O-linked glycosylation in maize-expressed human IgA1.
O-Linked glycans vary between eukaryotic cell types and play an important role in determining a glycoprotein's properties, including stability, target recognition, and potentially immunogenicity. We describe O-linked glycan structures of a recombinant human IgA1 (hIgA1) expressed in transgenic maize. Up to six proline/hydroxyproline conversions and variable amounts of arabinosylation (Pro/Hyp + Ara) were found in the hinge region of maize-expressed hIgA1 heavy chain (HC) by using a combination of matrix-assisted laser-desorption ionization mass spectrometry (MALDI MS), chromatography, and amino acid analysis. Approximately 90% of hIgA1 was modified in this way. An average molar ratio of six Ara units per molecule of hIgA1 was revealed. Substantial sequence similarity was identified between the HC hinge region of hIgA1 and regions of maize extensin-family of hydroxyproline-rich glycoproteins (HRGP). We propose that because of this sequence similarity, the HC hinge region of maize-expressed hIgA1 can become a substrate for posttranslational conversion of Pro to Hyp by maize prolyl-hydroxylase(s) with the subsequent arabinosylation of the Hyp residues by Hyp-glycosyltransferase(s) in the Golgi apparatus in maize endosperm tissue. The observation of up to six Pro/Hyp hydroxylations combined with extensive arabinosylation in the hIgA1 HC hinge region is well in agreement with the Pro/Hyp hydroxylation model and the Hyp contiguity hypothesis suggested earlier in literature for plant HRGP. For the first time, the extensin-like Hyp/Pro conversion and O-linked arabinosylation are described for a recombinant therapeutic protein expressed in transgenic plants. Our findings are of significance to the field of plant biotechnology and biopharmaceutical industry-developing transgenic plants as a platform for the production of recombinant therapeutic proteins.
Karnoup
Anton S
AS
Analytical Sciences, The Dow Chemical Company, 1897 Building, Midland, MI 48667, USA. askarnoup@dow.com
Turkelson
Virgil
V
Anderson
W H Kerr
WH
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D006801:Humans; D007070:Immunoglobulin A / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D003313:Zea mays / Q000235:genetics / Q000378:metabolism*
D007070:Immunoglobulin A; D011994:Recombinant Proteins