Transglycosylation Activity of Glycosynthase Mutants of Endo-β-<i>N</i>-Acetylglucosaminidase from <i>Coprinopsis cinerea</i>

<div><p>Endo-β-<i>N</i>-acetylglucosaminidase (ENGase), which catalyzes hydrolysis of <i>N</i>-linked oligosaccharides, is a useful tool for analyzing oligosaccharide contents of glycoproteins. However, there are only a few known ENGases that can catalyze the hydrolysis of human complex type oligosaccharides, and although commercially available, they are expensive. Here, we report the cloning of two ENGase encoding cDNAs from the basidiomycete fungus <i>Coprinopsis cinerea</i>, Endo-CC1 and Endo-CC2. We successfully expressed recombinant His₆-tagged Endo-CC1 and Endo-CC2 in <i>Escherichia coli</i> and purified them for enzymatic characterization. Both Endo-CC1 and Endo-CC2 showed hydrolytic activity on high-mannose and complex type oligosaccharides. Since Endo-CC1 could be prepared more easily than Endo-CC2 from <i>E</i>. <i>coli</i> cultures, we examined the enzymatic properties of Endo-CC1 in detail. Our results showed that Endo-CC1 acted on both <i>N</i>-linked high-mannose type and sialobiantennary type complex oligosaccharides of glycoproteins RNase B and human transferrin, respectively, but not on the sialotriantennary type complex oligosaccharide of glycoprotein fetuin. Examination of the transglycosylation activity of Endo-CC1 revealed that the wild-type Endo-CC1 could not transfer the sialobiantennary type complex oligosaccharide onto the deglycosylated RNase B. To obtain an Endo-CC1 mutant with desired transglycosylation activity, we performed mutation analysis of the active site residue Asn 180 (N180), known to be important for catalysis, by individually replacing it with the remaining 19 amino acid residues. Transglycosylation analyses of these mutants led us to identify one mutant, namely Endo-CC1^N180H, which exhibited the desired transglycosylation activity. Taken together, we suggest that Endo-CC1 would potentially be a valuable tool for analyzing oligosaccharides on glycoproteins, as large quantities of it could be made available more easily and less expensively than the currently used enzyme, Endo-M.</p></div

SDS-PAGE analysis of purified Endo-CCs.

<p>Endo-CC1 and Endo-CC2, expressed in <i>E</i>. <i>coli</i>, were purified and then 1 μg of these protein samples were loaded onto a 10% acrylamide gel. The band marked with an asterisk in lane 2 is likely a contaminating <i>E</i>. <i>coli</i> protein. Lane M, molecular weight markers; lane 1, purified recombinant Endo-CC1; lane 2, purified recombinant Endo-CC2.</p

Transglycosylation activity of Endo-CC1^N180X.

<p>SDS-PAGE analysis of transglycosylation reaction mixtures that contained deglycosylated RNase B, indicated purified Endo-CC1^N180X mutant (where X is the altered amino acid residue in the point mutant) and complex type oligosaccharide derived from SGP: 1 h incubation (A) and 12 h incubation (B). The single letter label on each lane of the gel indicates the point mutant that was used in the transglycosylation assay; thus, lane labeled N is the wild-type Endo-CC1. Mw, molecular weight markers.</p

Hydrolase activity of Endo-CC1: effect on glycoproteins.

<p>SDS-PAGE analysis of reaction mixtures containing 1 μg of RNase B (A), human transferrin (B) and fetuin (C) treated with or without Endo-CC1. The double bands seen in the lane 1 of (A) are likely due to different modifications of <i>N</i>-glycosylation on RNaseB. The protein band marked with an asterisk in (B) represents Endo-CC1. Lane M, molecular weight markers; lane 1, negative control (without Endo-CC1); lane 2, reaction product (with Endo-CC1).</p

SDS-PAGE analyses of purified Neo-RNase B.

<p>(A) Neo-RNase B was purified with a Con A column. Lane M, molecular weight markers; lane 1, before purification; lane 2, after purification. (B) Lane M, molecular weight markers; lane 1, native RNase B (control); lane 2, native RNase B treated with Endo-A; lane 3, native RNase B treated with Endo-CC1. Note that the double bands seen in the lane 1 are likely due to different modifications of <i>N</i>-glycosylation on RNaseB. (C) Lane M, molecular weight markers; lane 1, purified Neo-RNase B (control); lane 2, purified Neo-RNase B treated with Endo-A; lane 3, purified Neo-RNase B treated with Endo-CC1.</p

Relative hydrolase activity of recombinant wild-type and mutant Endo-CC1s.

<p>The Asn 180 (N180) residue of the wild-type (WT) Endo-CC1 was replaced individually with the one of the remaining 19 amino acid residues (indicated above using single-letter code for each amino acid residue) and the relative hydrolase activity of each mutant was determined using purified recombinant mutant and Dns-DG as the substrate.</p><p>^a The relative activity of the WT Endo-CC1 was set as 100.</p><p>^b ND, not detectable.</p><p>Relative hydrolase activity of recombinant wild-type and mutant Endo-CC1s.</p

Transglycosylation of deglycosylated RNase B by Endo-CC1^N180H and End-CC1^N180Q at various SGP concentration.

<p>SDS-PAGE analysis of transglycosylation reaction mixtures that contained deglycosylated RNase B, purified Endo-CC1 mutant (Endo-CC1^N180H or EndoCC1^N180Q) and different amounts of complex type oligosaccharide derived from SGP: 1 h incubation (A) and 12 h incubation (B). Amount of SGP used in the reaction mixture (μg): Lane 1, 62.5; lane 2, 125; lane 3, 250; lane 4, 500; lane 5, 1000. Note that in lanes 5, mobility shifts were seen likely due to a large amount of SGP included in the samples.</p

Analyses of hydrolase activity of Endo-CCs by TLC.

<p>Reaction mixtures containing Dns-Man₅GlcNAc₂Asn or Dns-sialylglyco-Asn and Endo-CC1 (A) or Endo-CC2 (B) were analyzed by TLC. Lane 1, Dns-sialylglyco-Asn without any added enzyme; lane 2, Dns-Asn-GlcNAc without any added enzyme; lane 3, Dns-Man₅GlcNAc₂Asn with added enzyme; lane 4, Dns-sialylglyco-Asn with added enzyme.</p

Sequence alignment of Endo-CCs and Endo-M.

<p>(A) Phylogenetic tree of ENGases belonging either to GH85 or to GH18 family was generated by the neighbor-joining method and using the MEGA 6.02 program [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132859#pone.0132859.ref016" target="_blank">16</a>]. Endo-CC1 and Endo-CC2 are assigned in the same clade with Endo-M. Note that Endo-CC1, Endo-CC2 and Endo-M are from fungi, whereas Endo-A, Endo-BH and Endo-D are from bacteria. Accession numbers of proteins used in this study are given in Materials and Methods. (B) Endo-CC1 and Endo-CC2 are consisted of 787 and 689 amino acid residues, and show 46% and 40% similarities to Endo-M, respectively. GH85 domain (gray box) is conserved among these proteins. (C) Alignment of amino acid sequences of Endo-CC1, Endo-CC2 and Endo-M around the predicted active site is shown. Glutamic acid (indicated with a closed triangle) and tryptophan (indicated with an open triangle) residues are known to be important for the catalysis and transglycosylation activities, respectively. The asparagine residue, indicated with an asterisk, was subjected to point mutation analyses.</p

The MicroRNA Expression Signature of Bladder Cancer by Deep Sequencing: The Functional Significance of the <i>miR-195/497</i> Cluster

<div><p>Current genome-wide microRNA (miRNA) expression signature analysis using deep sequencing technologies can drive the discovery of novel cancer pathways regulated by oncogenic and/or tumor suppressive miRNAs. We determined the genome-wide miRNA expression signature in bladder cancer (BC) by deep sequencing technology. A total of ten small RNA libraries were sequenced (five BCs and five samples of histologically normal bladder epithelia (NBE)), and 13,190,619 to 18,559,060 clean small RNA reads were obtained. A total of 933 known miRNAs and 17 new miRNA candidates were detected in this analysis. Among the known miRNAs, a total of 60 miRNAs were significantly downregulated in BC compared with NBE. We also found that several miRNAs, such as <i>miR-1/133a</i>, <i>miR-206/133b</i>, <i>let-7c/miR-99a</i>, <i>miR-143/145</i> and <i>miR-195/497</i>, were located close together at five distinct loci and constituted clustered miRNAs. Among these clustered miRNAs, we focused on the <i>miR-195/497</i> cluster because this clustered miRNA had not been analyzed in BC. Transfection of mature <i>miR-195</i> or <i>miR-497</i> in two BC cell lines (BOY and T24) significantly inhibited cancer cell proliferation, migration and invasion, suggesting that the <i>miR-195/497</i> cluster functioned as tumor suppressors in BC. Regarding the genes targeted by the <i>miR-195/497</i> cluster, the TargetScan algorithm showed that 6,730 genes were putative <i>miR-195/497</i> targets, and 113 significantly enriched signaling pathways were identified in this analysis. The “Pathways in cancer” category was the most enriched, involving 104 candidate target genes. Gene expression data revealed that 27 of 104 candidate target genes were actually upregulated in BC clinical specimens. Luciferase reporter assays and Western blotting demonstrated that <i>BIRC5</i> and <i>WNT7A</i> were directly targeted by <i>miR-195/497</i>. In conclusion, aberrant expression of clustered miRNAs was identified by deep sequencing, and downregulation of <i>miR-195/497</i> contributed to BC progression and metastasis. Tumor suppressive miRNA-mediated cancer pathways provide new insights into the potential mechanisms of BC oncogenesis.</p></div