Purification and characterization of two forms of N-acetylglucosaminidase from Candida albicans showing widely different outer chain glycosylation

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Abstract
Two forms of N-acetylglucosaminidase were purified to homogeneity by ion exchange (TSK DEAE-3SW, Aquapore CX-300) and gel filtration (TSK G4000 SW) HPLC of Candida albicans ATCC 10261 culture filtrates. Synthesis and secretion of N-acetylglucosaminidase were induced by incubating starved yeast cells at 37 °C in medium containing N-acetylglucosamine (GlcNAc). The form of the enzyme depended on the cell growth and starvation conditions before GlcNAc induction. N-Acetylglucosaminidase A (32% total carbohydrate, Mr 85000 subunit) was isolated from cells grown in glucose/salts/biotin medium, and N-acetylglucosaminidase B (56% carbohydrate, Mr 132000 subunit) was isolated from cells grown in yeast extract/peptone/dextrose. The estimated relative molecular masses of the native enzymes, based on Sephacryl S-300 gel filtration were: A form, 350000; B form, 600000; A and B forms after endoglycosidase H (endo H) treatment, 180 000. The purified enzymes migrated on SDS polyacrylamide gels as heterogeneous glycoproteins of Mr centred at ~ 100 000 (A) and ~ 150 000 (B) but were reduced to a single 58 000 band after denaturation with SDS and cleavage of asparagine-linked sidechains by endo H. When the native glycoproteins were treated with endo H, both enzyme forms had three oligosaccharide sidechains of Mr ~ 3000 that were endo H resistant. Therefore the difference in the size of N-acetylglucosaminidase A and B was due to variations in outer chain glycosylation of endo H-sensitive inner core structures. N-Acetylglucosaminidase was active and stable over a broad pH range with maximum activity against both p-nitrophenylGIcNAc (pNPGIcNAc) and pNPGalNAc at pH 4.0. The kinetic parameters kcat (s-1) and Km (mM) of N-acetylglucosaminidase A using the following substrates were, respectively: pNPGIcNAc, 740, 0.77; pNPGalNAc, 910, 1.26; N,N'-diacetylchitobiose 620, 0.20; and N,N',N-triacetylchitotriose, 170, 0.044. The enzyme showed substrate inhibition with all substrates above 0.5 mM except for pNPGalNAc.

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