title: Production of glycosylated soluble amyloid precursor protein alpha (sAPPalpha) in Leishmania tarentolae.
abstract: Soluble amyloid precursor protein alpha (sAPPalpha) is a cleavage product of the amyloid precursor protein (APP), the etiologic agent in Alzheimer's disease (AD). Reduced expression of sAPPalpha was previously found in the brains of AD patients, and it was suggested that sAPPalpha might counteract neurotoxic effects of Abeta, another APP cleavage product with enhanced abundance in Alzheimer's diseased brains. However, little is known about the biological functions of sAPPalpha. Thus, efficient production of this protein is a prerequisite for further studies. The unicellular eukaryotic parasite Leishmania tarentolae has recently emerged as a promising expression system for eukaryotic proteins due to its ability to posttranslationally modify proteins combined with easy cultivation and high protein yield. Interestingly, sAPPalpha produced in L. tarentolae was biologically active and glycosylated. In contrast to nonglycosylated sAPPalpha expressed in Eschericha coli, it also featured higher stability against enzymatic degradation. Detailed analysis of the glycosylation pattern of sAPPalpha produced in L. tarentolae by PGC-LC-ESI-MS/MS N-glycan analysis identified among eukaryotic species the highly conserved core pentasaccharide (Man3GlcNAc2) as being attached to Asn467 of sAPPalpha. Using oxonium ion scanning of CID-MS/MS spectra in combination with ETD fragmentation, we also identified two peptides (peptides 269-288 and 575-587) modified with N-acetyl hexosamine (HexNAc) residues. One of these O-glycosylation sites could be unambiguously assigned to Thr576 of sAPPalpha. This is the first time that O-glycosylation of a recombinant protein expressed in L. tarentolae has been demonstrated. Together, human sAPPalpha produced in L. tarentolae was N- and O-glycosylated on similar sites as described for mammalian-expressed sAPPalpha and showed similar biological activity. This demonstrates that L. tarentolae is a very suitable and simple to handle expression system for mammalian glycoproteins.
authors.affiliation: Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Klatt
authors.forename: Michael
authors.initials: M
authors.lastname: Rohe
authors.forename: Kathirvel
authors.initials: K
authors.lastname: Alagesan
authors.forename: Daniel
authors.initials: D
authors.lastname: Kolarich
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Konthur
authors.forename: Daniela
authors.initials: D
authors.lastname: Hartl
mesh_terms: D000544:Alzheimer Disease / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D000682:Amyloid / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D001921:Brain / Q000378:metabolism / Q000473:pathology; D004926:Escherichia coli; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007891:Leishmania / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D053719:Tandem Mass Spectrometry
chemical_list: C545487:APP protein, human; D000682:Amyloid; D016564:Amyloid beta-Protein Precursor; D010446:Peptide Fragments; D011994:Recombinant Proteins