Dr. Steve Evans

 

Research of Dr. Stephen V. Evans
PhD British Columbia

Biosynthesis and Recognition of Oligosaccharides

The human blood group A and B antigens are trisaccharides derived by the addition of single sugar to the human blood group O disaccharide by specific glycosyltransferase (GT) enzymes, where GTA transfers GalNAc from UDP-GalNAc to the O antigen and GTB transfers Gal. This means that the A and B antigens differ only in the substitution of an hydroxyl group for an acetamido, yet in a mis-matched blood transfusion or organ donation this small change can result in death. This makes the human A and B blood group antigens a paradigm for specificity in oligosaccharide biosynthesis and immune recognition. Our direct objective is to understand mechanisms of specificity and to exploit them to create new carbohydrate molecules. In the case of antibodies we seek to design better therapeutic and diagnostic agents, and in the case of enzymes we seek ultimately to create molecules of altered regio- and stereo-selectivity to produce robust chemosynthetic tools.

Mammalian glycoconjugates are largely composed from nine monosaccharides, and it is estimated that hundreds of GTs utilize these nine sugars in various combinations to synthesize the observed arrays of natural structures. GTs act in a highly regio- and stereospecific manner, which permits the assembly of complex specific oligosaccharides in the absence of a template. Despite the fact the GTs are one of the largest groups of functionally related enzymes, relatively little is known about structure-function relationships and the recognition of the substrates at the molecular level. With a few exceptions, there is little amino acid sequence similarity between most GTs, even if they use the same oligosaccharide acceptor or donor substrates.
We have solved the structures of the human blood group ABO(H) A and B glycosyltransferases to high resolution, which we are using as a model system to explore GT mechanisms and specificity.

Recognition of Carbohydrate Structures by Germline Antibodies

The specific recognition of oligosaccharides by immunoglobulins is a critical aspect of immune surveillance for microbial pathogenesis and diseased tissue. The initial antibody response to a new immunogen arises largely from antibodies constructed from germline gene segments, which are subject to natural selection based on pathogen prevalence. This is a critical consideration for antibodies specific for carbohydrate antigens, which generally do not undergo class switching and affinity maturation. We have selected from a large library of monoclonal antibody clones several near-germline antibodies of high sequence homology that recognize different carbohydrate epitopes based on 3-deoxy-D-manno-2-octulosonic acid (Kdo), which is found in bacterial pathogens. We have crystallized Fabs from some of these antibodies unliganded and in the presence of several different Kdo epitopes, which requires the antibody to utilize the same binding-site amino acid residues in different modalities in the various complexes. These structures demonstrate how the murine genome can code for a general antibody-binding site that serves to recognize a range of different epitopes based on one bacterial sugar.

Scientific Visualization of Macromolecules

My group is continuing to develop 80,000 line application SETOR for computer-aided scientific visualization of protein and nucleic acid macromolecules. SETOR is becoming increasingly popular because of its highly developed user-interface and rendering capability. This is demonstrated by its citation by many groups in their publications describing new macromolecular structures, including papers in high-impact journals like Science and Nature. SETOR is distributed on a non-profit basis and is in use in more than 500 crystallographic, NMR, and modelling groups in North and South America, Europe, Australia and Asia. SETOR was named in the January 23, 1995 issue of The Scientist as a "Hot New Paper" in scientific visualization for 1994, based on the highest number of citations for a paper in this field less than 2 years old, according to data in the Science Citation Index. SETOR continues to enjoy wide use, and a new version is about to be released which will allow complete visualization of protein-nucleic acid complexes. In 2002 the original paper describing SETOR was named by Thompson-ISI as one of the top 15 papers cited world wide over the past 10 years at the interface of Computer Science and Medicine.

Selected Recent Publications

Evans, D. W., Müller-Loennies, S., Brooks, C.L., Brade, L., Kosma, P., Brade, H. & Evans, S.V. (2011) Structural insights into parallel strategies for germline antibody recognition of LPS from Chlamydia, Glycobiology, 21, 1049-1059.

Blackler, R. J., Müller-Loennies, S., Brooks, C.L., Evans, D.W., Brade, L., Kosma, P., Brade, H. & Evans, S.V. (2011) A common NH53K single point mutation in the combining site of antibodies raised against Chlamydial LPS glycoconjugates significantly increases avidity, Biochemistry, 50, 3357-3368.

Schuman, B., Fisher, S.Z., Kovalevsky, A., Borisova, S.N., Palcic, M.M., Coates, L., Langan, P. & Evans, S.V. (2011) Preliminary joint neutron time-of-flight and X-ray crystallographic study of human ABO(H) blood group A glycosyltransferase, Acta Crystallogr. F., 67, 258-262.

Schuman, B., Persson, M., Landry, R.C., Polakowski, R., Weadge, J.T., Seto, N.O.L., Borisova, S.N., Palcic, M.M. & Evans, S.V. (2010) Cysteine-to-Serine Mutants Dramatically Reorder the Active Site of Human ABO(H) Blood Group B Glycosyltransferase without Affecting Activity: Structural Insights into Cooperative Substrate Binding, J. Mol. Biol. 402, 399-411.

Gerstenbruch, S., Brooks, C.L., Kosma, P., Brade, L., Mackenzie, C.R., Evans, S.V., Brade, H. & Müller-Loennies, S. (2010) Analysis of cross-reactive and specific anti-carbohydrate antibodies against lipopolysaccharide from Chlamydophila psittaci, Glycobiology 20, 461-472.

Brooks, C.L., Schietinger, A., Borisova, S.N., Kufer, P., Okon, M., Hirama, T., MacKenzie, C.R., Wang, L.X., Schreiber, H. & Evans, S.V. (2010) Antibody recognition of a unique tumor-specific glycopeptide antigen, Proc. Natl Acad. Sci. USA 107, 10056-10061.

Brooks, C.L., Müller-Loennies, S., Borisova, S.N., Brade, L., Kosma, P., Hirama, T., Mackenzie, C.R., Brade, H. & Evans, S.V. (2010) Antibodies raised against chlamydial lipopolysaccharide antigens reveal convergence ingermline gene usage and differential epitope recognition, Biochemistry 49, 570-581.

Brooks, C.L., Blackler, R.J., Sixta, G., Kosma, P., Müller-Loennies, S., Brade, L., Hirama, T., MacKenzie, C.R., Brade, H. & Evans, S.V. (2010) The role of CDR H3 in antibody recognition of a synthetic analog of a lipopolysaccharide antigen, Glycobiology 20, 138-147.

Brooks, C.L., Müller-Loennies, S., Brade, L., Kosma, P., Hirama, T., MacKenzie, C.R., Brade, H. & Evans, S.V. (2008) Exploration of specificity in germline monoclonal antibody recognition of a range of natural and synthetic epitopes, J. Mol. Biol. 377, 450-468.

Brooks, C.L., Blackler, R.J., Gerstenbruch, S., Kosma, P., Müller-Loennies, S., Brade, H. & Evans, S.V. (2008) Pseudo-symmetry and twinning in crystals of homologous antibody Fv fragments, Acta Crystallogr. D Biol. Crystallogr. 64, 1250-1258.

Alfaro, J.A., Zheng, R.B., Persson, M., Letts, J.A., Polakowski, R., Bai, Y., Borisova, S.N., Seto, N.O.L., Lowary, T.L., Palcic, M.M. & Evans, S.V.(2008) ABO(H) blood group A and B glycosyltransferases recognize substrate via specific conformational changes, J. Biol. Chem. 283, 10097-10108.

Persson, M., Letts, J.A., Hosseini-Maaf, B., Borisova, S.N., Palcic, M.M., Evans, S.V. & Olsson, M.L. (2007) Structural effects of naturally occurring human blood group B galactosyltransferase mutations adjacent to the DXD motif, J. Biol. Chem. 282, 9564-9570.

Letts, J.A., Persson, M., Schuman, B., Borisova, S.N., Palcic, M.M. & Evans, S.V.(2007) The effect of heavy atoms on the conformation of the active-site polypeptide loop in human ABO(H) blood-group glycosyltransferase B, Acta Crystallogr. D Biol. Crystallogr. 63, 860-865.

Hosseini-Maaf, B., Letts, J.A., Persson, M., Smart, E., LePennec, P.Y., Hustinx, H., Zhao, Z.H., Palcic, M.M., Evans, S.V., Chester, M. A.& Olsson, M. L. (2007) Structural basis for red cell phenotypic changes in newly identified, naturally occurring subgroup mutants of the human blood group B glycosyltransferase, Transfusion 47, 864-875.