Multivalent scaffolds induce galectin-3 aggregation into nanoparticles

Summary Galectin-3 meditates cell surface glycoprotein clustering, cross linking, and lattice formation. In cancer biology, galectin-3 has been reported to play a role in aggregation processes that lead to tumor embolization and survival. Here, we show that lactose-functionalized dendrimers interact with galectin-3 in a multivalent fashion to form aggregates. The glycodendrimer–galectin aggregates were characterized by dynamic light scattering and fluorescence microscopy methodologies and were found to be discrete particles that increased in size as the dendrimer generation was increased. These results show that nucleated aggregation of galectin-3 can be regulated by the nucleating polymer and provide insights that improve the general understanding of the binding and function of sugar-binding proteins.


Expression and purification of galectin-3
Following previously published methods [1], an 80 mL culture of E. coli transformed with pGEX-6p-galectin-3 was grown overnight (37 °C, 250 rpm) in YT/AMP media then transferred to 1 L YT/AMP media and grown until the OD 600 previously reported [1,2]. Purity of the galectin-3 product was verified by running a 15% SDS-PAGE gel on protein samples. Figure S1 shows the purified galectin-3 removed from the GS beads, the two additional GS bead washes and combined, dialyzed product (1.3 mg/mL). A single band is observed between 25 and 30 kDa, as expected for galectin-3 ( Figure S1).

Dynamic light scattering theory, sample preparation and results
Theory: The autocorrelation function (g(t)) is fit assuming particles are spherical and can be generally defined by the equation below: where G() is the distribution function, t refers to the time delay between measurements and  is the product of the translational diffusion coefficient (D) and the scattering efficiency of the molecule (q). Mathematically,  is defined as: where k B is Boltzmann's constant, T is temperature,  is the solvent viscosity (0.89 cP), d is the particle diameter, n is the index of refraction (1.33),  is the is S6 the scattering angle (90°) and  is the laser wavelength (633 nm). The method of cumulants uses a Taylor series expansion to express the exponential given in equation 1 as a polynomial, then fits the correlation function to this polynomial.
The first coefficient, or moment (), is equal to  and used to determine the effective diameter. The second term is proportional to the variance of the weighted diffusion coefficient distribution (G()) and can be used to calculate a reduced second moment ( 2 ') that is defined as the relative variance in this paper (in the referenced paper and conventional DLS it is referred to as the polydispersity).
Further detail is given by Koppel [3]. Representative fitted data is shown in Figure S7 for compounds 4 and 5.
Solution preparation: Prior to mixing, all solutions were filtered with 0.22 mm Millipore Millex ® GP filter units to eliminate dust interferences. Galectin-3 concentration was determined by measuring the absorbance at 280 nm using  1% =6.1 ml/mg [4,5]. Glycodendrimers 2-5 were prepared by dissolving the lyophilized powder in filtered Millipore water to a final concentration of 100 and 10 M. Mannose-functionalized, generation 4 dendrimer was synthesized and characterized as described previously [6]. Varying amounts of glycodendrimer (0.14, 3.3 and 11.5 M final concentration) were added to a galectin-3 solution (final concentration 31 M) and incubated for 1 h at rt. The reported data is the S7 average of triplicate measurements on 1-6 samples. High relative variances were obtained for samples where the particle size was too small or too few particles were present to be detected (kcps < 50). High relative variances (>1) are characteristic of highly polydisperse samples and could not be fit well using the method of cumulants. This was observed at the highest concentration of dendrimer (no galectin-3 added).

Fluorescence microscopy image calibration and sample preparation
Protocols for preparation of samples for fluorescence microscopy are provided below. 0.9956 a Y represents particle diameter, and x is the number of pixels in the particle perimeter as quantitated by Pixcavator. b 190 nm standard could not be detected and was not included in the fit. was dissolved in 20 L DMSO and immediately added to 1 mL of the above galectin-3 solution. The reaction was rotated for 1 h at rt and was purified via dialysis (Spectrumlabs, MWCO 3500). The degree of labeling was determined according to the labeling protocol provided [7]. The solution of purified, Alexa-488 labeled galectin-3 was filtered and diluted to a final concentration of 31 M.

S10
Glycodendrimer-galectin samples were prepared as described for DLS (0.14 M glycodendrimer, 1 h incubation at rt). To verify addition of dye molecule did not affect aggregation, particle sizes were verified by DLS prior to viewing. A 5 L aliquot was mounted on a glass slide and covered with a coverslip. Average aggregate diameter and polydispersity (Table S6) were determined from data converted from pixels to diameter using the calibration curves.