Karnoup Anton S, Turkelson Virgil, Anderson W H Kerr
Analytical Sciences, The Dow Chemical Company, 1897 Building, Midland, MI 48667, USA.
Glycobiology. 2005 Oct;15(10):965-81. doi: 10.1093/glycob/cwi077. Epub 2005 May 18.
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.
O-连接聚糖在真核细胞类型之间存在差异，并且在决定糖蛋白的性质（包括稳定性、靶标识别以及潜在的免疫原性）方面发挥着重要作用。我们描述了在转基因玉米中表达的重组人IgA1（hIgA1）的O-连接聚糖结构。通过结合基质辅助激光解吸电离质谱（MALDI MS）、色谱法和氨基酸分析，发现在玉米表达的hIgA1重链（HC）的铰链区中存在多达六个脯氨酸/羟脯氨酸转化以及不同量的阿拉伯糖基化（Pro/Hyp + Ara）。大约90%的hIgA1以这种方式被修饰。揭示了每分子hIgA1平均含有六个阿拉伯糖单元的摩尔比。在hIgA1的HC铰链区与玉米富含羟脯氨酸糖蛋白（HRGP）的伸展蛋白家族区域之间鉴定出了显著的序列相似性。我们提出，由于这种序列相似性，玉米表达的hIgA1的HC铰链区可以成为玉米脯氨酰羟化酶将Pro翻译后转化为Hyp的底物，随后高尔基体中的Hyp糖基转移酶会对玉米胚乳组织中Hyp残基进行阿拉伯糖基化。在hIgA1 HC铰链区观察到多达六个Pro/Hyp羟基化以及广泛的阿拉伯糖基化，这与文献中先前针对植物HRGP提出的Pro/Hyp羟基化模型和Hyp邻接假说高度一致。首次描述了转基因植物中表达的重组治疗性蛋白的类伸展蛋白Hyp/Pro转化和O-连接阿拉伯糖基化。我们的发现对于植物生物技术和生物制药行业——将转基因植物开发为生产重组治疗性蛋白的平台这一领域具有重要意义。
