Norgard-Sumnicht K, Varki A
Glycobiology Program, University of California, San Diego, La Jolla 92093, USA.
J Biol Chem. 1995 May 19;270(20):12012-24. doi: 10.1074/jbc.270.20.12012.
We earlier reported calcium-dependent, heparin-like L-selectin ligands in cultured bovine endothelial cells (Norgard-Sumnicht, K. E., Varki, N. M., and Varki, A. (1993) Science 261,480-483). Here we show that these are heparan sulfate proteoglycans (HSPGs) associated either with the cultured cells or secreted into the medium and extracellular matrix. Activation of the endothelial cells with bacterial lipopolysaccharide (LPS) does not markedly alter the amount or distribution of this material. A major portion of the glycosaminoglycan (GAG) chains released from these HSPGs by alkaline beta-elimination rebinds to L-selectin in the presence of calcium, indicating that these saccharides alone can mediate the high affinity recognition. Heparin lyase digestions indicate that these GAG chains are enriched in heparan sulfate, not heparin sequences. Current understanding of the biosynthesis of heparan sulfate chains indicates that all glucosamine amino groups must be either N-acetylated or N-sulfated. However, nitrous acid deamination at pH 4.0 suggests the presence of some unsubstituted amino groups in these L-selectin-binding GAG chains from endothelial cell HSPGs. This is confirmed by chemical N-reacetylation and by reactivity with sulfo-N-hydroxysuccinimide-biotin. These unsubstituted amino groups are also found on HSPGs from human umbilical vein endothelial cells, but are not detected in those from Chinese hamster ovary cells. In both bovine and human endothelial cells, these novel groups are enriched for in the HS-GAG chains which bind to L-selectin. Despite this, studies with N-reacetylation and nitrous acid deamination do not show conclusive evidence for the direct involvement of the unsubstituted amino groups in L-selectin binding. This may be because the chemical reactions used to modify the amino groups do not go to completion. Alternatively, the unsubstituted amino groups may only be indirectly involved in generating binding, by dictating the biosynthesis of another critical group. Regardless, these studies shown that HSPGs from cultured endothelial cells which can bind to L-selectin are enriched with unsubstituted amino groups on their GAG chains. The possible biochemical mechanisms for generation of these novel groups are discussed.
我们先前报道了培养的牛内皮细胞中存在钙依赖性、类肝素的L-选择素配体（诺加德-萨姆尼希特，K.E.，瓦尔基，N.M.，和瓦尔基，A.（1993年）《科学》261卷，480 - 483页）。在此我们表明，这些配体是与培养细胞相关联或分泌到培养基和细胞外基质中的硫酸乙酰肝素蛋白聚糖（HSPGs）。用细菌脂多糖（LPS）激活内皮细胞并不会显著改变这种物质的量或分布。通过碱性β-消除从这些HSPGs释放的糖胺聚糖（GAG）链的一大部分在有钙存在的情况下会重新结合到L-选择素上，这表明仅这些糖类就能介导高亲和力识别。肝素酶消化表明这些GAG链富含硫酸乙酰肝素，而非肝素序列。目前对硫酸乙酰肝素链生物合成的理解表明，所有葡糖胺氨基必须要么被N-乙酰化要么被N-硫酸化。然而，在pH 4.0下用亚硝酸脱氨基表明，来自内皮细胞HSPGs的这些与L-选择素结合的GAG链中存在一些未被取代的氨基。化学N-重新乙酰化以及与磺基-N-羟基琥珀酰亚胺-生物素的反应性证实了这一点。在人脐静脉内皮细胞的HSPGs上也发现了这些未被取代的氨基，但在仓鼠卵巢细胞的HSPGs中未检测到。在牛和人内皮细胞中，这些新基团在与L-选择素结合的HS-GAG链中都很丰富。尽管如此，用N-重新乙酰化和亚硝酸脱氨基进行的研究并未显示出未被取代的氨基直接参与L-选择素结合的确凿证据。这可能是因为用于修饰氨基的化学反应没有完全完成。或者，未被取代的氨基可能只是通过决定另一个关键基团的生物合成而间接参与产生结合。无论如何，这些研究表明，来自培养内皮细胞的能与L-选择素结合的HSPGs在其GAG链上富含未被取代的氨基。本文讨论了产生这些新基团的可能生化机制。
