Jack Goldstein

Cloning and functional expression of a cDNA encoding coffee bean α-galactosidase

Abstract Purified coffee bean α-galactosidase (αGal) has been used for removing terminal α-galactose residues from the glycoconjugates at the red cell surface, in studies of blood group conversion. Here, we report the isolation and sequence of the full-length clone for coffee bean αGal by using the polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) techniques. The cDNA clone (1.4 kb) contains a single open reading frame which encodes a protein of 378 amino acids (aa). Its authenticity is confirmed by perfect alignment of aa sequences obtained from purified coffee bean αGal, and by immune reaction with the antibody raised against the enzyme. Furthermore, the protein produced in insect cells shows enzymatic activity towards a synthetic αGal substrate, p -nitro-phenyl- α -galactopyranoside.

Conversion of ABO Blood Groups

Progress is being made toward producing erythrocytes similar to native group O cells from A and B donors. Blood group A and B antigens are known to be carbohydrate in nature. The antigenicity is conferred by different terminal sugars. Removal of these sugars by specific exoglycosidases produces the H antigenic structure that is the determinant found on group O cells. Conditions have been developed that allow for the removal of these antigens while maintaining the metabolic and membrane viability of the red cell. Following successful autologous transfusions with gibbons, appropriately treated human group B erythrocytes are now being used in preclinical studies with normal healthy human volunteers. Results indicate that such treated cells have normal in vivo life spans in both group A and O recipients. However, the latter exhibit a transitory increase in anti-B antibody titer, the significance of which is not yet known. Similar exoglycosidic treatment of group A erythrocytes does not remove all serologically detectable A antigens. This is probably due to the presence of a second internal A antigenic site adjacent to the usual terminal A antigen on some structures. Several approaches are being used to address this problem, including projected treatment with a combination of pertinent exoglycosidases and a search for an endoglycosidase that will cleave the polysaccharide chain at a site upstream from both A antigens.

Transfusions to group O subjects of 2 units of red cells enzymatically converted from group B to group O

Background: It has previously been shown that full‐unit (200 mL) transfusions of red cells (RBCs) enzymatically converted from group B to group O by treatment with α‐galactosidase (ECO RBCs) are both safe and efficacious for normal group O or A subjects.

Role of A and B blood group antigens in the expression of adhesive activity of von Willebrand factor

ABO (H) blood group antigens are covalently linked to the oligosaccharide side‐chains of von Willebrand factor (VWF). In this study, we investigated the role of the A and B antigens in the expression of VWF adhesive activity. VWF of type A, B or O was purified from fresh frozen plasma. Presence of A or B antigen on the VWF was confirmed by enzyme‐linked immunosorbent assay (ELISA) and by immunoblotting with monoclonal anti‐A or anti‐B. The A or B antigen was also detected in the 48/52‐kDa fragment of the respective VWF after trypsin digestion. Removal of A antigen with α‐N‐acetylgalactosaminidase or B antigen with α‐galactosidase did not affect its multimer size or antigenic level, but decreased the ristocetin cofactor (RCoF) activity of the respective VWF by 33–39% (P < 0·01–0·002). Removal of A or B antigen from VWF did not affect the binding of the VWF to immobilized type III collagen. A and B antigens were not detected in platelet VWF. These results indicate that AB structures play a role in platelet aggregating activity of VWF.

Further Evidence for the Presence of A Antigen on Group B Erythrocytes through the Use of Specific Exoglycosidases

Abstract. Certain antibodies have shown the ability to detect small amounts of A antigenic structures on certain group B cells. These rare cells that reverse group as B, are designated here as B(A) cells. Among the anti‐A antibodies capable of detecting these cells are MHO4 (an IgM murine monoclonal antibody) and polyclonal anti‐A derived from blood group O donors. The latter (anti‐A, B) have been adsorbed exhaustively with normal B cells, to deplete the serum of antibodies to group B antigen. The cell specificity detected by these antibodies can be removed only by an α‐Nacetylgalactosaminidase (A‐zyme) but not by an α‐galactosidase (B‐zyme). Inhibition studies show that these reactions can be inhibited by A secretor saliva and cannot be inhibited by B secretor saliva. Moreover, papain treatment of normal group B cells not previously agglutinable with these antibodies, now causes these cells to become reactive, and this specificity, too, is removed only by A‐zyme. These results suggest that low levels of blood group A antigen are being recognized by these antibodies and that these structures can exist not only on B(A) cells but on all group B erythrocytes.

Identification of tyrosine 108 in coffee bean α-galactosidase as an essential residue for the enzyme activity

The cDNA for coffee bean alpha-galactosidase (alpha-Gal) has been cloned and expressed in a baculovirus expression system. An early study of coconut alpha-Gal by chemical modification suggested that one tyrosine residue is at or near the active site. In order to identify such a critical residue, we replaced two tyrosine residues (positions 108 and 158) with phenylalanine by site-directed mutagenesis. The mutated DNA strands, as well as the wild-type ones, were subcloned into pVL vector and transformed into Sf9 insect cells for intracellular expression. The replacement of Tyr-158 with phenylalanine resulted in a mutant alpha-Gal (Y158F) which retained approx. 88% of the activity of wild-type enzyme. However, the substitution of Tyr-108 by phenylalanine (Y108F) almost abolished the enzymatic activity (1.8% of wild-type activity). The Vmax/Km value for the mutant Y108F was 0.027, which was over a 1000-fold lower than that of wild-type alpha-Gal. Our data suggest that Tyr-108 is critical for the enzymatic activity of alpha-Gal.

Distribution of 5-S RNA in erythroid cells

Abstract 1. 1. The distribution and proportion of 5-S RNA in erythroid cell ribosomes were studied by Sephadex-gel filtration and polyacrylamide-gel electrophoresis in an attempt to find some correlation between 5-S RNA content of ribosomes and cell maturation. 2. 2. Ribosomes were isolated from phenylhydrazine-induced reticulocytes. Both free and membrane-bound ribosomes were examined. These ribosomes all contained 5-S RNA as a constant proportion of the total rRNA. Ribosomes isolated from normal circulating erythroid cells and reticulocytes of varying ages also contained the same proportion of 5-S RNA. 3. 3. It is concluded that one cannot distinguish between ribosomes from cells of varying age and presumably of different functional capabilities on the basis of gross 5-S RNA content.

α-d-Galactosidase immobilized on a soluble polymer

α-d-Galactosidase (α-d-galactoside galactohydrolase, EC 3.2.1.22) from green coffee beans has been immobilized by attachment to cyanogen bromide-activated Dextran T-70. Since this represents the first reported example of the preparation of a water-soluble derivative of an enzyme showing substrate inhibition, the kinetic properties, thermal stability and pH optima were investigated and compared with those of the free enzyme. The Km, Ks, Ki, Vmax, optimum substrate concentration and optimum pH were all lower than those of free enzyme. The enzyme conjugate showed greater resistance than the free enzyme to thermal inactivation. These data, although obtained with the synthetic substrate 4-nitrophenyl-α-d-galactoside, suggest some advantages in using the enzyme conjugate for the removal of terminal α-d-galactopyranosyl groups from the erythrocyte cell surface.

Cloning, functional expression and purification of endo-β-galactosidase from Flavobacterium keratolyticus

Endo-beta-galactosidase (EC 3.2.1.103) is an enzyme that hydrolyzes internal endo-beta-galactosyl linkages in keratan sulfate, and glycoconjugates with N-acetyl-lactosamine repeating units. Here, we report the cloning of the endo-beta-galactosidase-encoding gene from Flavobacterium keratolyticus, its expression in Escherichia coli and the purification of the enzyme. The enzyme was purified over 15000-fold to apparent homogeneity. The purified endo-beta-galactosidase consists of a single band of about 43kDa on SDS-PAGE and has a specific activity of 148micro/mg. Based on peptide sequences derived from the purified enzyme, a full-length clone encoding endo-beta-galactosidase was isolated from F. keratolyticus genomic DNA. The gene contains a single open reading frame coding for a protein of 422 amino acid residues with a putative N-terminal signal peptide. Its authenticity was confirmed by colinearity of deduced amino acid sequences with the peptide sequences, and synthesis of enzyme in E. coli.

Separation of H‐Activity from Isolated Glycoproteins of Human O Erythrocyte Membranes

Abstract. H activity can be dissociated from human erythrocyte membrane glycoproteins prepared from OMM or ONN erythrocytes utilizing Phaseolus vulgaris phytohemagglutinin coupled to Sepharose 4B or by precipitation of the glycoproteins with soluble phytohemagglutinin. The three glycoprotein components of the erythrocyte membrane, as resolved by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, bind specifically to phytohemagglutinin. Although such glycoproteins no longer exhibit H activity, they do retain M or N activity. H activity can be recovered in a fraction that does not bind to phytohemagglutinin coupled to Sepharose or to soluble phytohemagglutinin, contains no detectable glycoprotein components, and binds to Ulex lectin coupled to Sepharose.