Structural Analysis of Free <i>N</i>-Glycans in α-Glucosidase Mutants of <i>Saccharomyces cerevisiae</i>: Lack of the Evidence for the Occurrence of Catabolic α-Glucosidase Acting on the <i>N</i>-Glycans

<div><p><i>Saccharomyces cerevisiae</i> produces two different α-glucosidases, Glucosidase 1 (Gls1) and Glucosidase 2 (Gls2), which are responsible for the removal of the glucose molecules from <i>N-g</i>lycans (Glc₃Man₉GlcNAc₂) of glycoproteins in the endoplasmic reticulum. Whether any additional α-glucosidases playing a role in catabolizing the glucosylated <i>N</i>-glycans are produced by this yeast, however, remains unknown. We report herein on a search for additional α-glucosidases in <i>S</i>. <i>cerevisiae</i>. To this end, the precise structures of cytosolic free <i>N</i>-glycans (FNGs), mainly derived from the peptide:<i>N</i>-glycanase (Png1) mediated deglycosylation of <i>N-</i>glycoproteins were analyzed in the endoplasmic reticulum α-glucosidase-deficient mutants. 12 new glucosylated FNG structures were successfully identified through 2-dimentional HPLC analysis. On the other hand, non-glucosylated FNGs were not detected at all under any culture conditions. It can therefore be safely concluded that no catabolic α-glucosidases acting on <i>N</i>-glycans are produced by this yeast.</p></div

Size-fractionation HPLC profiles of PA-labeled FNGs derived from the glucosidase mutant cells in stationary phase.

<p>Peaks 'l-p' produced in <i>gls1</i>Δ and <i>gls1</i>Δ <i>gls2</i>Δ cells, correspond to Glc₃Man_4-8GlcNAc₂-FNGs, whereas peaks 'q-u' produced in <i>gls2</i>Δ cells correspond to Glc₂Man_4-8GlcNAc₂-FNGs. The arrowheads indicate the elution position of PA-glucose oligomer for elution standards. The arrow with the asterisk shows the elution position of Man₁GlcNAc₂.</p

Reversed-phase HPLC profiles of Glc₃Man_4-8GlcNAc₂ in the stationary phase of <i>gls1</i>Δ cells.

<p>Peaks 'l-p' of <i>gls1</i>Δ cells were isolated by size fractionation HPLC and re-injected in reversed-phase HPLC to separate the isomers of each FNG. Asterisks (*) indicate the structures with ManNAc residue at the reducing ends. The arrowheads indicate the elution position of PA-glucose oligomer for elution standards. The contaminating peak indicated by × was observed in almost all samples as a very minor component (not seen under normal analytical conditions and only visible when much more sample was injected to detect the very minor FNGs).</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Enzymes that act on <i>N</i>-glycans in <i>S</i>. <i>cerevisiae</i>.

<p>The enzymes in the top panel (shaded light blue) are located in the ER. α and β indicates glycosidic linkages. Mns1, ER (α-1,2) mannosidase 1; Htm1, homologous to mannosidase 1.</p

Structures and quantities of PA-labeled FNGs in the log and stationary phases of <i>gls2</i>Δ cells.

<p>*: Structure with ManNAc at the reducing terminus. ^#: Structure was confirmed as standard PA-glycans are available.</p

Digestion of PA-labeled glucosylated FNGs with Gls1-only and/or Gls2-only microsomes.

<p>Size-fractionation HPLC profiles are shown. The arrowheads indicate the elution position of PA-glucose oligomer for elution standards; (top panel) profile of PA-G3M9A’. (2^nd panel) profile of PA-G3M9A’ digested with Gls1-only microsomes. Arrow shows the elution position of PA-G2M9A’. (3^rd panel) profile of PA-G3M9A’ digested with Gls1-only and Gls2-only microsomes. Arrow shows the elution position of PA-M9A’. (4^th panel) profile of PA-G2M9A’. (5^th panel) profile of PA-G2M9A’ digested with Gls2-only microsomes. Arrow shows the elution position of PA-M9A’.</p

Reversed-phase HPLC profile of tri- and di- glucosylated Man₆GlcNAc₂ (A), Man₇GlcNAc₂ (B), and Man₈GlcNAc₂ (C).

<p>The tri- and di- glucosylated FNGs (peaks 'n, o, p' and peaks 's, t, u' respectively) produced in the stationary phase of <i>gls1</i>Δ and <i>gls2</i>Δ cells respectively, were isolated by size fractionation HPLC and structural isomers of each were further separated in reversed-phase HPLC. Asterisks (*) indicate the structures with ManNAc residues at the reducing end which are produced by GlcNAc-to-ManNAc epimerization of the <i>N</i>-acetyl group during PA-labeling reaction. The arrowheads indicate the elution position of PA-glucose oligomer for elution standards.</p

Structures and quantities of PA-labeled FNGs extracted from <i>gls1</i>Δ and <i>gls1</i>Δ <i>gls2</i>Δ cells.

<p>The nomenclatures of the FNGs are essentially according to those suggested by Yanagida <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151891#pone.0151891.ref034" target="_blank">34</a>]. The Och1-modified FNGs (with the letters 'H' and 'I') were named according to Hirayama <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151891#pone.0151891.ref012" target="_blank">12</a>]. n.d.: not determined (<0.01 pmol/10⁶ cells). *: Structure with ManNAc at the reducing terminus. ^#1: No standard PA-glycans were available; the structures were deduced based on the GU values. ^#2: Structure was determined based on the GU value of the non-glucosylated glycans reported previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151891#pone.0151891.ref012" target="_blank">12</a>].</p

Reversed-phase HPLC profiles of Glc₃Man_6-9GlcNAc₂ in the log phase of <i>gls1</i>Δ cells.

<p>Peaks 'd-g' of <i>gls1</i>Δ cells were isolated by size fractionation HPLC and re-injected in reversed-phase HPLC to separate the isomers of each FNG. Asterisks (*) indicate the structures containing ManNAc residue at their reducing ends. Those ManNAc-containing PA-glycans are produced by GlcNAc-to-ManNAc epimerization of the <i>N</i>-acetyl group during PA-labeling reaction. The arrowheads indicate the elution position of PA-glucose oligomer for elution standards. The contaminating peak indicated by × was observed in almost all samples as a very minor component (not seen under normal analytical conditions and only visible when much more sample was injected to detect the very minor FNGs).</p