilic Granules into Pathogen-Infected Sputum

“…Outside the vertebrate subphylum, invertebrates such as Caenorhabditis elegans (16,18,(22)(23)(24) and Drosophila melanogaster (15,17,25,26), plants (27), and other "lower" organisms (28) abundantly produce protein paucimannosylation. Paucimannose synthesis in these species is facilitated by high ␤-Nacetylhexosaminidase activity, allowing partial suppression of the complex N-glycan biosynthetic route.…”

β‐N‐Acetylhexosaminidase in Peripheral Blood Lymphocytes and Monocytes in the Different Forms and Stages of Multiple Sclerosis

“…Enrichment of Hex B and B-like Hex isoenzymes was also described in the PM fraction of HL 60 leukemic cell line (Martino et al, 1995), where its activity is modulated by cell differentiation (Emiliani et al, 1995c). Altmann et al (1995) demonstrated that in Sf-i insect cells Hex exists in a soluble and a membrane-bound form and that the latter is probably involved in the processing of Nglycan proteins.…”