Molecular Cloning and Characterization of Chick SPACRCAN*
Molecular Cloning and Characterization of Chick SPACRCAN*
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DOI：
10.1074/jbc.m508161200
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发表时间：
2003-05
影响因子：
4.8
通讯作者：
Y. Inoue;M. Yoneda;Jinsong Zhao;O. Miyaishi;A. Ohno-Jinno;Takuya Kataoka;Z. Isogai;K. Kimata;M. Iwaki;M. Zako
中科院分区：
文献类型：
--
作者：
Y. Inoue;M. Yoneda;Jinsong Zhao;O. Miyaishi;A. Ohno-Jinno;Takuya Kataoka;Z. Isogai;K. Kimata;M. Iwaki;M. Zako
MY-174是一种单克隆抗体，可与鸡SPACR（与视锥细胞和视杆细胞相关的唾液蛋白）的特异性唾液酸化O-连接糖缀合物反应，也可识别另一种300 kDa的分子。在这里，我们证实，这300 kDa的分子是鸡SPACRCAN（唾液蛋白聚糖与锥和杆），另一个成员的一个新的interphotoreceptor基质分子家族。使用鸡SPACR的探针通过噬斑杂交进行鸡SPACRCAN的筛选。针对鸡SPACRCAN产生的特异性多克隆抗体用于以下实验。为了确定MY-174检测到的300-kDa分子是否与300-kDa鸡SPACRCAN相同，在各种糖苷酶消化后检查这些条带的迁移。此外，在视网膜发育过程中的表达水平进行了测量，并与鸡SPACR。结果表明，MY-174识别的300 kDa分子是鸡SPACRCAN，我们进一步鉴定它是一种具有硫酸软骨素链的蛋白聚糖。SPACRCAN具有高度唾液酸化的N-和O-连接的糖缀合物，其MY-174抗原性在神经氨酸酶处理后被O-聚糖酶处理消除，如在鸡SPACR中观察到的。在视网膜发育过程中，mRNA和核心蛋白的表达水平，MY-174抗原性，透明质酸结合能力的SPACRCAN达到高峰，胚胎17天左右，然后逐渐下降，而相应的表达水平的SPACR只是增加，但不是其透明质酸结合能力。与新生儿视网膜相比，成人视网膜中SPACRCAN的MY-174反应性降低，而SPACR的MY-174反应性升高。SPACR的透明质酸结合减少是由成年期过量唾液酸的抑制作用引起的。因此，具有相似的核心蛋白质结构和特异性唾液酸化糖缀合物但不同的硫酸软骨素链，SPACRCAN和SPACR可能由于其在发育期间的不同表达谱而在视网膜中具有不同的作用。
MY-174, a monoclonal antibody that reacts with specific sialylated O-linked glycoconjugates of chick SPACR (sialoprotein associated with cones and rods), also recognizes another molecule of 300 kDa. Here, we verified that this 300-kDa molecule is chick SPACRCAN (sialoproteoglycan associated with cones and rods), another member of a novel interphotoreceptor matrix molecule family. Screening for chick SPACRCAN was carried out by plaque hybridization using a probe for chick SPACR. Specific polyclonal antibodies raised against chick SPACRCAN were used for the following experiments. To determine whether the 300-kDa molecule detected by MY-174 was identical to 300-kDa chick SPACRCAN, the migrations of these bands were examined after various glycosidase digestions. Furthermore, the expression levels were measured during retinal development and compared with those of chick SPACR. The results demonstrated that the 300-kDa molecule recognized by MY-174 was chick SPACRCAN, and we further identified it as a proteoglycan with chondroitin sulfate chains. SPACRCAN had heavily sialylated N- and O-linked glycoconjugates, and its MY-174 antigenicity was abolished by O-glycanase treatment after neuraminidase treatment, as observed for chick SPACR. During retinal development, the mRNA and core protein expression levels, MY-174 antigenicity, and hyaluronan binding ability of SPACRCAN peaked around embryonic day 17 and then gradually decreased, whereas the corresponding expression levels of SPACR simply increased, but not its hyaluronan binding ability. The MY-174 reactivity of SPACRCAN in the adult retina was decreased compared with that in the newborn retina, whereas that of SPACR was increased. The decreased hyaluronan binding of SPACR was induced by an inhibitory effect of the excess of sialic acids in the adult stage. Thus, with similar core protein structures and specific sialylated glycoconjugates but distinct chondroitin sulfate chains, SPACRCAN and SPACR may have separate roles in the retina due to their differing expression profiles during development.