Evidence for an essential deglycosylation-independent activity of PNGase in Drosophila melanogaster.
BACKGROUND
Peptide:N-glycanase (PNGase) is an enzyme which releases N-linked glycans from glycopeptides/glycoproteins. This enzyme plays a role in the ER-associated degradation (ERAD) pathway in yeast and mice, but the biological importance of this activity remains unknown.


RESULTS
In this study, we characterized the ortholog of cytoplasmic PNGases, PNGase-like (Pngl), in Drosophila melanogaster. Pngl was found to have a molecular weight of approximately 74K and was mainly localized in the cytosol. Pngl lacks a CXXC motif that is critical for enzymatic activity in other species and accordingly did not appear to possess PNGase activity, though it still retains carbohydrate-binding activity. We generated microdeletions in the Pngl locus in order to investigate the functional importance of this protein in vivo. Elimination of Pngl led to a serious developmental delay or arrest during the larval and pupal stages, and surviving mutant adult males and females were frequently sterile. Most importantly, these phenotypes were rescued by ubiquitous expression of Pngl, clearly indicating that those phenotypic consequences were indeed due to the lack of functional Pngl. Interestingly, a putative "catalytic-inactive" mutant could not rescue the growth-delay phenotype, indicating that a biochemical activity of this protein is important for its biological function.


CONCLUSIONS
Pngl was shown to be inevitable for the proper developmental transition and the biochemical properties other than deglycosylation activity is important for its biological function.
Funakoshi
Yoko
Y
Glycometabolome Team, Systems Glycobiology Research Group, RIKEN Advanced Science Institute, Wako, Saitama, Japan. yokofuna@riken.jp
Negishi
Yuki
Y
Gergen
J Peter
JP
Seino
Junichi
J
Ishii
Kumiko
K
Lennarz
William J
WJ
Matsuo
Ichiro
I
Ito
Yukishige
Y
Taniguchi
Naoyuki
N
Suzuki
Tadashi
T
D000595:Amino Acid Sequence; D000818:Animals; D050260:Carbohydrate Metabolism; D003600:Cytosol / Q000201:enzymology; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D017353:Gene Deletion; D018507:Gene Expression Regulation, Developmental; D006031:Glycosylation; D006720:Homozygote; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D019076:Transgenes / Q000235:genetics
D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase