Influence of protein (human galectin-3) design on aspects of lectin activity

The concept of biomedical significance of the functional pairing between tissue lectins and their glycoconjugate counterreceptors has reached the mainstream of research on the flow of biological information. A major challenge now is to identify the principles of structure–activity relationships that underlie specificity of recognition and the ensuing post-binding processes. Toward this end, we focus on a distinct feature on the side of the lectin, i.e. its architecture to present the carbohydrate recognition domain (CRD). Working with a multifunctional human lectin, i.e. galectin-3, as model, its CRD is used in protein engineering to build variants with different modular assembly. Hereby, it becomes possible to compare activity features of the natural design, i.e. CRD attached to an N-terminal tail, with those of homo- and heterodimers and the tail-free protein. Thermodynamics of binding disaccharides proved full activity of all proteins at very similar affinity. The following glycan array testing revealed maintained preferential contact formation with N-acetyllactosamine oligomers and histo-blood group ABH epitopes irrespective of variant design. The study of carbohydrate-inhibitable binding of the test panel disclosed up to qualitative cell-type-dependent differences in sections of fixed murine epididymis and especially jejunum. By probing topological aspects of binding, the susceptibility to inhibition by a tetravalent glycocluster was markedly different for the wild-type vs the homodimeric variant proteins. The results teach the salient lesson that protein design matters: the type of CRD presentation can have a profound bearing on whether basically suited oligosaccharides, which for example tested positively in an array, will become binding partners in situ. When lectin-glycoconjugate aggregates (lattices) are formed, their structural organization will depend on this parameter. Further testing (ga)lectin variants will thus be instrumental (i) to define the full range of impact of altering protein assembly and (ii) to explain why certain types of design have been favored during the course of evolution, besides opening biomedical perspectives for potential applications of the novel galectin forms

Galectin-glycan interaction: guideline for monitoring by 77Se NMR spectroscopy and solvent (H2O/D2O) impact on binding

10 p.-6 fig.-3 tb.Functional pairing between cellular glyco-con-ju-gates and tissue lectins like galectins has wide (patho)physio-logical significance. Their study is facilitated by non-hydrolyzable deri-va-tives of the natural O-glycans, such as S- and Se-glycosides. The latter en-able extensive analyses by specific 77 Se NMR spectroscopy, but still remain under-explored. By the example of seleno--digalac-to-side (SeDG) and the human galectins 1 and 3, we evaluate diverse 77 Se NMR detec-tion methods and pro-pose selec-tive 1 H, 77 Se hetero-nuclear Hartmann-Hahn transfer for efficient use in com-peti-tive NMR screening against a seleno-glycoside spy ligand. By fluores-cence aniso-tropy, circular dichroism, and isothermal titra-tion calori-metry (ITC) we show that affinity and thermodynamics of SeDG bind-ing by galectins are similar to thiodi-galac-toside (TDG) and N-acetyl-lactos-amine (LacNAc), con-firm-ing that Se substitution has no major impact. ITC data in D 2 O vs. H 2 O are similar for TDG and LacNAc binding by both galectins, but a solvent effect, indicating solvent rearrangement at the binding site, is hinted for SeDG and clearly observed for LacNAc dimers with extend-ed chain length.We are much indebted to L. Szilágyi (University of Debrecen,Hungary) for kindly providing selenoglycosides for this study. We are grateful for inspiring discussions to Drs. B. Friday, A. Leddoz and A. W. L. Nose as well as for generous funding from the Spanish Ministry of Economy and Competitiveness (grant BFU2016-77835-R; to A.R.), the Department of Industry, Tourism and Trade of the Government of the Autonomous Community of the Basque Country (Elkartek BG2019; to T.D.), the Severo Ochoa Excellence Accreditation from MCIU (SEV-2016-0644; to T.D.), the Science Foundation of Ireland (grants 13/IA/1959 and 16/RC/3889;to S.O.), National Institute of Health (NIH) grant CA242351 to M.Cudic, and the COST Action CA 18103 (InnoGly; to H.-J.G.).Peer reviewe

Influence of protein (human galectin-3) design on aspects of lectin activity

The concept of biomedical significance of the functional pairing between tissue lectins and their glycoconjugate counterreceptors has reached the mainstream of research on the flow of biological information. A major challenge now is to identify the principles of structure-activity relationships that underlie specificity of recognition and the ensuing post-binding processes. Toward this end, we focus on a distinct feature on the side of the lectin, i.e. its architecture to present the carbohydrate recognition domain (CRD). Working with a multifunctional human lectin, i.e. galectin-3, as model, its CRD is used in protein engineering to build variants with different modular assembly. Hereby, it becomes possible to compare activity features of the natural design, i.e. CRD attached to an N-terminal tail, with those of homo- and heterodimers and the tail-free protein. Thermodynamics of binding disaccharides proved full activity of all proteins at very similar affinity. The following glycan array testing revealed maintained preferential contact formation with N-acetyllactosamine oligomers and histo-blood group ABH epitopes irrespective of variant design. The study of carbohydrate-inhibitable binding of the test panel disclosed up to qualitative cell-type-dependent differences in sections of fixed murine epididymis and especially jejunum. By probing topological aspects of binding, the susceptibility to inhibition by a tetravalent glycocluster was markedly different for the wild-type vs the homodimeric variant proteins. The results teach the salient lesson that protein design matters: the type of CRD presentation can have a profound bearing on whether basically suited oligosaccharides, which for example tested positively in an array, will become binding partners in situ. When lectin-glycoconjugate aggregates (lattices) are formed, their structural organization will depend on this parameter. Further testing (ga)lectin variants will thus be instrumental (i) to define the full range of impact of altering protein assembly and (ii) to explain why certain types of design have been favored during the course of evolution, besides opening biomedical perspectives for potential applications of the novel galectin forms.Open Access funding provided by Projekt DEAL. We gratefully acknowledge inspiring discussions with Drs. B. Friday, A. Leddoz and A. W. L. Nose, valuable input during the review process and generous fnancial support by an NIH Grant (No. CA242351; to M.C.)

Imitating evolution’s tinkering by protein engineering reveals extension of human galectin-7 activity

Wild-type lectins have distinct types of modular design. As a step to explain the physiological importance of their special status, hypothesis-driven protein engineering is used to generate variants. Concerning adhesion/growth-regulatory galectins, non-covalently associated homodimers are commonly encountered in vertebrates. The homodimeric galectin-7 (Gal-7) is a multifunctional context-dependent modulator. Since the possibility of conversion from the homodimer to hybrids with other galectin domains, i.e. from Gal-1 and Gal-3, has recently been discovered, we designed Gal-7-based constructs, i.e. stable (covalently linked) homo- and heterodimers. They were produced and purified by affinity chromatography, and the sugar-binding activity of each lectin unit proven by calorimetry. Inspection of profiles of binding of labeled galectins to an array-like platform with various cell types, i.e. sections of murine epididymis and jejunum, and impact on neuroblastoma cell proliferation revealed no major difference between natural and artificial (stable) homodimers. When analyzing heterodimers, acquisition of altered properties was seen. Remarkably, binding properties and activity as effector can depend on the order of arrangement of lectin domains (from N- to C-termini) and on the linker length. After dissociation of the homodimer, the Gal-7 domain can build new functionally active hybrids with other partners. This study provides a clear direction for research on defining the full range of Gal-7 functionality and offers the perspective of testing applications for engineered heterodimers