Morrison A I, Keeble S, Watt F M
Max-Planck-Institut für Systemphysiologie, Dortmund, Federal Republic of Germany.
Exp Cell Res. 1988 Aug;177(2):247-56. doi: 10.1016/0014-4827(88)90459-4.
Peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes, both in intact epidermis and in culture. In order to investigate the significance of this change in cell-surface carbohydrate we have identified the PNA-binding glycoproteins of cultured human keratinocytes and raised antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of [14C]galactose- or [14C]mannose- and [14C]glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to Pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium (0.1 mM calcium ions) were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification.
已知花生凝集素（PNA）在完整表皮和培养物中，与正在进行终末分化的角质形成细胞的结合比与增殖的角质形成细胞的结合更强。为了研究细胞表面碳水化合物这种变化的意义，我们鉴定了培养的人角质形成细胞的PNA结合糖蛋白，并制备了针对它们的抗体。用半乳糖从PNA亲和柱上洗脱的[¹⁴C]半乳糖、[¹⁴C]甘露糖和[¹⁴C]葡糖胺标记的细胞提取物经聚丙烯酰胺凝胶电泳（PAGE）分离出两条高度糖基化的条带，分子量分别为110 kDa和250 kDa。在转移到硝酸纤维素膜上的未标记细胞提取物的PNA印迹上也检测到了较高分子量的条带。两条带都对链霉蛋白酶消化敏感，但只有250 kDa的条带能被胰蛋白酶消化。我们制备（抗PNA-gp）的兔抗血清能从细胞提取物中免疫沉淀这两条带。与PNA不同，抗PNA-gp与增殖细胞和终末分化细胞的结合程度相同，这表明PNA结合糖蛋白的一些表位在终末分化之前就存在于细胞表面。当在低钙培养基（0.1 mM钙离子）中单层生长的角质形成细胞换成含有2 mM钙离子的培养基以诱导桥粒形成和分层时，PNA结合糖蛋白发生了显著的重新分布，它们集中在细胞之间的边界处。这可能表明这些糖蛋白在分层过程中的细胞间相互作用中发挥作用。
