Cloning, sequence, and expression of a blood group B active recombinant α-D-Galactosidase from pinto bean (Phaseolus vulgaris)

Melissa O. Davis, D. Jane Hata, Scott A. Johnson, Darin E. Jones, Michael A. Harmata, Maria L. Evans, John C. Walker, Daniel S. Smith

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

A cDNA encoding pinto bean α-D-galactosidase [E.C. 3.2.1.22] was obtained by amplification of cDNA using highly conserved sequences found in eucaryotic α-D-galactosidases. Subsequently a full length Phaseolus cDNA clone was obtained that is 1537 nt long and contains untranslated 5' and 3' sequences. The nucleotide sequence of the cDNA has a high degree of homology with other eucaryotic α-D-galactosidase genes. The recombinant α-D-galactosidase (rGal) was expressed in Escherichia coli and purified by ion exchange and affinity chromatography. Purified rGal was homogeneous by SDS-PAGE and had relative masses of 40.1 and 45.4 kDa under nonreducing and reducing conditions, respectively. The N-terminal sequence of the expressed protein contained the sequence GNGLGQTPPMG corresponding to that deduced from the cDNA sequence. The native molecular weight for rGal was determined to be 32.18 kDa by Sephacryl S-200 chromatography. The specific activity of the rGal was 349 μmoles of PNP-α-D-galactopyranoside hydrolyzed per mg of pure rGal per min. rGal was highly specific for α-D-galactosyl residues and degraded B oligosaccharide. No detectable hemagglutinin or protease activity was present in the preparations. Furthermore, rGal was active against the blood group B antigen on native human erythrocytes in cell suspension assays. The only detectable RBC phenotypic change was loss of the B and P1 epitopes. Recombinant Phaseolus vulgan's α-D-galactosidase may have useful biotechnical applications in the potential mass production of enzymatically converted, universally transfusable type O RBCs. α-D-galactosidase [E.C. 3.2.1.22] has been purified from a variety of procaryotic and eucaryotic species (1,2,3,4). Most α-D-galactosidases have similar low molecular weight substrate specificities, but activity against high molecular weight substrates is variable (5,6). Terminal α-D-galactoside residues are present in glycoproteins and glycolipids (7). Some α-D-galactosidases have activity against α-D-galactosyl residues on cell membrane glycoconjugates. Glycosidases with this property are useful for carbohydrate structural studies and biotechnical applications (8,9). Enzymes free of other glycosidase activities with activity near neutral pH are particularly useful for membrane modification studies on native cells. Complex sugar chains in glycolipids and glycoproteins have often been implicated in the growth and development of eucaryotes (10). In particular, complex sugar chains play an important role in the recognition of self in the immune system (11). Some α-D-galactosidases can modify certain carbohydrate membrane epitopes, thereby modulating the immune response. For example, the blood group B epitope expressed on erythrocytes contains a terminal α-D-galactosyl residue. Individuals lacking this antigen produce naturally occurring complement fixing antibodies to the B epitope (12). Hydrolysis of this terminal saccharide destroys the antigenic activity of the B determinant producing H antigen (blood type O) on erythrocytes (13). Only rare individuals produce clinically significant antibodies to the H antigen, and therefore, type O red blood cells are 'universally' compatible and in great demand (14). Dhar purified α-D-galactosidase isozymes from Phaseolus vulgaris and characterized their activity (15). To our knowledge, our laboratory, in a brief report, is the first to describe the cloning of the gene and the use of recombinant enzyme for seroconverting blood type B to O cells (16). This paper describes the cloning, sequence, expression, purification, and characterization of recombinant α-D-galactosidase. Activity of the recombinant enzyme on the native human erythrocyte blood group B epitope is shown.

Original languageEnglish (US)
Pages (from-to)453-467
Number of pages15
JournalBiochemistry and Molecular Biology International
Volume42
Issue number3
DOIs
StatePublished - Jul 1997
Externally publishedYes

Keywords

  • Recombinant α-D-galactosidase
  • Type O red blood cells

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Genetics

Fingerprint Dive into the research topics of 'Cloning, sequence, and expression of a blood group B active recombinant α-D-Galactosidase from pinto bean (Phaseolus vulgaris)'. Together they form a unique fingerprint.

  • Cite this