Robert Polakowski

A Single Point Mutation Reverses the Donor Specificity of Human Blood Group B-synthesizing Galactosyltransferase

Blood group A and B antigens are carbohydrate structures that are synthesized by glycosyltransferase enzymes. The final step in B antigen synthesis is carried out by an α1–3 galactosyltransferase (GTB) that transfers galactose from UDP-Gal to type 1 or type 2, αFuc1→2βGal-R (H)-terminating acceptors. Similarly the A antigen is produced by an α1–3N-acetylgalactosaminyltransferase that transfersN-acetylgalactosamine from UDP-GalNAc to H-acceptors. Human α1–3 N-acetylgalactosaminyltransferase and GTB are highly homologous enzymes differing in only four of 354 amino acids (R176G, G235S, L266M, and G268A). Single crystal x-ray diffraction studies have shown that the latter two of these amino acids are responsible for the difference in donor specificity, while the other residues have roles in acceptor binding and turnover. Recently a novelcis-AB allele was discovered that produced A and B cell surface structures. It had codons corresponding to GTB with a single point mutation that replaced the conserved amino acid proline 234 with serine. Active enzyme expressed from a synthetic gene corresponding to GTB with a P234S mutation shows a dramatic and complete reversal of donor specificity. Although this enzyme contains all four “critical” amino acids associated with the production of blood group B antigen, it preferentially utilizes the blood group A donor UDP-GalNAc and shows only marginal transfer of UDP-Gal. The crystal structure of the mutant reveals the basis for the shift in donor specificity.

ABO(H) blood group A and B glycosyltransferases recognize substrate via specific conformational changes.

The final step in the enzymatic synthesis of the ABO(H) blood group A and B antigens is catalyzed by two closely related glycosyltransferases, an α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and an α-(1→3)-galactosyltransferase (GTB). Of their 354 amino acid residues, GTA and GTB differ by only four “critical” residues. High resolution structures for GTB and the GTA/GTB chimeric enzymes GTB/G176R and GTB/G176R/G235S bound to a panel of donor and acceptor analog substrates reveal “open,” “semi-closed,” and “closed” conformations as the enzymes go from the unliganded to the liganded states. In the open form the internal polypeptide loop (amino acid residues 177-195) adjacent to the active site in the unliganded or H antigen-bound enzymes is composed of two α-helices spanning Arg180-Met186 and Arg188-Asp194, respectively. The semi-closed and closed forms of the enzymes are generated by binding of UDP or of UDP and H antigen analogs, respectively, and show that these helices merge to form a single distorted helical structure with alternating α-310-α character that partially occludes the active site. The closed form is distinguished from the semi-closed form by the ordering of the final nine C-terminal residues through the formation of hydrogen bonds to both UDP and H antigen analogs. The semi-closed forms for various mutants generally show significantly more disorder than the open forms, whereas the closed forms display little or no disorder depending strongly on the identity of residue 176. Finally, the use of synthetic analogs reveals how H antigen acceptor binding can be critical in stabilizing the closed conformation. These structures demonstrate a delicately balanced substrate recognition mechanism and give insight on critical aspects of donor and acceptor specificity, on the order of substrate binding, and on the requirements for catalysis.

Labeling effects on the isoelectric point of green fluorescent protein

We studied the effects of fluorescent labeling on the isoelectric points (pI values) of proteins using capillary isoelectric focusing with laser-induced fluorescence detection (cIEF-LIF). Specifically, we labeled green fluorescent protein (GFP) from the jellyfish Aequorea victoria with the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). cIEF-LIF was used to monitor the native fluorescence of GFP and showed pI changes in GFPs FQ-labeled products. Multiple labeling of GFP with FQ produced a series of products with pI values shifted towards a low pH. We verified cIEF-LIF results with traditional slab gel IEF. Our cIEF-LIF technique can routinely detect 10(-11) M of FQ-labeled protein, whereas traditional slab gel IEF with silver stain detection gives detection limits of 10(-7) M in the same samples.

The Donor Cross-Specificity of Human Blood Group Aand B-Synthesizing Glycosyltransferases

The human ABO blood group is the most clinically significant blood group system and is certainly the best known. At its foundation are the genes coding for two glycosyltransferases which catalyze the transfer of distinct monosaccharides to a pre-existing acceptor structure (H) to form carbohydrate determinants distinguishing the A, B, and O(H) blood groups. Since the genes are thought to be co-dominant, the existence of the rare AB blood group has mainly been ascribed to the inheritance of both enzymes, a so-called trans-AB genotype. As the techniques of blood grouping have become more advanced however, the unusual genotypes cis-AB and B(A) have raised some interest, and the enzymes expressed by them are providing great opportunities for functionstructure studies on this highly important system.