Enzymatic properties and subcellular localization of Arabidopsis -N-acetylhexosaminidases (correction)

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1 Enzymatic properties and subcellular localization of Arabidopsis β-n-acetylhexosaminidases (correction) Richard Strasser, Jayakumar Singh Bondili, Jennifer Schoberer, Barbara Svoboda, Eva Liebminger, Josef Glössl, Friedrich Altmann, Herta Steinkellner, and Lukas Mach Institute of Applied Genetics and Cell Biology (R.S., J.S., B.S., E.L., J.G., H.S., L.M.) and Department of Chemistry (J.S.B., F.A.), BOKU-University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria Results and Discussion Heterologous Expression of Arabidopsis HEXO3 Protein in Insect Cells In the course of other studies, we have recently detected a previously unnoticed inadvertent deletion of a single base pair close to the 3 -end of the HEXO3 coding region affecting the construct previously used for expression of this enzyme in insect cells. The ensuing frame-shift changed the last five amino acids of recombinant HEXO3 and resulted in the elongation of the protein by 27 amino acids (Fig. 1). In particular, the mutation removed a residue (Cys 532 ) required for proper function of this class of β-nacetylhexosaminidases (Lemieux et al., 2006). These findings prompted us to re-investigate the enzymatic properties of HEXO3. For this, we have generated a new pvtbac-his-1 expression construct encoding the authentic HEXO3 sequence and expressed it in Spodoptera frugiperda Sf21 cells as previously described (Strasser et al., 2007). Biochemical Characterization of the Recombinant Arabidopsis HEXO3 Protein The secreted recombinant protein was purified from culture supernatants of HEXO3-producing Sf21 cells by means of nickel-chelate affinity chromatography. Immunoblot analysis of recombinant HEXO3 revealed a major immunoreactive band of 63 kd. Peptide N-glycosidase F digestion led to a 59 kd band. The detected molecular mass of deglycosylated HEXO3 is in good agreement with the calculated one (61.0 kd; Fig. 2). Purified recombinant HEXO3 protein was assayed using different synthetic substrates (pnp-glcnac, pnp-galnac, MU-GlcNAc, MU-GlcNAc-6SO 4 ). For HEXO3, pnp-glcnac was the preferred substrate over pnp-galnac as also observed for the other two HEXO proteins (Table I). HEXO3 was also highly active on the fluorogenic MU-GlcNAc substrate, but its sulfated derivative (MU-GlcNAc- 6SO 4 ) was hydrolyzed similarly poorly by HEXO3 as by HEXO1 and HEXO2 (Table I). The ph optimum for pnp-glcnac hydrolysis was 4.0 for HEXO3 (Fig. 3), and the apparent K m and V max values for this substrate were found to be 0.5 mm and 118 µmol min -1 mg -1. These values are in the same range as previously reported for HEXO1 (Strasser et al., 2007). To address the question whether complex N-glycans are substrates for HEXO3, pyridylaminated GnGn (GnGn-PA) and other N-glycan substrates were analyzed (Table II). The PA-oligosaccharide substrates were incubated with purified recombinant HEXO3, and the reaction products were analyzed 1

2 by reverse-phase HPLC (Fig. 4). With all tested substrates, release of GlcNAc was clearly detected thus demonstrating that HEXO3 lacks any pronounced branch specificity. In general, the rate of GlcNAc release was similar to HEXO1 and much higher than that by HEXO2. The specific activity of HEXO3 with pyridylaminated chitotriose ((GlcNAc) 3 -PA) was found to be higher than that of HEXO1 and HEXO2. Hydrolysis of pyridylaminated chitobiose ((GlcNAc) 2 -PA) by HEXO3 was detectable, but significantly lower than that by HEXO1 (Table II). Our results suggest that HEXO1 and HEXO3 account for the generation of paucimannosidic structures in plants, since these two enzymes are far more efficient in hydrolyzing N-glycan substrates than HEXO2. HEXO3 and HEXO1 have been localized at the plasma membrane and in the vacuole, respectively (Strasser et al., 2007). Hence, HEXO3 could be responsible for the removal of terminal GlcNAc residues from plasma membrane proteins and secreted glycoproteins, whereas HEXO1 is probably involved in the degradation of vacuolar glycoproteins. Materials and Methods Expression in Insect Cells The N-terminal deletion construct of HEXO3 containing amino acids was generated by PCR using primers AtHex3-Xba-R 5 -TATATCTAGAGAGAGGTTGAGGATTTGGC-3 and AtHex3- Not-R 5 -AATAGCGGCCGCATCACTGAGCGAGACAAGAACCTGG-3 and cloned into pvtbac- His-1 baculovirus transfer vector. Expression in Spodoptera frugiperda Sf21 cells, purification and quantification of recombinant HEXO1 and HEXO3 as well as enzymatic deglycosylation using peptide N-glycosidase F (PNGase F, Roche, Mannheim, Germany) was performed as described previously (Bencúr et al., 2005; Strasser et al., 2007). Immunoblot detection of the recombinant HEXO forms was done using antibodies recognizing the enterokinase cleavage sequence (Invitrogen). Assays with Synthetic and Pyridylaminated Substrates All assays were performed as previously described (Strasser et al., 2007). Acknowledgments We thank Ulrike Vavra for cloning of HEXO3 into pvtbac-his-1, Thomas Dalik and Karin Polacsek for providing N-glycan substrates and Martin Pabst for assisting with HPLC analysis (all from University of Natural Resources and Applied Life Sciences). References Bencúr P, Steinkellner H, Svoboda B, Mucha J, Strasser R, Kolarich D, Hann S, Köllensperger G, Glössl J, Altmann F, Mach L (2005) Arabidopsis thaliana beta1,2-xylosyltransferase: an unusual glycosyltransferase with the potential to act at multiple stages of the plant N- glycosylation pathway. Biochem J 388:

3 Lemieux M, Mark B, Cherney M, Withers S, Mahuran D, James M (2006) Crystallographic structure of human beta-hexosaminidase A: interpretation of Tay-Sachs mutations and loss of GM2 ganglioside hydrolysis. J Mol Biol 359: Strasser R, Bondili JS, Schoberer J, Svoboda B, Liebminger E, Glössl J, Altmann F, Steinkellner H, Mach L (2007) Enzymatic properties and subcellular localization of Arabidopsis beta-nacetylhexosaminidases. Plant Physiol 145: 5-16 Table I. Activity of HEXO proteins and jack bean β-hexosaminidase (Sigma) with synthetic substrates. Activities were determined at substrate concentrations of 5 mm (pnp substrates) or 1 mm (MU substrates) in 50 mm sodium citrate buffer (ph 4.6) at 37 C. *, data taken from Strasser et al. (2007). µmol min -1 mg -1 enzyme protein (HEXO) or total protein (jack bean) Substrate HEXO1* HEXO2* HEXO3 jack bean* pnp-glcnac pnp-galnac MU-GlcNAc MU-GlcNAc-6SO Table II. Activity of HEXO proteins and jack bean β-hexosaminidase (Sigma) with N-glycan and chitooligosaccharide substrates. Substrate (5 µm) was incubated with HEXO and jack bean enzymes in 0.1 M sodium citrate/phosphate buffer (ph 5.0) for different times at 30 C. The samples were then analyzed by reverse-phase HPLC. For GnGn-PA and GnGnXF-PA, the hydrolysis rates shown are derived from assays where less than 5% of the substrate was converted into MM-PA or MMXF-PA, respectively. *, data taken from Strasser et al. (2007). pmol min -1 mg -1 enzyme protein (HEXO) or total protein (jack bean) Substrate Products HEXO1* HEXO2* HEXO3 jack bean* GnGn-PA GnM-PA GnGn-PA MGn-PA MGn-PA MM-PA GnM-PA MM-PA GnGnXF-PA GnMXF-PA GnGnXF-PA MGnXF-PA Man5Gn-PA Man5-PA (GlcNAc) 3 -PA (GlcNAc) 2 -PA (GlcNAc) 2 -PA GlcNAc-PA <

4 HEXO3: VTTRLAHFRCLLNQRGVAAAPLVGGGRVVPFEPGSCLAQ HEXO3_old: VTTRLAHFRCLLNQRGVAAAPLVGGGRVVPFEPGLVSLSDAAALANSVPTLLKRRKFSLKFPWCSK Figure 1. Sequence comparison between authentic HEXO3 and the mutated HEXO3 form (HEXO3_old). Only the last 40 amino acids of HEXO3 are shown. Identical residues are shaded in grey. The position of Cys 532 is highlighted in black. Figure 2. Western blot of heterologously expressed HEXO enzymes. Purified recombinant HEXO proteins were incubated overnight in the absence (-) or presence (+) of PNGase F, analyzed by SDS- PAGE under reducing conditions and subjected to Western blot analysis with antibodies to the enterokinase cleavage sequence. 4

5 Figure 3. Influence of ph on the activity of recombinant HEXO enzymes. The ability of HEXO1 ( ), HEXO2 ( ), HEXO3 ( ) and jack bean β-hexosaminidase (x) to hydrolyze pnp-glcnac (A), GnGn- PA (B) and (GlcNAc) 3 -PA (C) was analyzed under different ph conditions. The data for HEXO1, HEXO2 and jack bean β-hexosaminidase are taken from Strasser et al. (2007). 5

6 Figure 4. Activity assays of recombinant HEXO3 with GnGn-PA (A) and GnGnXF-PA (B) as substrates. The samples were analyzed by reverse-phase HPLC with fluorescence detection. Pyridylaminated oligosaccharides were incubated with 15 ng of HEXO3 for 2 hours. The elution positions of standards (MGn-, MM-, GnGn-, GnM-PA and MGnXF-, MMXF-, GnGnXF-, GnMXF- PA) are shown (S). 6