Human Factor IX is a highly post-translationally modified protein that is an important clotting factor in the blood coagulation cascade. Functional deficiencies in Factor IX result in the bleeding disorder haemophilia B, which is treated with plasma-derived or recombinant Factor IX concentrates. Here, we investigated the post-translational modifications of human serum-derived Factor IX and report previously undescribed O-linked monosaccharide compositions at serine 141 and a novel site of glycosylation. At serine 141 we observed two monosaccharide compositions, with HexNAc₁Hex₁NeuAc₂ dominant and a low level of HexNAc₁Hex₁NeuAc₁. This O-linked site lies N-terminal to the first cleavage site for the activation peptide, an important region of the protein that is removed to activate Factor IX. The novel site is an N-linked site in the serine protease domain with low occupancy in a non-canonical consensus motif at asparagine 258, observed with a HexNAc₄Hex₅NeuAc₂ monosaccharide composition attached. This is the first reported instance of a site of modification in the serine protease domain. The description of these glycosylation events provides a basis for future functional studies and contributes to structural characterisation of native Factor IX for the production of effective therapeutic biosimilars and biobetters.

中文翻译：

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鉴定人血清来源的因子IX的新型糖基化事件。

人因子IX是高度翻译后修饰的蛋白，是凝血级联反应中的重要凝血因子。因子IX的功能缺陷导致出血性疾病B型血友病，可用血浆或重组的因子IX浓缩物治疗。在这里，我们研究了人类血清来源的因子IX的翻译后修饰，并报告了丝氨酸141和糖基化的新位点先前未描述的O-联单糖组成。在丝氨酸141处，我们观察到两种单糖成分，其中HexNAc ₁ Hex ₁ NeuAc ₂占优势，而HexNAc ₁ Hex ₁ NeuAc ₁含量低。这个O-连接位点位于激活肽的第一个切割位点的N-末端，激活肽是被去除以激活因子IX的蛋白质的重要​​区域。该新位点是丝氨酸蛋白酶结构域中的N-连接位点，在天冬酰胺258的非经典共有基序中具有低占有率，用附着的HexNAc ₄ Hex ₅ NeuAc ₂单糖组合物观察到。这是丝氨酸蛋白酶结构域中修饰位点的第一个报道实例。这些糖基化事件的描述为将来的功能研究提供了基础，并有助于天然因子IX的结构表征，以生产有效的治疗性生物仿制药和生物制剂。