Identification and characterization of peptide: N-glycanase from Dictyostelium discoideum.
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.
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
Anuradha
A
Gosain
Rakhee
R
Lohia
Anju
A
Shrivastava
Shweta
S
Saran
D000595:Amino Acid Sequence; D000818:Animals; D004023:Dictyostelium / Q000201:enzymology*; D055786:Gene Knockout Techniques; D006031:Glycosylation; D051379:Mice; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D016415:Sequence Alignment; D017385:Sequence Homology
D015800:Protozoan Proteins; D012313:RNA; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase