Towards defining the role of glycans as hardware in information storage and transfer: Basic principles, experimental approaches and recent progress

The term `code' in biological information transfer appears to be tightly and hitherto exclusively connected with the genetic code based on nucleotides and translated into functional activities via proteins. However, the recent appreciation of the enormous coding capacity of oligosaccharide chains of natural glycoconjugates has spurred to give heed to a new concept: versatile glycan assembly by the genetically encoded glycosyltransferases endows cells with a probably not yet fully catalogued array of meaningful messages. Enciphered by sugar receptors such as endogenous lectins the information of code words established by a series of covalently linked monosaccharides as fetters for example guides correct intra- and intercellular routing of glycoproteins, modulates cell proliferation or migration and mediates cell adhesion. Evidently, the elucidation of the structural frameworks and the recognition strategies within the operation of the sugar code poses a fascinating conundrum. The far-reaching impact of this recognition mode on the level of cells, tissues and organs has fueled vigorous investigations to probe the subtleties of protein-carbohydrate interactions. This review presents information on the necessarily concerted approach using X-ray crystallography, molecular modeling, nuclear magnetic resonance spectroscopy, thermodynamic analysis and engineered ligands and receptors. This part of the treatise is flanked by exemplarily chosen insights made possible by these techniques. Copyright (C) 2001 S. Karger AG, Basel

Prognostic significance of endogenous adhesion/growth-regulatory lectins in lung cancer

Objective: To determine the expression of endogenous adhesion/growth-regulatory lectins and their binding sites using labeled tissue lectins as well as the binding profile of hyaluronic acid as an approach to define new prognostic markers. Methods: Sections of paraffin-embedded histological material of 481 lungs from lung tumor patients following radical lung excision processed by a routine immunohistochemical method (avidin-biotin labeling, DAB chromogen). Specific antibodies against galectins-1 and - 3 and the heparin-binding lectin were tested. Staining by labeled galectins and hyaluronic acid was similarly visualized by a routine protocol. After semiquantitative assessment of staining, the results were compared with the pT and pN stages and the histological type. Survival was calculated by univariate and multivariate methods. Results: Binding of galectin-1 and its expression tended to increase, whereas the parameters for galectin-3 decreased in advanced pT and pN stages at a statistically significant level. The number of positive cases was considerably smaller among the cases with small cell lung cancer than in the group with non-small-cell lung cancer, among which adenocarcinomas figured prominently with the exception of galectin-1 expression. Kaplan-Meier computations revealed that the survival rate of patients with galectin-3-binding or galectin-1-expressing tumors was significantly poorer than that of the negative cases. In the multivariate calculations of survival lymph node metastases ( p < 0.0001), histological type ( p = 0.003), galectin-3-binding capacity ( p = 0.01), galectin-3 expression ( p = 0.03) and pT status ( p = 0.003) proved to be independent prognostic factors, not correlated with the pN stage. Conclusion: The expression and the capacity to bind the adhesion/growth regulatory galectin-3 is defined as an unfavorable prognostic factor not correlated with the pTN stage. Copyright (C) 2005 S. Karger AG, Basel

Horizontal gene transfer contributed to the evolution of extracellular surface structures

The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem β-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment

Crystallization of a human galectin-3 variant with two ordered segments in the shortened N-terminal tail

Among members of the family of adhesion/growth-regulatory galectins, galectin-3 (Gal-3) bears a unique modular architecture. A N-terminal tail (NT) consisting of the N-terminal segment (NTS) and nine collagen-like repeats is linked to the canonical lectin domain. In contrast to bivalent protoand tandem-repeat-type galectins, Gal-3 is monomeric in solution, capable to self-associate in the presence of bi-to multivalent ligands, and the NTS is involved in cellular compartmentalization. Since no crystallographic information on Gal-3 beyond the lectin domain is available, we used a shortened variant with NTS and repeats VII-IX. This protein crystallized as tetramers with contacts between the lectin domains. The region from Tyr101 (in repeat IX) to Leu114 (in the CRD) formed a hairpin. The NTS extends the canonical beta-sheet of F1-F5 strands with two new beta-strands on the F face. Together, crystallographic and SAXS data reveal a mode of intramolecular structure building involving the highly flexible Gal-3's NT

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

Exploring functional pairing between surface glycoconjugates and human galectins using programmable glycodendrimersomes

Precise translation of glycan-encoded information into cellular activity depends critically on highly specific functional pairing between glycans and their human lectin counter receptors. Sulfoglycolipids, such as sulfatides, are important glycolipid components of the biological membranes found in the nervous and immune systems. The optimal molecular and spatial design aspects of sulfated and nonsulfated glycans with high specificity for lectin-mediated bridging are unknown. To elucidate how different molecular and spatial aspects combine to ensure the high specificity of lectin-mediated bridging, a bottom-up toolbox is devised. To this end, negatively surface-charged glycodendrimersomes (GDSs), of different nanoscale dimensions, containing sulfo-lactose groups are self-assembled in buffer from a synthetic sulfatide mimic: Janus glycodendrimer (JGD) containing a 3′-O-sulfo-lactose headgroup. Also prepared for comparative analysis are GDSs with nonsulfated lactose, a common epitope of human membranes. These self-assembled GDSs are employed in aggregation assays with 15 galectins, comprising disease-related human galectins, and other natural and engineered variants from four families, having homodimeric, heterodimeric, and chimera architectures. There are pronounced differences in aggregation capacity between human homodimeric and heterodimeric galectins, and also with respect to their responsiveness to the charge of carbohydrate-derived ligand. Assays reveal strong differential impact of ligand surface charge and density, as well as lectin concentration and structure, on the extent of surface cross-linking. These findings demonstrate how synthetic JGD-headgroup tailoring teamed with protein engineering and network assays can help explain how molecular matchmaking operates in the cellular context of glycan and lectin complexity

Human Galectins Induce Conversion of Dermal Fibroblasts into Myofibroblasts and Production of Extracellular Matrix: Potential Application in Tissue Engineering and Wound Repair

Members of the galectin family of endogenous lectins are potent adhesion/growth-regulatory effectors. Their multi-functionality opens possibilities for their use in bioapplications. We studied whether human galectins induce the conversion of human dermal fibroblasts into myofibroblasts (MFBs) and the production of a bioactive extracellular matrix scaffold is suitable for cell culture. Testing a panel of galectins of all three subgroups, including natural and engineered variants, we detected activity for the proto-type galectin-1 and galectin-7, the chimera-type galectin-3 and the tandem-repeat-type galectin-4. The activity of galectin-1 required the integrity of the carbohydrate recognition domain. It was independent of the presence of TGF-beta 1, but it yielded an additive effect. The resulting MFBs, relevant, for example, for tumor progression, generated a matrix scaffold rich in fibronectin and galectin-1 that supported keratinocyte culture without feeder cells. Of note, keratinocytes cultured on this substratum presented a stem-like cell phenotype with small size and keratin-19 expression. In vivo in rats, galectin-1 had a positive effect on skin wound closure 21 days after surgery. In conclusion, we describe the differential potential of certain human galectins to induce the conversion of dermal fibroblasts into MFBs and the generation of a bioactive cell culture substratum. Copyright (C) 2011 S. Karger AG, Base