Kure S, Yoshie O
J Immunol. 1986 Dec 15;137(12):3900-8.
We have isolated a syngeneic monoclonal antibody (HepSS-1) reactive to a murine methylcholanthrene-induced fibrosarcoma, Meth-A. HepSS-1 also bound to a wide variety of established and fresh normal cells derived from not only mice but also other species such as human, monkey, rat, hamster, and chicken. Immunoprecipitation of surface iodinated Meth-A cell extract with HepSS-1, as well as Sepharose 4B gel chromatography of Meth-A cell extract and detection of antigens recognized by HepSS-1 by a sandwich-type radioimmunoassay revealed that the HepSS-1 antigens were composed of several molecular species, with one as large as approximately 10(6) daltons. The following evidence indicates that HepSS-1 specifically recognizes an epitope present in heparan sulfate glycosaminoglycan (HS-GAG). First, treatment of Meth-A cells with heparitinase or heparinase, but not with chondroitinase ABC or hyaluronidase, resulted in the loss of HepSS-1 binding. Second, HS-GAG but not seven other types of GAG (hyaluronic acid, heparin, chondroitin, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and keratan sulfate) inhibited HepSS-1 binding to Meth-A cells. Third, HepSS-1 bound with HS-GAG but not with the seven other types of GAG. From the binding analysis of HepSS-1 to various modified HS-GAG and whale omega-heparin, it is additionally suggested that HepSS-1 recognizes an epitope closely related to O-sulfated and N-acetylated glucosamine. We found that NIH 3T3 cells expressed more HepSS-1 epitopes at a low cell density than at confluency and in G2 + M than in G1, whereas NIH 3T3 cells transformed with Kirsten-ras oncogene or SV-40 expressed high levels of HepSS-1 epitopes and ceased to show the density-dependent change in the amount of HepSS-1 epitopes. These observations were also reproduced by using NIH 3T3 cells transformed with a temperature sensitive Kirsten murine sarcoma virus maintained at permissive and non-permissive temperatures. Thus HepSS-1 is a first monoclonal antibody to HS-GAG and seems to be useful to elucidate changes in cell surface HS-GAG in normal cell growth and cell transformation.
我们分离出了一种同基因单克隆抗体（HepSS - 1），它能与小鼠甲基胆蒽诱导的纤维肉瘤Meth - A发生反应。HepSS - 1还能与多种已建立的以及新鲜的正常细胞结合，这些细胞不仅来源于小鼠，还包括其他物种，如人类、猴子、大鼠、仓鼠和鸡。用HepSS - 1对表面碘化的Meth - A细胞提取物进行免疫沉淀，以及对Meth - A细胞提取物进行琼脂糖4B凝胶层析，并通过夹心型放射免疫测定法检测HepSS - 1识别的抗原，结果显示HepSS - 1抗原由几种分子组成，其中一种分子量约为10⁶道尔顿。以下证据表明HepSS - 1能特异性识别硫酸乙酰肝素糖胺聚糖（HS - GAG）中存在的一个表位。首先，用硫酸乙酰肝素酶或肝素酶处理Meth - A细胞，而非用软骨素酶ABC或透明质酸酶处理，会导致HepSS - 1结合丧失。其次，HS - GAG而非其他七种类型的糖胺聚糖（透明质酸、肝素、软骨素、硫酸软骨素4 - 硫酸酯、硫酸软骨素6 - 硫酸酯、硫酸皮肤素和硫酸角质素）能抑制HepSS - 1与Meth - A细胞的结合。第三，HepSS - 1与HS - GAG结合，而非与其他七种类型的糖胺聚糖结合。从HepSS - 1与各种修饰的HS - GAG和鲸ω - 肝素的结合分析来看，还表明HepSS - 1识别一个与O - 硫酸化和N - 乙酰化葡糖胺密切相关的表位。我们发现，NIH 3T3细胞在低密度时比汇合时表达更多的HepSS - 1表位，在G2 + M期比在G1期表达更多；而用 Kirsten - ras癌基因或SV - 40转化的NIH 3T3细胞则高水平表达HepSS - 1表位，且不再显示HepSS - 1表位数量的密度依赖性变化。使用在允许温度和非允许温度下培养的温度敏感型 Kirsten 小鼠肉瘤病毒转化的NIH 3T3细胞，也重现了这些观察结果。因此，HepSS - 1是第一种针对HS - GAG的单克隆抗体，似乎有助于阐明正常细胞生长和细胞转化过程中细胞表面HS - GAG的变化。
