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

Shaodong Zhang; Ralph-Olivier Moussodia; Sabine Vértesy; Sabine André; Michael L. Klein; Hans-Joachim Gabius; Virgil Percec

doi:10.1073/pnas.1506220112

Contributed by Michael L. Klein, March 31, 2015 (sent for review March 5, 2015; reviewed by Ricardo Riguera Vega and Peter H. Seeberger)

Significance

Lectins are endogenous sugar receptors involved in diverse physiological and disease-associated processes. The functional consequences of naturally occurring single-nucleotide polymorphism and alternative splicing in lectins has been explored using glycodendrimersomes, a versatile test system with programmable glycan (complex carbohydrates) display. Importantly, glycodendrimersomes facilitate quantitative determination of lectin-mediated cross-linking, a hallmark of their activity. Threshold and kinetic effects measured for a human galectin associated with autoimmune disease document the sensitivity of the test system and highlight its potential as a new and highly versatile supramolecular sensor for biomedical applications.

Abstract

Surface-presented glycans (complex carbohydrates) are docking sites for adhesion/growth-regulatory galectins within cell–cell/matrix interactions. Alteration of the linker length in human galectin-8 and single-site mutation (F19Y) are used herein to illustrate the potential of glycodendrimersomes with programmable glycan displays as a model system to reveal the functional impact of natural sequence variations in trans recognition. Extension of the linker length slightly reduces lectin capacity as agglutinin and slows down aggregate formation at low ligand surface density. The mutant protein is considerably less active as agglutinin and less sensitive to low-level ligand presentation. The present results suggest that mimicking glycan complexity and microdomain occurrence on the glycodendrimersome surface can provide key insights into mechanisms to accomplish natural selectivity and specificity of lectins in structural and topological terms.

The sociology of cells critically depends on selective interactions between cells and the extracellular matrix. Taking advantage of the capacity of carbohydrates to store biological information, glycans are a versatile means to generate the required cell-surface recognition (1 ⇓–3). These molecular signals are docking sites for tissue receptors (lectins), which translate the glycan-based information into adhesion and bridging, often 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 using cells (2, 7, 8). Questions on sugar specificity and density leading to the detection of threshold phenomena were resolved this way for hepatocyte and macrophage adhesion (7, 9). Extending this conceptual approach to cell models, we recently introduced glycodendrimersomes with programmable glycan display and established their bioactivity by testing lectins for their capacity in bridging (trans interactions) (10, 11). Having thus validated design and suitability for application of the test system, it can now be applied to resolve questions on physiological structure–activity correlation for tissue lectins, thereby merging supramolecular chemistry with biomedicine. The present study focuses on two such issues for the class of adhesion/growth regulatory galectins (5, 12, 13).

One mode of structural design for cross-linking is the tandem-repeat display of two-carbohydrate recognition domains (CRDs) connected by a linker peptide shown for galectin-8 (Gal-8) in Fig. 1. As expected, this bivalent protein is a potent mediator of cell adhesion, referred to as a “novel type of matricellular protein” (14 ⇓–16). Physiologically, alternative splicing facilitates its occurrence in two isoforms with different linker lengths, termed 8S and 8L (Fig. 1; for details of amino acid sequences, see Fig. S1) (17, 18). Because the functional impact of this change has not yet been defined, the expression pattern appears to be stochastic in nature (17). Notably, the presence of Gal-8 is conserved in other branches of the phylogenetic tree, with further variation in the linker length, e.g., in the chicken. This ortholog, i.e., Gal-8S (CG-8S), has a linker of only nine amino acids (Fig. 1) (19). Because the linker is susceptible to proteolytic cleavage (20), the two CRDs can be separated from each other and present as free proteins. Equally interesting is the fact that Gal-8 is the first case of a single amino acid polymorphism in the coding region with medical relevance, i.e., associated with rheumatoid arthritis (21). The F19Y substitution causes a displacement of ∼1.5 Å of the positions of N-terminal amino acids 11–15 and a shift of the β-strand F0 in the vicinity of the linker, with impact on thermal stability and enthalpic/entropic contributions to ligand binding (22). Thus, questions on relative bioactivity levels depending on linker length and sugar density on the glycodendrimersomes can be answered on a proof-of-principle basis, as can be done for the natural variant (F19Y). The respective experiments reveal a conspicuous significance of the examined parameter changes and complete switch-off by linker cleavage.

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)

Results and Discussion

Topological Aspects of Ligand Reactivity.

Distinct topological modes of ligand presentation have been assessed by their reactivity to Gal-8S; this can lead to bridging between glycodendrimersomes, which experimentally is followed by an increase in absorbance at 450 nm. To exclude carbohydrate-independent binding, glycodendrimersomes presenting the noncognate d-mannose (Man) on their surface were first tested and found to be inert (Fig. 2). Using the common galectin ligand d-lactose (Lac) as headgroup, three different amphiphiles for glycodendrimersomes were tested comparatively under identical conditions (Fig. 2). Obviously, a spacing between Lac units turned out to be favorable (Fig. 2). The agglutination level with the mono- or bivalent structures was less than for the twin-mixed design. Absence of autoagglutination (by carbohydrate–carbohydrate recognition) and the dependence of the extent of cross-linking from the concentration of Gal-8S underscored the potency of this galectin to establish firm trans contacts (Fig. S2). To address whether cleavage in the linker, a physiological process, will impair this activity, the separate CRDs and their mixture were tested and found to be negative (Fig. 3). Given the documented lack of Man reactivity to Gal-8S, the effect of density could further be examined by systematically varying the ratio of Man/Lac-presenting twin-mixed amphiphilic Janus dendrimers.

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. 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).

Notably, a low density of ligand (i.e., 10%) was not sufficient for agglutination (Fig. 4). Whereas a low-level absorbance change was recorded at 15%, a larger increase occurred at 25%, revealing a marked influence of density, as a threshold. The presence of an equimolar ratio of Man/Lac resulted in nearly plateau level (Fig. 4). Thus, Gal-8S is a strong mediator of interparticle adhesion; its activity is susceptible to modulation by changes in ligand density, as monomer and as constituent of a mixture, here with a steep increase between 15% and 25% Lac presence. The presented data set the stage for the comparative analysis on impact of linker length and single-site mutation.

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).

Effect of Linker Length on Cross-Linking Activity.

The testing of the two natural forms of Gal-8 with longer (Gal-8L) or shorter linker (CG-8S), as shown in Fig. 1, disclosed a minor (Gal-8L) and a clear difference (CG-8C) (Fig. S3). By running agglutination assays, varying the Lac/Man surface ratio, a further difference to Gal-8S was delineated. In the case of Gal-8L, the kinetics of absorbance increase was considerably slowed down, because increases were still seen after the standard period of 1,000 s (Fig. 5A). The plateau level was reached after an eightfold prolonged period, and these values were in the same range irrespective of the Lac/Man ratio (Fig. 5B). The linker-length extension apparently lets the bivalent lectin sense ligand presence at low densities, a time-consuming process, to accomplish trans connections. In contrast, length reduction did not affect the kinetics (Fig. S3). A ratio of Lac/Man at 0.25 was already very effective for a change in absorbance.

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).

Effect of Single-Site (F19Y) Mutation.

To detect an influence of the structural consequences of the presence of the hydroxyl group in position 19, Gal-8S (F19Y) was tested in parallel with the wild-type protein. A significant decrease in extent of agglutination was measured (Fig. S3). In fact, the activity appeared to be ∼50% lower than that of Gal-8S. The sensitivity to Lac presence on the surface of the glycodendrimersome was also lowered, activity increases requiring a higher Lac/Man ratio than for the wild-type protein (Fig. 4). The rather small structural deviation in the natural variant can apparently make its presence felt noticeably when determining cross-linking activity, an indication of a functional relationship to the association with autoimmune diseases.

There is a growing realization of the importance of the specific sugar–protein interactions (1 ⇓–3), which has directed attention to the elucidation of the structures of glycans and lectins and the principles of their interaction. In principle, lectins were assumed to associate with available cognate sites, irrespective of the context. On the cellular level, however, endogenous lectins were found to target distinct counterreceptors, posing the question of the underlying causes for the selectivity (23, 24). For example, suitably glycosylated T-cell glycoproteins CD7, CD43, and CD45; laminin or carcinoembryonic antigen on colon carcinoma cells; and the α₅β₁-integrin (fibronectin receptor) upon tumor suppressor expression in pancreatic carcinoma cells are the main binding partner and the functional counter receptors for Gal-1/Gal-3 (25 ⇓–27). Finding out why, despite the abundance of cell surface β-galactosides, tissue galectins exhibit this preference poses a major challenge. Obviously, model systems with programmable surface properties will be valuable to resolve this conundrum. Equally important is the fact that the in-depth study of structural features of endogenous lectins has delineated physiological variations. If a single-nucleotide polymorphism, one route to such changes on the genetic level that is seen in populations, causes occurrence of an early stop and thus a defective receptor, then the emerging loss of a function is readily interpreted. This case is possible for occurrence of Y238X in the gene for the β-glucan receptor dectin-1 and its association to fungal infections in patients (28, 29). At this early stage of analyzing such variants, case studies on single-nucleotide polymorphisms have either revealed relevance for quaternary structure (for a serum collectin) and processing by proteolysis (for Gal-3) or no effect (for E-selectin) (30 ⇓–32). A readily applicable robust functional assay would considerably help track down implications of the sequence variation. In this study, we apply a highly versatile chemical platform with cell-like features to investigate whether and how the length of the linker in a tandem repeat-type galectin and the natural single-site mutation affect the protein’s activity as mediator of trans contacts.

Following the demonstration of carbohydrate/protein concentration dependence, wild-type Gal-8S is shown to be a potent agglutinin of Lac-presenting glycodendrimersomes. The linker of this lectin, shown by NMR spectroscopy to be rather unstructured when connecting the two CRDs of Gal-1 (33), appears to present the two binding sites of Gal-8S capable for bridging vesicles. When probing the mode of Lac presentation, a hybrid with only one sugar headgroup in a bivalent setting accessible on the glycodendrimersome surface results in higher signals than a denser Lac presentation by the mono- or bivalent analogs. Extension of the linker, mechanistically by alternative splicing, leads to reduced extent of agglutination. Mimicking glycome complexity in simple terms by mixing Lac/Man-presenting building blocks, two further differences were disclosed with linker-length extension: (i) diminution of extent of agglutination at 100% Lac presentation and (ii) retarded absorbance increase at low-level Lac presentation. It is also interesting to note that structural manifestation of the single-nucleotide polymorphism significantly impairs agglutination activity, at full-scale Lac presentation and in titrations of the Lac/Man surface ratio. Thus, Gal-8 being a part of the galectin network and these data revealing different activities, measurement of this galectin’s expression profile should be expanded to the tested parameters of structural variability. Pursuing the network concept, the phenomenologically proposed functional antagonism between CG-8 (Fig. 6) and the prototype CG-1A in formation of precartilage mesenchymal condensations in the developing limb could now be dissected on the mechanistic level (34) to give a further example of arising biomedical applications.

Fig. 6.

Agglutination assays between CG-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).

Conclusions

In general terms, the presented results document proof-of-principle evidence for the sensitivity of the functionalized glycodendrimersomes with programmable glycan display as versatile tool to unravel structure–activity relationships. Importantly, besides varying the ligand density, any chemical alteration of the headgroup, such as 3′-sulfation/sialylation, can be implemented, a route to simulate the physiological glycome complexity. Also, multivalency can be mimicked by local clusters as in branched glycans (35 ⇓⇓⇓⇓⇓⇓–42). Parallel experiments on monolayers to characterize cis interactions plus consideration of presence of cholesterol and sphingolipids for microdomain generation would further broaden the scope, together with consideration of secondary effects (43). In this way, structurally programmed amphiphilic Janus glycodendrimers or mixtures of respective preparations have the enormous potential to shed light on fundamental aspects of structure–activity relationships within the process of how specific carbohydrate–lectin interactions are realized and enhanced. Also, as indicated previously (44), rigorous testing of lectin variants could come up with robust structural improvements for biomedical applications.

Methods

The full-length tandem repeat-type Gal-8 proteins and the N-terminal CRD were obtained by recombinant production and affinity chromatography as described (19, 22). Due to lack of binding to lactosylated Sepharose 4B, Gal-8C was made available by following fusion protein approach: the C-terminal CRD (226–359) was cloned in the pGEX-6P-2 plasmid vector (GE Healthcare) using the BamHI and SalI restriction sites to produce a GST fusion protein. To substitute the amino acids of the BamHI cleavage site, the QuikChange Site-Directed Mutagenesis protocol (Stratagene) was used. Protein was expressed in Escherichia coli cells BL21 (DE3) pLysS (Promega) induced by addition of isopropylβ-d-1-thiogalactopyranoside (100 μM) followed by overnight incubation at 22 °C. Cells were collected by centrifugation and lysed by sonification. The fusion protein was purified using glutathione Sepharose 4B (GE Healthcare) and the GST-tag was removed by proteolysis with human rhinovirus 3C protease followed by separation of Gal-8C from GST and the protease. All proteins were ascertained for purity by 1D and 2D gel electrophoresis and mass-spectrometric analysis (peptic fingerprint) and for activity by hemagglutination, solid-phase, and cell growth assays as described (19, 22).

Glycodendrimersomes self-assembled from amphiphilic Janus dendrimers of different topology for ligand presentation (11, 44) were prepared by the injection method as follows (45, 46). A stock solution was prepared by dissolving the required amount of amphiphilic Janus glycodendrimers in freshly distilled THF. Glycodendrimersomes were then generated by injection of 100 μL of the stock solution into 2 mL Millipore water or PBS or Hepes buffer, followed by 5-s vortexing to give the final concentration of glycodendrimers of 0.5–1 mg × mL⁻¹ in water or in buffer. Any exceptions to this protocol are noted in the respective figure captions.

Agglutination assays of glycodendrimersomes and lectins were monitored in 1.50 mL semimicro disposable cuvettes at 23 °C at single wavelength λ = 450 nm by using a Shimadzu UV-VIS Spectrophotometer UV-1601 with Shimadzu/UV Probe software with kinetic mode. The solution of lectin (100 μL) was injected into 900 μL of solution of glycodendrimersomes. The mixture was shaken for 2 s before recording the absorbance change in time. The agglutination assays were carried out in PBS (1×) buffer. The concentration of lectins and glycodendrimer solutions can be found in the caption of each figure. The solutions of glycodendrimersome were freshly prepared via injection method from THF solution into PBS (1×) buffer. The same buffer was used to bring the lectins into solutions that were used immediately. The solutions of lectin were kept at 0 °C during the agglutination assays.

Acknowledgments

We thank Prof. B. Friday and Dr. G. Notelecs for inspiring discussions. Financial support was provided by National Science Foundation Grants DMR-1066116 and DMR-1120901 (to V.P.) and DMR-1120901 (to M.L.K.), the P. Roy Vagelos Chair at the University of Pennsylvania (to V.P.), and the European Commission Seventh Framework Programme Grant 317297; GLYCOPHARM (to H.-J.G.).

Footnotes

↵¹To whom correspondence may be addressed. Email: mlklein{at}temple.edu or percec{at}sas.upenn.edu.

Author contributions: H.-J.G. and V.P. designed research; S.Z., R.-O.M., S.V., and S.A. performed research; S.Z., S.A., M.L.K., H.-J.G., and V.P. analyzed data; and S.Z., S.A., M.L.K., H.-J.G., and V.P. wrote the paper.
Reviewers: R.R.V., University of Santiago de Compostela; and P.H.S., Max Planck Institute of Colloids and Interfaces.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1506220112/-/DCSupplemental.

Freely available online through the PNAS open access option.

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[1] ↵
Roseman S
(1970) The synthesis of complex carbohydrates by multiglycosyltransferase systems and their potential function in intercellular adhesion. Chem Phys Lipids 5(1):270–297
.
OpenUrl CrossRef PubMed

[2] Roseman S

[3] ↵
Brandley BK,
Schnaar RL
(1986) Cell-surface carbohydrates in cell recognition and response. J Leukoc Biol 40(1):97–111
.
OpenUrl Abstract

[4] Brandley BK,

[5] Schnaar RL

[6] ↵
Gabius HJ
, ed (2009) The Sugar Code. Fundamentals of Glycosciences (Wiley, Weinheim, Germany)
.

[7] Gabius HJ

[8] ↵
Barondes SH
(1984) Soluble lectins: A new class of extracellular proteins. Science 223(4642):1259–1264
.
OpenUrl Abstract/FREE Full Text

[9] Barondes SH

[10] ↵
Lis H,
Sharon N
(1998) Lectins: Carbohydrate-specific proteins that mediate cellular recognition. Chem Rev 98(2):637–674
.
OpenUrl CrossRef PubMed

[11] Lis H,

[12] Sharon N

[13] ↵
André S,
Kaltner H,
Manning JC,
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Unraveling functional significance of natural variations of a human galectin by glycodendrimersomes with programmable glycan surface

Significance

Abstract

Results and Discussion

Topological Aspects of Ligand Reactivity.

Effect of Linker Length on Cross-Linking Activity.

Effect of Single-Site (F19Y) Mutation.

Conclusions

Methods

Acknowledgments

Footnotes

References

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Significance

Abstract

Results and Discussion

Topological Aspects of Ligand Reactivity.

Effect of Linker Length on Cross-Linking Activity.

Effect of Single-Site (F19Y) Mutation.

Conclusions

Methods

Acknowledgments

Footnotes

References

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