Glycosylation of proteins in Aspergillus niger: isolation of the N-acetylglucosamine-phosphate-tranferase gene (GPT)

Tine Kring Sorensen, Paul Dyer, Ulrike Laube, and John Peberdy.

Abstract: 

Filamentous fungi secrete a range of enzymes, the majority of which are hydrolytic and play an important role in fungal nutrition. Some fungi are capable of secreting high levels of proteins and have consequently attracted attention as potential producers of heterologous proteins, but so far yields obtained have been very low. Not only are improved yields required, but also the proteins should be highly authentic, thereby preserving functionality and avoiding antigenic reactions. Many proteins secreted by fungi are glycosylated. Most information on glycosylation has come from studies on mammalian and yeast systems, but only little is known about the process in filamentous fungi. It is therefore desirable to better understand the events of protein secretion in fungi, thus making it possible to control the process and thereby tailor the glycans produced on secreted proteins. Studies are in progress to investigate the biochemical and molecular controls of glycosylation in Aspergillus niger. One aspect of this involves the ER-located enzyme UDP-N-acetyl glucosamine:dolichyl phosphate N-acetyl glucosamine-l-phosphate transferase (GPT), catalyzing the first step of the N-glycosylation pathway: the synthesis of N-acetylglucosamine-dolichol phosphate. The GPT-gene has now been cloned from a genomic library of A.niger by screening with a PCR derived homologous fragment (470 bp) isolated from genomic DNA. The ORF was 1.4 kb with 2 introns (58, 91 bp). The sequence from A.niger showed about 46% identity with the GPT gene cloned from yeasts and 35-37% identity with rodents and human. Northern blotting, using a homologous probe, produced a 2.4 kb mRNA transcript. Expression studies are in progress. Acknowledgement of funding sources: The Danish Research Academy and The Danish Research Council.
1999

abstract No: 

Fungal Genet. Newsl. 46 (Supl):

Full conference title: 

Fungal Genetics Conference 20th
    • Fungal Genetics Conference 20th (1999)