Human interleukin-3 (hIL-3) is a clinically important cytokine used to treat hematological malignancies, bone marrow transplantation, cytopenias, and immunological disorders. The cloning of hIL-3 gene was previously reported by our group, where its expression was optimized under methanol-inducible AOX1 promoter having N-terminal α mating factor signal sequence from Saccharomyces cerevisiae. This study investigated the role of glycosylation pattern on its molecular stability, secretion efficiency, and biological activity using the mutagenesis approach. The two N-linked glycosylation positions at N15th (Asn¹⁵) and N70th (Asn⁷⁰) were sequentially mutated to generate three recombinant hIL-3 variants, i.e., N15A, N70A, and N15/70A. Asparagine at these positions was replaced with non-polar alanine amino acid (Ala, A). The alteration of N-linked glycosylation sites was disadvantageous to its efficient secretion in Pichia pastoris, where a 52.32%, 36.48%, 71.41% lower production was observed in N15A, N70A, and N15/70A mutants, respectively, as compared to native control. The fully glycosylated native hIL-3 protein showed higher thermal stability over its deglycosylated counterparts. The biological activity of native, N15A, N70A, and N15/70A hIL-3 protein was evaluated, where N15/70A mutant showed slightly higher proliferation efficacy than other combinations.

人白介素-3 (hIL-3) 是一种临床上重要的细胞因子，用于治疗血液恶性肿瘤、骨髓移植、血细胞减少症和免疫性疾病。我们小组先前报道了hIL-3基因的克隆，其中其表达在具有来自酿酒酵母的N端α交配因子信号序列的甲醇诱导的AOX1启动子下进行了优化。本研究利用诱变方法研究了糖基化模式对其分子稳定性、分泌效率和生物活性的作用。^{N15位(Asn 15} )和N70位(Asn ⁷⁰ )处的两个N联糖基化位置连续突变以产生三种重组hIL-3变体，即N15A、N70A和N15/70A。这些位置的天冬酰胺被非极性丙氨酸（Ala，A）取代。N-连接糖基化位点的改变不利于其在毕赤酵母中的有效分泌，与天然对照相比，在N15A、N70A和N15/70A突变体中观察到产量分别降低了52.32%、36.48%、71.41% 。完全糖基化的天然 hIL-3 蛋白比其去糖基化的对应物表现出更高的热稳定性。评估了天然、N15A、N70A和N15/70A hIL-3蛋白的生物活性，其中N15/70A突变体表现出比其他组合略高的增殖功效。