Jalkanen M, Rapraeger A, Bernfield M
Department of Pediatrics, Stanford University School of Medicine, California 94305.
J Cell Biol. 1988 Mar;106(3):953-62. doi: 10.1083/jcb.106.3.953.
Cultured mouse mammary (NMuMG) cells produce heparan sulfate-rich proteoglycans that are found at the cell surface, in the culture medium, and beneath the monolayer. The cell surface proteoglycan consists of a lipophilic membrane-associated domain and an extracellular domain, or ectodomain, that contains both heparan and chondroitin sulfate chains. During culture, the cells release into the medium a soluble proteoglycan that is indistinguishable from the ectodomain released from the cells by trypsin treatment. This medium ectodomain was isolated, purified, and used as an antigen to prepare an affinity-purified serum antibody from rabbits. The antibody recognizes polypeptide determinants on the core protein of the ectodomain of the cell surface proteoglycan. The reactivity of this antibody was compared with that of a serum antibody (BM-1) directed against the low density basement membrane proteoglycan of the Englebarth-Holm-Swarm tumor (Hassell, J. R., W. C. Leyshon, S. R. Ledbetter, B. Tyree, S. Suzuki, M. Kato, K. Kimata, and H. Kleinman. 1985. J. Biol. Chem. 250:8098-8105). The BM-1 antibody recognized a large, low density heparan sulfate-rich proteoglycan in the cells and in the basal extracellular materials beneath the monolayer where it accumulated in patchy deposits. The affinity-purified anti-ectodomain antibody recognized the cell surface proteoglycan on the cells, where it is seen on apical cell surfaces in subconfluent cultures and in fine filamentous arrays at the basal cell surface in confluent cultures, but detected no proteoglycan in the basal extracellular materials beneath the monolayer. The amino acid composition of the purified medium ectodomain was substantially different from that reported for the basement membrane proteoglycan. Thus, NMuMG cells produce at least two heparan sulfate-rich proteoglycans that contain distinct core proteins, a cell surface proteoglycan, and a basement membrane proteoglycan. In newborn mouse skin, these proteoglycans localize to distinct sites; the basement membrane proteoglycan is seen solely at the dermal-epidermal boundary and the cell surface proteoglycan is seen solely at the surfaces of keratinocytes in the basal, spinous, and granular cell layers. These results suggest that although heparan sulfate-rich proteoglycans may have similar glycosaminoglycan chains, they are sorted by the epithelial cells to different sites on the basis of differences in their core proteins.
培养的小鼠乳腺（NMuMG）细胞产生富含硫酸乙酰肝素的蛋白聚糖，这些蛋白聚糖存在于细胞表面、培养基中以及单层细胞之下。细胞表面蛋白聚糖由一个亲脂性膜相关结构域和一个细胞外结构域（或胞外结构域）组成，胞外结构域同时含有硫酸乙酰肝素和硫酸软骨素链。在培养过程中，细胞向培养基中释放一种可溶性蛋白聚糖，它与经胰蛋白酶处理后从细胞释放的胞外结构域无法区分。这种培养基胞外结构域被分离、纯化，并用作抗原，从兔中制备亲和纯化的血清抗体。该抗体识别细胞表面蛋白聚糖胞外结构域核心蛋白上的多肽决定簇。将这种抗体的反应性与针对恩格尔巴思 - 霍尔姆 - 斯沃姆肿瘤低密度基底膜蛋白聚糖的血清抗体（BM - 1）的反应性进行了比较（哈塞尔，J. R.，W. C. 莱肖恩，S. R. 莱德贝特，B. 泰里，S. 铃木，M. 加藤，K. 木田，和 H. 克莱曼。1985 年。《生物化学杂志》250:8098 - 8105）。BM - 1 抗体识别细胞内以及单层细胞之下基底细胞外物质中的一种大的、低密度的富含硫酸乙酰肝素的蛋白聚糖，它在这些部位呈斑片状沉积。亲和纯化的抗胞外结构域抗体识别细胞表面的蛋白聚糖，在亚汇合培养物中它出现在顶端细胞表面，在汇合培养物中出现在基底细胞表面的细丝状阵列中，但在单层细胞之下的基底细胞外物质中未检测到蛋白聚糖。纯化的培养基胞外结构域的氨基酸组成与报道的基底膜蛋白聚糖的氨基酸组成有很大不同。因此，NMuMG 细胞产生至少两种富含硫酸乙酰肝素的蛋白聚糖，它们含有不同的核心蛋白，一种是细胞表面蛋白聚糖，另一种是基底膜蛋白聚糖。在新生小鼠皮肤中，这些蛋白聚糖定位于不同部位；基底膜蛋白聚糖仅见于真皮 - 表皮边界，而细胞表面蛋白聚糖仅见于基底细胞层、棘细胞层和颗粒细胞层的角质形成细胞表面。这些结果表明，尽管富含硫酸乙酰肝素的蛋白聚糖可能具有相似的糖胺聚糖链，但上皮细胞根据它们核心蛋白的差异将它们分选到不同的部位。
