Gleeson P A, McNamara M, Wettenhall R E, Stone B A, Fincher G B
Department of Biochemistry, La Trobe University, Bundoora, Vic., Australia.
Biochem J. 1989 Dec 15;264(3):857-62. doi: 10.1042/bj2640857.
An arabinogalactan-protein (AGP) purified from the filtrate of liquid-suspension-cultured Italian-ryegrass (Lolium multiflorum) endosperm cells by affinity chromatography on myeloma protein J539-Sepharose was deglycosylated with trifluoromethanesulphonic acid to remove polysaccharide chains that are covalently associated with hydroxyproline residues in the peptide component of the proteoglycan. The protein core, which accounts for less than 10% (w/w) of the intact proteoglycan, was purified by h.p.l.c. It has an apparent Mr of 35,000, but reacts very poorly with both Coomassie Brilliant Blue R and silver stains. Amino-acid-sequence analysis of the N-terminus of the h.p.l.c.-purified protein core and of tryptic peptides generated from the unpurified protein reveals a high content of hydroxyproline and alanine. These are sometimes arranged in short (Ala-Hyp) repeat sequences of up to six residues. Polyclonal antibodies raised against the protein core do not cross-react with native AGP, the synthetic peptide (Ala-Hyp)4, poly-L-hydroxyproline or poly-L-proline. The results suggest that the polysaccharide chains in the native AGP render the protein core of the proteoglycan inaccessible to the antibodies and that the immunodominant epitopes include domains of the protein other than those rich in Ala-Hyp repeating units.
通过在骨髓瘤蛋白J539-琼脂糖凝胶上进行亲和层析，从意大利黑麦草（多花黑麦草）胚乳细胞的液体悬浮培养滤液中纯化出一种阿拉伯半乳聚糖蛋白（AGP），并用三氟甲磺酸对其进行去糖基化处理，以去除与蛋白聚糖肽成分中羟脯氨酸残基共价结合的多糖链。占完整蛋白聚糖不到10%（w/w）的蛋白核心通过高效液相色谱法进行纯化。它的表观分子量为35,000，但与考马斯亮蓝R和银染的反应都很差。对高效液相色谱法纯化的蛋白核心的N端以及未纯化蛋白产生的胰蛋白酶肽进行氨基酸序列分析，发现羟脯氨酸和丙氨酸含量很高。这些氨基酸有时会排列成长度达六个残基的短（Ala-Hyp）重复序列。针对蛋白核心产生的多克隆抗体与天然AGP、合成肽（Ala-Hyp）4、聚-L-羟脯氨酸或聚-L-脯氨酸均无交叉反应。结果表明，天然AGP中的多糖链使蛋白聚糖的蛋白核心无法与抗体接触，并且免疫显性表位包括蛋白中除富含Ala-Hyp重复单元的结构域之外的其他结构域。
