Compositions and methods for modifying blood cell carbohydrates
View Patent ↗This invention relates to enzymatic removal of type A and B antigens from blood group A, B, and AB reactive cells in blood products, and thereby converting these to non-A and non-B reactive cells. The invention further relates to using unique αN-acetylgalactosaminidases and α-galactosidases with superior kinetic properties for removing the immunodominant monosaccharides of the blood group A and B antigens and improved performance in enzymatic conversion of red blood cells. The preferred unique α-N-acetylgalactosaminidases and α-galactosidases exhibit the following characteristics: (i) exclusive, preferred or no less than 10% substrate specificity for the type A and B branched polysaccharide structures relative to measurable activity with simple mono- and disaccharide structures and aglycon derivatives hereof; (ii) optimal performance at neutral pH with blood group oligosaccharides and in enzymatic conversion of cells; and (iii) a favorable kinetic constant K m with mono- and oligosaccharide substrates. The conversion methods of the invention use significantly lower amounts of recombinant glycosidase enzymes than previous and result in complete sero-conversion of all blood group A and B red cells.
1. A method for removing immunodominant α-galactose monosaccharides from blood group B or AB reactive cells in a blood product, said method comprising the steps of:
(a) contacting said blood product with an α-galactosidase enzyme under pH conditions ranging from 6 to 8, thereby cleaving the immunodominant α-galactose monosaccharides from the B or AB reactive cells,
(b) removing said enzyme and the immunodominant α-galactose monosaccharides from said blood product, and
(c) assaying the enzyme contacted blood product for serotype B or AB reactivity, wherein a lack of B or AB serotype indicates removal of the immunodominant monosaccharides,
wherein said α-galactosidase enzyme has the following characteristics:
(i) active in red blood cell conversion at neutral pH, and
(ii) isolated and purified from a non-recombinant strain of Streptomyces griseoplanus , wherein said α-galactosidase enzyme is isolatable by a method comprising the steps of:
(1) fermentation culturing of an α-galactosidase producing Streptomyces griseoplanus strain;
(2) disrupting the cultured Streptomyces griseoplanus strain of step (1);
(3) isolating an α-galactosidase-containing supernatant fraction from the disrupted Streptomyces griseoplanus strain of step (2) by centrifugation;
(4) treating the α-galactosidase-containing supernatant fraction of step (3) with ammonium sulfate to yield a 20 to 60 percent ammonium sulfate fraction enriched in the α-galactosidase;
(5) purifying the α-galactosidase from the 20 to 60 percent ammonium sulfate fraction of step (4) by anion exchange chromatography followed by cation exchange chromatography to yield an ion exchange purified α-galactosidase; and
(6) fractionating the ion exchange purified α-galactosidase of step (5) by size-exclusion chromatography to yield a purified α-galactosidase which elutes with a molecular weight in the range of 40-80 kD.
2. A method for converting type B or AB erythrocytes to non-B erythrocytes, said method comprising the steps of:
(a) contacting said blood product with an α-galactosidase enzyme, under pH conditions ranging from 6 to 8, thereby cleaving the immunodominant α-galactose monosaccharides from the B or AB reactive cells,
(b) removing said enzyme and the immunodominant α-galactose monosaccharides from said blood product, and
(c) assaying the enzyme contacted blood product for serotype B or AB reactivity wherein a lack of B or AB serotype indicates removal of the immunodominant monosaccharides,
wherein said α-galactosidase enzyme has the following characteristics:
(i) active in red blood cell conversion at neutral pH, and
(ii) isolated and purified from a non-recombinant strain of Streptomyces griseoplanus , wherein said α-galactosidase enzyme is isolatable by a method comprising the steps of:
(1) fermentation culturing of an α-galactosidase producing Streptomyces griseoplanus strain;
(2) disrupting the cultured Streptomyces griseoplanus strain of step (1);
(3) isolating an α-galactosidase-containing supernatant fraction from the disrupted Streptomyces griseoplanus strain of step (2) by centrifugation;
(4) treating the α-galactosidase-containing supernatant fraction of step (3) with ammonium sulfate to yield a 20 to 60 percent ammonium sulfate fraction enriched in the α-galactosidase;
(5) purifying the α-galactosidase from the 20 to 60 percent ammonium sulfate fraction of step (4) by anion exchange chromatography followed by cation exchange chromatography to yield an ion exchange purified α-galactosidase; and
(6) fractionating the ion exchange purified α-galactosidase of step (5) by size-exclusion chromatography to yield a purified α-galactosidase which elutes with a molecular weight in the range of 40-80 kD.
3. The method of claim 1 , wherein the strain of Streptomyces griseoplanus is S. griseoplanus , ATCC Deposit No. PTA-4077.
4. The method of claim 2 , wherein the strain of Streptomyces griseoplanus is S. griseoplanus , ATCC Deposit No. PTA-4077.