Expression of the fJ subunit of spectrin in nonerythroid cells
Expression of the fJ subunit of spectrin in nonerythroid cells
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血影蛋白fJ亚基在非红系细胞中的表达
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E. Lazarides
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作者：
E. Lazarides
针对电泳纯化的鸡红细胞血影蛋白亚基（称为“1-血影蛋白”）产生的抗体已用于证明该多肽的免疫反应形式在非红细胞组织中的存在。免疫放射自显影显示，在鸡红细胞中，这种抗血清与 P-血影蛋白 (Mr 220,000) 和另一种多肽 (Mr 230,000) 发生反应，通过二维胰蛋白酶肽分析，该多肽与 P-血影蛋白具有广泛的同源性，但与血影蛋白的 a 亚基（称为“e-血影蛋白”）不具有广泛的同源性。用这种抗血清对各种鸡组织进行免疫放射自显影和免疫沉淀显示，表达了 f-血影蛋白的一种变体或两种变体。间接免疫荧光显示抗血清与红细胞和一些非红细胞的质膜相关成分发生反应。在骨骼肌和心肌细胞中观察到特别强的荧光，其中 (3-血影蛋白似乎沿着肌膜内表面形成网格状网络。a 和 (-血影蛋白变体的不协调分布表明它们的表达可能根据不同细胞中质膜的功能需求进行定制。膜的流体镶嵌模型是从红细胞膜的早期研究中发展而来的 (1, 2)，已扩展到包括蛋白质的横向流动性的概念。膜平面受到这些蛋白质和细胞骨架之间相互作用的限制 (3, 4)，尽管事实上在哺乳动物红细胞中涉及多种膜相关蛋白，但这些相互作用的性质仍然是一个未解决的问题，肌动蛋白通过一种高分子量蛋白质血影蛋白附着在质膜上，该蛋白质存在于质膜的内表面：它由两个不同的亚基多肽组成。 “a-血影蛋白”（Mr 240,000）和“/-血影蛋白”（Mr 220,000）（有关评论，请参阅参考文献 5-7），血影蛋白与肌动蛋白丝 (8, 9) 形成交联网络，并通过 8 亚基与锚蛋白 (10, 11) 的结合与膜连接。因此，在红细胞中，血影蛋白似乎在连接质膜和细胞骨架以及限制某些跨膜多肽的流动性方面发挥着关键作用。过去，未能检测到非红细胞组织中血影蛋白的免疫反应形式 (12, 13)，但最近，一些实验室已经确定了血影蛋白在高度专业化的红细胞中的作用。非红细胞中的血影蛋白 (14-20)；该蛋白显示出与红细胞中的 α-血影蛋白 (18-20) 常见的几种生化特性，因为红细胞中血影蛋白的功能构象似乎是 (a,/3)2 四聚体（参考文献 21 和 22；为了回顾，请参阅参考文献 7），因此确定 β-血影蛋白是否也在非红细胞组织中表达非常重要。在这方面，应该注意的是，在非红细胞中检测到了免疫反应形式的锚蛋白 (23)，我们证明针对禽类红细胞 B3 血影蛋白产生的抗体与多种非红细胞组织中的质膜相关蛋白发生反应。 抗体的制备如所述 (24) 一样进行，并通过以下方法分离其蛋白质。基于经修改和描述的Laemmli(25)系统(26)的NaDodSO 4 /10％或12.5％聚丙烯酰胺凝胶电泳将对应于鸡(-血影蛋白(24))的条带从凝胶中切下，均质化，并重新施加到NaDodSO 4 /10％聚丙烯酰胺凝胶上。再电泳后(参见图1A，泳道a)，/3血影蛋白条带被分离。从考马斯亮蓝染色的凝胶中切下并在 0.15 M 磷酸钠（pH 7.4）中平衡。在玻璃匀浆器（Potter-Elvehjem）中用电机驱动的聚四氟乙烯杵将凝胶切片（约 20-30 g 蛋白质）匀浆，并用弗氏完全佐剂乳化，用于新西兰白兔的免疫接种（27）；分别在初次免疫后 29 天和 39 天，在 4°C 下用 50% 饱和度的硫酸铵沉淀来收集血液。本研究使用来自第一次采血的血清：在免疫放射自显影之前从同一只兔子中获取免疫前血清。将鸡切碎，并在 20 mM Tris HCl、pH 7.2/150mM NaCl 中洗涤，按照所述方法 (29) 在 NaDodSO4 样品缓冲液中煮沸，以增强免疫反应性多肽的免疫沉淀。基本上按照Lingappa 等人(30) 的方法(由Levine 和Willard (15) 修改) 进行，不同之处在于每次免疫沉淀使用10 ul 抗血清(20)，不同之处在于使用1:10 血清稀释液的初级血清，切除含有a 或83-血影蛋白的考马斯蓝染色条带。从鸡红细胞质膜蛋白凝胶中分离出蛋白质，并通过电泳将蛋白质从凝胶中洗脱到透析管中，将洗脱的蛋白质用 100 mM 碳酸氢铵进行广泛透析，冻干，在 -20°C 下用丙酮/HCl 提取，并在稀释的 (1:100) 83-血影蛋白中孵育。抗血清；离心后，上清液用于免疫荧光或免疫沉淀。363 因此，根据 18 U.S.C. §1734，本文必须标记为“广告”，以表明这一事实。按照 Elder 等人 (31) 的描述，用 NaDodSO4 凝胶切片进行脱固定和染色的肽，并通过在 11.4:10:379（体积/体积）的乙酸/甲酸/水中，在 pH 1.9 下在第一维上电泳 40 分钟，在纤维素 TLC 板（Eastman）上分离洗脱的肽。 2小时并在丁醇/吡啶/乙酸/水5.5:3.3:1:3（体积/体积）中进行色谱分离，在第二维中。
Antibodies raised against electrophoretically purified chicken erythrocyte fi subunit of spectrin, called "1-spectrin," have been used to demonstrate the presence of an immunoreactive form of this polypeptide in nonerythroid tissues. Immunoautoradiography shows that, in chicken erythrocytes, this antiserum reacts with P-spectrin (Mr 220,000) and another polypeptide (Mr 230,000) that, by two-dimensional tryptic peptide analysis, shows extensive homology with P-spectrin but not with the a subunit of spectrin, called "e-spectrin." Immunoautoradiography and immunoprecipitation of various chicken tissues with this antiserum shows that either one variant or both variants of f-spectrin are expressed. Indirect immunofluorescence reveals that the antiserum reacts with a plasma membrane-associated component of erythroid and some nonerythroid cells. Particularly strong fluorescence is observed in skeletal and cardiac muscle cells where (3-spectrin appears to form a grid-like network along the inner surface ofthe sarcolemma. The noncoordinated distribution of aand (-spectrin variants indicates that their expression may be tailored to the functional requirements of the plasma membrane in different cells. The fluid mosaic model for membranes, which was developed from early studies on the erythrocyte membrane (1, 2), has been extended to include the notion that the lateral mobility of proteins in the plane of the membrane is restricted by interactions between these proteins and the cytoskeleton (3, 4). The nature of these interactions remains, to a great extent, an unresolved problem despite the fact that several membrane-associated proteins have been implicated in this process. In mammalian erythrocytes, actin is attached to the plasma membrane through a high molecular weight protein, spectrin, which is found on the inner surface of the plasma membrane: it is composed of two nonidentical subunit polypeptides herein termed "a-spectrin" (Mr 240,000) and "/-spectrin" (Mr 220,000) (for reviews, see refs. 5-7). Spectrin forms a crosslinked network with actin filaments (8, 9) and is linked to the membrane through the association of the 8 subunit with ankyrin (10, 11). Ankyrin associates with the membrane by binding to a subset of the transmembrane anion transporters (5, 7). Thus, in the erythrocyte, spectrin appears to play a pivotal role in linking the plasma membrane to the cytoskeleton and in restricting the mobility of certain transmembrane polypeptides. In the past, the failure to detect immunoreactive forms of spectrin in nonerythroid tissues (12, 13) has limited generalizations regarding the role of spectrin in the highly specialized erythrocyte. Recently, however, several laboratories have identified a spectrinlike protein in nonerythroid cells (14-20); the protein shows several biochemical properties common with a-spectrin from erythrocytes (18-20). As the functional conformation ofspectrin in the erythrocyte appears to be the (a,/3)2 tetramer (refs. 21 and 22; for review, see ref. 7) it is important to determine whether ,B-spectrin is also expressed in nonerythroid tissues. In this respect it should be noted that immunoreactive forms ofankyrin have been detected in nonerythroid cells (23). In the studies reported here, we demonstrate that antibodies raised against avian erythrocyte ,B3spectrin react with a plasma membrane-associated protein in a variety of nonerythroid tissues. MATERIALS AND METHODS Production of Antibody. Chicken erythrocyte membranes were prepared as described (24), and their proteins were separated by NaDodSO4/10% or 12.5% polyacrylamide gel electrophoresis based on the system of Laemmli (25) as modified and described (26). The band corresponding to chicken (-spectrin (24) was excised from the gel, homogenized, and reapplied to a NaDodSO4/10% polyacrylamide gel. After reelectrophoresis (see Fig. IA, lane a), the /3spectrin band was excised from the Coomassie blue-stained gel and equilibrated in 0.15 M sodium phosphate (pH 7.4). The gel slices (ca. 20-30 ,g of protein) were homogenized with a motor-driven Teflon pestle in a glass homogenizer (Potter-Elvehjem) and emulsified with Freund's complete adjuvant for the immunization of a New Zealand White rabbit (27); booster injections contained Freund's incomplete adjuvant and were administered 29 days and 39 days, respectively, after the initial immunization. Blood was collected 9 days after the second and third injections. The IgG fraction was partially purified by precipitation with ammonium sulfate at 50% saturation at 4°C. Serum from the first bleed was used in this study: preimmune serum was obtained from the same rabbit prior to antigen injection. Immunoautoradiography. Various tissues were removed from adult White Leghorn chickens, minced, and washed in 20 mM Tris HCl, pH 7.2/150mM NaCl. The samples were boiled in NaDodSO4 sample buffer (26). Immunoautoradiography (28) was performed as described (29). Intensifying screens were not used during film exposure to enhance the resolution of the immunoreactive polypeptides. Immunoprecipitation. The immunoprecipitation ofpolypeptides from various chicken tissues with antisera against chicken ,B3spectrin was performed essentially by the method of Lingappa et al. (30) as modified by Levine and Willard (15) except that 10 ,ul of antisera was used per immunoprecipitation. Immunofluorescence. Indirect immunofluorescence was performed as described (20) except that the primary serum was used at 1:10 serum dilution. For immunoadsorption ofantisera, Coomassie blue-stained bands containing either aor 83-spectrin were excised from gels ofchicken erythrocyte plasma membrane proteins, and the proteins were eluted electrophoretically from the gel into dialysis tubing. The eluted proteins were extensively dialyzed against 100 mM ammonium bicarbonate, lyophilized, extracted with acetone/HCl at -20°C, and relyophilized. The samples of aand 83-spectrin were incubated in diluted (1: 100) 83-spectrin antiserum; after centrifugation, the supernatant was used either for immunofluorescence or immunoprecipitation. 363 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 364 Biochemistry: Nelson and Lazarides Two-Dimensional Tryptic Peptide Mapping. Radioiodination offixed and stained proteins with NaDodSO4 gel slices and tryptic release of peptides for mapping were performed as described by Elder et al. (31). Eluted peptides were separated on cellulose TLC plates (Eastman) by electrophoresis in the first dimension at 800 V for 40 min in acetic acid/formic acid/ water, 11.4:10:379 (vol/vol) at pH 1.9. The plates were dried for 2 hr and chromatographed in butanol/pyridine/acetic acid/ water 5.5:3.3:1:3 (vol/vol), in the second dimension.