Garone L, Edmunds T, Hanson E, Bernasconi R, Huntington J A, Meagher J L, Fan B, Gettins P G
Department of Structural Protein Chemistry, Genzyme Corporation, Framingham, Massachusetts 01701-9322, USA.
Biochemistry. 1996 Jul 9;35(27):8881-9. doi: 10.1021/bi960542m.
The two human plasma antithrombin isoforms, alpha and beta, differ in glycosylation at asparagine 135. Only the alpha form carries carbohydrate at this position and has lower affinity for heparin than the beta form. We previously found additional heterogeneity in a recombinant N135Q antithrombin variant, evidenced by two isoforms with a 2-fold difference in heparin affinity [Turko, I. V., Fan, B., & Gettins, P. G. W. (1993) FEBS Lett. 335, 9-12]. To test whether this heterogeneity of heparin affinity results from specific glycosylation differences, we have determined the carbohydrate composition at the three remaining glycosylation sites, asparagine residues 96, 155, and 192, in each of the two N135Q isoforms, by a combination of peptide fragmentation and electrospray mass spectrometry. Patterns of glycosylation at residues 96 and 192 were similar for each isoform and showed the presence of mono-, bi-, and triantennary complex carbohydrate, as well as fucosylation of all types of chains. At position 155, however, there was a marked difference between the isoforms, with the form with lower heparin affinity being 97% fucosylated at this position, whereas the form with higher affinity for heparin was not fucosylated. Other differences in carbohydrate type showed no strong correlation between the two isoforms. We conclude that formation of the two heparin-affinity isoforms of N135Q antithrombin results from the specific difference in fucosylation at residue 155, which may result in different structural properties of the carbohydrate. Consistent with these findings was the elimination of heparin-affinity heterogeneity in a double N135Q-N155Q variant antithrombin. It is possible that fucosylation of antithrombin may occur in vivo as a means of modifying the physiological properties of the antithrombin through alteration of the amount of antithrombin bound to surface heparin-like species.
两种人血浆抗凝血酶亚型，α和β，在天冬酰胺135处的糖基化有所不同。只有α型在该位置带有碳水化合物，并且与肝素的亲和力低于β型。我们之前在重组N135Q抗凝血酶变体中发现了额外的异质性，表现为两种亚型对肝素的亲和力相差2倍[Turko, I. V., Fan, B., & Gettins, P. G. W. (1993) FEBS Lett. 335, 9 - 12]。为了测试这种肝素亲和力的异质性是否源于特定的糖基化差异，我们通过肽片段化和电喷雾质谱联用，确定了两种N135Q亚型中其余三个糖基化位点，即天冬酰胺残基96、155和192处的碳水化合物组成。每种亚型在残基96和192处的糖基化模式相似，显示存在单天线、双天线和三天线复合碳水化合物，以及所有类型链的岩藻糖基化。然而，在位置155处，亚型之间存在明显差异，肝素亲和力较低的亚型在该位置97%被岩藻糖基化，而对肝素亲和力较高的亚型未被岩藻糖基化。碳水化合物类型的其他差异在两种亚型之间没有显示出强相关性。我们得出结论，N135Q抗凝血酶的两种肝素亲和力亚型的形成是由于残基155处岩藻糖基化的特定差异，这可能导致碳水化合物具有不同的结构特性。与这些发现一致的是，双N135Q - N155Q变体抗凝血酶中肝素亲和力的异质性消除。抗凝血酶的岩藻糖基化有可能在体内发生，作为通过改变与表面类肝素物质结合的抗凝血酶量来改变抗凝血酶生理特性的一种方式。
