Fractionation, Purification, And Structural Analysis Of PH-modified Citrus Pectin And Its Interaction With Galectin-3

Galectin-3, a β-galactose-binding protein, is associated with the metastasis of primary cancer cells. Given its apparent central role in cancer, galectin-3 has become a molecular target in the development of anti-cancer therapeutics. The pH-modified citrus pectin(MCP) is produced from citrus pectin via pH. Recent studies demonstrated that MCP has shown strong anti-tumor activities both in vivo and in vitro. Galectin-3 seems to be an important target of MCP. It has been proposed that MCP interacts directly with galectin-3 to inhibit various cellular processes in cancer, e.g. galectin-3-mediated tumor cells proliferation and metastasis. Up till now, it is unclear how many bioactive pectins are in MCP, and what their structure-activity relationships are. Therefore, we focused on fractionating MCP, analyzing the chemical structures of the resulting fractions, and assessing structure-activity relationships.Firstly, to study the bioactive components of MCP, five assays were developed and compared to detect the interactions between galectin-3 and inhibitors. The results show that,(1) surface plasmon resonance(SPR), bio-layer interferometry(BLI), competitive fluorescence-linked immunosorbent assays(cFLISA), and galectin-3-mediated hemagglutination(G3H) are all suitable to assess pectin galectin-3 binding;(2) FP and G3 H assays are both suitable to assess small inhibitors galectin-3 binding, and depend greatly on the selection of the probe;(3) although KD, IC50, and MIC values do differ to some extent, data derived from these methods are generally in agreement;(4) conclusions obtained by different assays could be more reliable.Secondly, we fractionated MCP into four fractions(MCP-1,-2,-3, and-4) and then sub-fractions of type-I rhamnogalacturonan-rich pectins(RG-Ⅰ: MCP-1a, MCP-2a, MCP-3Sa and MCP-4a) and homogalacturonan-rich pectins(HG: MCP-2b, MCP-3Sb, MCP-3P, MCP-4b) by a combination of DEAE-cellulose ion-exchange and Sepharose CL-6B gel permeation chromatographies. RG-Ⅰ sub-fractions contain(1"4)-linked β-D-galactan and(1"5)-linked α-L-arabinans or type-I arabinogalactan side-chains. We used galectin-3-mediated hemagglutination assay to define which MCP fraction best promotes anti-galectin-3 activity. Our results demonstrate that MCP-2 was the most inhibitory(ten-fold more potent than parent MCP), and RG-Ⅰ-rich pectins with(1"4)-linked β-D-galactan side-chains were more active than the other RG-Ⅰ-rich and HG-rich pectins. In addition, our results suggested that RG-Ⅰand HG-type pectins may act synergistically, because their activities were significantly reduced upon fractionation.Thirdly, we investigated MCP-fractioned RG-Ⅰ-rich pectin MCP-2a and HG-rich pectins MCP-2b and MCP-3P by using several assays(G3H, cFLISA, BLI and galectin-3-mediated T-cell apoptosis), and found that in combination(MCP-2a : MCP-2b = 1 : 5, MCP-2a : MCP-3P = 1 : 8, m/m) they could synergistically antagonize galectin-3-mediated hemagglutination and T-cell apoptosis; BLI results also showed that combination of MCP-2a with HG fractions significantly decreased the KD values, consistent with the synergistic inhibitory effects found in the cell assays. The mechanisms of the synergistic effects for RG-Ⅰ and HG were studied by several assays. The results showed that,(1) HG could interact with galectin-3, and HG with the molecular weight about 5 kDa exhibited the strongest inhibition effect;(2) fragments(HG) larger than 5 kDa(1 to 5) clearly demonstrated synergistic effects in combination with MCP-2a;(3) HG significantly decreased intrinsic fluorescence intensity of galectin-3, indicating that the environment around one or more of Trp residues in galectin-3 is modified by HG binding event;(4) MCP-2a-binding was sensitive to lactose treatment, while HG-binding was not, suggesting that MCP-2a and HG exhibited different binding modes;(5) the particle size of the mixture was different from either MCP-2a or HG pectin alone, suggesting that MCP-2a and HG pectin act as complexes or microaggregates with both MCP-2a and HG being attached to galectin-3.In summary, we fractionated MCP into several pectin fractions which showed different strength inhibition effects on galectin-3, and MCP-2 was the strongest one among them; RG-Ⅰ and HG could synergistically antagonize galectin-3, and the synergistical mechanism was studied. All the results contributes to better understanding of how complex pectins interact with galectin-3 and to the development of galectin-3-targeted herbal medicines.