A NOVEL HYDROXYPROLINE-DEFICIENT ARABINOGALACTAN PROTEIN SECRETED BY SUSPENSION-CULTURED CELLS OF DAUCUS-CAROTA - PURIFICATION AND PARTIAL CHARACTERIZATION
A NOVEL HYDROXYPROLINE-DEFICIENT ARABINOGALACTAN PROTEIN SECRETED BY SUSPENSION-CULTURED CELLS OF DAUCUS-CAROTA - PURIFICATION AND PARTIAL CHARACTERIZATION
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DOI：
10.1104/pp.103.1.115
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发表时间：
1993-09-01
期刊：
影响因子：
7.4
通讯作者：
ROBERTS, K
中科院分区：
文献类型：
--
作者：
BALDWIN, TC;MCCANN, MC;ROBERTS, K
阿拉伯半乳聚糖蛋白 (AGP) 是分泌型或膜相关糖蛋白，在操作上被定义为与 β-葡萄糖基 Yariv 人工抗原结合，富含阿拉伯糖和半乳糖，并含有高水平的丙氨酸、丝氨酸和羟脯氨酸。使用与溴化氰激活的 Sepharose 4B 结合的抗 AGP 单克隆抗体 (MAC 207)，我们通过免疫亲和层析从悬浮培养的胡萝卜 (Daucus carota) 细胞的培养基中纯化了细胞外 AGP。通过十二烷基硫酸钠-聚丙烯酰胺凝胶判断，这种高度糖基化的蛋白多糖的表观分子量为 70 至 100 kD。尽管其糖分析、β-葡萄糖基 Yariv 结合以及高丙氨酸、丝氨酸和脯氨酸含量与其 AGP 一致，但氨基酸组成出人意料地显示该分子没有可检测到的羟脯氨酸。这表明这种糖蛋白不是“经典”AGP，而是代表了新一类缺乏羟脯氨酸的 AGP 的第一个例子。用无水氟化氢对 AGP 进行去糖基化，结果表明纯化的蛋白多糖可能含有表观分子质量为 30 kD 的单个核心蛋白。在电子显微镜中直接观察天然 AGP 显示出椭圆形的假定 AGP 单体，大约 25 nm x 15 nm，显示出自组装成更高阶结构的强烈倾向。干燥后，糖基化的 AGP 形成光学显微镜下可见的旁晶阵列。这些阵列的偏振傅里叶变换红外显微光谱显示在这些条件下糖部分具有高度偏振。这些结果对当前的 AGP 结构模型提出了可能的限制；讨论了这些新型 AGP 作为果胶结合蛋白的假定作用。
Arabinogalactan proteins (AGPs) are secreted or membrane-associated glycoproteins that have been operationally defined as binding to beta-glucosyl Yariv artificial antigen, being rich in arabinose and galactose, and containing high levels of alanine, serine, and hydroxyproline. Using an anti-AGP monoclonal antibody (MAC 207) bound to cyanogen bromide-activated Sepharose 4B, we have purified by immunoaffinity chromatography an extracellular AGP from the culture medium of suspension-cultured cells of carrot (Daucus carota). The apparent molecular mass of this highly glycosylated proteoglycan is 70 to 100 kD as judged by sodium dodecyl sulfate-polyacrylamide gels. Although its sugar analysis, beta-glucosyl Yariv binding, and high alanine, serine, and proline content are consistent with it being an AGP, the amino acid composition unexpectedly revealed this molecule to have no detectable hydroxyproline. This suggests that this glycoprotein is not a ''classical' AGP, but represents the first example of a new class of hydroxyproline-poor AGPs. Deglycosylation of the AGP with anhydrous hydrogen fluoride revealed that the purified proteoglycan contains probably a single core protein with an apparent molecular mass of 30 kD. Direct visualization of the native AGP in the electron microscope showed ellipsoidal putative AGP monomers, approximately 25 nm by 15 nm, that showed a strong tendency to self assemble into higher-order structures. Upon desiccation, the glycosylated AGP formed paracrystalline arrays visible in the light microscope. Polarized Fourier transform infrared microspectroscopy of these arrays demonstrated a high degree of polarization of the sugar moieties under these conditions. These results put possible constraints on current models of AGP structure; a putative role for these novel AGPs as pectin-binding proteins is discussed.