International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita-shi, Osaka, 565, Japan; Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University, 2-1 Yamada-oka, Suita-shi, Osaka, 565-0871, Japan.
Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8572, Japan; Tsukuba-Plant Innovation Research Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8572, Japan.
Plant Physiol Biochem. 2022 May 1;178:70-79. doi: 10.1016/j.plaphy.2022.02.026. Epub 2022 Mar 2.
Miracle fruit, Synsepalum dulcificum, produces a unique taste-modifying protein, miraculin (MIR), which has an attractive potential for commercial application as a novel low-calorie sweetener. To establish a stable supply system for MIR, a previous study established a platform for recombinant MIR (rMIR) production in tomato plants and demonstrated that native miraculin from miracle fruit (nMIR) and rMIR were almost identical in their protein modifications with N-glycan. However, neither N-glycosylation nor the influence of fruit maturation on the structural changes of N-glycan have been fully characterized in detail. Here, with a focus on N-glycosylation and the contribution of fruit maturation to N-glycan, we reanalyzed the N-glycosylation of the natural maturation stages of nMIR and rMIR, and then compared the N-glycan structures on MIRs prepared from the fruit at two different maturation stages. The detailed peptide mapping and N-glycosylation analysis of MIRs provided evidence that MIRs have variants, which were derived mainly from the differences in the N-glycan structure in nMIR and the N-glycosylation in rMIR and not from the cleavage of the peptide backbone. N-Glycan analysis of MIRs from the maturation stage of fruits demonstrated that N-glycan structures were similar among nMIRs and rMIRs at every maturation stage. These results indicated that the heterogeneously expressed rMIRs had the same characteristics in post-translational modifications, especially N-glycosylation and N-glycan structures, throughout the maturation stages. This study demonstrated the potential of recombinant protein expressed in tomato plants and paves the way for the commercial use of rMIR.
奇迹果，Synsepalum dulcificum，产生一种独特的味觉修饰蛋白， miraculin（MIR），具有作为新型低热量甜味剂的商业应用的吸引力。为了建立 MIR 的稳定供应系统，先前的研究在番茄植物中建立了重组 MIR（rMIR）生产的平台，并证明来自奇迹果的天然 miraculin（nMIR）和 rMIR 在其蛋白质修饰方面与 N-糖几乎相同。然而，N-糖基化和果实成熟对 N-糖基化结构变化的影响都没有得到充分详细的表征。在这里，我们重点研究了 N-糖基化和果实成熟对 N-糖基化的影响，重新分析了 nMIR 和 rMIR 的自然成熟阶段的 N-糖基化，然后比较了来自两个不同成熟阶段的果实制备的 MIR 的 N-糖基化结构。MIR 的详细肽图和 N-糖基化分析提供了证据，表明 MIR 有变体，主要来源于 nMIR 中 N-糖基化结构的差异和 rMIR 中的 N-糖基化差异，而不是肽主链的裂解。MIR 的 N-糖基化分析表明，在每个成熟阶段，nMIR 和 rMIR 之间的 MIRs 的 N-糖基化结构相似。这些结果表明，在整个成熟阶段，异源表达的 rMIR 在翻译后修饰方面具有相同的特征，特别是 N-糖基化和 N-糖基化结构。本研究展示了在番茄植物中表达的重组蛋白的潜力，为 rMIR 的商业应用铺平了道路。
