Nishimori I, Johnson N R, Sanderson S D, Perini F, Mountjoy K, Cerny R L, Gross M L, Hollingsworth M A
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805.
J Biol Chem. 1994 Jun 10;269(23):16123-30.
Synthetic peptides (30 and 20 residues long) corresponding to the native MUC1 tandem repeat sequence (20 residues long) were glycosylated in vitro using UDP-[3H]GalNAc and lysates from the human breast tumor cell line MCF7. Purified glycopeptides were sequenced on a gas-phase sequenator, and glycosylated positions were determined by measuring the incorporated radioactivity in fractions collected following each round of Edman degradation. The results showed that 2 of 3 threonines on the MUC1 tandem repeat peptides were glycosylated at the following positions: GVTSAPDTRPAPGSTAPPAH (underlined Thr residues indicate positions of GalNAc attachment); no glycosylation of serine residues was detected. Determination of the mass of the glycopeptides by mass spectrometry showed that a maximum of two molecules of GalNAc were covalently linked to each 20-residue repeat unit in the peptides. The influence of substrate primary amino acid sequence in determining the substrate specificity of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyl-transferase activity was evaluated using as acceptor substrates a series of overlapping 9-residue peptides that represent a moving set through the tandem repeat of the MUC1 mucin. In addition, the influence of primary amino acid sequence on acceptor substrate activity was evaluated using several peptides that contained single or double amino acid substitutions (relative to the native human MUC1 sequence). These included substitutions in the residues that were glycosylated and substitutions in the surrounding primary amino acid sequence. This study demonstrates that primary amino acid sequence, length, and relative position of the residue to be glycosylated dramatically affect the ability of peptides to serve as acceptor substrates for UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase.
使用UDP-[3H]GalNAc和人乳腺肿瘤细胞系MCF7的裂解物，在体外对与天然MUC1串联重复序列（20个残基长）相对应的合成肽（30和20个残基长）进行糖基化。纯化后的糖肽在气相测序仪上进行测序，并通过测量每轮埃德曼降解后收集的馏分中掺入的放射性来确定糖基化位置。结果表明，MUC1串联重复肽上3个苏氨酸中的2个在以下位置被糖基化：GVTSAPDTRPAPGSTAPPAH（带下划线的苏氨酸残基表示GalNAc附着位置）；未检测到丝氨酸残基的糖基化。通过质谱测定糖肽的质量表明，每个20个残基的重复单元中最多有两个GalNAc分子与肽共价连接。使用一系列重叠的9个残基肽作为受体底物，评估底物一级氨基酸序列对UDP-N-乙酰半乳糖胺：多肽N-乙酰半乳糖胺基转移酶活性底物特异性的影响，这些肽代表了贯穿MUC1粘蛋白串联重复序列的移动序列。此外，使用几个含有单或双氨基酸取代（相对于天然人MUC1序列）的肽评估一级氨基酸序列对受体底物活性的影响。这些取代包括糖基化残基的取代以及周围一级氨基酸序列的取代。这项研究表明，待糖基化残基的一级氨基酸序列、长度和相对位置会显著影响肽作为UDP-N-乙酰半乳糖胺：多肽N-乙酰半乳糖胺基转移酶受体底物的能力。
