Galactofuranoic‐oligomannose N‐linked glycans of α‐galactosidase A fromAspergillus niger

Abstract

Extracellular a-galactosidase A was purified from the culture filtrate of an over-producing strain of Aspergillus niger containing multiple copies of the encoding aglA gene under the control of the glucoamylase (glaA) promoter. Endoglycosidase digestion followed by SDS/PAGE, lectin and immunoblotting suggested that glycosylation accounted for < 25% of the molecular size of the purified protein. Monosaccharide analysis showed that this was composed of N-acetyl glucosamine, mannose and galactose. Mild acid hydrolysis, mild methanolysis, immunoblotting and exoglycosidase digestion indicated that the majority of the galactosyl component was in the furanoic conformation (b-d-galactofuranose, Galf). At least 20 different N-linked oligosaccharides were fractionated by high-pH anion-exchange chromatography following release from the polypeptide by peptide-N-glycosidase F. The structures of these were subsequently determined by fast atom bombardment mass spectrometry to be a linear series of Hex 7226 HexHAc 2 . Indicating that oligosaccharides from GlcNAc 2 Man 7 , increasing in molecular size up to GlcNAc 2-Man 24 were present. Each of these were additionally substituted with up to three b-Galf residues. Linkage analysis confirmed the presence of mild acid labile terminal hexofuranose residues. These results show that filamentous fungi are capable of producing a heterogeneous mixture of high molecular-size N-linked glycans substituted with galactofuranoic residues, on a secreted glycoprotein.

N-Glycan Modification in Aspergillus Species

“…However, we cannot rule out the possibility of other hexoses. For example, in A. niger N-linked glycans, the presence of galactose in a galactofuranose conformation has been established (44,46). In this work, we did not analyze for their appearance by digestion with alternative mannosidases, such as an ␣-1,2/3/6-mannosidase.…”

“…Some of the glycans found resemble mammalian high-mannose glycans [Man (6)(7)(8)(9) GlcNAc 2 ]. In addition, typical "fungal-type" glycans that are structurally different from the mammalian glycans have been identified on different Aspergillus glycoproteins [Man (5)(6)(7)(8)(9)(10)(11)(12) GlcNAc 2 ] (1, 29,44,45).…”

Characterization of Humanized Antibodies Secreted byAspergillus niger

“…These were the average values from two determinations. Glycans of filamentous fungi are known to be of the high-mannose type, and terminal galactose, in the furanose form (Galf), has been observed on N-linked glycans from A. niger (29).…”

“…In particular, the presence of terminal Galf may be dependent on the culture conditions and may also be affected by the strain of A. niger used (18). It has been suggested that terminal Galf may be antigenic (29); therefore, elimination of this residue from glycan by an appropriate choice of a strain or manipulation of a strain would be beneficial. Since approximately half of the heavy chains were shown to be nonglycosylated, it is likely that some fraction, conceivably a major fraction, of assembled antibodies had one nonglycosylated heavy chain and one glycosylated heavy chain.…”

Lepidopteran cells, an alternative for the production of recombinant antibodies?

“…This isomer of galactose is absent in mammals and may be highly immunogenic in humans. 126 For a long time, plants were considered low-cost alternative for the production of recombinant proteins. In 1989, Hiatt and colleagues 127 were the first to demonstrate the production of a functional recombinant IgG1 in transgenic tobacco.…”