Sweatt A J, Balsamo J, Lilien J
Department of Ophthalmology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem North Carolina 27157.
Invest Ophthalmol Vis Sci. 1995 Jan;36(1):163-73.
To determine the exact location of a cell surface glycosyltransferase (N-acetylgalactosaminylphosphotransferase, (GalNAcPTase) immunochemically identified in mammalian rod outer segments (ROS), to determine whether anti-GalNAcPTase antibody recognizes retinal molecules that possess transferase activity and to characterize ROS transferase enzyme activity and acceptors. The GalNAcPTase is known to be associated with the adhesion molecule N-cadherin in embryonic avian retinas and with E-cadherin in mammalian pancreatic islet cells.
Purified, fixed ROS were reacted with anti-chick GalNAcPTase antibody followed by secondary antibody conjugated to colloidal gold and were examined by electron microscopy. Fractions of retinal and ROS proteins enriched in the transferase were obtained through batch adsorption on Sepharose, separated by gel electrophoresis, transferred to nitrocellulose, and either reacted with anti-GalNAcPTase antibody or assayed for transferase activity. Interphotoreceptor matrix (IPM) was examined for the presence of immunoreactive GalNAcPTase by gel electrophoresis and immunoblot. The kinetics and endogenous acceptors of the cow ROS transferase were characterized.
ROS are specifically labeled by anti-GalNAcPTase antibody at the cell surface. The immunogold label was associated with the cell surface and with flocculent material adherent to the cell surface. In addition, soluble and particulate fractions of the IPM showed GalNAcPTase-like immunoreactivity. The transferase appears as single immunoreactive band at or near 220 kd. Transferase enzyme activity was present at this position on Western transfers of retinal and ROS proteins. In whole ROS, transferase activity was directed toward endogenous acceptors of very high molecular mass.
The GalNAcPTase is localized on ROS in association with the cell surface and with components of the IPM. The molecule recognized by the anti-GalNAcPTase antibody possesses transferase activity toward itself and a few other proteins, but mostly toward very large molecules that may be IPM proteoglycans. It is not yet known whether the enzyme of the adult retina specifically transfers sugar or sugar-phosphate groups to its acceptors. It is proposed that the ROS GalNAcPTase is involved in the modulation of adhesive phenomena between or within photoreceptors or between photoreceptors and the interphotoreceptor matrix.
确定在哺乳动物视杆外段（ROS）中通过免疫化学鉴定的细胞表面糖基转移酶（N - 乙酰半乳糖胺磷酸转移酶，GalNAcPTase）的确切位置，确定抗GalNAcPTase抗体是否识别具有转移酶活性的视网膜分子，并表征ROS转移酶的酶活性和受体。已知GalNAcPTase在胚胎鸡视网膜中与粘附分子N - 钙粘蛋白相关，在哺乳动物胰岛细胞中与E - 钙粘蛋白相关。
纯化、固定的ROS与抗鸡GalNAcPTase抗体反应，然后与胶体金偶联的二抗反应，并通过电子显微镜检查。通过在琼脂糖上的批量吸附获得富含转移酶的视网膜和ROS蛋白组分，通过凝胶电泳分离，转移到硝酸纤维素膜上，然后与抗GalNAcPTase抗体反应或测定转移酶活性。通过凝胶电泳和免疫印迹检查光感受器间基质（IPM）中免疫反应性GalNAcPTase的存在。表征了牛ROS转移酶的动力学和内源性受体。
ROS在细胞表面被抗GalNAcPTase抗体特异性标记。免疫金标记与细胞表面以及附着在细胞表面的絮状物质相关。此外，IPM的可溶性和颗粒性组分显示出GalNAcPTase样免疫反应性。转移酶在220 kd或其附近表现为单一免疫反应条带。在视网膜和ROS蛋白的Western转移中，该位置存在转移酶活性。在整个ROS中，转移酶活性针对非常高分子量的内源性受体。
GalNAcPTase定位于ROS上，与细胞表面和IPM的成分相关。抗GalNAcPTase抗体识别的分子对自身和其他一些蛋白质具有转移酶活性，但主要针对可能是IPM蛋白聚糖的非常大的分子。尚不清楚成年视网膜的酶是否特异性地将糖或糖磷酸基团转移到其受体上。有人提出，ROS GalNAcPTase参与光感受器之间或内部、或光感受器与光感受器间基质之间粘附现象的调节。
