Sutherland D R, Marsh J C, Davidson J, Baker M A, Keating A, Mellors A
Oncology Research Laboratory, Toronto Hospital, Ontario, Canada.
Exp Hematol. 1992 Jun;20(5):590-9.
Our previous studies have shown that a unique glycoprotease from Pasteurella haemolytica specifically cleaves only proteins containing sialylated O-linked glycans. The hematopoietic progenitor cell antigen, CD34, which is heavily glycosylated with both N- and O-linked glycans, is readily cleaved by this protease. In this study, we demonstrate that the epitopes detected by five of the seven CD34 monoclonal antibodies are removed by the glycoprotease. The differential sensitivity of the CD34 epitopes to cleavage with either neuraminidase and/or glycoprotease establishes three classes of epitopes: 1) (class I) those identified by MY10, B1.3C5, 12.8, and ICH3 that are differentially affected by neuraminidase and removed by the glycoprotease; 2) (class II) the epitope detected by QBEND 10 that is removed only by the glycoprotease; and 3) (class III) those identified by TUK3 and 115.2 that are not removed by either enzyme. Cleavage of the 110-kd CD34 structure by the glycoprotease generates a major cell-bound fragment of about 75 kd, identified by the class III antibodies. We have also used the enzyme to improve the rapid recovery of CD34+ cells selected by immunomagnetic affinity techniques. In a preclinical model, we separated CD34+ KG1 cells with high yield (90%-95%) and high purity (94%-98%) from sham mixtures containing 50% CD34- cells. We also separated CD34+ blast cells from a patient in megakaryoblastic crisis of chronic myelogenous leukemia. In this case, the purity and yield were 93% and 94%, respectively. Enzyme treatment had no detrimental effect on cell viability, and the treated cells showed a normal quantitative expression and distribution of CD34 antigen as assessed with class III antibodies. We conclude that the P. haemolytica glycoprotease has potential to improve the isolation, from human bone marrow, of primitive hematopoietic cells that carry the CD34 antigen.
我们之前的研究表明，溶血巴斯德菌的一种独特糖蛋白酶仅特异性切割含有唾液酸化O-连接聚糖的蛋白质。造血祖细胞抗原CD34高度糖基化，同时含有N-连接和O-连接聚糖，很容易被这种蛋白酶切割。在本研究中，我们证明7种CD34单克隆抗体中的5种所检测到的表位被该糖蛋白酶去除。CD34表位对神经氨酸酶和/或糖蛋白酶切割的不同敏感性确定了三类表位：1）（I类）由MY10、B1.3C5、12.8和ICH3识别的表位，它们受神经氨酸酶的影响不同并被糖蛋白酶去除；2）（II类）由QBEND 10检测到的表位，仅被糖蛋白酶去除；3）（III类）由TUK3和115.2识别的表位，两种酶均不能将其去除。糖蛋白酶对110-kd CD34结构的切割产生了一个约75 kd的主要细胞结合片段，由III类抗体识别。我们还使用该酶提高了通过免疫磁亲和技术选择的CD34+细胞的快速回收率。在一个临床前模型中，我们从含有50% CD34-细胞的假混合物中以高产率（90%-95%）和高纯度（94%-98%）分离出CD34+ KG1细胞。我们还从一名慢性粒细胞白血病巨核细胞危象患者中分离出CD34+原始细胞。在这种情况下，纯度和产率分别为93%和94%。酶处理对细胞活力没有有害影响，并且用III类抗体评估时，处理后的细胞显示CD34抗原的正常定量表达和分布。我们得出结论，溶血巴斯德菌糖蛋白酶有潜力改善从人骨髓中分离携带CD34抗原的原始造血细胞的方法。
