The Study On Human RBCs Blood Group Conversion From A To O

Group O red blood cells (RBCs) contain neither A nor B antigens and can be safely transfused into recipients of any ABO blood groups. So, group O RBCs are considered"universal RBCs". Blood group conversion from group A, B and AB to group O plays an important role in clinical transfusion and military medicine. Universal RBCs could eliminate the risk for ABO-incompatible transfusion errors, enhance the transfusion safety, eliminate the shortage of units with specific ABO blood groups, reduce wastage of excessive donation of the less needed groups, reduce cost for logistics of blood group inventory management and for repeated typing of RBCs units, improve RBC supply in case of emergency. If universal RBCs were successful, it could be the main form of RBCs in clinical transfusion in the future. There are two methods for producing the universal RBCs: PEG shielding and glycosidase digestion. The former method is expensive due to high dose. Moreover, PEG antibodies have been found in some people recently. The later method is efficient, specific, safe and cheap. The key point is to find suitableα-galactosidase (αGAL) andα-N-acetylgalactosaminidase (αNAGA).Goldstein pioneered the field of enzymatic conversion of blood group B to O usingα-GAL from coffea beans in 1982. Since that time a lot of progress has been made. Several suitableαGALs andαNAGAs have been found by Zymequest company in America. The clinical trials are being performed smoothly. Our laboratory first clonedαGAL gene from Catimor caffea bean in China and caught out the blood group conversion from B to O. The clinical trial has been approved by SFDA. Blood conversion from A to O is difficult because of the A antigens'complex structure. The goal of this research is to find suitableαNAGA which can cleave A and A1 antigens to H antigens and convert blood group from A to O.One novelαNAGA gene (Genebank accession number: EU495239) was cloned by PCR from Elizabethkingia meningosepticum isolated from a domestic clinical sample. TheαNAGA gene consisted of 1335 bp and encoded 444 amino acid residues. The sequence identities of DNA and protein compared withαNAGA from ATCC 13253 (1335 bp) were 92.6% and 94.6% respectively. The pET-22b-αNAGA plasmid was used in prokaryotic expression system. In the condition of 25℃, low rotation speed, 0.1 mM IPTG induction, LB medium and growth for 12 h, recombinantαNAGA was expressed with high yield. The recombinantαNAGA protein consisted of 436 amino acid residues with a calculated molecular mass of 49.6 kD with a 6×His tag at the C-terminus. Highly pureαNAGA was obtained by protein purification. Reverse phase HPLC showed that the purification of purifiedαNAGA was about 97%. The recombinantαNAGA was identified by SDS-PAGE, Western blot and enzyme activity test.The optimal temperature ofαNAGA was 15℃～45℃and it was stable at 4℃～25℃. The optimal pH was 6⁷ and it was stable at pH 3⁸. Metal ion including K+,Li+,Na+,Co²⁺,Ca²⁺,Mg²⁺,Fe3+ and glucose, citric, adenine, manicol, imidazole, EDTA and so on didn't affect enzyme specific activity. Fe²⁺, Ni²⁺ and Zn²⁺ inhibited specific activity slightly, while Mn²⁺ enhanced specific activity greatly. In the method of Lineweaver-Burk double reciprocal plot, Km was 63μM,Kcat was 7.47 s-1, Vmax was 768μmol/(L·min) and Kcat/Km was 118.5 (s·μΜ)-1. The specific activity of substrate pNP-N-acetyl-α-D-galactosamine was presumed to be 100%, the specific activities of other pNP-linked monosaccharide conjugates were less than 0.01%. These data showed thatαNAGA was highly selective for terminal N-acetyl-α-D-galactosamine residues. In reaction mixtures of 400μL with 50 mM Na2HPO4, 50 mM NaH2PO4, 50 mM NaCl (pH 6.8), containing 0.034μg of enzyme and appropriable pNP-GalNAc one unit of enzyme activity was defined as the amount to cleave 1μmole of pNP-GalNAc substrate per minute at 25℃. The specific activity ofαNAGA was 3.36 U/mg.Buffers, hematocrit and temperature greatly affected enzyme activity. The activity ofαNAGA was poor in saline or PBS solution but high in 250 mM glycine (pH 6.8). The optimal hematocrit was 20%～40% and the activity decreased rapidly at hematocrit above 50%. In reaction mixtures of 1 mL with 0.02 U enzyme and 40% hematocrit, it took 30 min to completely remove A and A1 antigens at 37℃or 25℃and 60 min at 15℃and 120 min at 4℃. The conversion process was linear with enzyme dose and time.αNAGA could cleave A antigens and A1 antigens on the surface of group A1, A2, A1B or A2B RBC. Standard enzymatic conversion reactions were performed in 1 mL reaction mixtures containing 250 mM glycine (pH 6.8) with 40% fresh packed RBCs and 6μg enzyme at 25℃for 1 h. After enzymatic conversion, group A1 or A2 RBCs didn't agglutinate with anti-A mAb, anti-A1 mAb or human type O serum, while agglutinated stronger with anti-H mAb. The shape of RBC didn't change after enzymatic conversion. RBCs from ten group A1 donors and sera from 5 group A1, B, A1B and O donors were collected. After treatment in the above methods, RBCs were mixed with sera of different ABO blood groups separately. The typing tests were performed using standard methods of saline, polyamine and anti-human globulin. Ten units of enzyme-converted group A1 RBCs showed no agglutination or hemalyzation after being mixed with 5 human serum samples with groups O, A1, B or A1B. It indicated thatαNAGA successfully converted human blood group A RBCs to universally transfusable group O RBCs without the risk of ABO-incompatible transfusion reactions. The main rare blood group antigens didn't change afterαNAGA treatment. In 1 mL reaction mixtures containing 250 mM glycine, pH 6.8 with 40% fresh packed RBCs and 6μg enzyme at 25℃for 1 h, A antigens on A2 and A2B subgroup RBCs disappeared almost completely after enzymatic treatment for 10 min, whereas A and A1 antigens on subgroup A1 and A1B RBCs disappeared completely after being treated for 60 min. FCM showed that the quantity of A antigens or H antigens of enzyme-converted group A or AB RBCs were as many as those of native group O RBCs. The enzymatic conversion was same for group A1 and A1B RBC. At 25℃and 40% hematocrit, 1.5 mg enzyme was required to convert 1 unit (¹00 mL packed RBCs in China) of group A1 RBCs. Conversion of the weak A2 subgroup, however, required only 0.4 mg enzyme. The different enzyme dose was ascribed to different structures and quantities of A and A1 antigens. Approximately 30 mg or 7.5 mgαNAGA derived from ATCC 13253 was required to completely convert 100 mL packed RBCs of group A1 or A2. After washing, the remaining sera, ACD-B RBC preservation solution, MAP RBC addition elements (citric acid, citric sodium, glucose, NaH2PO4, adenine, manicol and so on) no longer affected enzyme activity.Combination ofαGAL from coffea bean andαNAGA could convert group A1B and A2B to O. Enzyme-converted group A1B or A2B RBCs didn't agglutinate with anti-A mAb, anti-A1 mAb, anti-B mAb or type O serum, while agglutinated stronger with anti-H mAb. FCM showed that A antigens, A1 antigens and B antigens disappeared and H antigens increased, which were similar with group O RBC.In summary,αNAGA cloned from Elizabethkingia meningosepticum was very suitable for enzymatic conversion of RBCs due to its excellent characteristics. It could convert subgroup A1 and A2 RBCs to group O and convert A1B and A2B subgroup RBCs to group B. The enzymatic conversion was efficient, specific and complete. The enzyme consumption was about one in a thousand of those of eukaryoticαNAGAs. The conversion process was convenient and cheap and was able to be performed at room temperature. It was promising in clinical use.