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: 9.67). 12/2006; 103(46):17214-9. DOI: 10.1073/pnas.0602954103
Source: PubMed


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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    • "This specific structure facilitates delivery of poly-ubiquitinated proteins to the proteasome [5]. The conserved UBL and UBA domains are flanking a more variable central region containing several Sti1 repeats, which confer chaperone-like activity [6]. Various interaction partners are known for ubiquilin 1, the most studied protein of the ubiquilin family, which was originally identified in a yeast two-hybrid screen as an interactor with the AD associated protein presenilin 1 and 2 [7]. "
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    ABSTRACT: Accumulation of aberrant proteins in inclusion bodies is a hallmark of many neurodegenerative diseases. Impairment of proteolytic systems is a common event in these protein misfolding diseases. Recently, mutations in the UBQLN 2 gene encoding ubiquilin 2 have been identified in X-linked amyotrophic lateral sclerosis (ALS). Furthermore, ubiquilin 2 is associated with inclusions in familial and sporadic ALS/dementia, synucleinopathies and polyglutamine diseases. Ubiquilin 2 exerts a regulatory role in proteostasis and thus it has been suggested that ubiquilin 2 pathology may be a common event in neurodegenerative diseases. Tauopathies, a heterogenous group of neurodegenerative diseases accompanied with dementia, are characterized by inclusions of the microtubule-binding protein tau. In the present study, we investigate whether ubiquilin 2 is connected with tau pathology in Alzheimer's disease (AD), supranuclear palsy (PSP) and Pick's disease (PiD) and familial cases with frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We show that ubiquilin 2 positive inclusions are absent in these tauopathies. Furthermore, we find decreased ubiquilin 2 protein levels in AD patients, but our results do not indicate a correlation with tau pathology. Our data show no evidence for involvement of ubiquilin 2 and indicate that other mechanisms underly the proteostatic disturbances in tauopathies.
    PLoS ONE 09/2013; 8(9):e76598. DOI:10.1371/journal.pone.0076598 · 3.23 Impact Factor
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    • "H6 α-helix is half the size in DdPNGase as compared to MmPNGase (14 aa). A helical region of 5 amino acids in the Zn binding region between S2 and S3 as shown in the tertiary structure model of MmPNGase (2f4mA) is also not mentioned in literature [28]. Even with the existing differences, we found that the cleft composed of H8 helix and six antiparallel β-strands to be completely superimposed. "
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    ABSTRACT: Background Peptide: N- glycanase (PNGase) enzyme cleaves oligosaccharides from the misfolded glycoproteins and prepares them for degradation. This enzyme plays a role in the endoplasmic reticulum associated degradation (ERAD) pathway in yeast and mice but its biological importance and role in multicellular development remain largely unknown. Results In this study, the PNGase from the cellular slime mold, Dictyostelium discoideum (DdPNGase) was identified based on the presence of a common TG (transglutaminase) core domain and its sequence homology with the known PNGases. The domain architecture and the sequence comparison validated the presence of probable functional domains in DdPNGase. The tertiary structure matched with the mouse PNGase. Here we show that DdPNGase is an essential protein, required for aggregation during multicellular development and a knockout strain of it results in small sized aggregates, all of which did not form fruiting bodies. The in situ hybridization and RT-PCR results show higher level of expression during the aggregate stage. The expression gets restricted to the prestalk region during later developmental stages. DdPNGase is a functional peptide:N-glycanase enzyme possessing deglycosylation activity, but does not possess any significant transamidation activity. Conclusions We have identified and characterized a novel PNGase from D. discoideum and confirmed its deglycosylation activity. The results emphasize the importance of PNGase in aggregation during multicellular development of this organism.
    BMC Biochemistry 06/2012; 13(1):9. DOI:10.1186/1471-2091-13-9 · 1.44 Impact Factor
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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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