Development of a colorimetric PNGase activity assay

Highlights

• A colorimetric method was established and optimized for testing PNGase activity.

• Released N-glycans could be quantified using a water-soluble formazan dye.

• Kinetic parameters of PNGase variants were determined using this optimized method.

Abstract

PNGases are crucial targets and valuable tools in analyzing asparagine-linked carbohydrate moieties (N-glycans) of glycoproteins. Activity tests of PNGases have been little improved since their discovery four decades ago, and still rely on observing deglycosylation patterns of glycoproteins or glycopeptides using SDS-PAGE or HPLC analysis. These techniques cannot be easily adapted for automated sampling and high-throughput procedures. Herein, we describe a PNGase activity assay which relies on the conversion of WST-1, a yellowish, water-soluble tetrazolium dye (sodium 2-(4-Iodophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolate), into a blue formazan dye. In this work, we showed that WST-1 could be reduced by N-glycans, which were enzymatically released from glycoprotein substrates. After optimization of the assay conditions, the robustness of the method was challenged by quantifying the activity of various PNGase isoforms at different purification stages using a microwell plate reader. Furthermore, the assay could be used to obtain steady-state kinetics of PNGase H⁺ wild-type and mutant variants, which showed significant differences in their enzymatic reaction rates. The simplicity and robustness of this method might be of benefit for the detection of PNGase activity in routine applications of large amounts of samples.

Introduction

Peptide-N(4)-(N-acetyl-β-glucosaminyl)asparagine amidases, also known as PNGases or N-glycanases, are widely used for the enzymatic release of N-linked carbohydrates from glycoproteins [[1], [2], [3]]. PNGases have been identified from bacteria and higher organisms, and can be classified according to their structural similarity and enzymatic properties into cytosolic PNGases, acidic PNGases and PNGase F-like N-glycanases [[4], [5], [6], [7]]. Cytosolic PNGases play a crucial role in the quality control of glycoprotein synthesis, namely the endoplasmic reticulum-associated degradation (ERAD) pathway [8]. The recent discovery of a human genetic disorder involving the NGLY1 gene has rekindled interest in this cytosolic PNGase [9,10]. Acidic PNGases, which are found in certain plant species, may play a role in fruit ripening [11,12]. The biological role of a recently discovered acidic PNGase of bacterial origin remains unclear [13]. Likewise, no definite in vivo physiological function of bacterial PNGase F-like N-glycanases has yet been described. Nevertheless, recombinant bacterial PNGases are considered to be an indispensable and useful tool for analyzing N-glycans. Beside these analytical applications, the deglycosylation capability of PNGases may also be used in biotechnological applications; the co-expression of bacterial PNGases in tobacco plants was considered to be a strategy of producing recombinant proteins free of N-glycans [14,15].

Although the first PNGase isoforms were described more than four decades ago [7], the methodologies for determining their enzymatic activity remain tedious. Initially, activity screens relied on radioisotope labeled glycopeptides, which were analyzed using paper chromatography or paper electrophoresis [16,17]. Later, glycopeptides bearing fluorescent or chromophoric tags, such as fluorenylmethyloxycarbonyl (Fmoc), 5-(dimethylamino)naphthalene-1-sulfonyl (dansyl) or 4-(dimethylamino)azobenzene-4′- sulfonyl (dabsyl), were used as substrates for High-Performance Liquid Chromatography (HPLC) analysis [[18], [19], [20]]. Using HPLC, these analytes can be detected with high sensitivity in low-pmol quantities [21]. However, both radioisotope and chemical labeling methods require the skillful preparation of purified glycopeptide substrates with several steps of gel filtration and anion-exchange chromatography [[22], [23], [24]]. Furthermore, kinetic parameters of PNGases can only be determined using these derivatized glycopeptide substrates. However, obtaining steady-state kinetics using native glycopeptides or glycoproteins as substrates would be highly desirable. Observing the deglycosylation of glycoproteins by using SDS-PAGE is currently the method of choice for assaying the activity of PNGase, but due to the limited resolution of this method, only semi-quantitative activity screens are presently achievable [25]. As a result of the limitations mentioned above, only a few kinetic parameters of PNGases have been reported [18], which impedes the comparison and functional evaluation of this class of enzymes.

The treatment of glycoproteins or glycopeptides by PNGase releases an oligosaccharide chain which, after water-mediated deamination, possesses a hemiacetal moiety located at the reducing terminus. This reducing end of the released N-glycan is highly reactive and can be derivatized using amines such as 2-aminobenzoic acid or 2-aminobenzamide [26,27]. Previous studies demonstrated that water-soluble tetrazolium salts, such as NBT (2,2‘,5,5‘-tetraphenyl-3,3‘-dimethoxy 4,4‘-biphenylene ditetrazolium chloride) or WST-1 (sodium 2-(4-Iodophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolate), two chromogenic reagents commonly used for the detection of superoxides in cell-based viability assays [[28], [29], [30]], can also be used for the colorimetric detection of carbohydrates. Jue et al. described the oxidation of simple sugars using NBT [31], and Hammond et al. developed a colorimetric assay for probing heparinase activity using WST-1 as a colorimetric dye [32]. Intrigued by the simplicity of the latter method, we decided to adapt and optimize this colorimetric assay for PNGase activity screens (Fig. 1).

In previous studies, we described the discovery of an unstudied bacterial PNGase, which is suitable for N-glycan analysis of glycoproteins of both plant and animal origin [13,33]. However, due to the absence of a suitable method for quantifying the activity of PNGases, no kinetic parameters for this type of enzymes have been reported. Herein, we demonstrate the practicality of the developed colorimetric assay for obtaining kinetic parameters of wild-type and mutant variants of PNGase H⁺.