新型α-N-乙酰半乳糖胺酶酶解红细胞A型抗原的机理研究

Group O red blood cells(O RBCs) are referred as 'universal blood' which are suitable for transfusion to recipients of group O, A, B and AB. Conversion of group A, B, or AB RBCs to O RBCs will increase the supply of universal RBCs. α-N-acetylgalactosaminidase(NAGA) is a hydrolase which can convert group A RBCs (A RBCs) to O RBCs. Because of the complexity of group A antigens, most NAGA isolated from different species could not convert A1 RBCs into O RBCs. Recently，a new NAGA which could efficiently convert both A1 RBC and A2 RBC to O RBC was cloned and expressed in our laboratory. However, the mechanism of new NAGA, especially its roles in hydrolysis of A1 antigen, is still unclear. For example, why the NAGA can cleave both A antigen and A1 antigen (more complicate than A antigen)? Does NAGA have endo-glycosidase activity? Is the antigens of enzymatic converted group O RBCs from A RBCs( A-ECO RBCs) same as that of O RBCs? Is there any new antigen produced after hydrolysis? The aim of this project is to study the relationship between the structure and function of NAGA, the products of A-like oligosaccharide substrates after NAGA digestion, the saccharides removed from A1 RBCs after NAGA treatment, the carbohydrate chain on the surface of A-ECO RBCs, as well as the interaction between A-ECO RBCs and the serum of group O and group B recipients, so as to eluciate the mechanism of new NAGA in hydrolysis of A1 antigen and the immunological characteristics of antigens on the surface of A-ECO RBCs. These results will provide date support for the safety of clinical application of A-ECO RBCs.

α-N-乙酰半乳糖胺酶（NAGA）是一种糖水解酶，可将A型红细胞（A RBC）转变为通用O型红细胞(O RBC)，但大多NAGA只能将A2 RBC转变成O RBC，无法将A1 RBC转变成O RBC。最近我们克隆表达了一种新型NAGA，可将A1、A2 RBC都转变为O RBC。但这种新型NAGA水解A型抗原的机理尚不清楚。如NAGA如何既作用于A抗原又作用于复杂的A1抗原？NAGA是否具备内切酶活性？NAGA酶解转变形成的O RBC(A-ECO RBC)的抗原结构是否与O RBC一致？是否暴露了新抗原表位（比如3型H抗原）？本课题将通过研究上述问题，明确NAGA的作用机理和A-ECO RBC的抗原特性。这一研究将为A-ECO RBC临床应用的安全性提供科学依据。

脑膜脓毒性金黄杆菌来源的α-N-乙酰半乳糖胺酶（NAGA）是一种糖水解酶，既可将含A抗原的A2 型红细胞（RBC）酶解转变成通用O型红细胞(A2 ECO RBC)， 又能将含复杂A1抗原的A1 RBC转变为A1 ECO RBC。NAGA如何既作用于A抗原又作用于复杂的A1抗原，NAGA是一种外切酶还是一种兼具内切和外切酶活性的新型糖苷水解酶是本项目需要解决的关键科学问题；另外A ECO RBC的抗原结构是否与O RBC一致，在临床上应用的安全性如何也是本项目需要回答的另一科学问题。.本项目通过对NAGA酶解A1 RBC获得的寡糖成分的分析，证实了产物为单糖N-乙酰半乳糖胺，流式结果证明A1 ECO RBC表面含有大量的3-H抗原，从而明确了a-N-乙酰半乳糖胺酶为外切酶，切掉A1型红细胞糖链末端的N-乙酰半乳糖胺将红细胞上的3型A抗原变成了3型H抗原。通过对红细胞洗涤机程序及耗材的改进建立了自动化制备ECO RBC的方法。通过对酶解转变的红细胞包括A ECO RBC，B ECO RBC，AB ECO RBC进行的结构功能及安全性评价，发现血型转变对红细胞的结构功能没有明显的影响，交叉配血实验证实B ECO RBC是安全的，但A ECO RBC，AB ECO RBC因为与O血清和B血清凝集的比例较高目前尚不能作为通用型RBC在临床上使用。