Glycan microarrays for screening sialyltransferase specificities
Glycan microarrays for screening sialyltransferase specificities
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DOI：
10.1007/s10719-007-9062-z
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发表时间：
2008-01-01
影响因子：
3
通讯作者：
Razi, Nahid
中科院分区：
文献类型：
--
作者：
Blixt, Ola;Allin, Kirk;Razi, Nahid
在这里，我们证明了葡聚糖微阵列可以用于各种重组唾液酸基转移酶的高通量受体特异性筛选。胞苷-5‘-单磷-N-乙酰神经氨酸(CMP-Neu5Ac)是在N-乙酰神经氨酸(Neu5Ac)的第9位用化学酶法合成的。活化的糖核苷酸被用作各种哺乳动物唾液酸转移酶的供体底物，这些酶同时将生物素化的唾液酸转移到固定在微阵列玻片上的葡聚糖受体上。用荧光素链霉亲和素偶联物检测生物素化葡聚糖，以生成每个酶的特异性图谱，既证实了先前已知的特异性，又揭示了额外的特异性信息。在我们的阵列系统中，人α2，6唾液酸转移酶-I(hST6Gal-I)也能唾液酸化甲壳二糖结构(GlcNAcβ1-4GlcNAc)(N)，大鼠α2，3唾液酸转移酶(rST3Gal-III)能耐受岩藻糖化受体，如Lewis(A)、人α2，3唾液酸转移酶-IV(hST3Gal-IV)广泛唾液酸化1 4型寡糖和猪α2，3唾液酸转移酶-I(pST3Gal-I)唾液酸神经节低聚糖和核心20-糖。这些唾液酸基转移酶中的几个执行取代反应并将唾液酸化的受体与生物素化的唾液酸交换，但仅限于最特定的受体底物。因此，该方法允许快速生成酶特异性信息，并可用于合成新的碳水化合物和扩展糖链阵列化合物文库。
Here we demonstrate that glycan microarrays can be used for high-throughput acceptor specificity screening of various recombinant sialyltransferases. Cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) was biotinylated at position 9 of N-acetylneuraminic acid (Neu5Ac) by chemoenzymatic synthesis generating CMP-9Biot-Neu5Ac. The activated sugar nucleotide was used as donor substrate for various mammalian sialyltranferases which transferred biotinylated sialic acids simultaneously onto glycan acceptors immobilized onto a microarray glass slide. Biotinylated glycans detected with fluorescein streptavidin conjugate to generate a specificity profile for each enzyme both confirming previously known specificities and reveal additional specificity information. Human alpha 2,6sialyltransferase-I (hST6Gal-I) also sialylates chitobiose structures (GlcNAc beta 1-4GlcNAc)(n) including N-glycans, rat alpha 2,3sialyltransferase (rST3Gal-III) tolerates fucosylated acceptors such as Lewis(a), human alpha 2,3sialyltransferase-IV (hST3Gal-IV) broadly sialylates oligosaccharides of types 1 4 and porcine alpha 2,3sialyltransferase-I (pST3Gal-I) sialylates ganglio-oligosaccharides and core 2 O-glycans in our array system. Several of these sialyltransferases perform a substitution reaction and exchange a sialylated acceptor with a biotinylated sialic acid but are restricted to the most specific acceptor substrates. Thus, this method allows for a rapid generation of enzyme specificity information and can be used towards synthesis of new carbohydrate compounds and expand the glycan array compound library.