β-N-Acetylhexosaminidase from a filamentous fungus Aspergillus oryzae is a secreted enzyme known to be an important component of the binary chitinolytic system. Cloning of the hexA gene and sequencing of the enzyme revealed its unique preproprotein structure. While the enzyme's zincin-like and catalytic domain had significant similarities with members of the glycohydrolase 20 family, the propeptide was unique for the fungal enzyme. Detailed pulse–chase and inhibition studies revealed that propeptide was processed during the biosynthesis of the enzyme. Moreover, the presence of propeptide was necessary for enzyme activation, dimerization and secretion. The catalytic unit was N-glycosylated, and the propeptide was O-glycosylated, both in their C-terminal parts. Deglycosylation experiments revealed that the N-glycosylation increased the stability and solubility of the enzyme. In contrast, O-glycosylated propeptide was necessary to attain the full enzymic activity.

Introduction

Fungal β-N-acetylhexosaminidases (chitobiases; EC 3.2.1.52) are the terminal components of the binary chitinolytic system that cleave chitobiose into the constituent monosaccharides (N-acetyl-D-glucosamine) [1]. An early report indicating the involvement of these enzymes in the formation of cell-wall elements [2] was recently supported by Kim et al. [3]. These authors observed poor growth of an hexAspergillus strain on a medium containing chitobiose as a carbon source. We reported the significant inducibility of β-N-acetylhexosaminidase in Aspergillus oryzae grown on media containing N-acetyl-D-glucosamine or the chitooligomers [4]. Good availability of the induced enzyme resulted in its frequent use in the synthesis of unique oligosaccharide sequences [5]. In the present study, we describe our recent results on the interesting glycosylation, and complex architecture of this enzyme.

N- and O-glycosylation occurs in distinct parts of the enzyme

Molecular cloning and sequencing of β-N-acetylhexosaminidase gene (GenBank® accession number AY091636) revealed the occurrence of signal peptide, propeptide, zincin-like domain, catalytic domain of glycosyl hydrolase 20 family [6] and a C-terminal segment. Both the propeptide and the catalytic domain were predicted to be glycosylated (Figure 1A). We found that the processed propeptide associated non-covalently and co-purified with the catalytic domain. The catalytic subunit could be separated from the propeptide only using HPLC under acidic conditions (0.1% TFA, Figure 1B). MALDI-MS (matrix-assisted laser-desorption ionization-mass spectrometry) of C-terminal part of the propeptide confirmed substitution by hexoses, suggesting a possibility for O-mannosylation (Figure 1C). This was supported by quantitative sugar analysis indicative of three attachment sites. Oligosaccharide analysis of the separated catalytic domain using HPAEC-PAD (high-performance anion-exchange chromatography with pulsed amperometric detection) profiling, α-mannosidase digestions and MALDI-MS revealed the presence of N-glycans of high mannose type attached to all the six predicted glycosylation sites (Figure 1D).

Domain structure and glycosylation of β-N-acetylhexosaminidase

Figure 1
Domain structure and glycosylation of β-N-acetylhexosaminidase

(A) Predicted domain structure of β-N-acetylhexosaminidase and its glycosylation. (B) Separation of propeptide (1) from the mature enzyme (2) on a reversed-phase (Vydac C-4) column. (C) O-glycosylation of propeptide; MALDI-MS of the C-terminal propeptide tryptic fragment containing hexose substitutions. (D) N-glycosylation of the catalytic domain; HPAEC-PAD analysis of N-linked glycans of high-mannose type oligosaccharides (M4–M11). The anticipated structures were confirmed by MALDI-MS in combination with α-mannosidase digestions.

Figure 1
Domain structure and glycosylation of β-N-acetylhexosaminidase

(A) Predicted domain structure of β-N-acetylhexosaminidase and its glycosylation. (B) Separation of propeptide (1) from the mature enzyme (2) on a reversed-phase (Vydac C-4) column. (C) O-glycosylation of propeptide; MALDI-MS of the C-terminal propeptide tryptic fragment containing hexose substitutions. (D) N-glycosylation of the catalytic domain; HPAEC-PAD analysis of N-linked glycans of high-mannose type oligosaccharides (M4–M11). The anticipated structures were confirmed by MALDI-MS in combination with α-mannosidase digestions.

Complex protein architecture of the fungal β-N-acetylhexosaminidase

Gel filtration of the native enzyme complex revealed its dimeric nature. However, its stoichiometry varied depending on prevalence of molecular forms produced under various conditions. Isolated monomeric catalytic subunits occurred only intracellularly as short-time, metastable, species devoid of enzymic activity. Most of the secreted enzyme contained dimers of the catalytic subunit associated with one or two molecules of the propeptide. Interestingly, the later molecular form was secreted with approximately double velocity compared with species containing a single propeptide.

The role of N-glycosylation in enzyme solubility and stability revealed by deglycosylation studies

Complete deglycosylation of the enzyme using N-glycanase resulted in precipitation. When β-N-acetylhexosaminidase was deglycosylated using endoglycosidase H, the deglycosylated enzyme remained fully enzymically active, but it was significantly less stable under acidic pH. The relative activity of the deglycosylated enzyme after incubation at pH 2.5 was only 45% of the native control.

Propeptide and its O-glycosylation are necessary for full enzymic activity

When the catalytic unit separated under acidic environment was renatured at optimum pH (pH 5.0) enzymic activity was restored only in the presence of the propeptide (Table 1). Interestingly, whereas the association of one catalytic unit with its propeptide was sufficient for dimerization, the resulting complex had only half specific activity when compared with the complex containing both the propeptides (Table 1). Moreover, pulse–chase labelling data indicated slow acquisition of enzymic activity: the enzyme became fully active only after 18 min when it passed to Golgi complex, and the synthesis of O-linked glycans was completed (α-galactosylation detected by Euonymus europaeus lectin).

Table 1
Reconstitution of the enzymic activity of β-N-acetylhexosaminidase using 1 nmol of the monomeric catalytic unit after 1 h incubation at pH 5.0
Propeptide addition Gel filtration analysis Specific activity (units/nmol of catalytic unit*) 
Native enzyme Dimeric 2.81±0.01 
Monomeric 0.07±0.01 
0.5 nmol Dimeric 1.38±0.11 
1.0 nmol Dimeric 2.90±0.05 
Propeptide addition Gel filtration analysis Specific activity (units/nmol of catalytic unit*) 
Native enzyme Dimeric 2.81±0.01 
Monomeric 0.07±0.01 
0.5 nmol Dimeric 1.38±0.11 
1.0 nmol Dimeric 2.90±0.05 
*

Activity was assayed using a continuous spectrophotometric method [7], and is expressed as the means±S.D. for three independent determinations.

Conclusions

Fungal β-N-acetylhexosaminidases contain a unique, large propeptide shown to be important for enzyme activation, dimerization and secretion. The processed peptide associated non-covalently with the catalytic subunits giving rise to molecular species composed of two catalytic subunits and one or two propeptides. However, the propeptide does not seem to be a typical intracellular chaperone: it keeps active only those catalytic units to which it remains bound. The dichotomy in the functional importance of N- and O-glycosylation shown here seems to be interesting. Further work is in progress to establish the more general occurrence of this phenomenon.

Structure Related to Function: Molecules and Cells: A Focus Topic at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by D. Alessi (Dundee, U.K.), T. Cass (Imperial College London, U.K.), T. Corfield (Bristol, U.K.), M. Cousin (Edinburgh, U.K.), A. Entwistle (Ludwig Institute for Cancer Research, London, U.K.), I. Fearnley (Cambridge, U.K.), P. Haris (De Montfort, Leicester, U.K.), J. Mayer (Nottingham, U.K.) and M. Tuite (Canterbury, U.K.).

Abbreviations

     
  • MALDI-MS

    matrix-assisted laser-desorption ionization-mass spectrometry

This work was supported by Ministry of Education of the Czech Republic grant (MSM113100001) by the Institutional Research Concept for the Academy of Science (AVOZ5020903) and by Grant Agency of the Czech Republic (grant 203/04/1045).

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