van Berkel P H, van Veen H A, Geerts M E, de Boer H A, Nuijens J H
Leiden Institute of Chemistry, Medical Biotechnology Department, Gorlaeus Laboratories, Leiden University, The Netherlands.
Biochem J. 1996 Oct 1;319 ( Pt 1)(Pt 1):117-22. doi: 10.1042/bj3190117.
Human lactoferrin (hLF) is a glycoprotein involved in the host defence against infection and excessive inflammation. Our objective was to determine to what extent each of the three sequons for N-linked glycosylation in hLF is actually used. Human kidney-derived 293(S) cell lines expressing recombinant hLF (rhLF) or glycosylation-site mutants were produced. The mutations involved replacement of asparagine residues with glutamine at one or more sequons for N-glycosylation (Asn138, Asn479 and Asn624). Comparative SDS/PAGE analyses of rhLF, mutated rhLF and human-milk-derived (natural) hLF led us to propose that glycosylation of hLF occurs at two sites (at Asn138 and Asn479) in approx. 85% of all hLF molecules. Glycosylation at a single site (Asn479) or at all three sites occurs in approx, 5% and 9% of hLF respectively. The extent of glycosylation at Asn624 was increased to approx. 29% and 40% of Asn479 and Asn138/479 mutant molecules respectively, which indicates that glycosylation at Asn624 in natural hLF might be limited by glycosylation at Asn479. The presence in supernatant of unglycosylated hLF (approx. 60% of the total) after mutations of Asn138 and Asn479 suggests that glycosylation of hLF is not an absolute requirement for its secretion. The pronounced degradation of unglycosylated hLF in supernatant after mutation at all three glycosylation sites (Asn138/479/624 mutant) but not after mutation at both Asn138 and Asn479 suggests that an altered conformation rather than the lack of glycosylation has rendered the Asn138/479/624 mutant susceptible to intra- and/or extra-cellular degradation.
人乳铁蛋白（hLF）是一种糖蛋白，参与宿主抵御感染和过度炎症反应。我们的目标是确定hLF中三个N-糖基化序列位点各自的实际利用程度。构建了表达重组hLF（rhLF）或糖基化位点突变体的人肾源293（S）细胞系。这些突变涉及在一个或多个N-糖基化序列位点（Asn138、Asn479和Asn624）用谷氨酰胺取代天冬酰胺残基。对rhLF、突变型rhLF和人乳来源（天然）hLF进行的SDS/PAGE比较分析使我们提出，hLF的糖基化发生在大约85%的所有hLF分子的两个位点（Asn138和Asn479）。单个位点（Asn479）或所有三个位点的糖基化分别发生在大约5%和9%的hLF中。Asn624位点的糖基化程度分别增加到Asn479和Asn138/479突变体分子的约29%和40%，这表明天然hLF中Asn624位点的糖基化可能受到Asn479位点糖基化的限制。Asn138和Asn479突变后上清液中未糖基化hLF（约占总量的60%）的存在表明，糖基化不是hLF分泌的绝对必要条件。在所有三个糖基化位点（Asn138/479/624突变体）突变后上清液中未糖基化hLF明显降解，但在Asn138和Asn479两者突变后未降解，这表明构象改变而非糖基化缺失使Asn138/479/624突变体易受细胞内和/或细胞外降解。
