Thayer White

Bacterial glycosidases for the production of universal red blood cells.

Enzymatic removal of blood group ABO antigens to develop universal red blood cells (RBCs) was a pioneering vision originally proposed more than 25 years ago. Although the feasibility of this approach was demonstrated in clinical trials for group B RBCs, a major obstacle in translating this technology to clinical practice has been the lack of efficient glycosidase enzymes. Here we report two bacterial glycosidase gene families that provide enzymes capable of efficient removal of A and B antigens at neutral pH with low consumption of recombinant enzymes. The crystal structure of a member of the α-N-acetylgalactosaminidase family reveals an unusual catalytic mechanism involving NAD+. The enzymatic conversion processes we describe hold promise for achieving the goal of producing universal RBCs, which would improve the blood supply while enhancing the safety of clinical transfusions.

Lysophosphatidic acid acyltransferase-β: a novel target for induction of tumour cell apoptosis

Phosphatidic acid (PA) is a component of cellular membranes that is also a mediator of certain cell signalling functions associated with oncogenesis. These include ras/raf/Erk and Akt/mTor [1-3]. The authors have investigated whether it would be possible to interrupt these known oncogenic pathways through the inhibition of lysophosphatidic acid acyltransferase (LPAAT), an enzyme that catalyses the biosynthesis of PA. The expression and activity of the LPAAT-β isoform are elevated in human tumours, and the respective gene displays transforming capacity when overexpressed in vitro. Inhibition by either genetic means or by isoform-specific small molecules results in a block to cell signalling pathways and apoptosis. Furthermore, the small-molecule inhibitors of LPAAT-β are not cytotoxic to a number of normal cell types, including primary bone marrow progenitors, indicating a differential dependence of tumour cells on LPAAT-β function. These discoveries indicate that LPAAT-β represents a potential novel cancer therapy target.

Identification of a GH110 subfamily of alpha 1,3-galactosidases - Novel enzymes for removal of the alpha 3Gal xenotransplantation antigen

In search of alpha-galactosidases with improved kinetic properties for removal of the immunodominant alpha1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of alpha-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454-464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Galalpha1-3(Fucalpha1-2)Gal, whereas linear oligosaccharides terminated by alpha1,3-linked galactose such as the immunodominant xenotransplantation epitope Galalpha1-3Galbeta1-4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific alpha1,3-galactosidases that act equally well on both branched blood group B and linear alpha1,3Gal structures. We determined by one-dimensional (1)H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known alpha-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant alpha3Gal xenotransplantation epitope.In search of α-galactosidases with improved kinetic properties for removal of the immunodominant α1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of α-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454–464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Galα1–3(Fucα1–2)Gal, whereas linear oligosaccharides terminated by α1,3-linked galactose such as the immunodominant xenotransplantation epitope Galα1–3Galβ1–4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific α1,3-galactosidases that act equally well on both branched blood group B and linear α1,3Gal structures. We determined by one-dimensional 1H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known α-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant α3Gal xenotransplantation epitope.

A new procedure for establishing functional monoclonal antibodies capable of inhibiting E- or P-selectin-dependent cell adhesion

Employing a new procedure, we established many monoclonal antibodies (mAbs) which inhibit E- or P-selectin-dependent cell adhesion. One of these mAbs is capable of staining selectin in paraffin-embedded histological sections. The procedure is based on immunization of BALB/c mice with irradiated mouse myeloma NS-1 cells (syngeneic HAT-sensitive fusion partner cells) transfected with cDNA encoding human E- or P-selectin. Resulting NS-1 transfectant cells permanently express human E- or P-selectin as immunogen. The mAbs are useful for detecting selectins by flow cytometric and immunohistological methods, and for inhibiting selectin-dependent adhesion in experimental models. In contrast, the majority of anti-selectin mAbs previously established do not have these capabilities. Abbreviations: Ig, immunoglobulin; mAb, monoclonal antibody

Characterization of two spliced variants of human phosphatidic acid phosphatase cDNAs that are differentially expressed in normal and tumor cells.

Phosphatidic acid (PA) and diacylglycerol (DG) are lipids involved in signal transduction and in structural membrane lipid biosynthesis in cells. Phosphatidic acid phosphatase (PAP) (EC 3.1.3.4) catalyzes the conversion of PA to DG. This enzyme is known to exist in at least two isoforms, one of which (PAP1) is presumed to be cytosolic and membrane associated and the other (PAP2), an integral membrane protein (Brindley and Waggoner, 1996). PAP1 has been implicated in glycerolipid biosynthesis; whereas PAP2 is suggested to play a role in signal transduction. In addition to dephosphorylating PA, purified PAP2 from rat liver has also been found to dephosphorylate lysophosphatidic acid (LPA), ceramide-1-phosphate (C-l-P), sphingosine-1-phosphate (S-l-P) (Waggoner et al., 1996), and DG pyrophosphate (Carman, 1997), suggesting the involvement of PAP2 in modulating the balance of a broad spectrum of bioactive lipids generated during cell signaling. A murine PAP2 cDNA (Kai et al., 1996) and two human cDNAs isoforms, hPAP-2a and hPAP-2b, coding for PAP2 have been identified (Kai et al., 1997). Homology search of the GenBank database using the murine PAP2 sequence probe has also enabled us to isolate several putative human isoenzymes. This paper reports the isolation and expression of two alternatively spliced variants termed PAP2-αl and PAP2-α2 encoded by the human PAP-2a gene and two other isoforms of PAP2 derived from separate genes.