Structural and biochemical studies of the C-terminal domain of mouse peptide-N-glycanase identify it as a mannose-binding module
- Xiaoke Zhou*,†,
- Gang Zhao*,†,
- James J. Truglio*,†,
- Liqun Wang*,†,
- Guangtao Li†,
- William J. Lennarz†, and
- Hermann Schindelin*,†,†,‡,§
- *Center for Structural Biology and
- †Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215; and
- ‡Rudolf Virchow Center for Experimental Biomedicine and Institute of Structural Biology, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany
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Edited by John Kuriyan, University of California, Berkeley, CA, and approved September 15, 2006 (received for review April 11, 2006)
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.
Footnotes
- §To whom correspondence should be addressed. E-mail: hermann.schindelin{at}virchow.uni-wuerzburg.de
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Author contributions: X.Z., W.J.L., and H.S. designed research; X.Z., G.Z., L.W., and H.S. performed research; G.Z. and G.L. contributed new reagents/analytic tools; J.J.T. and H.S. analyzed data; and X.Z., W.J.L., and H.S. wrote the paper.
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The authors declare no conflict of interest.
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This article is a PNAS direct submission.
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Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 2G9F (intein-tagged protein), 2G9G (His-tagged protein), and 2I74 (complex)].
- Abbreviations:
- PNGase,
- peptide-N-glycanase;
- Z-VAD-fmk,
- carbobenzyloxy-Val-Ala-Asp-α-fluoromethyl ketone;
- ER,
- endoplasmic reticulum;
- ITC,
- isothermal titration calorimetry.
- © 2006 by The National Academy of Sciences of the USA
