Fishing for lectins from diverse sequence libraries by yeast surface display – An exploratory study

Glycobiology vol. 18 no. 2 pp. 137–144, 2008 doi:10.1093/glycob/cwm131 Advance Access publication on December 17, 2007 Fishing for lectins from diverse sequence libraries by yeast surface display – An exploratory study Stefan Ryckaert2,3,4 , Nico Callewaert1,5,6 , Pieter P. Jacobs3,4 , Sylviane Dewaele3,4 , Isabelle Dewerte5,6 , and Roland Contreras3,4 3 Department for Molecular Biomedical Research, Unit for Fundamental and Applied Molecular Biology, VIB; 4 Department of Molecular Biology, Ghent University; 5 Department for Molecular Biomedical Research, Unit for Molecular Glycobiology, VIB; and 6 Department of Biochemistry, Physiology and Microbiology, Ghent University, B-9052 Ghent, Belgium Received on May 9, 2007; revised on November 28, 2007; accepted on November 30, 2007 The establishment of a robust technology platform for the expression cloning of carbohydrate-binding proteins remains a key challenge in glycomics. Here we explore the utility of using yeast surface display (YSD) technology in the interaction-based lectin cloning from complete cDNA libraries. This should pave the way for more detailed studies of protein–carbohydrate interactions. To evaluate the performance of this system, lectins representing three different subfamilies (galectins, siglecs, and C-type lectins) were successfully displayed on the surface of Saccharomyces cerevisiae and Pichia pastoris as a-agglutinin and/or αagglutinin fusions. The predicted carbohydrate-binding activity could be detected for three out of five lectins tested (galectin-1, galectin-3, and siaoadhesin). For galectin-4 and E-selectin, no specific carbohydrate-binding activity could be detected. We also demonstrate that proteins with carbohydrate affinity can be specifically isolated from complex metazoan cDNA libraries through multiple rounds of FACS sorting, employing multivalent, fluorescent-labeled polyacrylamide-based glycoconjugates. Keywords: expression cloning/lectin/Pichia pastoris/Saccharomyces cerevisiae/yeast surface display Introduction Lectins are carbohydrate-binding proteins involved in numerous biological processes (Gabius et al. 2002). Moreover, they are essential tools in the emerging field of glycomics, allowing specific detection of target analytes, hence fulfilling functions analogous to that of antibodies in proteomics. Phage display represents a major advance in the antibodyengineering field, but is of limited use in the interaction cloning of proteins with carbohydrate-binding activity (Yamamoto et al. 1999; Ravn et al. 2004). First, because protein-carbohydrate interactions depend largely on multivalency (Ravn et al. 2004), 1 To whom correspondence should be addressed: Tel: +32-9-331-3617; Fax: +32-9-331-3502; e-mail: 2 Both first authors contributed equally. the monovalent display format, which is applied in most cases, may not be optimal for dealing with carbohydrate antigens. Second, a prokaryotic expression host has an unpredictable but frequently strong expression bias against many eukaryotic proteins. To complement the lectin cloning tools offered by phage display with a eukaryotic counterpart, we rely on yeast surface display (YSD) (Schreuder et al. 1993; Boder and Wittrup 1997). This technology was originally developed to enhance secretion efficiency, stability, and affinity of proteins (Boder et al. 2000; Holler et al. 2000; Shusta et al. 2000), but has not been used to identify proteins with carbohydrate affinity. The multivalent YSD format intrinsically mimics the natural multivalent presentation of lectins on cells (Figure 1A), and could largely overcome the technical problems observed with other (prokaryotic) protein display platforms that have been used for lectin cloning (Ravn et al. 2004). We build on the successes of the YSD format that were obtained in the antibody-engineering field. Here it proved successful in both the isolation of scFv’s from large nonimmune libraries (Feldhaus et al. 2003) and the maturation of these scFv’s toward higher affinities. Currently, the yeast display method has yielded the highest affinity (48 fM) for any antibody (Boder et al. 2000). We foresee similar application for YSD in the cloning and engineering of lectins. Results and discussion To evaluate the possibility of using YSD to study proteincarbohydrate interactions, we displayed on the surface of Saccharomyces cerevisiae three galectins from different subfamilies and possessing different natural oligomerization properties (galectin-1: dimeric, galectin-3 and -4: monomeric) (system outline: Figure 1A). Galectins are soluble β-galactosidebinding lectins (Barondes et al. 1994) that are expressed as surface receptors when introduced in the YSD system. Likewise, when expressing cDNA libraries, all soluble receptors become cell surface localized. Hereby, the AGA1-AGA2 complex links these proteins to the cell wall. Using fluorescenceactivated cell sorting (FACS) analysis with a fluorescein isothiocyanate (FITC)-labeled anti-V5-tag mAb, we determined that a large fraction of cells displayed high levels of the fullsize proteins (mean fluorescence intensity is up to 100-fold higher than that of nonexpressing cells) (Figure 2A–C). Subsequently, by FACS analysis we demonstrated the interaction between the surface-displayed galectins and multivalent, LacNAc-containing, fluorescent-labeled polyacrylamide-based glycoconjugates (LacNAc-PAA-FITC) (Galanina et al. 1998). We demonstrated that galectin-1 and -3 interact specifically with LacNAc-PAA-FITC (Figure 2F and G). However, only a few cells displaying galectin-4 showed binding to this conjugate (Figure 2H), which concurs with the notion that galectin-4 has c The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail:
137 S Ryckaert et al. Fig. 1. System outline and general strategy for interaction-based lectin cloning using yeast surface display. (A) Multivalent display of fusion proteins on the surface of S. cerevisiae mimics natural cell-surface lectin presentation and allows avidity-stabilized interaction with multivalent probes of the type sugar-PAA-FITC. Schematic representation of a- and α-agglutinin-based YSD systems. a-agglutinin: the gene of interest is fused to AGA2. We adapted the display vector to permit fusion of cDNA libraries in all three reading frames. Upon induction, the Aga2p-fusion protein forms a disulfide-mediated heterodimer with Aga1p, and is targeted to the outer glucan layers of the cell wall, where it becomes covalently attached. α-agglutinin: the gene of interest is fused to the SAG1 part encoding the last 320 amino acids and containing the GPI anchor attachment site. Cell surface anchorage is like that of a-agglutinin fusions. In S. cerevisiae, both systems are placed under the control of the GAL1 promoter, whereas in P. pastoris, the AOX1 promoter is used. For both systems, immunofluorescent labeling of a surface displayed protein is shown using FITC-labeled anti- (...truncated)