Endo-beta-N-acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol

Abstract

Formation of oligosaccharides occurs both in the cytosol and in the lumen of the endoplasmic reticulum (ER). Luminal oligosaccharides are transported into the cytosol to ensure that they do not interfere with proper functioning of the glycan-dependent quality control machinery in the lumen of the ER for newly synthesized glycoproteins. Once in the cytosol, free oligosaccharides are catabolized, possibly to maximize the reutilization of the component sugars. An endo-beta-N-acetylglucosaminidase (ENGase) is a key enzyme involved in the processing of free oligosaccharides in the cytosol. This enzyme activity has been widely described in animal cells, but the gene encoding this enzyme activity has not been reported. Here, we report the identification of the gene encoding human cytosolic ENGase. After 11 steps, the enzyme was purified 150,000-fold to homogeneity from hen oviduct, and several internal amino acid sequences were analyzed. Based on the internal sequence and examination of expressed sequence tag (EST) databases, we identified the human orthologue of the purified protein. The human protein consists of 743 aa and has no apparent signal sequence, supporting the idea that this enzyme is localized in the cytosol. By expressing the cDNA of the putative human ENGase in COS-7 cells, the enzyme activity in the soluble fraction was enhanced 100-fold over the basal level, confirming that the human gene identified indeed encodes for ENGase. Careful gene database surveys revealed the occurrence of ENGase homologues in Drosophila melanogaster, Caenorhabditis elegans, and Arabidopsis thaliana, indicating the broad occurrence of ENGase in higher eukaryotes. This gene was expressed in a variety of human tissues, suggesting that this enzyme is involved in basic biological processes in eukaryotic cells.

Figure 1

Analysis of purified ENGase fractions from hen oviduct by SDS/PAGE. The purified ENGase samples (fractions II and Ib, 10 μl each after precipitation with 10% TCA) were analyzed by 10% SDS/PAGE gel. Protein bands were visualized by Coomassie brilliant blue staining. The protein band for ENGase (≈55 kDa) from each fraction was subjected to amino acid sequence analysis.

Figure 2

Identification of ENGase activity in COS-7 cells transfected with human ENGase cDNA. Analysis of the reaction products produced by incubation of ¹⁴C-labeled substrate (¹⁴C-GP IVB) with the extract of COS-7 cells transfected with or without prior transfection with the human ENGase (hENGase) was carried out by paper chromatography. Lane 1, COS-7 cell extract with vector alone. Lane 2, COS-7 cells with hENGase-expression vector. Lane 3, Authentic reaction product produced by incubation of the substrate with Endo H.

Figure 3

Distribution in various tissues of human ENGase mRNA. FirstChoice Northern Blots (Ambion) was used for Northern blot analysis, and positions for RNA sizing marker were indicated.

Figure 4

Comparison of sequences of human ENGase and its homologues in other eukaryotes. hENGase, human ENGase (GenBank accession no. AF512564); DmENGase, D. melanogaster ENGase (AE003505; protein no. CG5613 in http://www.fruitfly.org); CeENGase, C. elegans ENGase (AB079783); AtENGase1/2, two ENGase orthologues found in A. thaliana genome (AB010692 and AC009991, protein nos. At5g05460 and At3g11040, respectively, in http://www.arabidopsis.org).