Yoshida A, Suzuki M, Ikenaga H, Takeuchi M
Central Laboratories for Key Technology, Kirin Brewery Co., Ltd., 1-13-5, Fukuura, Kanazawa-ku, Yokohama 236, Japan.
J Biol Chem. 1997 Jul 4;272(27):16884-8. doi: 10.1074/jbc.272.27.16884.
The consensus primary amino acid sequence for mucin-type O-glycosylation sites has not been identified. To determine the shortest motif sequence required for high level mucin-type O-glycosylation, we prepared more than 100 synthetic peptides and assayed in vitro O-GalNAc transfer to serine or threonine in these peptides using a bovine colostrum UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyl transferase (O-GalNAcT). We chose the sequence PDAASAAP from human erythropoietin (hEPO) for further systematic substitutions because it accepted GalNAc and was a fairly simple sequence consisting only of four kinds of amino acids. Several substitutions showed that threonine is approximately 40-fold better than serine as the glycosylated amino acid and a proline at position +3 on the C-terminal side is very important. To define the effect of proline residues around the glycosylation site, we analyzed a series of peptides containing one to three proline residues in a parent peptide AAATAAA. The results clearly indicated that prolines at positions +1 and +3 had a positive effect. The O-GalNAc transfer level of AAATPAP was increased approximately 90-fold from AAATAAA. The deletion of amino acids from the N-terminal side of the glycosylation site suggested that five amino acids from position -1 to +3 were especially important for glycosylation. Moreover, the influence of all 20 amino acids at positions -1, +2, and +4 was analyzed. Uncharged amino acids were preferred at position -1, and small or positively charged amino acids were preferred at position +2. No preference was observed at position +4. We propose a mucin-type O-glycosylation motif, XTPXP, which may be suitable as a signal for protein O-glycosylation. The features observed in this study also appear to be very useful for prediction of mucin-type O-glycosylation sites in glycoproteins.
粘蛋白型O-糖基化位点的共有一级氨基酸序列尚未确定。为了确定高水平粘蛋白型O-糖基化所需的最短基序序列，我们制备了100多种合成肽，并使用牛初乳UDP-N-乙酰半乳糖胺：多肽N-乙酰半乳糖胺基转移酶（O-GalNAcT）在体外检测这些肽中丝氨酸或苏氨酸的O-半乳糖胺转移。我们选择了来自人促红细胞生成素（hEPO）的序列PDAASAAP进行进一步的系统替换，因为它能接受GalNAc，且是一个仅由四种氨基酸组成的相当简单的序列。几次替换表明，作为糖基化氨基酸，苏氨酸比丝氨酸的效果约好40倍，且C末端一侧+3位的脯氨酸非常重要。为了确定糖基化位点周围脯氨酸残基的作用，我们分析了一系列在母肽AAATAAA中含有一至三个脯氨酸残基的肽。结果清楚地表明，+1和+3位的脯氨酸有积极作用。AAATPAP的O-GalNAc转移水平比AAATAAA提高了约90倍。从糖基化位点的N末端一侧缺失氨基酸表明，从-1位到+3位的五个氨基酸对糖基化尤为重要。此外，还分析了-1、+2和+4位所有20种氨基酸的影响。-1位优选非带电氨基酸，+2位优选小的或带正电荷的氨基酸。在+4位未观察到偏好。我们提出了一个粘蛋白型O-糖基化基序XTPXP，它可能适合作为蛋白质O-糖基化的信号。本研究中观察到的特征似乎对预测糖蛋白中粘蛋白型O-糖基化位点也非常有用。
