Callaghan J M, Toh B H, Pettitt J M, Humphris D C, Gleeson P A
Department of Pathology and Immunology, Monash University Medical School, Melbourne, Victoria, Australia.
J Cell Sci. 1990 Apr;95 ( Pt 4):563-76. doi: 10.1242/jcs.95.4.563.
The cytoplasmic tubulovesicular and canalicular membranes of gastric parietal cells are intimately involved in hydrochloric acid secretion. To characterise the glycoproteins of these membranes, we examined a panel of lectins for reactivity with parietal cells in paraffin sections of rat, dog and pig stomach. The poly-N-acetyllactosamine-specific lectin from Lycopersicon esculentum (tomato) and from Solanum tuberosum (potato), and the galactose-specific lectin Ricinus communis agglutinin (RCA120), showed strong cytoplasmic binding of parietal cells of all three species, with a pattern indicative of an intracellular membrane network. Binding to parietal cells was confirmed by double-labelling studies with parietal cell auto-antibodies from patients with autoimmune gastritis. Mucous cells and mucin also bound these lectins strongly. Other gastric cell types did not stain with either tomato or potato lectin, but stained weakly with RCA120. Electron-microscopic examination of lectin binding sites using biotinylated tomato lectin or RCA120 and streptavidin-gold, revealed specific binding to the luminal face of parietal cell tubulovesicular and canalicular membranes as well as the contents of mucous cell secretory granules. Tomato lectin and RCA120 reacted by lectin blotting with a major species of apparent molecular weight 60-90 X 10(3) Mr from rat, dog and pig gastric membranes. A tubulovesicular membrane fraction, enriched 10-fold for K(+)-dependent phosphatase activity, was also enriched three-fold for tomato lectin binding as assessed by a solid-phase lectin assay. The 60-90K (K = 10(3) Mr) component, in 125I-labelled detergent extracts of dog tubulovesicular membranes, bound to an affinity support of tomato lectin-Sepharose and was specifically eluted with N,N',N'-triacetylchitotriose. Digestion with N-glycanase collapsed the 60-90K component into a sharp 35K band. We conclude that: (1) a 60-90K membrane glycoprotein localised on the luminal face of tubulovesicles and canaliculi of parietal cells interacts strongly with tomato lectin and RCA120; and (2) the glycoprotein is composed of a 35K core protein glycosylated with N-glycans probably containing poly-N-acetyllactosamine sequences with terminal galactosyl residues. The properties of this 60-90K glycoprotein are identical to a major parietal cell autoantigen recognised by sera of patients with autoimmune gastritis.(ABSTRACT TRUNCATED AT 400 WORDS)
胃壁细胞的细胞质微管泡状膜和小管状膜与盐酸分泌密切相关。为了鉴定这些膜的糖蛋白，我们检测了一组凝集素与大鼠、狗和猪胃石蜡切片中壁细胞的反应性。来自番茄（Lycopersicon esculentum）和马铃薯（Solanum tuberosum）的多聚N-乙酰乳糖胺特异性凝集素，以及半乳糖特异性凝集素蓖麻凝集素（RCA120），在所有三种动物的壁细胞胞质中均显示出强烈结合，其模式表明存在细胞内膜网络。用自身免疫性胃炎患者的壁细胞自身抗体进行双标记研究，证实了这些凝集素与壁细胞的结合。黏液细胞和黏液也能与这些凝集素强烈结合。其他胃细胞类型用番茄或马铃薯凝集素染色均无反应，但用RCA120染色呈弱阳性。使用生物素化的番茄凝集素或RCA120以及链霉亲和素-金对凝集素结合位点进行电子显微镜检查，发现其特异性结合于壁细胞微管泡状膜和小管状膜的腔面以及黏液细胞分泌颗粒的内容物。番茄凝集素和RCA120通过凝集素印迹法与大鼠、狗和猪胃膜中一种表观分子量为60 - 90×10³ Mr的主要蛋白发生反应。通过固相凝集素测定评估，一个富含K⁺依赖性磷酸酶活性10倍的微管泡状膜组分，其番茄凝集素结合能力也富集了3倍。在狗微管泡状膜的¹²⁵I标记去污剂提取物中，60 - 90K（K = 10³ Mr）组分与番茄凝集素-琼脂糖亲和支持物结合，并被N,N',N'-三乙酰壳三糖特异性洗脱。用N-聚糖酶消化后，60 - 90K组分塌缩成一条清晰的35K条带。我们得出以下结论：（1）一种定位于壁细胞微管泡和小管腔面的60 - 90K膜糖蛋白与番茄凝集素和RCA120强烈相互作用；（2）该糖蛋白由一个35K核心蛋白组成，其N-聚糖糖基化，可能含有带有末端半乳糖基残基的多聚N-乙酰乳糖胺序列。这种60 - 90K糖蛋白的特性与自身免疫性胃炎患者血清识别的一种主要壁细胞自身抗原相同。（摘要截短于400字）
