CONTROL OF MUCIN SYNTHESIS - THE PEPTIDE PORTION OF SYNTHETIC O-GLYCOPEPTIDE SUBSTRATES INFLUENCES THE ACTIVITY OF O-GLYCAN CORE-1 UDPGALACTOSE-N-ACETYL-ALPHA-GALACTOSAMINYL-R BETA-3-GALACTOSYLTRANSFERASE
CONTROL OF MUCIN SYNTHESIS - THE PEPTIDE PORTION OF SYNTHETIC O-GLYCOPEPTIDE SUBSTRATES INFLUENCES THE ACTIVITY OF O-GLYCAN CORE-1 UDPGALACTOSE-N-ACETYL-ALPHA-GALACTOSAMINYL-R BETA-3-GALACTOSYLTRANSFERASE
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DOI：
10.1021/bi00496a008
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发表时间：
1990-11-06
期刊：
影响因子：
2.9
通讯作者：
PAULSEN, H
中科院分区：
文献类型：
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作者：
BROCKHAUSEN, I;MOLLER, G;PAULSEN, H
以含有一个或两个GalNAc残基的人工合成O-糖肽为底物，研究了肽结构对猪胃、猪和大鼠结肠粘膜UDP-Gal：GalNAcα-Rβ3-Gal-转移酶粗制品和大鼠肝脏部分纯化酶制剂活性的影响。用高效液相色谱分离酶产物表明，氨基末端带有乙酰基、羧基末端带有叔丁基或酰胺基的未改变糖肽对粗品中的蛋白水解性具有抵抗力。β3-半乳糖基转移酶的活性随底物多肽部分的序列和长度、保护基团的存在、GalNAc的附着位置和底物中GalNAc残基的数量而变化。Pro的存在和位置对酶活性影响不大，GalNAc单元附近的电离基团对酶活性有干扰。由于许多O-糖肽中的GalNAc-Thr基团具有相似的刚性构象，因此酶活性的变化表明β3-半乳糖基转移酶既识别底物的多肽部分，又识别底物的碳水化合物部分。大鼠和猪结肠粘膜匀浆含有β3-和β6-GlcNAc-转移酶，分别合成O-糖链核心3(GlcNAc.Beta.3GalNac.α-R)和核心4[GlcNAc.beta.6(GlcNAc.beta.3)GalNac.-R]。这些酶在不同的多肽结构中也表现出不同的活性变化；这些作用与β-3-半乳糖基转移酶观察到的不同。结果表明，糖蛋白中的多肽部分影响作用于GalNAc.α-Ser(Thr)-X的三种糖基转移酶的活性。据预测，个别O-糖基化位点携带独特的O-糖链核心结构。
Synthetic O-glycopeptides containing one or two GalNAc residues attached to Ser or Thr were used as substrates to investigate the effect of peptide structure on the activity of crude preparations of UDP-Gal:GalNAc.alpha.-R .beta.3-Gal-transferase from pig stomach and pig and rat colonic mucosa and of a partially purified enzyme preparation from rat liver. High-performance liquid chromatography used to separate enzyme products revealed the unchanged glycopeptides with an acetyl group at the amino-terminal end and a tertiary butyl or an amide group at the carboxy-terminal end were resistant to proteolysis in crude preparations. The activity of .beta.3-Gal-transferase varied with the sequence and length of the peptide portion of the substrate, the presence of protecting groups, the attachment site of GalNAc, and the number of GalNAc residues in the substrate. The presence and position of Pro had little effect on enzyme activity; ionizing groups near the GalNAc unit interfered with enzyme activity. Since the GalNAc-Thr moeities in many of these O-glycopeptides have been shown to assume similar rigid conformations, the variation in enzyme activity indicates that the .beta.3-Gal-transferase recognizes both the peptide and carbohydrate moieties of the substrate. Rat and pig colonic mucosal homogenates contain .beta.3- and .beta.6-GlcNAc-transferases that synthesize respectively O-glycan core 3 (GlcNAc.beta.3GalNAc.alpha.-R) and core 4 [GlcNAc.beta.6(GlcNAc.beta.3) GalNAc.alpha.-R]. These enzymes also showed variations in activity with different peptide structures; these effects did not parallel those observed with .beta.3-Gal-transferase. The results show that the peptide portion in glycoprotein affects the activities of three glycosyltransferases which act on GalNAc.alpha.-Ser(Thr)-X. It is predicted that individual O-glycosylation sites carry characteristic O-glycan core structures.