Inoue Yoko, Yoneda Masahiko, Zhao Jinsong, Miyaishi Osamu, Ohno-Jinno Akiko, Kataoka Takuya, Isogai Zenzo, Kimata Koji, Iwaki Masayoshi, Zako Masahiro
Department of Ophthalmology, Aichi Medical University, Nagakute-cho, Aichi-gun, Aichi-ken 480-1195, Japan.
J Biol Chem. 2006 Apr 14;281(15):10381-8. doi: 10.1074/jbc.M508161200. Epub 2006 Feb 9.
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.
MY-174是一种单克隆抗体，可与鸡的视锥和视杆相关唾液酸化O-连接糖缀合物（SPACR，视锥和视杆相关唾液蛋白）发生反应，它还能识别另一种300 kDa的分子。在此，我们证实这个300 kDa的分子是鸡的视锥和视杆相关唾液蛋白聚糖（SPACRCAN），它是新型光感受器间基质分子家族的另一个成员。使用针对鸡SPACR的探针通过噬菌斑杂交法筛选鸡SPACRCAN。针对鸡SPACRCAN制备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的鸡SPACRCAN相同，在进行各种糖苷酶消化后检测这些条带的迁移情况。此外，还测定了视网膜发育过程中的表达水平，并与鸡SPACR的表达水平进行比较。结果表明，MY-174识别的300 kDa分子是鸡SPACRCAN，我们进一步将其鉴定为带有硫酸软骨素链的蛋白聚糖。SPACRCAN具有高度唾液酸化的N-连接和O-连接糖缀合物，并且与鸡SPACR一样，经神经氨酸酶处理后再用O-聚糖酶处理可消除其MY-174抗原性。在视网膜发育过程中，SPACRCAN的mRNA和核心蛋白表达水平、MY-174抗原性以及透明质酸结合能力在胚胎第17天左右达到峰值，然后逐渐下降，而SPACR的相应表达水平只是升高，但其透明质酸结合能力并未增加。与新生视网膜相比，成年视网膜中SPACRCAN的MY-174反应性降低，而SPACR的反应性增加。成年期过量唾液酸的抑制作用导致SPACR的透明质酸结合能力下降。因此，SPACRCAN和SPACR具有相似的核心蛋白结构和特异性唾液酸化糖缀合物，但硫酸软骨素链不同，由于它们在发育过程中不同的表达谱，可能在视网膜中发挥不同的作用。
