Unraveling functional significance of natural variations of a human galectin by glycodendrimersomes with programmable glycan surface

Shaodong Zhanga, Ralph-Olivier Moussodiaa, Sabine Vértesyb, Sabine Andréb, Michael L. Kleinc,1, Hans-Joachim Gabiusb, and Virgil Perceca,1

aRoy & Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323; bInstitute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, 80539 Munich, Germany; and cInstitute of Computational Molecular Science, Temple University, Philadelphia, PA 19122

Contributed by Michael L. Klein, March 31, 2015 (sent for review March 5, 2015; reviewed by Ricardo Riguera Vega and Peter H. Seeberger) Surface-presented glycans (complex carbohydrates) are docking conserved in other branches of the phylogenetic tree, with fur- sites for adhesion/growth-regulatory galectins within cell–cell/ ther variation in the linker length, e.g., in the chicken. This matrix interactions. Alteration of the linker length in human galectin- ortholog, i.e., Gal-8S (CG-8S), has a linker of only nine amino 8 and single-site mutation (F19Y) are used herein to illustrate acids (Fig. 1) (19). Because the linker is susceptible to proteolytic the potential of glycodendrimersomes with programmable glycan cleavage (20), the two CRDs can be separated from each other displays as a model system to reveal the functional impact of nat- and present as free proteins. Equally interesting is the fact that ural sequence variations in trans recognition. Extension of the Gal-8 is the first case of a single amino acid polymorphism in the linker length slightly reduces lectin capacity as agglutinin and coding region with medical relevance, i.e., associated with rheu- slows down aggregate formation at low ligand surface density. matoid arthritis (21). The F19Y substitution causes a displace- The mutant protein is considerably less active as agglutinin and ment of ∼1.5 Å of the positions of N-terminal amino acids 11–15 less sensitive to low-level ligand presentation. The present results and a shift of the β-strand F0 in the vicinity of the linker, with suggest that mimicking glycan complexity and microdomain occur- impact on thermal stability and enthalpic/entropic contributions CHEMISTRY rence on the glycodendrimersome surface can provide key insights to ligand binding (22). Thus, questions on relative bioactivity into mechanisms to accomplish natural selectivity and specificity levels depending on linker length and sugar density on the of lectins in structural and topological terms. glycodendrimersomes can be answered on a proof-of-principle basis, as can be done for the natural variant (F19Y). The re- adhesion | agglutination | glycobiology | membrane mimic | self-assembly spective experiments reveal a conspicuous significance of the examined parameter changes and complete switch-off by linker he sociology of cells critically depends on selective inter- cleavage. Tactions between cells and the extracellular matrix. Taking advantage of the capacity of carbohydrates to store biological Results and Discussion information, glycans are a versatile means to generate the re- Topological Aspects of Ligand Reactivity. Distinct topological modes – quired cell-surface recognition (1 3). These molecular signals of ligand presentation have been assessed by their reactivity to are docking sites for tissue receptors (lectins), which translate Gal-8S; this can lead to bridging between glycodendrimersomes, the glycan-based information into adhesion and bridging, often which experimentally is followed by an increase in absorbance at triggering outside-in signaling (3–6). Key insights into these processes have been obtained by teaming up synthetic chemistry, which delivers a defined custom-made matrix, with bioassays Significance using cells (2, 7, 8). Questions on sugar specificity and density leading to the detection of threshold phenomena were resolved Lectins are endogenous sugar receptors involved in diverse this way for hepatocyte and macrophage adhesion (7, 9). Ex- physiological and disease-associated processes. The functional tending this conceptual approach to cell models, we recently consequences of naturally occurring single-nucleotide poly- introduced glycodendrimersomes with programmable glycan dis- morphism and alternative splicing in lectins has been explored play and established their bioactivity by testing lectins for their using glycodendrimersomes, a versatile test system with pro- capacity in bridging (trans interactions) (10, 11). Having thus grammable glycan (complex carbohydrates) display. Importantly, validated design and suitability for application of the test system, glycodendrimersomes facilitate quantitative determination of it can now be applied to resolve questions on physiological lectin-mediated cross-linking, a hallmark of their activity. Thresh- structure–activity correlation for tissue lectins, thereby merging old and kinetic effects measured for a human galectin associated supramolecular chemistry with biomedicine. The present study with autoimmune disease document the sensitivity of the test focuses on two such issues for the class of adhesion/growth system and highlight its potential as a new and highly versatile regulatory galectins (5, 12, 13). supramolecular sensor for biomedical applications. One mode of structural design for cross-linking is the tandem- Author contributions: H.-J.G. and V.P. designed research; S.Z., R.-O.M., S.V., and S.A. repeat display of two-carbohydrate recognition domains (CRDs) performed research; S.Z., S.A., M.L.K., H.-J.G., and V.P. analyzed data; and S.Z., S.A., connected by a linker peptide shown for galectin-8 (Gal-8) in M.L.K., H.-J.G., and V.P. wrote the paper. Fig. 1. As expected, this bivalent protein is a potent mediator of Reviewers: R.R.V., University of Santiago de Compostela; and P.H.S., Max Planck Institute cell adhesion, referred to as a “novel type of matricellular pro- of Colloids and Interfaces. tein” (14–16). Physiologically, alternative splicing facilitates its The authors declare no conflict of interest. occurrence in two isoforms with different linker lengths, termed Freely available online through the PNAS open access option. 8S and 8L (Fig. 1; for details of amino acid sequences, see Fig. 1To whom correspondence may be addressed. Email: [email protected] or percec@sas. S1) (17, 18). Because the functional impact of this change upenn.edu. has not yet been defined, the expression pattern appears to be This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. stochastic in nature (17). Notably, the presence of Gal-8 is 1073/pnas.1506220112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1506220112 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Fig. 1. The different physiological forms of hGal-8 differing in linker length (hGal-8L and hGal-8S) and presence of a single-site sequence deviation at position 19 (labeled by an asterisk). Schematic of the architectures of the proteins with two different carbohydrate recognition domains (A), their fold (B), and sequences (C). Arrows in B denote site of contact for the ligand (galactose)

450 nm. To exclude carbohydrate-independent binding, glyco- equimolar ratio of Man/Lac resulted in nearly plateau level (Fig. dendrimersomes presenting the noncognate D-mannose (Man) 4). Thus, Gal-8S is a strong mediator of interparticle adhesion; its on their surface were first tested and found to be inert (Fig. 2). activity is susceptible to modulation by changes in ligand density, Using the common galectin ligand D-lactose (Lac) as headgroup, as monomer and as constituent of a mixture, here with a steep three different amphiphiles for glycodendrimersomes were tested increase between 15% and 25% Lac presence. The presented data comparatively under identical conditions (Fig. 2). Obviously, a set the stage for the comparative analysis on impact of linker length spacing between Lac units turned out to be favorable (Fig. 2). and single-site mutation. The agglutination level with the mono- or bivalent structures was less than for the twin-mixed design. Absence of autoagglutina- Effect of Linker Length on Cross-Linking Activity. The testing of the – tion (by carbohydrate carbohydrate recognition) and the de- two natural forms of Gal-8 with longer (Gal-8L) or shorter linker pendence of the extent of cross-linking from the concentration of (CG-8S), as shown in Fig. 1, disclosed a minor (Gal-8L) and a Gal-8S underscored the potency of this galectin to establish firm clear difference (CG-8C) (Fig. S3). By running agglutination trans contacts (Fig. S2). To address whether cleavage in the assays, varying the Lac/Man surface ratio, a further difference to linker, a physiological process, will impair this activity, the sepa- Gal-8S was delineated. In the case of Gal-8L, the kinetics of rate CRDs and their mixture were tested and found to be nega- tive (Fig. 3). Given the documented lack of Man reactivity to Gal- absorbance increase was considerably slowed down, because in- 8S, the effect of density could further be examined by systemat- creases were still seen after the standard period of 1,000 s (Fig. A ically varying the ratio of Man/Lac-presenting twin-mixed am- 5 ). The plateau level was reached after an eightfold prolonged phiphilic Janus dendrimers. period, and these values were in the same range irrespective of Notably, a low density of ligand (i.e., 10%) was not sufficient the Lac/Man ratio (Fig. 5B). The linker-length extension ap- for agglutination (Fig. 4). Whereas a low-level absorbance change parently lets the bivalent lectin sense ligand presence at low was recorded at 15%, a larger increase occurred at 25%, revealing densities, a time-consuming process, to accomplish trans con- a marked influence of density, as a threshold. The presence of an nections. In contrast, length reduction did not affect the kinetics

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1506220112 Zhang et al. Downloaded by guest on September 29, 2021 Fig. 2. Agglutination assays between hGal-8S (2 mg·mL−1 in 100 μL of PBS) and Lac-containing glycodendrimersomes (mmol·L−1 in 900 μL of PBS) of different topological modes to present the binding partner for the lectin.

(Fig. S3). A ratio of Lac/Man at 0.25 was already very effective wild-type protein (Fig. 4). The rather small structural deviation in CHEMISTRY for a change in absorbance. the natural variant can apparently make its presence felt noticeably when determining cross-linking activity, an indication of a func- Effect of Single-Site (F19Y) Mutation. To detect an influence of the tional relationship to the association with autoimmune diseases. structural consequences of the presence of the hydroxyl group in There is a growing realization of the importance of the specific position 19, Gal-8S (F19Y) was tested in parallel with the wild- sugar–protein interactions (1–3), which has directed attention to type protein. A significant decrease in extent of agglutination the elucidation of the structures of glycans and lectins and the was measured (Fig. S3). In fact, the activity appeared to be principles of their interaction. In principle, lectins were assumed ∼50% lower than that of Gal-8S. The sensitivity to Lac presence to associate with available cognate sites, irrespective of the on the surface of the glycodendrimersome was also lowered, context. On the cellular level, however, endogenous lectins were activity increases requiring a higher Lac/Man ratio than for the found to target distinct counterreceptors, posing the question

− Fig. 3. Agglutination assays between twin-mixed Lac-presenting (Lac) glycodendrimersomes (0.2 mmol·L 1 in 900 μL of PBS) and hGal-8 with solely C or N − domain or mixture of the C + N domains (2.0 mg·mL 1 in 100 μL of PBS).

Zhang et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 − Fig. 4. Agglutination assays between hGal-8S (2 mg·mL 1 in 100 μL of PBS) and mixtures of twin-mixed Man-containing (Man) and Lac-containing (Lac) − glycodendrimersomes to produce different surface compositions (Man + Lac = 0.2 mmol·L 1 in 900 μL of PBS).

of the underlying causes for the selectivity (23, 24). For exam- main binding partner and the functional counter receptors for ple, suitably glycosylated T-cell glycoproteins CD7, CD43, and Gal-1/Gal-3 (25–27). Finding out why, despite the abundance CD45; laminin or carcinoembryonic antigen on colon carcinoma of cell surface β-galactosides, tissue galectins exhibit this pref- cells; and the α5β1-integrin (fibronectin receptor) upon tumor erence poses a major challenge. Obviously, model systems with suppressor expression in pancreatic carcinoma cells are the programmable surface properties will be valuable to resolve this

Fig. 5. Agglutination assays between hGal-8L (2 mg·mL−1 in 100 μL of PBS) and mixtures of twin-mixed Man-containing (Man) and Lac-containing (Lac) glycodendrimersomes to produce different surface compositions (Man + Lac = 0.2 mmol·L−1 in 900 μL of PBS) for 1,500 s (A) and 8,000 s (B).

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1506220112 Zhang et al. Downloaded by guest on September 29, 2021 · −1 μ Fig. 6. Agglutination assays between CG-8S (2 mg mL in 100 L of PBS) and mixtures of twin-mixed Man-containing (Man) and Lac-containing (Lac) CHEMISTRY glycodendrimersomes to produce different surface compositions (Man + Lac = 0.2 mmol·L−1 in 900 μL of PBS).

conundrum. Equally important is the fact that the in-depth study of titrations of the Lac/Man surface ratio. Thus, Gal-8 being a part of structural features of endogenous lectins has delineated physio- the galectin network and these data revealing different activities, logical variations. If a single-nucleotide polymorphism, one route measurement of this galectin’s expression profile should be ex- to such changes on the genetic level that is seen in populations, panded to the tested parameters of structural variability. Pursuing causes occurrence of an early stop and thus a defective receptor, the network concept, the phenomenologically proposed functional then the emerging loss of a function is readily interpreted. This antagonism between CG-8 (Fig. 6) and the prototype CG-1A in case is possible for occurrence of Y238X in the gene for the formation of precartilage mesenchymal condensations in the de- β-glucan receptor dectin-1 and its association to fungal infections in veloping limb could now be dissected on the mechanistic level (34) patients (28, 29). At this early stage of analyzing such variants, case to give a further example of arising biomedical applications. studies on single-nucleotide polymorphisms have either revealed relevance for quaternary structure (for a serum collectin) and Conclusions processing by proteolysis (for Gal-3) or no effect (for E-selectin) In general terms, the presented results document proof-of-prin- (30–32). A readily applicable robust functional assay would con- ciple evidence for the sensitivity of the functionalized glycoden- siderably help track down implications of the sequence variation. In drimersomes with programmable glycan display as versatile tool this study, we apply a highly versatile chemical platform with cell- to unravel structure–activity relationships. Importantly, besides like features to investigate whether and how the length of the linker varying the ligand density, any chemical alteration of the head- ′ in a tandem repeat-type galectin and the natural single-site muta- group, such as 3 -sulfation/sialylation, can be implemented, a route tion affect the protein’sactivityasmediatoroftrans contacts. to simulate the physiological glycome complexity. Also, multi- Following the demonstration of carbohydrate/protein concen- valency can be mimicked by local clusters as in branched glycans – cis tration dependence, wild-type Gal-8S is shown to be a potent (35 42). Parallel experiments on monolayers to characterize agglutinin of Lac-presenting glycodendrimersomes. The linker of interactions plus consideration of presence of cholesterol and this lectin, shown by NMR spectroscopy to be rather unstructured sphingolipids for microdomain generation would further broaden when connecting the two CRDs of Gal-1 (33), appears to present the scope, together with consideration of secondary effects (43). In the two binding sites of Gal-8S capable for bridging vesicles. this way, structurally programmed amphiphilic Janus glycoden- When probing the mode of Lac presentation, a hybrid with only drimers or mixtures of respective preparations have the enormous – one sugar headgroup in a bivalent setting accessible on the gly- potential to shed light on fundamental aspects of structure activity – codendrimersome surface results in higher signals than a denser relationships within the process of how specific carbohydrate Lac presentation by the mono- or bivalent analogs. Extension of lectin interactions are realized and enhanced. Also, as indicated the linker, mechanistically by alternative splicing, leads to re- previously (44), rigorous testing of lectin variants could come up duced extent of agglutination. Mimicking glycome complexity in with robust structural improvements for biomedical applications. simple terms by mixing Lac/Man-presenting building blocks, two Methods further differences were disclosed with linker-length extension: (i) diminution of extent of agglutination at 100% Lac pre- The full-length tandem repeat-type Gal-8 proteins and the N-terminal CRD ii were obtained by recombinant production and affinity chromatography as sentation and ( ) retarded absorbance increase at low-level Lac described (19, 22). Due to lack of binding to lactosylated Sepharose 4B, Gal- presentation. It is also interesting to note that structural mani- 8C was made available by following fusion protein approach: the C-terminal festation of the single-nucleotide polymorphism significantly im- CRD (226–359) was cloned in the pGEX-6P-2 plasmid vector (GE Healthcare) pairs agglutination activity, at full-scale Lac presentation and in using the BamHI and SalI restriction sites to produce a GST fusion protein. To

Zhang et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 substitute the amino acids of the BamHI cleavage site, the QuikChange Site- Agglutination assays of glycodendrimersomes and lectins were moni- Directed Mutagenesis protocol (Stratagene) was used. Protein was expressed tored in 1.50 mL semimicro disposable cuvettes at 23 °C at single wavelength in Escherichia coli cells BL21 (DE3) pLysS (Promega) induced by addition of λ = 450 nm by using a Shimadzu UV-VIS Spectrophotometer UV-1601 with isopropylβ-D-1-thiogalactopyranoside (100 μM) followed by overnight in- Shimadzu/UV Probe software with kinetic mode. The solution of lectin cubation at 22 °C. Cells were collected by centrifugation and lysed by soni- (100 μL) was injected into 900 μL of solution of glycodendrimersomes. The fication. The fusion protein was purified using glutathione Sepharose 4B (GE mixture was shaken for 2 s before recording the absorbance change in time. Healthcare) and the GST-tag was removed by proteolysis with human rhi- The agglutination assays were carried out in PBS (1×) buffer. The concen- novirus 3C protease followed by separation of Gal-8C from GST and the tration of lectins and glycodendrimer solutions can be found in the caption protease. All proteins were ascertained for purity by 1D and 2D gel electro- of each figure. The solutions of glycodendrimersome were freshly prepared phoresis and mass-spectrometric analysis (peptic fingerprint) and for activity via injection method from THF solution into PBS (1×)buffer.Thesame by hemagglutination, solid-phase, and cell growth assays as described (19, 22). buffer was used to bring the lectins into solutions that were used im- Glycodendrimersomes self-assembled from amphiphilic Janus dendrimers mediately. The solutions of lectin were kept at 0 °C during the aggluti- of different topology for ligand presentation (11, 44) were prepared by the nation assays. injection method as follows (45, 46). A stock solution was prepared by dis- solving the required amount of amphiphilic Janus glycodendrimers in freshly ACKNOWLEDGMENTS. We thank Prof. B. Friday and Dr. G. Notelecs for distilled THF. Glycodendrimersomes were then generated by injection of μ inspiring discussions. Financial support was provided by National Science 100 L of the stock solution into 2 mL Millipore water or PBS or Hepes Foundation Grants DMR-1066116 and DMR-1120901 (to V.P.) and DMR- buffer, followed by 5-s vortexing to give the final concentration of glyco- − 1120901 (to M.L.K.), the P. Roy Vagelos Chair at the University of Pennsylvania dendrimers of 0.5–1mg× mL 1 in water or in buffer. Any exceptions to this (to V.P.), and the European Commission Seventh Framework Programme protocol are noted in the respective figure captions. Grant 317297; GLYCOPHARM (to H.-J.G.).

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