Identification of proteins that interact with mammalian peptide:N-glycanase and implicate this hydrolase in the proteasome-dependent pathway for protein degradation

Abstract

Peptide:N-glycanase (PNGase) cleaves oligosaccharide chains from glycopeptides and glycoproteins. Recently the deduced amino acid sequence of a cytoplasmic PNGase has been identified in various eukaryotes ranging from yeast to mammals, suggesting that deglycosylation may play a central role in some catabolic process. Several lines of evidence indicate that the cytoplasmic enzyme is involved in the quality control system for newly synthesized glycoproteins. Two-hybrid library screening by using mouse PNGase as the target yielded several PNGase-interacting proteins that previously had been implicated in proteasome-dependent protein degradation: mHR23B, ubiquitin, a regulatory subunit of the 19S proteasome, as well as a protein containing an ubiquitin regulatory motif (UBX) and an ubiquitin-associated motif (UBA). These findings by using the two-hybrid system were further confirmed either by in vitro binding assays or size fractionation assays. These results suggest that PNGase may be required for efficient proteasome-mediated degradation of misfolded glycoproteins in mammalian cells.

Figure 1

Comparison of the deduced amino acid sequences of yPng1p and mPng1p. yPng1p has a 363-aa ORF whereas mPng1p contains a 651-aa ORF. The conserved middle domains contain unique transglutaminase-like motif (26).

Figure 2

Anti-mPng1p antibody specifically recognizes mammalian PNGase. (A) Western blot analysis of mouse tissue extracts by using a polyclonal anti-mPng1p antibody. Mouse liver, kidney, and heart were homogenized in B88 buffer and subjected to Western blot analysis. Each mouse tissue revealed the presence of an ≈65-kDa band reacting with the anti-mPng1p antibody. (B) Western blot analysis of COS-1 cells expressing mPng1-GFP fusion protein. Lysates of COS-1 cells were analyzed for expression of mPng1-GFP. (B Left) Western blot using anti-mPng1p antibody against COS-1 lysates (untransfected, GFP-transfected, and mPng1-GFP transfected). Each lane shows endogenous COS-1 Png1p with an approximate M_r of 65-kDa and two right lanes show that transfected mPng1p-GFP is expressed (≈80-kDa band). (B Right) Western blot using an anti-GFP mAb. GFP (24 kDa) and mPng1-GFP (80-kDa) bands are detected in COS1/GFP and COS-1/mPng1-GFP, respectively.

Figure 3

PNGase assay on various mPng1p fragments expressed in E. coli demonstrates that the N-terminal domain and the middle domain of mPng1p are necessary for the enzyme activity. E. coli BL21(DE3) lysates expressing various fragment were tested for PNGase enzyme activity. Each reaction was subjected to a paper chromatography as described (21). A, The starting substrate ([¹⁴C]-Leu-Asn(GlcNac₅Man₃Gal₃)-Asn-Ser-Arg); B, the de-N-glycosylated product ([¹⁴C]-Leu-Asp-Asn-Ser-Arg).

Figure 4

Localization of mPng1-GFP in COS-1 cells. COS-1 cells transfected with pEGFP and pmPng1-GFP were subjected to a fluorescent microscope to detect expression of GFP proteins. Left, The expression of control GFP protein (GFP localized mostly at nucleus). Right, The expression pattern of mPng1-GFP. mPng1-GFP appeared to localize mostly at the cytoplasm, around the nuclear envelope.

Figure 5

In vitro binding assay for mPng1p-GST-mHR23B interaction. E. coli DH5α cells expressing GST or GST-mHR23B (amino acid 244–416) were lysed and subjected to glutathione-agarose binding. E. coli BL21(DE3) cell lysates expressing full-length mPng1p was used for in vitro binding. Lane 1, GST + mPng1p; lanes 2 and 3, GST-mHR23B + mPng1p, and lane 4, mPng1p only (input). For lanes 1–3, glutathione agarose-bound GST or GST-mHR23B was incubated with BL21(DE3) lysates expressing mPng1p and beads were washed and subjected to SDS/PAGE. For lane 4, BL21(DE3) lysates expressing mPng1p was used.

Figure 6

Gel filtration assays on mammalian cell lysates show that mammalian Png1p co-migrates with the proteasome. (A) Sephacryl 400 column for COS-1 lysates. (B) Sephacryl 400 column for NIH 3T3 lysates in the absence of ATP. Mammalian cell lysates in 0.5-ml lysis buffer were applied on Sephacryl 400 column with or without ATP. Proteasome assays were done in the presence of ATP (A and B). Nonspecific bands in A (for anti-S4 antibody) were sometimes seen but clearly distinct from real S4 bands.

Figure 7

Model for protein–protein interactions of mPng1p. mPng1p might act as a “platform” to recruit various ubiquitin pathway-related proteins and present them to the 26S proteasome.