Biological Applications of a Chimeric Probe for the Assessment of Galectin-3 Ligands

Abstract

(RC) S U M M A R Y b1-6 branching of N-linked oligosaccharides has been correlated with the progression of different cancers. The leukoagglutinins of Phaseolus vulgaris (L-PHA) have been used to study this pattern of glycosylation whose biological significance is incompletely understood. The animal lectin, galectin-3, also binds to structures recognized by L-PHA. To develop a functional tool for the in situ identification of this pattern of glycosylation, human galectin-3 was fused to bacterial alkaline phosphatase (gal3/AP). Gal3/AP recognized both A and B blood group saccharides (B.A) and lactosamine derivatives. Gal3/AP recognition depended at least in part on the N-linked oligosaccharides of different glycoproteins. The presence and distribution of galectin-3 ligands were analyzed in both murine and human normal and tumor samples. Loss of apical expression of galectin-3 ligands was commonly found in carcinomas. Endothelial and inflammatory cells were enriched in galectin-3 ligands as compared with tumor cells; thus, gal3/AP is a suitable tool for studying tumor microenvironments. Comparative analysis of both gal3/AP and L-PHA binding patterns indicated that although similar, these patterns are not identical. The probe developed was useful for several immunoenzymatic assays and will allow the physiological and clinical significance of the expression pattern of galectin-3 ligands to be established. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 55:1015-1026, 2007

Sialylation of 1 Integrins Blocks Cell Adhesion to Galectin-3 and Protects Cells against Galectin-3-induced Apoptosis

“…Similarly, T H 2 cells, which are resistant to gal-1-induced apoptosis, express higher levels of ␣2-6 sialylation than T H 1 cells (which are gal-1 sensitive), and T H 2 gal-1 susceptibility can be restored by the enzymatic removal of ␣2-6, but not ␣2-3, sialic acids (46) In accordance with studies of cell surface glycans, Hirabayashi's work indicated that ␣2-3 sialylation of synthetic ␤-galactosides was tolerated by some galectins, but none of the galectins studied could bind to ␣2-6 sialylated ␤-galactosides (20). A somewhat different finding was reported by de Melo et al (25). This group did observe some binding of gal-3 to synthetic oligosaccharides with ␣2-6 sialylated ␤-galactosides; however, the level of binding was markedly lower than that observed with ␣2-3 sialylated oligosaccharides.…”

“…Precipitated proteins were resolved by on 7% SDS-PAGE and transferred to PVDF membranes. Membranes were exposed to alkaline phosphatase (AP)-conjugated gal-3 and developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (25).…”

O-glycan sialylation alters galectin-3 subcellular localization and decreases chemotherapy sensitivity in gastric cancer

“…Tissue fragments were immunostained with anti-sialyl-Tn antibody ((TKH2 antibody [75]), anti-galectin-3 (M3/38, ATCC TIB166) or incubated with hrGal-3/AP [76]. The tissue were classified using a 0-to-3 scale: 0 for 0–5% positive tumor cells, 1 for 6–50% positive tumor cells, 2 for >50% positive tumor cells.…”

Genetically modified proteins: functional improvement and chimeragenesis

“…The synthesis of hybrid proteins by domain fusion or the ligation of chemically or biologically synthesized peptides improves apparently a functional unit utility and is generally used for biological probing approach such as biosensors and bioassays, molecular imaging, targeted cancer or antiinflammatory therapy, regulated drug delivery. 41,42,43,44,78,79,80,81 Hybrid proteins may possess two or more functional activities realized by different modes of action indicating that the molecules of hybrid act as several distinct pharmacophores. Many pharmaceutically or industrially relevant enzymatic processes can also utilize hybrid enzymes with multiple domains.…”

“…Such molecules with a narrow specificity, which are able to bind selectively to carbohydrates, have also a key importance in the development of research related with mechanism of cancerogenesis or inflammation at the molecular level as well as for designing drugs targeted to a relevant molecule. 44,78,79,81 To develop a less complex chimeric fusion protein then the native human lectin MBL with similar ligand recognition and enhanced effector functions, the MBL carbohydrate recognition domain and L-ficolin (L-FCN) collagenous domain were fused and applied to reduce more significantly infection by wild type Ebola virus (Table 1). 79 A method for producing a highly carbohydrate-specific bifunctional hybrid holothurian lectin MBL-AJ, whose expression and purification were monitored by the alkaline phosphatase CmAP activity, has been developed for improving the enzyme-linked MBL-AJ-based assay (ELLA) for cervical cancer diagnosis (Table 1).…”