Human RBCs Blood Group Conversion From a to O Using a Novel a-N-Acetylgalactosaminidase of High Specific Activity

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  Chinese Science Bulletin © 2008 SCIENCE IN CHINA PRESS  Springer | | Chinese Science Bulletin | July 2008 | vol. 53 | no. 13 | 2008-2016   Human RBCs blood group conversion from A to O using a novel -N-acetylgalactosaminidase of high specific activity YU ChengYu 1 , XU Hua 2 , WANG LiSheng 3 , ZHANG JianGeng 2  & ZHANG YangPei 1 †   1 Department of Blood Molecular Biology, Institute of Military Transfusion, Academy of Military Medical Science, Beijing 100850, China; 2 Shaanxi Blood Centre, Xi’an 710061, China; 3 Department of Experimental Hematology, Institute of Radiation Medicine, Academy of Military Medical Science, Beijing 100850, China -N-acetylgalactosaminidase ( α NAGA) can convert group A human red blood cells (RBCs) to group O. One novel α  NAGA  gene was cloned by PCR from Elizabethkingia meningosepticum  isolated from a domestic clinical sample. Pure recombinant α NAGA was obtained by genetic engineering and protein purification with a calculated molecule of 49.6 kD. α NAGA was selective for terminal α -N-acetylgalacto- samine residue with a high specific activity. α NAGA could completely remove A antigens of 1 U (about 100 mL) group A 1  or A 2  RBCs in 1 h at pH 6.8 and 25   with a consumption of 1.5 or 0.4 mg recombinant enzyme. Enzyme-converted group A RBCs did not agglutinate after being mixed with monoclonal anti-A or sera of groups A, B, AB and O. Other blood group antigens except ABO had no change. FCM analy-sis showed that A antigens and A 1  antigens disappeared while H antigens increased. It indicated that α NAGA successfully converted human blood group A RBCs to universally transfusable group O RBCs without the risk of ABO-incompatible transfusion reactions. This α NAGA was suitable for producing universal RBCs to increase clinical transfusion safety, improve the RBCs supply, and to decrease transfusion cost and support transfusion service in case of emergency. universal RBCs, blood group conversion, α -N-acetylgalactosaminidase, transfusion, A antigen, A 1  antigen Transfusion safety and shortage of units with specific ABO blood groups are two challenges to the providers of blood in the world. Some subjective and objective causes such as incorrect typing and identifications bring on high mistransfusion rate for half a century. The mis-transfusion rate is about one in 12000 units in the USA, while one in 33000 units is ABO-incompatible. The mortality due to ABO errors is one per 800000 blood units, compared with about one per 2000000 transfu-sions for transmission of HIV, HBV or HCV [1,2] . Inad-vertent mismatching of RBCs for ABO blood groups remains the most common and persistent cause of seri-ous and sometimes fatal adverse events following trans-fusion. Because the shelf time of RBCs is about 6 weeks, shortage of units with specific ABO blood groups often occurs, whereas units with other ABO blood groups are at the risk of outdating and discarding. Many domestic  blood centers had the experience of seeking donors with specific ABO groups. Group O RBCs contain neither A nor B antigens and can be safely transfused into recipi-ents of every ABO blood groups. Thus, group O RBCs are considered “universal RBCs”. One possible solution to the above two problems is to produce universally Received February 21, 2008; accepted April 12, 2008 doi: 10.1007/s11434-008-0248-y † Corresponding author (email: Supported by the National Basic Research Program of China (Grant No. 2002CB713804), National High Technology Research and Development Program of China (Grant No. 102-09-04-02) and the PLA Research Program (Grant No. 2000252910)    YU ChengYu et al. Chinese Science Bulletin  | July 2008 | vol. 53 | no. 13 | 2008-2016   2009      A   R   T   I   C   L   E   S    M   E   D   I   C   A   L   B   I   O   C   H   E   M   I   S   T   R   Y transfusable RBCs, the A and/or B antigens of which have been eliminated by glycosidase. The ABO blood group system, discovered in 1900 by Landsteiner, is of great importance in transfusion medi-cine. ABO system is based on the presence or absence of A and/or B antigens. The ABO blood group structures are glycoproteins or glycolipids essentially, whose specificity is determined by the nature and linkage of monosaccharides at the ends of the carbohydrate chains [3] . Group A RBCs contain A antigens. Similarly, group B RBCs contain B antigens. Group AB RBCs contain both antigens and group O RBCs contain neither antigen. The immunodominant monosaccharide deter-mining group B specificity is a terminal α 1-3 linked galactose, while the corresponding monosaccharide determining group A specificity is an α 1-3 linked α -N-acetylgalactosamine. Group A is divided into two major subgroups: A 1  and A 2 . Subgroup A 1  RBCs have more A antigens than subgroup A 2  and have glycolipids with a repeated A structure, which in A 2  RBCs mainly exists as an A-associated H structure. Subgroup A 1  adds up to >99% in China. If perfect α  NAGA was obtained that could remove α -N-acetylgalactosamine residues of A and A 1  antigens to expose the underlying H antigens,  blood group conversion from A to O would be real-ized [4] . Goldstein pioneered the field of enzymatic conversion of blood group B to O using α -galactosidase from coffee  beans [5 ― 7] . It is difficult to convert group A RBCs be-cause the biochemistry of A 1  antigen is more complex than that of B antigen. A variety of α  NAGAs derived from human beings, chicken liver, sea squirt, yeast, ni-ger and so on have been cloned, but the specificity and efficiency were low [8 ― 11] . The α  NAGA and endo- β ga- lactosidase from could convert group A 2  RBCs to O, but they could not convert group A 1  RBCs [12,13] . The poor biochemistry characteristics of these glycosidases were the major obstacle to clinical implementation. In 2002 an α  NAGA was found from spp. by NEB Co. which could selec-tively cleave α -N-acetylgalactosamine [14] . In this re-search one novel α   gene was cloned by PCR from isolated in domestic clinical sample and the conversion of blood group A and AB was explored. 1 Materials and methods 1.1 Strains and reagents The was isolated from domestic clinical sample. The DH5 α  and BL21(DE3) were conserved in our laboratory. Plasmid pET-22b(+) was a gift from research fellow Zhang. I, I and T 4  DNA ligase were purchased from NEB. LA Taq DNA polymerase and pMD-18T were purchased from Takara. FITC-conjugated mono-clonal anti-H and anti-A were purchased from Sigma and Becton Dickinson respectively. Monoclonal anti-D was purchased from Celliance. Monoclonal anti-H and anti-A 1  were purchased from Dominion. Monoclonal anti-A and anti-B were purchased from Changchun Bode Co. Monoclonal anti-C, anti-c, anti-E, anti-e were pur-chased from Shanghai Blood Biological Co. Monoclonal anti-Jk  a , anti-Jk   b , anti-P 1 , anti-Le a , anti-Le  b , anti-M, anti-N, anti-Fy a , anti-Fy  b  and anti-k were purchased from Sanquin Plesmanlaan. Monoclonal anti-6×His was  purchased from Tiangen company. Goat anti-mouse μ -chain specific alkaline phosphatase conjugate was  purchased from Jackson Co (USA). The -nitrophenyl (NP), NP-N-acetyl- α -D-galactosaminide and other  NP-linked monosaccharide substrates were purchased from Sigma. Protein purification system AKTA FPLC, chromatography resins Ni 2+  Sepharose 6 FF and Phenyl Sepharose 6 FF were purchased from Pharmacia. Hu-man RBCs and sera were obtained from Shaanxi Blood Centre and were less than 21 d old. IPTG was purchased from Amresco. Synthesis of primes and sequencing was completed by Aoke Biology Co. Other materials were of analysis purity grade. 1.2 Culture conditions The was cultured in a medium composed of 5 g/L yeast extract, 5 g/L tryptone, 5 g/L NaCl, 3 g/L KH 2 PO 4  (pH 7.2) at 35 ℃  for 24 h shaken at 200 r/min. The culture was centrifuged at 13201× for 5 min. Cell pellets were collected for ge-nomic DNA purification. 1.3 Cloning of α  NAGA  gene The entire coding region of α  was amplified by PCR from genomic DNA using the high fidelity PCR system with primers F1(5 ′ -ATGGGCGCCTTAATTC- CC-3 ′ ) and R1: (5 ′ -TTAGTAGTCGTCATTTATTGC- 3 ′ ) [14] . PCR was run for 30 cycles consisting of 30 s of    2010   YU ChengYu et al. Chinese Science Bulletin  | July 2008 | vol. 53 | no. 13 | 2008-2016   denaturation at 95 ℃ , 30 s of annealing at 50 ℃ , and 90 s of extension at 72 ℃ . The product was ligated into  pMD-18T vector and sequenced. 1.4 Construction of plasmid pET-22b-    NAGA  and induction expression A truncated construct lacking the N-terminal signal pep-tide (residues 1 ― 17) was amplified by PCR from  pMD-18T- α   using the following primers/restric- tion sites: F2, 5 ′ -ATCATATGCCAAAAAAAGTAAG- AATTGC-3 ′ ( I); R2, 5 ′ -TGCTCGAGGTAGTCG- TCATTTATTGC-3 ′ ( I). PCR was run for 30 cycles consisting of 30 s of denaturation at 95 ℃ , 30 s of an-nealing at 43 ℃ , and 90 s of extension at 72 ℃ . The PCR  product was digested with I/ I, ligated into  pET-22b(+) vector, sequenced, and named pET-22b- α  . This plasmid was transferred into BL21 (DE3). A single colony was chosen to inoculate a 10-mL starter culture in LB containing 100 μ g/mL ampicillin. Following incubation at 37 ℃  with shaking at 200 r/min for 12 h, the culture was added to 1-L culture and incu- bation was continued with shaking at 25 ℃ . When 600  was 0.8, IPTG was added into the culture to a final con-centration of 0.2 mmol/L. Following a further 12-h in-cubation with shaking, the cells were harvested by cen-trifugation. 1.5 Purification of .NAGA According to the instructions of Amersham, each gram  pellet was resuspended in 10 mL buffer A (10 mmol/L  Na 2 HPO 4 , 10 mmol/L NaH 2 PO 4 , 0.5 mol/L NaCl, 20 mmol/L imidazole, pH 7.4). Then 0.2 mg/mL lysozyme, 20 μ g/mL DNase I, 1 mmol/L MgCl 2  and 1 mmol/L PMSF were added and the solution was stirred for 30 min at 25 ℃ . The cells were sonicated at 0 ℃ . After cen-trifugation at 8603 g for 20 min, the supernatant was applied to an Ni 2+  Sepharose 6 FF column equilibrated with buffer A. The column was washed with 10 column volumes of the same buffer. The bound proteins were eluted gradiently with buffer B (10 mmol/L Na 2 HPO 4 , 10 mmol/L NaH 2 PO 4 , 0.5 mol/L NaCl, 500 mmol/L im-idazole, pH 7.4). Fractions containing enzyme activity were concentrated and dialyzed against buffer C (25 mmol/L Na 2 HPO 4 , 25 mmol/L NaH 2 PO 4 , 0.5 mol/L  NaCl, 1 mol/L (NH 4 ) 2 SO 4 , pH 7.4). The proteins were then applied to a Phenyl Sepharose 6 FF column which  previously equilibrated with buffer C. This column was washed with 10 column volumes of buffer C and the  bound proteins were eluted gradiently with buffer D (25 mmol/L Na 2 HPO 4 , 25 mmol/L NaH 2 PO 4 , 0.5 mol/L  NaCl, pH 7.4). Fractions containing enzyme activity were collected and applied to Sepharose G-25 column to desalt. The protein concentration was determined with BCA protein quantification kit. Immunoblotting was  performed with the purified α  NAGA resolved by 12% SDS-PAGE and transferred to a PVDF membrane by using primary antibody against 6×His (1:1000 dilution) and the secondary anti-mouse IgG antibody (1:2000 di-lution). Alkaline phosphatase activity was detected with ECL. 1.6  K  m ,  K  cat  and substrate specificity of .NAGA Assays with chromogenic GalNAc α  NP substrate were carried out at 25 ℃  for 10 min in reaction mixtures of 400 μ L with 50 mmol/L Na 2 HPO 4 , 50 mmol/L NaH 2 PO 4 , 50 mmol/L NaCl (pH 6.8), containing 0.034 μ g of en-zyme and 1000 μ mol/L, 500 μ mol/L, 200 μ mol/L, 100 μ mol/L, 50 μ mol/L or 20 μ mol/L GalNAc α  NP re-spectively [15] . Reactions were terminated by adding 600 μ L, 1 mol/L Na 2 CO 3  and NP formation was quantified at 405 nm. m , cat  and m / cat  were calculated. One unit of enzyme activity was defined as the amount to cleave 1 μ mole of GalNAc α -NP substrate per minute under the above conditions. Similarly, assays with other chromogenic NP substrates were carried out under the same conditions, including NP- α L-fucopyranoside,  NP- α -D-galacto-pyranoside, NP- α -D-manno-pyrano- side, NP- α -D-gluco-pyranoside, NP- β -D-fucopyra- noside, NP- β -D-mannopyranoside, NP- β -D-galacto-  pyranoside, NP- β -D-glucopyranoside, NP-N-acetyl- α -D-glucosaminide, NP-N-acetyl- β -D-glucosaminide and NP-N-acetyl- β -D-galactosaminide. 1.7 Enzymatic conversion of RBCs Standard enzymatic conversion reactions were per-formed in 1 mL reaction mixtures containing 250 mmol/L glycine, pH 6.8 with 40% fresh packed RBCs and 6 μ g enzyme at 25 ℃  for 1 h. Packed RBCs of groups A 1 , A 2 , A 1 B, A 2 B and O were obtained from Shaanxi blood centre. RBCs were prewashed with saline once and with 250 mmol/L glycine twice. The RBCs were incubated for 1 h at 25 ℃  with gently and com- pletely mixing every 15 min, followed by 2 repeated washing cycles with 1 : 4 (/) of saline by centrifugation    YU ChengYu et al. Chinese Science Bulletin  | July 2008 | vol. 53 | no. 13 | 2008-2016   2011      A   R   T   I   C   L   E   S    M   E   D   I   C   A   L   B   I   O   C   H   E   M   I   S   T   R   Y at 1500 r/min. After washing enzyme-treated RBCs were ABO-typed using licensed monoclonal antibody (mAb) reagents. The main rare blood group antigens were de-tected, too [16] . 150 mL saline was added to 1 U RBCs. After gently mixing, RBCs were centrifugated at 2000 r/min for 8 min and the supernatant was discarded. The RBCs were washed with conversion buffer twice and 150 mL con-version buffer and determined enzyme was added finally. Following incubation at 25 ℃  for 1 h, RBCs were ABO-typed using anti-A mAb. 1.8 Flow cytometry Flow cytometry analysis of native and enzyme-con- verted RBCs was performed using an FACScan flow cytometer (Becton Dickinson) with FITC-conjugated monoclonal anti-A and anti-H. Briefly, 200000 RBCs were prewashed with saline twice and resuspended in 100 μ L saline. Then 2 μ L of undiluted primary antibody was added and incubated for 60 min in darkness at 25 ℃ . RBCs were analyzed after 4 washes and resuspension in 500 μ L saline. A total of 10000 events were evaluated. 1.9 Main side cross-match tests (recipients’ sera+ donors’ RBCs) RBCs from ten group A 1  donors and sera from 5 group A 1 , B, A 1 B and O donors were collected. After treatment with 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 [16] . Similarly, en-zyme-converted group A 1 B RBCs were mixed with sera of groups B and A 1 B separately. 2 Results 2.1 Cloning of α  NAGA gene and expression plasmid construction The α  gene derived from consisted of 1335 bp (Figure 1). GenBank accession number was EU495239. This gene encoded 444 amino acid residues. The (G+C) content was 39%. The sequence identities of DNA and protein compared with α   from ATCC 13253 were 92.6% and 94.6% respectively. The difference was dispersive. A truncated construct lacking the N-terminal signal peptide (residues 1 ― 17) was amplified by PCR using F2 and R2 primes. The restriction endoenzyme reactions and sequencing indicated that the sequence of pET-22b- α   was correct. The recombinant α  NAGA 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. Figure 1 Gene cloning of α  gene by PCR. 1, molecular mass marker; 2, α  gene from domestic ; 3, α  gene from ATCC 13253; 4, negative control. 2.2 Expression and purification of .NAGA With the plasmid pET-22b- α  , recombinant α  NAGA was expressed as a soluble protein at a level of about 100 mg/L of culture. α  NAGA accounted for about 16% of cytoplasmic proteins (Figure 2). The purified α  NAGA moved as a single protein band of about 50 kD upon SDS-PAGE, which was in agreement with its cal-culated molecular mass from the nucleotide sequence (Figure 3(a)). Reverse HPLC showed that the purifica-tion of purified α  NAGA was 97%. α  NAGA appeared as a single band upon Western blot, which agreed well with its molecular mass (Figure 3(b)). Dialysis, dilution or chromatography was not able to recover the activity of inclusion. Figure 2 SDS-PAGE of recombinant α  NAGA purification. 1, molecular mass marker; 2, total proteins of pET-22b; 3, uninduced total proteins of  pET-22b- α  ; 4, induced total proteins of pET-22b- α  ; 5, su- pernatant of cell culture medium; 6, supernatant of lysates; 7, inclusions. 2.3  K  m ,  K  cat  and substrate specificity of NAGA In the method of Lineweaver-Burk double reciprocal  plot, m  was 63 μ mol/L, cat  was 7.47 s − 1 , max  was 768
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