Byrd J C, Lamport D T, Siddiqui B, Kuan S F, Erickson R, Itzkowitz S H, Kim Y S
Gastrointestinal Research Laboratory, V.A. Medical Center, San Francisco, CA 94121.
Biochem J. 1989 Jul 15;261(2):617-25. doi: 10.1042/bj2610617.
Mucin from xenografts of LS174T human colon cancer cells was treated with anhydrous HF for 1 h at 0 degree C to give a product (HFA) with over 80% of the glucosamine and hexose removed, but retaining some galactosamine, and for 3 h at room temperature to give a product (HFB) devoid of carbohydrate. Rabbit antibodies against HFA bound to HFA much more than to HFB, and bound to native mucin to an intermediate extent. Antibodies to HFB bound to HFB more than to HFA, and did not bind to native mucin. Both HFA and native mucin bound a number of lectins, but HFB did not. By SDS/polyacrylamide-gel electrophoresis and size-exclusion h.p.l.c., native mucin and HFA are of apparent molecular mass greater than 400 kDa, whereas HFB is heterogeneous and of low molecular mass. On Western blots, antibody to HFA detected both high-molecular-mass mucin and a 90 kDa protein in homogenates of LS174T cells. Antibody to HFB detected a major 70 kDa band as well as higher-molecular-mass species. In tissue sections of normal colon and colon cancers, antibody to HFA showed both cytoplasmic and extracellular staining, whereas antibody to HFB generally stained only cytoplasmic antigens. These results indicate that anti-HFB antibody is specific for apo-mucin, whereas anti-HFA antibody is specific for GalNAc-apo-mucin.
将LS174T人结肠癌细胞异种移植瘤中的粘蛋白在0℃下用无水HF处理1小时，得到一种产物（HFA），其中80%以上的氨基葡萄糖和己糖被去除，但仍保留一些半乳糖胺；在室温下处理3小时，得到一种无碳水化合物的产物（HFB）。抗HFA的兔抗体与HFA的结合程度远高于与HFB的结合程度，与天然粘蛋白的结合程度介于两者之间。抗HFB的抗体与HFB的结合程度高于与HFA的结合程度，且不与天然粘蛋白结合。HFA和天然粘蛋白都能结合多种凝集素，但HFB不能。通过SDS/聚丙烯酰胺凝胶电泳和尺寸排阻高效液相色谱法分析，天然粘蛋白和HFA的表观分子量大于400 kDa，而HFB具有异质性且分子量较低。在蛋白质免疫印迹中，抗HFA抗体在LS174T细胞匀浆中检测到高分子量粘蛋白和一种90 kDa的蛋白质。抗HFB抗体检测到一条主要的70 kDa条带以及更高分子量的条带。在正常结肠和结肠癌的组织切片中，抗HFA抗体显示出胞质和细胞外染色，而抗HFB抗体通常仅对胞质抗原染色。这些结果表明，抗HFB抗体对脱辅基粘蛋白具有特异性，而抗HFA抗体对GalNAc-脱辅基粘蛋白具有特异性。
