van Berkel P H, Geerts M E, van Veen H A, Kooiman P M, Pieper F R, de Boer H A, Nuijens J H
Leiden Institute of Chemistry, Medical Biotechnology Department, Gorlaeus Laboratories, Leiden University, The Netherlands.
Biochem J. 1995 Nov 15;312 ( Pt 1)(Pt 1):107-14. doi: 10.1042/bj3120107.
We studied the role of N-glycosylation of human lactoferrin (hLF) with respect to properties that are relevant to its antibacterial and anti-inflammatory activities. A human kidney-derived 293(S) cell line that constitutively expresses recombinant hLF (rhLF) was produced. The reactivity towards various antibodies of rhLF that had been expressed in the absence or presence of tunicamycin (which blocks N-linked glycosylation) did not differ from that of natural (human milk-derived) hLF. Cation-exchange chromatography and N-terminal protein sequencing showed identical cationic properties and an intact N-terminal sequence for rhLF and natural hLF. SDS/PAGE of rhLF expressed in the presence of tunicamycin revealed a protein with the same M(r) as that of enzymically deglycosylated natural hLF. Both glycosylated and unglycosylated rhLF appeared to be completely saturated with iron. The affinity of natural hLF, glycosylated and non-glycosylated rhLF for both human lysozyme (Kd 4.5 x 10(-8) M) and bacterial lipopolysaccharide did not differ. SDS/PAGE of hLF species subjected to trypsin indicated that unglycosylated rhLF was much more susceptible to degradation. Furthermore, this analysis suggests that N-glycosylation heterogeneity in natural hLF and rhLF resides in the C-lobe. Thus our results provide no argument for differential antibacterial and/or anti-inflammatory activity of natural and (glycosylated) rhLF and suggest that a major function of glycosylation in hLF is to protect it against proteolysis.
我们研究了人乳铁蛋白（hLF）的N-糖基化作用与其抗菌和抗炎活性相关特性之间的关系。构建了一种可组成型表达重组hLF（rhLF）的人肾源293（S）细胞系。在有无衣霉素（其可阻断N-连接糖基化）的情况下表达的rhLF，其对各种抗体的反应性与天然（人乳来源）hLF并无差异。阳离子交换色谱法和N端蛋白质测序表明，rhLF和天然hLF具有相同的阳离子特性和完整的N端序列。在衣霉素存在的情况下表达的rhLF经SDS/PAGE分析显示，其蛋白质分子量与经酶法去糖基化的天然hLF相同。糖基化和未糖基化的rhLF似乎都完全被铁饱和。天然hLF、糖基化和非糖基化rhLF对人溶菌酶（解离常数Kd为4.5×10⁻⁸ M）和细菌脂多糖的亲和力并无差异。经胰蛋白酶处理的hLF种类的SDS/PAGE分析表明，未糖基化的rhLF更容易被降解。此外，该分析表明天然hLF和rhLF中的N-糖基化异质性存在于C叶中。因此，我们的结果并未支持天然hLF和（糖基化）rhLF在抗菌和/或抗炎活性上存在差异的观点，并表明hLF中糖基化的主要功能是保护其免受蛋白水解作用。
