B to O erythrocyte conversion by the recombinant alpha-galactosidase.
BACKGROUND
Human group O red blood cells have great benefit in specialized transfusion areas such as armed conflict and natural calamity. The group B antigen differs structurally from group O antigen only by the addition of one terminal alpha-linked galactose residue. In this study we aimed to remove the terminal galactose from group B red blood cell to get group O red blood cell.


METHODS
alpha-galactosidase cDNA was cloned by RT-PCR from Catimor coffee beans grown on Hainan Island of China. The vector for alpha-galactosidase cDNA expression was constructed and transferred into Pichia pastoris cells by electroporation. The transgenic cells were cloned by fermentation and the recombinant alpha-galactosidase was purified by ion exchange chromatography. After studying the biochemical characters of alpha-galactosidase, we have used it in converting human erythrocytes from group B to group O.


RESULTS
The purity of recombinant alpha-galactosidase was higher than 96%, which was thought to be suitable for the use of blood conversion. Enzymatically converted human group O red blood cells (ECHORBC) exhibited membrane integrity, metabolic integrity, normal cell deformation and morphology. There were no coagulation between ECHORBC and any group of human blood. The ECHORBC will keep normal structure and function for a period of 21 days at 4 degrees C in monoammoniumphosphate nutrient solution. Experiments with Rhesus monkeys and gibbons showed that transfusion of enzymatically converted erythrocytes was safe.


CONCLUSIONS
ECHORBC can be easily obtained from group B red blood cell by alpha-galactosidase digestion. This study suggests that ECHORBC could be transfused to patients safely and efficiently.
Zhang
Yang-pei
YP
Institute of Blood Transfusion Medicine, Academy of Military Medical Sciences, Beijing 100850, China. zhangyp@nic.bmi.ac.cn
Gong
Feng
F
Bao
Guo-qiang
GQ
Gao
Hong-wei
HW
Ji
Shou-ping
SP
Tan
Ying-xia
YX
Li
Su-bo
SB
Li
Li-li
LL
Wang
Ying-li
YL
Xu
Hua
H
Xu
Li-juan
LJ
Tian
Shu-guang
SG
Zhang
Zhi-xin
ZX
Lü
Qiu-shuang
QS
Qiu
Yan
Y
Bai
Jian-shi
JS
Chen
Ji-ting
JT
D000017:ABO Blood-Group System / Q000145:classification / Q000378:metabolism*; D000818:Animals; D001803:Blood Transfusion; D003001:Cloning, Molecular; D003069:Coffee / Q000201:enzymology; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D008253:Macaca mulatta; D011786:Quality Control; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D000519:alpha-Galactosidase / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology* / Q000633:toxicity
D000017:ABO Blood-Group System; D003069:Coffee; D011994:Recombinant Proteins; D000519:alpha-Galactosidase