Sweatt AJ et al. (1991), Immunolocalization of N-acetylgalactosaminylpho...

XB-ART-24514

Exp Eye Res 1991 Oct 01;534:479-87.

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Immunolocalization of N-acetylgalactosaminylphosphotransferase in the adult retina and subretinal space.

Sweatt AJ , Balsamo J , Lilien J .

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The cell surface N-acetylgalactosaminylphosphotransferase (GalNAcPTase) modulates N-cadherin-mediated adhesion among embryonic chick retinal cells (Balsamo et al., 1990). We are investigating the potential role of this transferase in modulating adhesive interactions in the adult retina. Using a previously characterized monoclonal anti-GalNAcPTase, we have used immunohistochemical and immunoblot techniques to localize and characterize the transferase in the retinas of the post metamorphic frog (Xenopus laevis), adult cow, and adult cynomolgus macaque. On frozen sections, anti-GalNAcPTase specifically labels the outer segments of photoreceptors in all species. Immunolabel appears at the surface of, or between rod outer segments in all species. The nerve fiber layer also shows high immunoreactivity in all species. Monkey cone outer segments are also highly immunoreactive. Photoreceptor inner segments are clearly immunoreactive in the cow retina. Immunoblots of purified cow rod outer segments show immunolabeled bands near 220 kDa, which is the molecular mass of the GalNAcPTase used as immunogen. Purified Xenopus rod outer segments are not immunoreactive on blots, while soluble interphotoreceptor matrix (IPM) shows immunoreactive bands principally at 113-130, and 166 kDa. Cow soluble IPM shows immunoreactivity at 180 kDa. Based on these findings, we propose that the GalNAcPTase, or a fragment thereof, is a component of the IPM, and perhaps of the photoreceptor outer segment as well.

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Species referenced: Xenopus laevis
Genes referenced: csgalnact2 ighx rasl12 rpe tbx2
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	FIG ;. 1. Fluorescence micrographs of frog eyecup. A, Rhodamine image of tissue section immunolabeled with anti-transfer MAb 7A2, followed by rhodamine-conjugated goat anti-mouse IgM. B, UV image of same section as in (A), showing Hoec nuclear dye. C. Rhodamine image of control section incubated with non-specific mouse IgM. followed by rhodamine-conjug; secon ldary antibody. GC, Ganglion cell layer ; INL, inner nuclear layer; ONL, outer nuclear layer ; RIS, rod inner segments ; R rod o uter segments; RPE, retinal pigmented epithelium. Bar = 25 pm.
	FIG. 5. Immunoblots of retinal homogenates from adult chicken (lanes 1,2), Xenopus (3,4), cow (5, 6), and macaque (7. 8). Lanes 1, 3, 5, and 7 were probed with non-specific mouse IgM, while lanes 2,4, 6, and 8 were probed with antitransferase MAb 7A2. Secondary antibody for all lanes was horseradish peroxidase-conjugated goat anti-mouse IgM. Arrows mark positions of bands recognized by MAb 7A2.
	FIG. 6. Silver-stained SDS gel of Xenopus purified ROS (lane 1) and soluble IPM (lane 2). This gel had not been subjected to electrophoretic transfer. Each lane represents protein derived from five retinas. Immunoblots of proteins of purified ROS (lanes 3 and 4) and of soluble IPM (lanes 5 and 6), transferred from gel lanes loaded with proteins derived from 10 retinas. Lanes 3 and 5 were probed with non-specific mouse IgM, while lanes 4 and 6 were probed with MAb 7A2. Secondary antibody was HRP-conjugated goat anti-mouse IgM. Arrows indicate positions of immunoreactive bands.