Structural and biochemical studies of the C-terminal domain of mouse peptide-N-glycanase identify it as a mannose-binding module.

Center for Structural Biology, Stony Brook University, Stony Brook, NY 11794-5215, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 12/2006; 103(46):17214-9. DOI: 10.1073/pnas.0602954103
Source: PubMed

ABSTRACT The inability of certain N-linked glycoproteins to adopt their native conformation in the endoplasmic reticulum (ER) leads to their retrotranslocation into the cytosol and subsequent degradation by the proteasome. In this pathway the cytosolic peptide-N-glycanase (PNGase) cleaves the N-linked glycan chains off denatured glycoproteins. PNGase is highly conserved in eukaryotes and plays an important role in ER-associated protein degradation. In higher eukaryotes, PNGase has an N-terminal and a C-terminal extension in addition to its central catalytic domain, which is structurally and functionally related to transglutaminases. Although the N-terminal domain of PNGase is involved in protein-protein interactions, the function of the C-terminal domain has not previously been characterized. Here, we describe biophysical, biochemical, and crystallographic studies of the mouse PNGase C-terminal domain, including visualization of a complex between this domain and mannopentaose. These studies demonstrate that the C-terminal domain binds to the mannose moieties of N-linked oligosaccharide chains, and we further show that it enhances the activity of the mouse PNGase core domain, presumably by increasing the affinity of mouse PNGase for the glycan chains of misfolded glycoproteins.

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    • "NMR structure of the luminal domain of malectin reveals that the Glcα1–3Glc moiety is captured by a cluster of aromatic rings located on the loops connecting the β-sheets which are arranged in β-jelly roll folds (Fig. 3A) [22]. Intriguingly, this CRD domain exhibits close structural similarity to the CRDs of PNGase and a cytosolic lectin Fbs1, despite low sequence identity (Fig. 3E, F) [64] [65] "
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    ABSTRACT: N-linked oligosaccharides operate as tags for protein quality control, consigning glycoproteins to different fates, i.e. folding in the endoplasmic reticulum (ER), vesicular transport between the ER and the Golgi complex, and ER-associated degradation of glycoproteins, by interacting with a panel of intracellular lectins in the early secretory pathway. This review summarizes the current state of knowledge regarding the molecular and structural basis for glycoprotein-fate determination in cells that is achieved through the actions of the intracellular lectins and its partner proteins. Cumulative frontal affinity chromatography (FAC) data demonstrated that the intracellular lectins exhibit distinct sugar-binding specificity profiles. The glycotopes recognized by these lectins as fate determinants are embedded in the triantennary structures of the high-mannose-type oligosaccharides and are exposed upon trimming of the outer glucose and mannose residues during the N-glycan processing pathway. Furthermore, recently emerged 3D structural data offer mechanistic insights into functional interplay between an intracellular lectin and its binding partner in the early secretory pathway. Structural biology approaches in conjunction with FAC methods provide atomic pictures of the mechanisms behind the glycoprotein-fate determination in cells. This article is a part of a Special issue entitled: Glycoproteomics.
    Biochimica et Biophysica Acta 01/2012; 1820(9):1327-37. DOI:10.1016/j.bbagen.2011.12.017 · 4.66 Impact Factor
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    • "Structurally and functionally distinct from the previously characterized PNGases in plants and prokaryotes (Norris et al. 1994; Altmann et al. 1998; Suzuki et al. 2002; Lee et al. 2005; Zhao et al. 2006), the cytoplasmic PNGase activity was first identified in Saccharomyces cerevisiae (Suzuki et al. 2000) and later found to be highly conserved from yeast to human (Suzuki et al. 2002). It is notable that in addition to the conserved catalytic core domain, PNGase in higher eukaryotes carries additional domains at both the N-terminus and C-terminus, which are involved in protein recognition (Park et al. 2001; Li et al. 2006) and binding to the oligo-mannose moiety of the N-linked glycan (Zhou et al. 2006), respectively. "
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    ABSTRACT: Peptide:N-glycanase (PNGase) is an important component of the endoplasmic reticulum-associated protein degradation pathway in which it de-glycosylates misfolded glycoproteins, thus facilitating their proteasomal degradation. PNGase belongs to the transglutaminase superfamily and features a Cys, His, and Asp catalytic triad, which is essential for its enzymatic activity. An elongated substrate-binding groove centered on the active site Cys191 was visualized in the crystal structure of apo-PNGase, whereas its complex with Z-VAD-fmk, a peptide-based inhibitor of PNGase, revealed that the inhibitor occupied one end of the substrate-binding groove while being covalently linked to the active site Cys. Recently, haloacetamidyl-containing carbohydrate-based inhibitors of PNGase were developed and shown to specifically label the active site Cys. In this study, we describe the crystal structure of yeast PNGase in complex with N,N'-diacetylchitobiose (chitobiose). We found that the chitobiose binds on the side opposite to the peptide binding site with the active site Cys191 being located approximately midway between the carbohydrate and peptide binding sites. Mutagenesis studies confirm the critical role of the chitobiose-interacting residues in substrate binding and suggest that efficient oligosaccharide binding is required for PNGase activity. In addition, the N-terminus of a symmetry-related PNGase was found to bind to the proposed peptide-binding site of PNGase. Together with the bound chitobiose, this enables us to propose a model for glycoprotein binding to PNGase. Finally, deleting the C-terminal residues of yeast PNGase, which are disordered in all structures of this enzyme, results in a significant reduction in enzyme activity, indicating that these residues might be involved in binding of the mannose residues of the glycan chain.
    Glycobiology 11/2008; 19(2):118-25. DOI:10.1093/glycob/cwn108 · 3.14 Impact Factor
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    ABSTRACT: During endoplasmic reticulum-associated degradation, the multifunctional AAA ATPase p97 is part of a protein degradation complex. p97 associates via its N-terminal domain with various cofactors to recruit ubiquitinated substrates. It also interacts with alternative substrate-processing cofactors, such as Ufd2, Ufd3, and peptide:N-glycanase (PNGase) in higher eukaryotes. These cofactors determine different fates of the substrates and they all bind outside of the N-terminal domain of p97. Here, we describe a cofactor-binding motif of p97 contained within the last 10 amino acid residues of the C terminus, which is both necessary and sufficient to mediate interactions of p97 with PNGase and Ufd3. The crystal structure of the N-terminal domain of PNGase in complex with this motif provides detailed insight into the interaction between p97 and its substrate-processing cofactors. Phosphorylation of p97's highly conserved penultimate tyrosine residue, which is the main phosphorylation site during T cell receptor stimulation, completely blocks binding of either PNGase or Ufd3 to p97. This observation suggests that phosphorylation of this residue modulates endoplasmic reticulum-associated protein degradation activity by discharging substrate-processing cofactors.
    Proceedings of the National Academy of Sciences 06/2007; 104(21):8785-90. DOI:10.1073/pnas.0702966104 · 9.81 Impact Factor
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